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Apoptotic effect of tannic acid on fatty acid synthase over-expressed human breast cancer cells.
Nie, Fangyuan; Liang, Yan; Jiang, Bing; Li, Xiabing; Xun, Hang; He, Wei; Lau, Hay Tong; Ma, Xiaofeng
Breast cancer is one of the most common cancers and is the second leading cause of cancer mortality in women worldwide. Novel therapies and chemo-therapeutic drugs are urgently needed to be developed for the treatment of breast cancer. Increasing evidence suggests that fatty acid synthase (FAS) plays an important role in breast cancer, for the expression of FAS is significantly higher in human breast cancer cells than in normal cells. Tannic acid (TA), a natural polyphenol, possesses significant biological functions, including bacteriostasis, hemostasis, and anti-oxidant. Our previous studies demonstrated that TA is a natural FAS inhibitor whose inhibitory activity is stronger than that of classical FAS inhibitors, such as C75 and cerulenin. This study further assessed the effect and therapeutic potential of TA on FAS over-expressed breast cancer cells, and as a result, TA had been proven to possess the functions of inhibiting intracellular FAS activity, down-regulating FAS expression in human breast cancer MDA-MB-231 and MCF-7 cells, and inducing cancer cell apoptosis. Since high-expressed FAS is recognized as a molecular marker for breast cancer and plays an important role in cancer prognosis, these findings suggest that TA is a potential drug candidate for treatment of breast cancer. PMID:26349913
PIK3CA mutations and EGFR overexpression predict for lithium sensitivity in human breast epithelial cells
Higgins, Michaela J; Beaver, Julia A; Wong, Hong yuen; Gustin, John P; Lauring, Josh D; Garay, Joseph P; Konishi, Hiroyuki; Mohseni, Morassa; Wang, Grace M; Cidado, Justin; Jelovac, Danijela; Cosgrove, David P; Tamaki, Akina; Park, Ben Ho
A high frequency of somatic mutations has been found in breast cancers within the gene encoding the catalytic p110α subunit of PI3K, PIK3CA. Using isogenic human breast epithelial cells, we have previously demonstrated that oncogenic PIK3CA "hotspot" mutations predict for response to the toxic effects of lithium. However, other somatic genetic alterations occur within this pathway in breast cancers, and it is possible that these changes may also predict for lithium sensitivity. We overexpressed the epidermal growth factor receptor (EGFR) into the non-tumorigenic human breast epithelial cell line MCF-10A, and compared these cells to isogenic cell lines previously created via somatic cell gene targeting to model Pten loss, PIK3CA mutations, and the invariant AKT1 mutation, E17K. EGFR overexpressing clones were capable of cellular proliferation in the absence of EGF and were sensitive to lithium similar to the results previously seen with cells harboring PIK3CA mutations. In contrast, AKT1 E17K cells and PTEN−/− cells displayed resistance or partial sensitivity to lithium, respectively. Western blot analysis demonstrated that lithium sensitivity correlated with significant decreases in both PI3K and MAPK signaling that were observed only in EGFR overexpressing and mutant PIK3CA cell lines. These studies demonstrate that EGFR overexpression and PIK3CA mutations are predictors of response to lithium, whereas Pten loss and AKT1 E17K mutations do not predict for lithium sensitivity. Our findings may have important implications for the use of these genetic lesions in breast cancer patients as predictive markers of response to emerging PI3K pathway inhibitors. PMID:21124076
Short-form RON overexpression augments benzyl isothiocyanate-induced apoptosis in human breast cancer cells.
Sehrawat, Anuradha; Singh, Shivendra V
Chemoprevention of breast cancer is feasible with the use of non-toxic phytochemicals from edible and medicinal plants. Benzyl isothiocyanate (BITC) is one such plant compound that prevents mammary cancer development in a transgenic mouse model in association with tumor cell apoptosis. Prior studies from our laboratory have demonstrated a role for reactive oxygen species (ROS)-dependent Bax activation through the intermediary of c-Jun N-terminal kinases in BITC-induced apoptosis in human breast cancer cells. The present study demonstrates that truncated Recepteur d'Origine Nantais (sfRON) is a novel regulator of BITC-induced apoptosis in breast cancer cells. Overexpression of sfRON in MCF-7 and MDA-MB-361 cells resulted in augmentation of BITC-induced apoptosis when the apoptotic fraction was normalized against vehicle control for each cell type (untransfected and sfRON overexpressing cells). ROS generation and G2 /M phase cell cycle arrest resulting from BITC treatment were significantly attenuated in sfRON overexpressing cells after normalization with vehicle control for each cell type. Increased BITC-induced apoptosis by sfRON overexpression was independent of c-Jun N-terminal kinase or p38 mitogen-activated protein kinase hyperphosphorylation. On the other hand, activation of Bax and Bak following BITC exposure was markedly more pronounced in sfRON overexpressing cells than in controls. sfRON overexpression also augmented apoptosis induction by structurally diverse cancer chemopreventive phytochemicals including withaferin A, phenethyl isothiocyanate, and D,L-sulforaphane. In conclusion, the present study provides novel mechanistic insights into the role of sfRON in apoptosis regulation by BITC and other electrophilic phytochemicals. © 2015 Wiley Periodicals, Inc. PMID:25857724
Overexpression of neogenin inhibits cell proliferation and induces apoptosis in human MDA-MB-231 breast carcinoma cells.
Zhang, Qingsong; Liang, Fang; Ke, Yang; Huo, Yanping; Li, Mingchuang; Li, Yanyan; Yue, Junmin
Neogenin has been documented as playing an important role in cancer development. Although an elevated expression of neogenin has been detected in human breast cancer, the role of neogenin in breast cancer cells is not clearly understood. In the present study, we investigated neogenin in breast cancer cell proliferation, migration and apoptosis. We found that neogenin overexpression markedly reduced the proliferation and migration of breast cancer cells (P<0.05). Neogenin overexpression resulted in a reduction in the apoptosis rate. Inhibition of neogenin expression by neogenin siRNA dramatically promoted the proliferation and migration of breast cancer cells, whereas it inhibited cell apoptosis. Furthermore, we found that BMP-2-induced phosphorylation of Smad1/5/8 which was inhibited by neogenin overexpression. The present study demonstrates that neogenin may be a tumor suppressor in breast cancer. Neogenin may serve as a potential diagnostic marker and therapeutic target for breast cancer. PMID:25998984
Nek8, a NIMA family kinase member, is overexpressed in primary human breast tumors.
Bowers, Alex J; Boylan, John F
The family of human Nek (NIMA Related Kinase) kinases currently contains 11 members. We have identified Nek8 as a new member of the Nek kinase family. For many of the Nek family members, primary tumor expression data and function have been limited. However, all of the Nek family proteins share considerable homology with the Never In Mitosis, gene A (NIMA) kinase from the filamentous fungus Aspergillus nidulans. NIMA, as well as its most closely related human ortholog, Nek2, are required for G(2)/M progression and promote centrosome maturation during mitosis. We isolated Nek8 from a primary human colon cDNA library, and found it to be highly homologous to murine Nek8. Recently, a previously named Nek8 sequence was renamed Nek9/Nercc1 in Genbank due to its lack of homology to murine Nek8 and its high homology to murine Nek9. Interestingly, in our study, phylogenetic analysis suggests that human Nek8 and Nek9 form a subfamily within the Nek family. Nek8 has high homology to the Nek family kinase domain as well as to a regulator of chromosome condensation domain (RCC1), which is also present in Nek9. The open reading frame of human Nek8 encodes a 692 amino-acid protein with a calculated molecular weight of 75 kDa. Nek8 is differently expressed between normal human breast tissue and breast tumors. Overexpression of a mutated kinase domain Nek8 in U2-0S cells led to a decrease in actin protein, and a small increase in the level of cdk1/cyclinB1. Our data demonstrate for the first time that Nek8 is a novel tumor associated gene, and shares considerable sequence homology with the Nek family of protein kinases and may be involved in G(2)/M progression. PMID:15019993
Human Epidermal Growth Factor Receptor Family-Targeted Therapies in the Treatment of HER2-Overexpressing Breast Cancer
Eroglu, Zeynep; Tagawa, Tomoko
Breast cancer characterized by overexpression of human epidermal growth factor receptor 2 (HER2) has been associated with more aggressive disease progression and a poorer prognosis. Although an improved understanding of breast cancer pathogenesis and the role of HER2 signaling has resulted in significant survival improvements in the past 20 years, resistance to HER2-targeted therapy remains a concern. A number of strategies to prevent or overcome resistance to HER2-targeted therapy in breast cancer are being evaluated. This article provides a comprehensive review of (a) the role of HER2 signaling in breast cancer pathogenesis, (b) potential receptor and downstream therapeutic targets in breast cancer to overcome resistance to HER2-targeted therapy, and (c) clinical trials evaluating agents targeting one or more members of the HER family and/or downstream pathways for the treatment of breast cancer, with a focus on metastatic disease. PMID:24436312
Identification of four novel human genes amplified and overexpressed in breast carcinoma and localized to the q11-q21.3 region of chromosome 17
Tomasetto, C.; Regnier, C.; Basset, P.
We have performed differential screening of a human metastatic lymph node cDNA library to identify genes possibly involved during breast cancer progression. We have identified four novel genes overexpressed in malignant tissues. They were all located between q11 and q21.3, a region known to contain the c-erbB-2 oncogene and the BRCA1 breast carcinomas, and overexpression of three of them was dependent on gene amplification in breast cancer cell lines. These findings further support the concept that human chromosome 17 specifically carries genes possibly involved in breast cancer progression. 61 refs., 3 figs., 4 tabs.
Protein O-glucosyltransferase 1 overexpression downregulates p16 in BT474 human breast cancer cells
Protein O-glucosyltransferase 1 (POGLUT1) is a novel gene that was initially isolated and identified from the bone marrow cells of patients with myelodysplastic syndrome/acute myeloid leukemia. Previous findings have suggested that POGLUT1 promotes the proliferation of U937 human tissue lymphoma cells. Furthermore, POGLUT1 has been identified in other tissues, including the mammary glands, lymph nodes, intestine, liver and spleen. In the present study, in order to investigate the function and target of POGLUT1 in BT474 breast cancer cells, the effect of POGLUT1 on cell proliferation, differentiation, apoptosis and key proteins in the transforming growth factor (TGF)-β1 signaling pathway was investigated in BT474 cells. The overexpression of POGLUT1 in the presence of TGF-β1 was found to significantly enhance cell viability. Flow cytometric and quantitative polymerase chain reaction analyses revealed that POGLUT1 had an effect on the cell cycle and inhibited the TGF-β1-induced transcriptional upregulation of p16, a major cyclin-dependent kinase inhibitor (CDKI). Furthermore, phosphorylated (p)-Smad3, which has a key role in mediating the TGF-β antiproliferative response, was greatly inhibited by exogenous POGLUT1, suggesting a role for POGLUT1 in the TGF-β1-mediated signaling pathway in the BT474 cell cycle. However, no significant changes were observed in the expression of other CDKIs or in cell apoptosis. The findings of the present study show that the increase in BT474 cell viabilty induced by POGLUT1 is associated with POGLUT1-induced inhibition of the transcriptional upregulation of p16 by TGF-β1, which may be a result of the inhibition of p-Smad3. PMID:25009645
Combined effects of lapatinib and bortezomib in human epidermal receptor 2 (HER2)-overexpressing breast cancer cells and activity of bortezomib against lapatinib-resistant breast cancer cells.
Ma, Chuandong; Niu, Xiuqing; Luo, Jianmin; Shao, Zhimin; Shen, Kunwei
Lapatinib and bortezomib are highly active against breast cancer cells. Breast cancer patients who initially respond to lapatinib may eventually manifest acquired resistance to this treatment. Thus, the identification of novel agents that may prevent or delay the development of acquired resistance to lapatinib is critical. In the current study, we show that the combination of lapatinib and bortezomib results in a synergistic growth inhibition in human epidermal receptor 2 (HER2)-overexpressing breast cancer cells and that the combination enhances apoptosis of SK-BR-3 cells. Importantly, we found that the combination of lapatinib plus bortezomib more effectively blocked activation of the HER2 pathway in SK-BR-3 cells, compared with monotherapy. In addition, we established a model of acquired resistance to lapatinib by chronically challenging SK-BR-3 breast cancer cells with increasing concentrations of lapatinib. Here, we showed that bortezomib notably induced apoptosis of lapatinib-resistant SK-BR-3 pools and further inhibited HER2 signaling in the resistant cells. Taken together, the current data indicate a synergistic interaction between lapatinib and bortezomib in HER2-overexpressing breast cancer cells and provide the rationale for the clinical evaluation of these two noncross-resistant targeted therapies. The combination of lapatinib and bortezomib may be a potentially novel approach to prevent or delay the onset of acquired resistance to lapatinib in HER2-overxpressing/estrogen receptor (ER)-negative breast cancers. PMID:20701607
Estrogen receptor alpha (ERα/ESR1) mediates the p53-independent overexpression of MDM4/MDMX and MDM2 in human breast cancer
Swetzig, Wendy M.; Wang, Jianmin; Das, Gokul M.
MDM2 and MDM4 are heterodimeric, non-redundant oncoproteins that potently inhibit the p53 tumor suppressor protein. MDM2 and MDM4 also enhance the tumorigenicity of breast cancer cells in in vitro and in vivo models and are overexpressed in primary human breast cancers. Prior studies have characterized Estrogen Receptor Alpha (ERα/ESR1) as a regulator of MDM2 expression and an MDM2- and p53-interacting protein. However, similar crosstalk between ERα and MDM4 has not been investigated. Moreover, signaling pathways that mediate the overexpression of MDM4 in human breast cancer remain to be elucidated. Using the Cancer Genome Atlas (TCGA) breast invasive carcinoma patient cohort, we have analyzed correlations between ERα status and MDM4 and MDM2 expression in primary, treatment-naïve, invasive breast carcinoma samples. We report that the expression of MDM4 and MDM2 is elevated in primary human breast cancers of luminal A/B subtypes and associates with ERα-positive disease, independently of p53 mutation status. Furthermore, in cell culture models, ERα positively regulates MDM4 and MDM2 expression via p53-independent mechanisms, and these effects can be blocked by the clinically-relevant endocrine therapies fulvestrant and tamoxifen. Additionally, ERα also positively regulates p53 expression. Lastly, we report that endogenous MDM4 negatively regulates ERα expression and forms a protein complex with ERα in breast cancer cell lines and primary human breast tumor tissue. This suggests direct signaling crosstalk and negative feedback loops between ERα and MDM4 expression in breast cancer cells. Collectively, these novel findings implicate ERα as a central component of the p53-MDM2-MDM4 signaling axis in human breast cancer. PMID:26909605
Prostate tumour overexpressed-1 promotes tumourigenicity in human breast cancer via activation of Wnt/β-catenin signalling.
Cui, Yanmei; Ma, Weifeng; Lei, Fangyong; Li, Qingyuan; Su, Yanhong; Lin, Xi; Lin, Chuyong; Zhang, Xin; Ye, Liping; Wu, Shu; Li, Jun; Yuan, Zhongyu; Song, Libing
Breast cancer is the most common malignancy in females. The presence of cancer stem cells (CSCs) is the main cause of local and distant tumour recurrence and is associated with poor outcome in breast cancer. However, the molecular mechanisms underlying the maintenance of CSCs remain largely unknown. This study demonstrates that prostate tumour overexpressed-1 (PTOV1) enhances the CSC population and augments the tumourigenicity of breast cancer cells both in vitro and in vivo. Moreover, PTOV1 suppresses transcription of Dickkopf-1 (DKK1) by recruiting histone deacetylases and subsequently reducing DKK1 promoter histone acetylation, followed by activation of Wnt/β-catenin signalling. Restoration of DKK1 expression in PTOV1-overexpressing cells counteracts the effects of PTOV1 on Wnt/β-catenin activation and the CSC population. Collectively, these results suggest that PTOV1 positively regulates the Wnt/β-catenin signalling pathway and enhances tumourigenicity in breast cancer; this novel mechanism may represent a therapeutic target for breast cancer. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:27060981
Methylation of PLCD1 and adenovirus-mediated PLCD1 overexpression elicits a gene therapy effect on human breast cancer
Mu, Haixi; Wang, Na; Zhao, Lijuan; Li, Shuman; Li, Qianqian; Chen, Ling; Luo, Xinrong; Qiu, Zhu; Li, Lili; Ren, Guosheng; Xu, Yongzhu; Zhou, Xiangyang; Xiang, Tingxiu
Our previous study showed that PLCD1 significantly decreases cell proliferation and affects cell cycle progression in breast cancer cells. In the present study, we aimed to investigate its functional and molecular mechanisms, and whether or not can become a new target for gene therapies. We found reduced PLCD1 protein expression in breast tumor tissues compared with paired surgical margin tissues. PLCD1 promoter CpG methylation was detected in 55 of 96 (57%) primary breast tumors, but not in surgical-margin tissues and normal breast tissues. Ectopic expression of PLCD1 inhibited breast tumor cell proliferation in vivo by inducing apoptosis and suppressed tumor cell migration by regulating cytoskeletal reorganization proteins including RhoA and phospho-cofilin. Furthermore, we found that PLCD1 induced p53 accumulation, increased p27 and p21 protein levels, and cleaved PARP. Finally, we constructed an adenoviral vector expressing PLCD1 (AdH5-PLCD1), which exhibited strong cytotoxicity in breast cancer cells. Our findings provide insights into the development of PLCD1 gene therapies for breast cancer and perhaps, other human cancers. - Highlights: • PLCD1 is downregulated via hypermethylation in breast cancer. • PLCD1 suppressed cell migration by regulating cytoskeletal reorganization proteins. • Adenovirus AdHu5-PLCD1 may be a novel therapeutic option for breast cancer.
Midline2 is overexpressed and a prognostic indicator in human breast cancer and promotes breast cancer cell proliferation in vitro and in vivo.
Wang, Lan; Wu, Jueheng; Yuan, Jie; Zhu, Xun; Wu, Hongmei; Li, Mengfeng
Midline2 (MID2) is an ubiquitin-conjugating E2 enzyme linked to tumor progression and a novel interacting partner of breast cancer 1, early-onset (BRCA1). However, the role of MID2 in breast cancer remains unknown. This study investigated the expression, prognostic value, and role of MID2 in breast cancer. The expression of MID2 mRNA and protein was significantly upregulated in breast cancer tissue and established cell lines compared with that in normal breast epithelial cells and paired adjacent non-tumor tissue (P < 0.001). Immunohistochemical analysis demonstrated that MID2 was overexpressed in 272 of 284 (95.8%) paraffinembedded, archived breast cancer tissue. Moreover, MID2 expression increased with advanced clinical stage (P < 0.001). High MID2 expression was significantly associated with advanced clinical stages and T, N, and M staging (all P < 0.05). Univariate and multivariate analyses indicated that high MID2 expression was an independent prognostic factor for poor overall survival in the entire cohort (93.73 vs. 172.1 months; P < 0.001, logrank test) and in subgroups with stages Tis + I + II and III + IV. Furthermore, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide colony formation, and anchorage-independent growth ability assays were conducted. Results showed that siRNA silencing of MID2 expression significantly reduced MCF-7 and MDA-MB-231 cell proliferation in vitro and blocked the growth of MDA-MB-231 cell xenograft tumors in vivo (P < 0.05). This study indicated that MID2 may be a novel prognostic marker and interventional target in breast cancer. PMID:26791755
UBE2Q1 in a Human Breast Carcinoma Cell Line: Overexpression and Interaction with p53.
Shafiee, Sayed Mohammad; Rasti, Mozhgan; Seghatoleslam, Atefeh; Azimi, Tayebeh; Owji, Ali Akbar
The p53 tumor suppressor protein is a principal mediator of growth arrest, senescence, and apoptosis in response to a broad array of cellular damage. p53 is a substrate for the ubiquitin-proteasome system, however, the ubiquitin-conjugating enzymes (E2s) involved in p53 ubiquitination have not been well studied. UBE2Q1 is a novel E2 ubiquitin conjugating enzyme gene. Here, we investigated the effect of UBE2Q1 overexpression on the level of p53 in the MDA-MB-468 breast cancer cell line as well as the interaction between UBE2Q1 and p53. By using a lipofection method, the p53 mutated breast cancer cell line, MDA-MB-468, was transfected with the vector pCMV6-AN-GFP, containing UBE2Q1 ORF. Western blot analysis was employed to verify the overexpression of UBE2Q1 in MDA-MB-468 cells and to evaluate the expression level of p53 before and after cell transfection. Immunoprecipitation and GST pull-down protocols were used to investigate the binding of UBE2Q1 to p53. We established MDA-MB-468 cells that transiently expressed a GFP fusion proteins containing UBE2Q1 (GFP-UBE2Q1). Western blot analysis revealed that levels of p53 were markedly lower in UBE2Q1 transfected MDA-MB-468 cells as compared with control MDA-MB-468 cells. Both in vivo and in vitro data showed that UBE2Q1 co-precipitated with p53 protein. Our data for the first time showed that overexpression of UBE2Q1can lead to the repression of p53 in MDA-MB-468 cells. This repression of p53 may be due to its UBE2Q1 mediated ubiquitination and subsequent proteasome degradation, a process that may involve direct interaction of UBE2Q1with p53. PMID:25987028
Increased regucalcin gene expression extends survival in breast cancer patients: Overexpression of regucalcin suppresses the proliferation and metastatic bone activity in MDA-MB-231 human breast cancer cells in vitro.
Yamaguchi, Masayoshi; Osuka, Satoru; Weitzmann, M Neale; Shoji, Mamoru; Murata, Tomiyasu
Human breast cancer is highly metastatic to bone and drives bone turnover. Breast cancer metastases cause osteolytic lesions and skeletal damage that leads to bone fractures. Regucalcin, which plays a pivotal role as an inhibitor of signal transduction and transcription activity, has been suggested to act as a suppressor of human cancer. In the present study, we compared the clinical outcome between 44 breast cancer patients with higher regucalcin expression and 43 patients with lower regucalcin expression. Prolonged relapse-free survival was identified in the patients with increased regucalcin gene expression. We further demonstrated that overexpression of full length, but not alternatively spliced variants of regucalcin, induces G1 and G2/M phase cell cycle arrest, suppressing the proliferation of MDA-MB-231 cells, a commonly used in vitro model of human breast cancer that metastasize to bone causing osteolytic lesions. Overexpression of regucalcin was found to suppress multiple signaling pathways including Akt, MAP kinase and SAPK/JNK, and NF-κB p65 and β-catenin along with increased p53, a tumor suppressor, and decreased K-ras, c-fos and c-jun. Moreover, we found that co-culture of regucalcin-overexpressing MDA-MB-231 cells with mouse bone marrow cells prevented enhanced osteoclastogenesis and suppressed mineralization in mouse bone marrow cells in vitro. Taken together, the present study suggests that regucalcin may have important anticancer properties in human breast cancer patients. Mechanistically, these effects are likely mediated through suppression of multiple signaling pathways, upregulation of p53 and downregulation of oncogenes leading to anti-proliferative effects and reduced metastases to bone, a phenotype associated with poor clinical outcome. PMID:27221776
A Novel Function for the nm23-Hl Gene: Overexpression in Human Breast Carcinoma Cells Leads to the Formation of Basement Membrane and Growth Arrest
Howlett, Anthony R; Petersen, Ole W; Steeg, Patricia S; Bissell, Mina J
We have developed a culture system using reconstituted basement membrane components in which normal human mammary epithelial cells exhibit several aspects of the development and differentiation process, including formation of acinar-like structures, production and basal deposition of basement membrane components, and production and apical secretion of sialomucins. Cell lines and cultures from human breast carcinomas failed to recapitulate this process. The data indicate the importance of cellular interactions with the basement membrane in the regulation of normal breast differentiation and, potentially, its loss in neoplasia. Our purpose was to use this assay to investigate the role of the putative metastasis suppressor gene nm23-H1 in mammary development and differentiation. The metastatic human breast carcinoma cell line MDA-MB-435, clones transfected with a control pCMVBamneo vector, and clones transfected with pCMVBamneo vector containing nm23-H1 complementary DNA (the latter of which exhibited a substantial reduction in spontaneous metastatic potential in vivo) were cultured within a reconstituted basement membrane. Clones were examined for formation of acinus-like spheres, deposition of basement membrane components, production of sialomucin, polarization, and growth arrest. In contrast to the parental cell line and control transfectants, MDA-MB-435 breast carcinoma cells overexpressing Nm23-H1 protein regained several aspects of the normal phenotype within reconstituted basement membrane. Nm23-H1 protein-positive cells formed organized acinus-like spheres, deposited the basement membrane components type IV collagen and, to some extent, laminin to the outside of the spheres, expressed sialomucin, and growth arrested. Growth arrest of Nm23-H1 protein-positive cells was preceded by and correlated with formation of a basement membrane, suggesting a causal relationship. The data indicate a previously unidentified cause-and-effect relationship between nm23-H1 gene
Hyaluronic acid-shelled acid-activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human breast tumor xenografts in vivo.
Zhong, Yinan; Goltsche, Katharina; Cheng, Liang; Xie, Fang; Meng, Fenghua; Deng, Chao; Zhong, Zhiyuan; Haag, Rainer
The therapeutic efficacy of nanoscale anticancer drug delivery systems is severely truncated by their low tumor-targetability and inefficient drug release at the target site. Here, we report the design and development of novel endosomal pH-activatable paclitaxel prodrug micelles based on hyaluronic acid-b-dendritic oligoglycerol (HA-dOG-PTX-PM) for active targeting and effective treatment of CD44-overexpressing human breast cancer xenografts in nude mice. HA-dOG-PTX-PM had a high drug content of 20.6 wt.% and an average diameter of 155 nm. The release of PTX was slow at pH 7.4 but greatly accelerated at endosomal pH. MTT assays, flow cytometry and confocal experiments showed that HA-dOG-PTX-PM possessed a high targetability and antitumor activity toward CD44 receptor overexpressing MCF-7 human breast cancer cells. The in vivo pharmacokinetics and biodistribution studies showed that HA-dOG-PTX-PM had a prolonged circulation time in the nude mice and a remarkably high accumulation in the MCF-7 tumor (6.19%ID/g at 12 h post injection). Interestingly, HA-dOG-PTX-PM could effectively treat mice bearing MCF-7 human breast tumor xenografts with little side effects, resulting in complete inhibition of tumor growth and a 100% survival rate over an experimental period of 55 days. These results indicate that hyaluronic acid-shelled acid-activatable PTX prodrug micelles have a great potential for targeted chemotherapy of CD44-positive cancers. PMID:26851390
p53 protein expression in human breast carcinoma: relationship to expression of epidermal growth factor receptor, c-erbB-2 protein overexpression, and oestrogen receptor.
Poller, D. N.; Hutchings, C. E.; Galea, M.; Bell, J. A.; Nicholson, R. A.; Elston, C. W.; Blamey, R. W.; Ellis, I. O.
The expression of p53 protein, oestrogen receptor protein, epidermal growth factor receptor (EGFR) and overexpression of the c-erbB-2 oncoprotein was examined in a series of 149 primary symptomatic breast carcinomas. Expression of p53 was present in 62 of 146 cases (42.5%) of the invasive carcinoma and one of three cases (33.3%) of ductal carcinoma in situ (DCIS) examined. Statistical associations of tumour oestrogen receptor positivity and lack of p53 protein expression, chi 2 = 19.78 (d.f. = 1), P less than 0.001, positive tumour p53 status and poor tumour grade; chi 2 = 14.1 (d.f. = 2), P less than 0.001, EGFR expression chi 2 = 7.07, (d.f. = 1), P less than 0.01 and tumour c-erbB-2 protein overexpression; chi 2 = 4.61 (d.f. = 1), P = 0.032 were identified. Expression of p53 is rare in invasive lobular carcinoma of classical type (8.3% of cases examined) in contrast to other common types of mammary carcinoma. Non-significant trends of p53 protein expression and increased regional tumour recurrence; chi 2 = 3.20 (d.f. = 1), P = 0.074 and also poorer patient survival; chi 2 = 3.76 (d.f. = 1), P = 0.053 were identified. p53 protein expression is a common event in human breast cancer and is present in both DCIS and invasive mammary carcinoma. Abnormal expression of p53 protein is a feature of both in situ and invasive breast carcinoma, implying that the abnormal p53 protein expression may be implicated in the early stages of mammary carcinoma progression. Images Figure 1 PMID:1355662
A Novel Subset of Human Tumors That Simultaneously Overexpress Multiple E2F-responsive Genes Found in Breast, Ovarian, and Prostate Cancers
Shackney, Stanley E; Chowdhury, Salim Akhter; Schwartz, Russell
Reasoning that overexpression of multiple E2F-responsive genes might be a useful marker for RB1 dysfunction, we compiled a list of E2F-responsive genes from the literature and evaluated their expression in publicly available gene expression microarray data of patients with breast cancer, serous ovarian cancer, and prostate cancer. In breast cancer, a group of tumors was identified, each of which simultaneously overexpressed multiple E2F-responsive genes. Seventy percent of these genes were concerned with cell cycle progression, DNA repair, or mitosis. These E2F-responsive gene overexpressing (ERGO) tumors frequently exhibited additional evidence of Rb/E2F axis dysfunction, were mostly triple negative, and preferentially overexpressed multiple basal cytokeratins, suggesting that they overlapped substantially with the basal-like tumor subset. ERGO tumors were also identified in serous ovarian cancer and prostate cancer. In these cancer types, there was no evidence for a tumor subset comparable to the breast cancer basal-like subset. A core group of about 30 E2F-responsive genes were overexpressed in all three cancer types. Thus, it appears that disorders of the Rb/E2F axis can arise at multiple organ sites and produce tumors that simultaneously overexpress multiple E2F-responsive genes. PMID:25392696
A Pretherapy Biodistribution and Dosimetry Study of Indium-111-Radiolabeled Trastuzumab in Patients with Human Epidermal Growth Factor Receptor 2-Overexpressing Breast Cancer
Raubitschek, Andrew; Yamauchi, Dave; Williams, Lawrence E.; Wu, Anna M.; Yazaki, Paul; Shively, John E.; Colcher, David; Somlo, George
Abstract Purpose The purposes of this study were to evaluate the organ biodistribution, pharmacokinetics, immunogenicity, and tumor uptake of 111Indium (111In)-MxDTPA-trastuzumab in patients with human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancers and to determine whether 90Y-MxDTPA-trastuzumab should be evaluated in subsequent clinical therapy trials. Experimental Design Patients with HER2-overexpressing breast cancers who were to undergo planned trastuzumab therapy first received unlabeled trastuzumab (4–8 mg/kg IV), followed 4 hours later by 5 mCi 111In-MxDTPA-trastuzumab (10 mg antibody). Serial blood samples, 24-hour urine collections, and nuclear scans were performed at defined time points for 7 days. Results Eight (8) patients received 111In-MxDTPA-trastuzumab, which was well tolerated with no adverse side-effects. Three (3) of 7 patients with known lesions demonstrated positive imaging on nuclear scans. No antiantibody responses were observed for 2 months postinfusion. Organ doses (cGy/mCi) assuming radiolabeling with 90Y were 19.9 for heart wall, 17.6 for liver, 4.6 for red marrow, and 2.8 for the whole body. Tumor doses ranged from 24 to 172 cGy/mCi. Conclusions In summary, results from this study indicate that 90Y-MxDTPA-trastuzumab is an appropriate agent to evaluate in therapy trials. No evidence of an immune response to 111In-MxDTPA-trastuzumab was detected, predicting for the ability to administer multiple cycles. With the exception of cardiac uptake, pharmacokinetics and organ biodistribution were comparable to other 90Y-labeled monoclonal antibodies previously evaluated in the clinic. Cardiac uptake was comparable to hepatic uptake and therefore predicted to not be prohibitively high as to result in dose-limiting cardiotoxicity. PMID:20707718
Overexpression of seven in absentia homolog 2 protein in human breast cancer tissues is associated with the promotion of tumor cell malignant behavior in in vitro.
Sun, Jie; Zhang, Xiaojuan; Han, Yanchun; Zhen, Juan; Meng, Yuan; Song, Min
Seven in absentia homolog 2 (SIAH2), a homologue of Drosophila seven in absentia (Sina), has emerged as an oncogene and plays important roles in cancer development and progression. This study further assessed the role of SIAH2 in breast cancer and the underlying molecular events. The data showed that SIAH2 protein was overexpressed in invasive breast cancer (IBC) compared to the expression noted in normal or ductal carcinoma in situ (DCIS) tissues, expression of which is associated with malignant behaviors. SIAH2 may function differently in different molecular subtypes (e.g., luminal- vs. basal-like type) of breast cancer. Manipulation of SIAH2 expression led to a 'cross-talk' of the ERK and PI3K pathway, which could be one of the mechanisms by which SIAH2 regulates viability, apoptosis, and invasion capacity in these breast cancer cell lines. PMID:27459914
Multidrug resistance-associated protein gene overexpression and reduced drug sensitivity of topoisomerase II in a human breast carcinoma MCF7 cell line selected for etoposide resistance.
Schneider, E; Horton, J K; Yang, C H; Nakagawa, M; Cowan, K H
A human breast cancer cell line (MCF7/WT) was selected for resistance to etoposide (VP-16) by stepwise exposure to 2-fold increasing concentrations of this agent. The resulting cell line (MCF7/VP) was 28-, 21-, and 9-fold resistant to VP-16, VM-26, and doxorubicin, respectively. MCF7/VP cells also exhibited low-level cross-resistance to 4'-(9-acridinylamino)-methanesulfon-m-anisidide, mitoxantrone, and vincristine and no cross-resistance to genistein and camptothecin. Furthermore, these cells were collaterally sensitive to the alkylating agents melphalan and chlorambucil. DNA topoisomerase II levels were similar in both wild-type MCF7/WT and drug-resistant MCF7/VP cells. In contrast, topoisomerase II from MCF7/VP cells appeared to be 7-fold less sensitive to drug-induced cleavable complex formation in whole cells and 3-fold less sensitive in nuclear extracts than topoisomerase II from MCF7/WT cells. Although this suggested that the resistant cells may contain a qualitatively altered topoisomerase II, no mutations were detected in either the ATP-binding nor the putative breakage/resealing regions of either DNA topoisomerase II alpha or II beta. In addition, the steady-state intracellular VP-16 concentration was reduced by 2-fold in the resistant cells, in the absence of detectable mdr1/P-gp expression and without any change in drug efflux. In contrast, expression of the gene encoding the MRP was increased at least 10-fold in resistant MCF7/VP cells as compared to sensitive MCF7/WT cells. These results suggest that resistance to epipodophyllotoxins in MCF7/VP cells is multifactorial, involving a reduction in intracellular drug concentration, possibly as a consequence of MRP overexpression, and an altered DNA topoisomerase II drug sensitivity. PMID:7903202
Inhibition of laminin-5 production in breast epithelial cells by overexpression of p300.
Miller, K A; Chung, J; Lo, D; Jones, J C; Thimmapaya, B; Weitzman, S A
The transcriptional coactivator p300 is essential for normal embryonic development and cellular differentiation. We have been studying the role of p300 in the transcription of a variety of genes, and we became interested in the role of this coactivator in the transcription of genes important in breast epithelial cell biology. From MCF-10A cells (spontaneously immortalized, nontransformed human breast epithelial cells), we developed cell lines that stably overexpress p300. These p300-overexpressing cells displayed reduced adhesion to culture dishes and were found to secrete an extracellular matrix deficient in laminin-5. Laminin-5 is the major extracellular matrix component produced by breast epithelium. Immunofluorescence studies, as well as experiments using normal matrix, confirmed that the decreased adhesion of p300-overexpressing cells is due to laminin-5-deficient extracellular matrix and not due to loss of laminin-5 receptors. Northern blots revealed markedly decreased levels of expression of two of the genes (designated LAMA3 and LAMC2) encoding the alpha3 and gamma2 chains of the laminin-5 heterotrimer in the cells that overexpress p300, whereas LAMB3 mRNA, encoding the third or beta3 chain of laminin-5, was not markedly reduced. Transient transfection experiments with a vector containing a murine LAMA3 promoter demonstrate that overexpressing p300 down-regulates the LAMA3 promoter. In summary, overexpression of p300 leads to down-regulation of laminin-5 production in breast epithelial cells, resulting in decreased adhesion. PMID:10713141
Her-2 overexpression increases the metastatic outgrowth of breast cancer cells in the brain.
Palmieri, Diane; Bronder, Julie L; Herring, Jeanne M; Yoneda, Toshiyuki; Weil, Robert J; Stark, Andreas M; Kurek, Raffael; Vega-Valle, Eleazar; Feigenbaum, Lionel; Halverson, Douglas; Vortmeyer, Alexander O; Steinberg, Seth M; Aldape, Kenneth; Steeg, Patricia S
Retrospective studies of breast cancer patients suggest that primary tumor Her-2 overexpression or trastuzumab therapy is associated with a devastating complication: the development of central nervous system (brain) metastases. Herein, we present Her-2 expression trends from resected human brain metastases and data from an experimental brain metastasis assay, both indicative of a functional contribution of Her-2 to brain metastatic colonization. Of 124 archival resected brain metastases from breast cancer patients, 36.2% overexpressed Her-2, indicating an enrichment in the frequency of tumor Her-2 overexpression at this metastatic site. Using quantitative real-time PCR of laser capture microdissected epithelial cells, Her-2 and epidermal growth factor receptor (EGFR) mRNA levels in a cohort of 12 frozen brain metastases were increased up to 5- and 9-fold, respectively, over those of Her-2-amplified primary tumors. Co-overexpression of Her-2 and EGFR was also observed in a subset of brain metastases. We then tested the hypothesis that overexpression of Her-2 increases the colonization of breast cancer cells in the brain in vivo. A subclone of MDA-MB-231 human breast carcinoma cells that selectively metastasizes to brain (231-BR) overexpressed EGFR; 231-BR cells were transfected with low (4- to 8-fold) or high (22- to 28-fold) levels of Her-2. In vivo, in a model of brain metastasis, low or high Her-2-overexpressing 231-BR clones produced comparable numbers of micrometastases in the brain as control transfectants; however, the Her-2 transfectants yielded 3-fold greater large metastases (>50 microm(2); P < 0.001). Our data indicate that Her-2 overexpression increases the outgrowth of metastatic tumor cells in the brain in this model system. PMID:17483330
Aluminium and human breast diseases.
Darbre, P D; Pugazhendhi, D; Mannello, F
The human breast is exposed to aluminium from many sources including diet and personal care products, but dermal application of aluminium-based antiperspirant salts provides a local long-term source of exposure. Recent measurements have shown that aluminium is present in both tissue and fat of the human breast but at levels which vary both between breasts and between tissue samples from the same breast. We have recently found increased levels of aluminium in noninvasively collected nipple aspirate fluids taken from breast cancer patients (mean 268 ± 28 μg/l) compared with control healthy subjects (mean 131 ± 10 μg/l) providing evidence of raised aluminium levels in the breast microenvironment when cancer is present. The measurement of higher levels of aluminium in type I human breast cyst fluids (median 150 μg/l) compared with human serum (median 6 μg/l) or human milk (median 25 μg/l) warrants further investigation into any possible role of aluminium in development of this benign breast disease. Emerging evidence for aluminium in several breast structures now requires biomarkers of aluminium action in order to ascertain whether the presence of aluminium has any biological impact. To this end, we report raised levels of proteins that modulate iron homeostasis (ferritin, transferrin) in parallel with raised aluminium in nipple aspirate fluids in vivo, and we report overexpression of mRNA for several S100 calcium binding proteins following long-term exposure of MCF-7 human breast cancer cells in vitro to aluminium chlorhydrate. PMID:22099158
18F-fluorodeoxy-glucose positron emission tomography (18FDG-PET) marks MYC-overexpressing human basal-like breast cancers
Palaskas, Nicolaos; Larson, Steven M.; Schultz, Nikolaus; Komisopoulou, Evangelia; Wong, Justin; Rohle, Dan; Campos, Carl; Yannuzzi, Nicolas; Osborne, Joseph R.; Linkov, Irina; Kastenhuber, Edward R.; Taschereau, Richard; Plaisier, Seema B.; Tran, Chris; Heguy, Adriana; Wu, Hong; Sander, Chris; Phelps, Michael E.; Brennan, Cameron; Port, Elisa; Huse, Jason T.; Graeber, Thomas G.; Mellinghoff, Ingo K.
In contrast to normal cells, cancer cells avidly take up glucose and metabolize it to lactate even when oxygen is abundant, a phenomenon referred to as the Warburg effect. This fundamental alteration in glucose metabolism in cancer cells enables their specific detection by Positron Emission Tomography (PET) following intravenous injection of the glucose analogue 18F-fluorodeoxy-glucose (18FDG). However, this useful imaging technique is limited by the fact that not all cancers avidly take up FDG. To identify molecular determinants of 18FDG-retention, we interogated the transcriptomes of human cancer cell lines and primary tumors for metabolic pathways associated with 18FDG radiotracer uptake. From 95 metabolic pathways that were interrogated, the glycolysis and several glycolysis-related pathways (pentose-phosphate, carbon fixation, aminoacyl-tRNA biosynthesis, one-carbon-pool by folate) showed the greatest transcriptional enrichment. This "FDG signature" predicted FDG-uptake in breast cancer cell lines and overlapped with established gene expression signatures for the "basal-like" breast cancer subtype and MYC-induced tumorigenesis in mice. Human breast cancers with nuclear MYC staining and high RNA expression of MYC target genes showed high 18FDG-PET uptake (p < 0.005). Presence of the FDG signature was similarly associated with MYC gene copy gain, increased MYC transcript levels, and elevated expression of metabolic MYC target genes in a human breast cancer genomic dataset. Together, our findings link clinical observations of glucose uptake with a pathologic and molecular subtype of human breast cancer. Further, they suggest related approaches to derive molecular determinants of radiotracer retention for other PET-imaging probes. PMID:21646475
Overexpression of kinesins mediates docetaxel resistance in breast cancer cells.
De, Sarmishtha; Cipriano, Rocky; Jackson, Mark W; Stark, George R
Resistance to chemotherapy remains a major barrier to the successful treatment of cancer. To understand mechanisms underlying docetaxel resistance in breast cancer, we used an insertional mutagenesis strategy to identify proteins whose overexpression confers resistance. A strong promoter was inserted approximately randomly into the genomes of tumor-derived breast cancer cells, using a novel lentiviral vector. We isolated a docetaxel-resistant clone in which the level of the kinesin KIFC3 was elevated. When KIFC3 or the additional kinesins KIFC1, KIF1A, or KIF5A were overexpressed in the breast cancer cell lines MDA-MB231 and MDA-MB 468, the cells became more resistant to docetaxel. The binding of kinesins to microtubules opposes the stabilizing effect of docetaxel that prevents cytokinesis and leads to apoptosis. Our finding that kinesins can mediate docetaxel resistance might lead to novel therapeutic approaches in which kinesin inhibitors are paired with taxanes. PMID:19789344
Complete response of brain metastases from breast cancer overexpressing Her-2/neu to radiation and concurrent Lapatinib and Capecitabine.
Abboud, Mirna; Saghir, Nagi S El; Salame, Joseph; Geara, Fady B
Breast cancers that overexpress the human epidermal growth factor receptor 2 (HER-2) have a predilection to metastasize to the brain. Therapeutic options for brain metastases with systemic therapy remain a challenge in those patients since targeted and chemotherapeutic agents have limited penetration through the blood-brain barrier. Here we report the case of a patient with brain metastases from breast cancer overexpressing HER-2 who achieved a complete radiologic response after treatment by radiation and concurrent Lapatinib and Capecitabine. PMID:21070441
Skp2 is over-expressed in breast cancer and promotes breast cancer cell proliferation.
Zhang, Wenwen; Cao, Lulu; Sun, Zijia; Xu, Jing; Tang, Lin; Chen, Weiwei; Luo, Jiayan; Yang, Fang; Wang, Yucai; Guan, Xiaoxiang
The F box protein Skp2 is oncogenic. Skp2 and Skp2B, an isoform of Skp2 are overexpressed in breast cancer. However, little is known regarding the mechanism by which Skp2B promotes the occurrence and development of breast cancer. Here, we determined the expression and clinical outcomes of Skp2 in breast cancer samples and cell lines using breast cancer database, and investigated the role of Skp2 and Skp2B in breast cancer cell growth, apoptosis and cell cycle arrest. We obtained Skp2 is significantly overexpressed in breast cancer samples and cell lines, and high Skp2 expression positively correlated with poor prognosis of breast cancer. Both Skp2 and Skp2B could promote breast cancer cell proliferation, inhibit cell apoptosis, change the cell cycle distribution and induce the increased S phase cells and therefore induce cell proliferation in breast cancer cells. Moreover, the 2 isoforms could both suppress PIG3 expression via independent pathways in the breast cancer cells. Skp2 suppressed p53 and inhibited PIG3-induced apoptosis, while Skp2B attenuated the function of PIG3 by inhibiting PHB. Our results indicate that Skp2 and Skp2B induce breast cancer cell development and progression, making Skp2 and Skp2B potential molecular targets for breast cancer therapy. PMID:27111245
Imaging features of HER2 overexpression in breast cancer: a systematic review and meta-analysis.
Elias, Sjoerd G; Adams, Arthur; Wisner, Dorota J; Esserman, Laura J; van't Veer, Laura J; Mali, Willem P Th M; Gilhuijs, Kenneth G A; Hylton, Nola M
Breast cancer imaging phenotype is diverse and may relate to molecular alterations driving cancer behavior. We systematically reviewed and meta-analyzed relations between breast cancer imaging features and human epidermal growth factor receptor type 2 (HER2) overexpression as a marker of breast cancer aggressiveness. MEDLINE and EMBASE were searched for mammography, breast ultrasound, magnetic resonance imaging (MRI), and/or [(18)F]fluorodeoxyglucose positron emission tomography studies through February 2013. Of 68 imaging features that could be pooled (85 articles, 23,255 cancers; random-effects meta-analysis), 11 significantly related to HER2 overexpression. Results based on five or more studies and robustness in subgroup analyses were as follows: the presence of microcalcifications on mammography [pooled odds ratio (pOR), 3.14; 95% confidence interval (CI), 2.46-4.00] or ultrasound (mass-associated pOR, 2.95; 95% CI, 2.34-3.71), branching or fine linear microcalcifications (pOR, 2.11; 95% CI, 1.07-4.14) or extremely dense breasts on mammography (pOR, 1.37; 95% CI, 1.07-1.76), and washout (pOR, 1.57; 95% CI, 1.11-2.21) or fast initial kinetics (pOR, 2.60; 95% CI, 1.43-4.73) on MRI all increased the chance of HER2 overexpression. Maximum [(18)F]fluorodeoxyglucose standardized uptake value (SUVmax) was higher upon HER2 overexpression (pooled mean difference, +0.76; 95% CI, 0.10-1.42). These results show that several imaging features relate to HER2 overexpression, lending credibility to the hypothesis that imaging phenotype reflects cancer behavior. This implies prognostic relevance, which is especially relevant as imaging is readily available during diagnostic work-up. PMID:24807204
LETM1 overexpression is correlated with the clinical features and survival outcome of breast cancer
Li, Nan; Zheng, Yahui; Xuan, Chouhui; Lin, Zhenhua; Piao, Longzhen; Liu, Shuangping
Background: Leucine zipper/EF hand-containing transmembrane-1 (LETM1) is a mitochondrial inner membrane protein that was first identified in Wolf-Hirschhorn syndrome. However, high-level expression of LETM1 has been correlated with multiple human malignancies, suggesting roles in carcinogenesis and tumor progression. This study is aimed to explore the clinicopathological characteristics and prognostic value of LETM1 overexpression in breast cancer. Methods: Immunohistochemical (IHC) staining, and immunofluorescence (IF) were performed to examine LETM1 expression in breast cancer cell line/tissues compared with adjacent normal tissues. Statistical analysis was applied to evaluate the correlation between LETM1 overexpression and the clinicopathological features of breast cancer. Survival rates were calculated using the Kaplan-Meier method, and the relationship between prognostic factors and patient survival was analyzed using the Cox proportional hazard models. Results: LETM1 protein showed cytoplasmic staining pattern in breast cancer. The strongly positive rate of LETM1 protein was 61.6% (98/159) in breast cancer, which was significantly higher than in DCIS (29.7%, 11/37), hyperplasia (16.7%, 3/18) and adjacent normal breast tissues (15.9%, 7/44). High-level expression of LETM1 protein was correlated with lymph node metastasis, poor differentiation, late clinical stage, disease-free survival (DFS) and overall survival (OS) rates in breast cancer. Moreover, multivariate analysis suggested that LETM1 emerged as a significant independent prognostic factor along with clinical stage of patients with breast cancer. Conclusions: LETM1 plays an important role in the progression of breast cancer. High level expression of LETM1 is an independent poor prognostic factor of breast cancer. PMID:26722481
Efficacy of TCH/TEC neoadjuvant chemotherapy for the treatment of HER-2-overexpressing breast cancer
The aim of the present study was to observe the efficacy of neoadjuvant trastuzumab combined with docetaxel and carboplatin (TCH), and docetaxel, epirubicin and cyclophosphamide (TEC) chemotherapy in human epidermal growth factor receptor-2 (HER-2)-overexpressing breast cancer. The total cohort of 64 cases of HER-2-overexpressing breast cancer patients was divided into two groups according to their treatment preferences: The TCH group, consisting of 39 patients, and the TEC group, consisting of 25 patients. The neoadjuvant chemotherapy was continued for six cycles prior to comparison of the treatment efficacy. The TCG and TEC groups exhibited an overall response rate of 94.9 and 72.0% (37/39 and 18/25 cases; P<0.05), respectively, and a pathological complete response (pCR; defined as the presence of no invasive or in situ residual tumors in the breast) rate of 69.2 and 32.0% (27/39 and 8/25 cases; P<0.05), respectively. Furthermore, no significant differences were identified between the two groups of patients in terms of adverse reactions, such as cardiac dysfunction, bone marrow suppression and liver function impairment. In the present study, the treatment of HER-2-overexpressing breast cancer patients with TCH neoadjuvant chemotherapy demonstrated more favorable efficacy and a higher pCR rate when compared with the TEC-treated group. PMID:25789069
p53 mutations and overexpression in locally advanced breast cancers.
Faille, A.; De Cremoux, P.; Extra, J. M.; Linares, G.; Espie, M.; Bourstyn, E.; De Rocquancourt, A.; Giacchetti, S.; Marty, M.; Calvo, F.
Alterations in the p53 gene were analysed in 39 patients with locally advanced breast cancers (LABCs) (stage III-IV) with inflammatory signs in most cases (UICC stage T4d = 32 patients) by molecular and immunohistochemical (IHC) approaches. All patients were included in the same therapy protocol. Using polymerase chain reaction (PCR) and a single-strand conformational polymorphism migration technique (SSCP), the presence of mutations in exons 2-11, covering the entire coding sequence of the p53 gene, was evaluated. Using the mouse specific anti-human p53 monoclonal antibody (PAb 1801), we also looked for overexpression of the p53 protein in tissue sections. In 16 cases shifted bands were reproducibly identified by PCR-SSCP, and all but one (localised to exon 10) were in exons 5-8, the usual mutational hotspots. Fifteen of these 16 samples were sequenced and 14 of the suspected mutations (36%) were confirmed. Most of them (12) were single nucleotide substitutions, and transitions were more frequent (eight cases) than transversions (four cases). Fourteen of the tumour samples were positively stained with the monoclonal antibody PAb 1801, 11 with nuclear staining only, two with mixed cytoplasmic and nuclear staining and one with cytoplasmic staining only. Staining patterns were very heterogeneous in terms of the percentage of positive cells (10-75%) and their distribution in the tissue section (isolated foci or dispersed cells). In 11 of the 14 mutated cases a positive immunostaining was observed. The presence of a p53 mutation was significantly associated with larger tumour diameter (chi 2 = 7.490, P = 0.0062) and the presence of clinical metastases (stage IV) (chi 2 = 10.113, P = 0.0015). A non-statistically significant trend of association was observed between p53 mutation, negative oestrogen receptors and lower response rate to therapy. Our results in this group of patients and the heterogeneity of the staining of tumour cells in tissue sections suggest that p53
Overexpressed ubiquitin ligase Cullin7 in breast cancer promotes cell proliferation and invasion via down-regulating p53
Guo, Hongsheng; Wu, Fenping; Wang, Yan; Yan, Chong; Su, Wenmei
Highlights: • Cullin7 is overexpressed in human breast cancer samples. • Cullin7 stimulated proliferation and invasion of breast cancer cells. • Inhibition of p53 contributes to Cullin7-induced proliferation and invasion. - Abstract: Ubiquitin ligase Cullin7 has been identified as an oncogene in some malignant diseases such as choriocarcinoma and neuroblastoma. However, the role of Cullin7 in breast cancer carcinogenesis remains unclear. In this study, we compared Cullin7 protein levels in breast cancer tissues with normal breast tissues and identified significantly higher expression of Cullin7 protein in breast cancer specimens. By overexpressing Cullin7 in breast cancer cells HCC1937, we found that Cullin7 could promote cell growth and invasion in vitro. In contrast, the cell growth and invasion was inhibited by silencing Cullin7 in breast cancer cell BT474. Moreover, we demonstrated that Cullin7 promoted breast cancer cell proliferation and invasion via down-regulating p53 expression. Thus, our study provided evidence that Cullin7 functions as a novel oncogene in breast cancer and may be a potential therapeutic target for breast cancer management.
SNEV overexpression extends the life span of human endothelial cells
Voglauer, Regina; Chang, Martina Wei-Fen; Dampier, Brigitta; Wieser, Matthias; Baumann, Kristin; Sterovsky, Thomas; Schreiber, Martin; Katinger, Hermann; Grillari, Johannes . E-mail: firstname.lastname@example.com
In a recent screening for genes downregulated in replicatively senescent human umbilical vein endothelial cells (HUVECs), we have isolated the novel protein SNEV. Since then SNEV has proven as a multifaceted protein playing a role in pre-mRNA splicing, DNA repair, and the ubiquitin/proteosome system. Here, we report that SNEV mRNA decreases in various cell types during replicative senescence, and that it is increased in various immortalized cell lines, as well as in breast tumors, where SNEV transcript levels also correlate with the survival of breast cancer patients. Since these mRNA profiles suggested a role of SNEV in the regulation of cell proliferation, the effect of its overexpression was tested. Thereby, a significant extension of the cellular life span was observed, which was not caused by altered telomerase activity or telomere dynamics but rather by enhanced stress resistance. When SNEV overexpressing cells were treated with bleomycin or bleomycin combined with BSO, inducing DNA damage as well as reactive oxygen species, a significantly lower fraction of apoptotic cells was found in comparison to vector control cells. These data suggest that high levels of SNEV might extend the cellular life span by increasing the resistance to stress or by improving the DNA repair capacity of the cells.
Aluminium and the human breast.
Darbre, P D
The human population is exposed to aluminium (Al) from diet, antacids and vaccine adjuvants, but frequent application of Al-based salts to the underarm as antiperspirant adds a high additional exposure directly to the local area of the human breast. Coincidentally the upper outer quadrant of the breast is where there is also a disproportionately high incidence of breast cysts and breast cancer. Al has been measured in human breast tissues/fluids at higher levels than in blood, and experimental evidence suggests that at physiologically relevant concentrations, Al can adversely impact on human breast epithelial cell biology. Gross cystic breast disease is the most common benign disorder of the breast and evidence is presented that Al may be a causative factor in formation of breast cysts. Evidence is also reviewed that Al can enable the development of multiple hallmarks associated with cancer in breast cells, in particular that it can cause genomic instability and inappropriate proliferation in human breast epithelial cells, and can increase migration and invasion of human breast cancer cells. In addition, Al is a metalloestrogen and oestrogen is a risk factor for breast cancer known to influence multiple hallmarks. The microenvironment is established as another determinant of breast cancer development and Al has been shown to cause adverse alterations to the breast microenvironment. If current usage patterns of Al-based antiperspirant salts contribute to causation of breast cysts and breast cancer, then reduction in exposure would offer a strategy for prevention, and regulatory review is now justified. PMID:26997127
HSET overexpression fuels tumor progression via centrosome clustering-independent mechanisms in breast cancer patients.
Pannu, Vaishali; Rida, Padmashree C G; Ogden, Angela; Turaga, Ravi Chakra; Donthamsetty, Shashikiran; Bowen, Nathan J; Rudd, Katie; Gupta, Meenakshi V; Reid, Michelle D; Cantuaria, Guilherme; Walczak, Claire E; Aneja, Ritu
Human breast tumors harbor supernumerary centrosomes in almost 80% of tumor cells. Although amplified centrosomes compromise cell viability via multipolar spindles resulting in death-inducing aneuploidy, cancer cells tend to cluster extra centrosomes during mitosis. As a result cancer cells display bipolar spindle phenotypes to maintain a tolerable level of aneuploidy, an edge to their survival. HSET/KifC1, a kinesin-like minus-end directed microtubule motor has recently found fame as a crucial centrosome clustering molecule. Here we show that HSET promotes tumor progression via mechanisms independent of centrosome clustering. We found that HSET is overexpressed in breast carcinomas wherein nuclear HSET accumulation correlated with histological grade and predicted poor progression-free and overall survival. In addition, deregulated HSET protein expression was associated with gene amplification and/or translocation. Our data provide compelling evidence that HSET overexpression is pro-proliferative, promotes clonogenic-survival and enhances cell-cycle kinetics through G2 and M-phases. Importantly, HSET co-immunoprecipitates with survivin, and its overexpression protects survivin from proteasome-mediated degradation, resulting in its increased steady-state levels. We provide the first evidence of centrosome clustering-independent activities of HSET that fuel tumor progression and firmly establish that HSET can serve both as a potential prognostic biomarker and as a valuable cancer-selective therapeutic target. PMID:25788277
HSET overexpression fuels tumor progression via centrosome clustering-independent mechanisms in breast cancer patients
Pannu, Vaishali; Rida, Padmashree C.G.; Ogden, Angela; Turaga, Ravi Chakra; Donthamsetty, Shashikiran; Bowen, Nathan J.; Rudd, Katie; Gupta, Meenakshi V.; Reid, Michelle D.; Cantuaria, Guilherme; Walczak, Claire E.; Aneja, Ritu
Human breast tumors harbor supernumerary centrosomes in almost 80% of tumor cells. Although amplified centrosomes compromise cell viability via multipolar spindles resulting in death-inducing aneuploidy, cancer cells tend to cluster extra centrosomes during mitosis. As a result cancer cells display bipolar spindle phenotypes to maintain a tolerable level of aneuploidy, an edge to their survival. HSET/KifC1, a kinesin-like minus-end directed microtubule motor has recently found fame as a crucial centrosome clustering molecule. Here we show that HSET promotes tumor progression via mechanisms independent of centrosome clustering. We found that HSET is overexpressed in breast carcinomas wherein nuclear HSET accumulation correlated with histological grade and predicted poor progression-free and overall survival. In addition, deregulated HSET protein expression was associated with gene amplification and/or translocation. Our data provide compelling evidence that HSET overexpression is pro-proliferative, promotes clonogenic-survival and enhances cell-cycle kinetics through G2 and M-phases. Importantly, HSET co-immunoprecipitates with survivin, and its overexpression protects survivin from proteasome-mediated degradation, resulting in its increased steady-state levels. We provide the first evidence of centrosome clustering-independent activities of HSET that fuel tumor progression and firmly establish that HSET can serve both as a potential prognostic biomarker and as a valuable cancer-selective therapeutic target. PMID:25788277
Evaluating treatment response using DW-MRI and DCE-MRI in trastuzumab responsive and resistant HER2-overexpressing human breast cancer xenografts
Whisenant, Jennifer G.; Sorace, Anna G.; McIntyre, J. Oliver; Kang, Hakmook; Sánchez, Violeta; Loveless, Mary E.; Yankeelov, Thomas E.
We report longitudinal diffusion-weighted magnetic resonance imaging (DW-MRI) and dynamic contrast enhanced (DCE)-MRI (7 T) studies designed to identify functional changes, prior to volume changes, in trastuzumab-sensitive and resistant HER2 + breast cancer xenografts. Athymic mice (N = 33) were subcutaneously implanted with trastuzumab-sensitive (BT474) or trastuzumab-resistant (HR6) breast cancer cells. Tumor-bearing animals were distributed into four groups: BT474 treated and control, HR6 treated and control. DW- and DCE-MRI were conducted at baseline, day 1, and day 4; trastuzumab (10 mg/kg) or saline was administered at baseline and day 3. Animals were sacrificed on day 4 and tumors resected for histology. Voxel-based DW- and DCE-MRI analyses were performed to generate parametric maps of ADC, Ktrans, and ve. On day 1, no differences in tumor size were observed between any of the groups. On day 4, significant differences in tumor size were observed between treated vs. control BT474, treated BT474 vs. treated HR6, and treated vs. control HR6 (P < .0001). On day 1, ve was significantly higher in the BT474 treated group compared to BT474 control (P = .002) and HR6 treated (P = .004). On day 4, ve and Ktrans were significantly higher in the treated BT474 tumors compared to BT474 controls (P = .0007, P = .02, respectively). A significant decrease in Ki67 staining reinforced response in the BT474 treated group compared to BT474 controls (P = .02). This work demonstrated that quantitative MRI biomarkers have the sensitivity to differentiate treatment response in HER2 + tumors prior to changes in tumor size. PMID:25500087
Frequent Nek1 overexpression in human gliomas.
Zhu, Jun; Cai, Yu; Liu, Pin; Zhao, Weiguo
Never in mitosis A (NIMA)-related kinase 1 (Nek1) regulates cell cycle progression to mitosis. Its expression and potential functions in human gliomas have not been studied. Here, our immunohistochemistry (IHC) assay and Western blot assay results showed that Nek1 expression was significantly upregulated in fresh and paraffin-embedded human glioma tissues. Its level in normal brain tissues was low. Nek1 overexpression in human gliomas was correlated with the proliferation marker (Ki-67), tumor grade, Karnofsky performance scale (KPS) and more importantly, patients' poor survival. Further studies showed that Nek1 expression level was also increased in multiple human glioma cell lines (U251-MG, U87-MG, U118, H4 and U373). Significantly, siRNA-mediated knockdown of Nek1 inhibited glioma cell (U87-MG/U251-MG) growth. Nek1 siRNA also sensitized U87-MG/U251-MG cells to temozolomide (TMZ), causing a profound apoptosis induction and growth inhibition. The current study indicates Nek1 might be a novel and valuable oncotarget of glioma, it is important for glioma cell growth and TMZ-resistance. PMID:27251576
Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer.
Camarda, Roman; Zhou, Alicia Y; Kohnz, Rebecca A; Balakrishnan, Sanjeev; Mahieu, Celine; Anderton, Brittany; Eyob, Henok; Kajimura, Shingo; Tward, Aaron; Krings, Gregor; Nomura, Daniel K; Goga, Andrei
Expression of the oncogenic transcription factor MYC is disproportionately elevated in triple-negative breast cancer (TNBC), as compared to estrogen receptor-, progesterone receptor- or human epidermal growth factor 2 receptor-positive (RP) breast cancer. We and others have shown that MYC alters metabolism during tumorigenesis. However, the role of MYC in TNBC metabolism remains mostly unexplored. We hypothesized that MYC-dependent metabolic dysregulation is essential for the growth of MYC-overexpressing TNBC cells and may identify new therapeutic targets for this clinically challenging subset of breast cancer. Using a targeted metabolomics approach, we identified fatty acid oxidation (FAO) intermediates as being dramatically upregulated in a MYC-driven model of TNBC. We also identified a lipid metabolism gene signature in patients with TNBC that were identified from The Cancer Genome Atlas database and from multiple other clinical data sets, implicating FAO as a dysregulated pathway that is critical for TNBC cell metabolism. We found that pharmacologic inhibition of FAO catastrophically decreased energy metabolism in MYC-overexpressing TNBC cells and blocked tumor growth in a MYC-driven transgenic TNBC model and in a MYC-overexpressing TNBC patient-derived xenograft. These findings demonstrate that MYC-overexpressing TNBC shows an increased bioenergetic reliance on FAO and identify the inhibition of FAO as a potential therapeutic strategy for this subset of breast cancer. PMID:26950360
Synaptojanin 2 is a druggable mediator of metastasis and the gene is overexpressed and amplified in breast cancer.
Ben-Chetrit, Nir; Chetrit, David; Russell, Roslin; Körner, Cindy; Mancini, Maicol; Abdul-Hai, Ali; Itkin, Tomer; Carvalho, Silvia; Cohen-Dvashi, Hadas; Koestler, Wolfgang J; Shukla, Kirti; Lindzen, Moshit; Kedmi, Merav; Lauriola, Mattia; Shulman, Ziv; Barr, Haim; Seger, Dalia; Ferraro, Daniela A; Pareja, Fresia; Gil-Henn, Hava; Lapidot, Tsvee; Alon, Ronen; Milanezi, Fernanda; Symons, Marc; Ben-Hamo, Rotem; Efroni, Sol; Schmitt, Fernando; Wiemann, Stefan; Caldas, Carlos; Ehrlich, Marcelo; Yarden, Yosef
Amplified HER2, which encodes a member of the epidermal growth factor receptor (EGFR) family, is a target of effective therapies against breast cancer. In search for similarly targetable genomic aberrations, we identified copy number gains in SYNJ2, which encodes the 5'-inositol lipid phosphatase synaptojanin 2, as well as overexpression in a small fraction of human breast tumors. Copy gain and overexpression correlated with shorter patient survival and a low abundance of the tumor suppressor microRNA miR-31. SYNJ2 promoted cell migration and invasion in culture and lung metastasis of breast tumor xenografts in mice. Knocking down SYNJ2 impaired the endocytic recycling of EGFR and the formation of cellular lamellipodia and invadopodia. Screening compound libraries identified SYNJ2-specific inhibitors that prevented cell migration but did not affect the related neural protein SYNJ1, suggesting that SYNJ2 is a potentially druggable target to block cancer cell migration. PMID:25605973
Lapatinib for the treatment of HER2-overexpressing breast cancer.
Jones, J; Takeda, A; Picot, J; von Keyserlingk, C; Clegg, A
This paper presents a summary of the evidence review group (ERG) report into the clinical effectiveness and cost-effectiveness of lapatinib for the treatment of advanced or metastatic HER2-overexpressing breast cancer based upon a review of the manufacturer's submission to the National Institute for Health and Clinical Excellence (NICE) as part of the single technology appraisal (STA) process. The scope included women with advanced, metastatic or recurrent HER2-overexpressing breast cancer who have had previous therapy that includes trastuzumab. Outcomes were time to progression, progression-free survival, response rates, overall survival, health-related quality of life and adverse effects. The submission's evidence came from one randomised controlled trial (RCT) of reasonable methodological quality, although it was not powered to detect a statistically significant difference in mean overall survival. Median time to progression was longer in the lapatinib plus capecitabine arm than in the capecitabine monotherapy arm {27.1 [95% confidence interval (CI) 17.4 to 49.4] versus 18.6 [95% CI 9.1 to 36.9] weeks; hazard ratio 0.57 [95% CI 0.43 to 0.77; p = 0.00013]}. Median overall survival was very similar between the groups [67.7 (95% CI 58.9 to 91.6) versus 66.6 (95% CI 49.1 to 75.0) weeks; hazard ratio 0.78 (95% CI 0.55 to 1.12; p = 0.177)]. Median progression-free survival was statistically significantly longer in the lapatinib plus capecitabine group than in the capecitabine monotherapy group [27.1 (95% CI 24.1 to 36.9) versus 17.6 (95% CI 13.3 to 20.1) weeks; hazard ratio 0.55 (95% CI 0.41 to 0.74); p = 0.000033]. The manufacturer's economic model to estimate progression-free and overall survival for patients with HER2-positive advanced/metastatic breast cancer who had relapsed following treatment with an anthracycline, a taxane and trastuzumab was appropriate for the disease area. The base-case incremental cost-effectiveness ratios (ICERs) for lapatinib plus
Human breast cancer cells harboring a gatekeeper T798M mutation in HER2 overexpress EGFR ligands and are sensitive to dual inhibition of EGFR and HER2
Rexer, Brent N.; Ghosh, Ritwik; Narasanna, Archana; Estrada, Mónica Valeria; Chakrabarty, Anindita; Song, Youngchul; Engelman, Jeffrey A.; Arteaga, Carlos L.
Purpose Mutations in receptor tyrosine kinase (RTK) genes can confer resistance to receptor-targeted therapies. A T798M mutation in the HER2 oncogene has been shown to confer resistance to the tyrosine kinase inhibitor (TKI) lapatinib. We studied the mechanisms of HER2-T798M-induced resistance to identify potential strategies to overcome that resistance. Experimental Design HER2-T798M was stably expressed in BT474 and MCF10A cells. Mutant cells and xenografts were evaluated for effects of the mutation on proliferation, signaling, and tumor growth after treatment with combinations of inhibitors targeting the EGFR-HER2-HER3-PI3K axis. Results A low 3% allelic frequency of the T798M mutant shifted10-fold the IC50 of lapatinib. In mutant-expressing cells, lapatinib did not block basal phosphorylation of HER2, HER3, AKT and ERK1/2. In vitro kinase assays showed increased autocatalytic activity of HER2-T798M. HER3 association with PI3K p85 was increased in mutant-expressing cells. BT474-T798M cells were also resistant to the HER2 antibody trastuzumab. These cells were sensitive to the pan-PI3K inhibitors BKM120 and XL147 and the irreversible HER2/EGFR TKI afatinib but not the MEK1/2 inhibitor CI-1040, suggesting continued dependence of the mutant cells on ErbB receptors and downstream PI3K signaling. BT474-T798M cells showed increased expression of the EGFR ligands EGF, TGFα, amphiregulin and HB-EGF. Addition of the EGFR neutralizing antibody cetuximab or lapatinib restored trastuzumab sensitivity of BT474-T798M cells and xenografts, suggesting increased EGFR ligand production was causally associated with drug resistance. Conclusions Simultaneous blockade of HER2 and EGFR should be an effective treatment strategy against HER2 gene-amplified breast cancer cells harboring T798M mutant alleles. PMID:23948973
Human Epidermal Growth Factor Receptor 2 (HER2) in Cancers: Overexpression and Therapeutic Implications
Iqbal, Nida; Iqbal, Naveed
Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor family having tyrosine kinase activity. Dimerization of the receptor results in the autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptors and initiates a variety of signaling pathways leading to cell proliferation and tumorigenesis. Amplification or overexpression of HER2 occurs in approximately 15–30% of breast cancers and 10–30% of gastric/gastroesophageal cancers and serves as a prognostic and predictive biomarker. HER2 overexpression has also been seen in other cancers like ovary, endometrium, bladder, lung, colon, and head and neck. The introduction of HER2 directed therapies has dramatically influenced the outcome of patients with HER2 positive breast and gastric/gastroesophageal cancers; however, the results have been proved disappointing in other HER2 overexpressing cancers. This review discusses the role of HER2 in various cancers and therapeutic modalities available targeting HER2. PMID:25276427
Kaur, Pavinder; Ward, Brenda; Saha, Baisakhi; Young, Lillian; Groshen, Susan; Techy, Geza; Lu, Yani; Atkinson, Roscoe; Taylor, Clive R.; Ingram, Marylou
Progress in our understanding of heterotypic cellular interaction in the tumor microenvironment, which is recognized to play major roles in cancer progression, has been hampered due to unavailability of an appropriate in vitro co-culture model. The aim of this study was to generate an in vitro 3-dimensional human breast cancer model, which consists of cancer cells and fibroblasts. Breast cancer cells (UACC-893) and fibroblasts at various densities were co-cultured in a rotating suspension culture system to establish co-culture parameters. Subsequently, UACC-893, BT.20, or MDA.MB.453 were co-cultured with fibroblasts for 9 days. Co-cultures resulted in the generation of breast cancer histoid (BCH) with cancer cells showing the invasion of fibroblast spheroids, which were visualized by immunohistochemical (IHC) staining of sections (4 µm thick) of BCH. A reproducible quantitative expression of C-erbB.2 was detected in UACC-893 cancer cells in BCH sections by IHC staining and the Automated Cellular Imaging System. BCH sections also consistently exhibited qualitative expression of pancytokeratins, p53, Ki-67, or E-cadherin in cancer cells and that of vimentin or GSTPi in fibroblasts, fibronectin in the basement membrane and collagen IV in the extracellular matrix. The expression of the protein analytes and cellular architecture of BCH were markedly similar to those of breast cancer tissue. PMID:22034518
Plumbagin induces apoptosis in Her2-overexpressing breast cancer cells through the mitochondrial-mediated pathway.
Kawiak, Anna; Zawacka-Pankau, Joanna; Lojkowska, Ewa
Breast cancer is the leading cause of death-related cancers in women. Approximately 30% of breast cancers overexpress the Her2 oncogene, which is associated with a poor prognosis and increased resistance to chemotherapy. Plumbagin (1), a constituent of species in the plant genera Drosera and Plumbago, displays antineoplastic activity toward various cancers. The present study was aimed at determining the anticancer potential of 1 toward Her2-overexpressing breast cancer cells and defining the mode of cell death induced in these cells. The results showed that 1 exhibited high antiproliferative activity toward the Her2-overexpressing cell lines SKBR3 and BT474. The antiproliferative activity of 1 was associated with apoptosis-mediated cell death, as revealed by caspase activation and an increase in the sub-G1 fraction of the cell cycle. Compound 1 increased the levels of the proapoptotic Bcl-2 family of proteins and decreased the level of the antiapoptotic Bcl-2 protein in SKBR3 and BT474 cells. Thus, these findings indicate that 1 induces apoptosis in Her2-overexpressing breast cancers through the mitochondrial-mediated pathway and suggest its potential for further investigation for the treatment of Her2-overexpressing breast cancer. PMID:22512718
Overexpression of miR-206 suppresses glycolysis, proliferation and migration in breast cancer cells via PFKFB3 targeting
Ge, Xin; Lyu, Pengwei; Cao, Zhang; Li, Jingruo; Guo, Guangcheng; Xia, Wanjun; Gu, Yuanting
miRNAs, sorting as non-coding RNAs, are differentially expressed in breast tumor and act as tumor promoters or suppressors. miR-206 could suppress the progression of breast cancer, the mechanism of which remains unclear. The study here was aimed to investigate the effect of miR-206 on human breast cancers. We found that miR-206 was down-regulated while one of its predicted targets, 6-Phosphofructo-2-kinase (PFKFB3) was up-regulated in human breast carcinomas. 17β-estradiol dose-dependently decreased miR-206 expression as well as enhanced PFKFB3 mRNA and protein expression in estrogen receptor α (ERα) positive breast cancer cells. Furthermore, we identified that miR-206 directly interacted with 3′-untranslated region (UTR) of PFKFB3 mRNA. miR-206 modulated PFKFB3 expression in MCF-7, T47D and SUM159 cells, which was influenced by 17β-estradiol depending on ERα expression. In addition, miR-206 overexpression impeded fructose-2,6-bisphosphate (F2,6BP) production, diminished lactate generation and reduced cell proliferation and migration in breast cancer cells. In conclusion, our study demonstrated that miR-206 regulated PFKFB3 expression in breast cancer cells, thereby stunting glycolysis, cell proliferation and migration. - Highlights: • miR-206 was down-regulated and PFKFB3 was up-regulated in human breast carcinomas. • 17β-estradiol regulated miR-206 and PFKFB3 expression in ERα+ cancer cells. • miR-206directly interacted with 3′-UTR of PFKFB3 mRNA. • miR-206 fructose-2,6-bisphosphate (F2,6BP) impeded production and lactate generation. • miR-206 reduced cell proliferation and migration in breast cancer cells.
Therapeutic strategies and mechanisms of tumorigenesis of HER2-overexpressing breast cancer.
Emde, Anna; Köstler, Wolfgang J; Yarden, Yosef
The receptor tyrosine kinase HER2 is overexpressed in approximately 25% of breast cancers. HER2 acts as a signal amplifier for its siblings, namely three different transmembrane receptors that collectively bind with 11 distinct growth factors of the EGF family. Thus, overexpression of HER2 confers aggressive invasive growth in preclinical models and in patients. Specific therapies targeting HER2 include monoclonal antibodies, antibody-drug conjugates, small molecule tyrosine kinase inhibitors, as well as heat shock protein and sheddase inhibitors. Two of these drugs have shown impressive - yet mostly transient - efficacy in patients with HER2 overexpressing breast cancer. We highlight the biological roles of HER2 in breast cancer progression, and overview the available therapeutic armamentarium directed against this receptor-kinase molecule. Focusing on the mechanisms that confer resistance to individual HER2 targeting agents, we envisage therapeutic approaches to delay or overcome the evolvement of resistance in patients. PMID:20951604
Body mass index and risk of luminal, HER2-overexpressing, and triple negative breast cancer.
Chen, Lu; Cook, Linda S; Tang, Mei-Tzu C; Porter, Peggy L; Hill, Deirdre A; Wiggins, Charles L; Li, Christopher I
Triple negative (TN, tumors that do not express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2)) and HER2-overexpressing (H2E, ER-/HER2+) tumors are two particularly aggressive subtypes of breast cancer. There is a lack of knowledge regarding the etiologies of these cancers and in particular how anthropometric factors are related to risk. We conducted a population-based case-case study consisting of 2659 women aged 20-69 years diagnosed with invasive breast cancer from 2004 to 2012. Four case groups defined based on joint ER/PR/HER2 status were included: TN, H2E, luminal A (ER+/HER2-), and luminal B (ER+/HER2+). Polytomous logistic regression was used to estimate odds ratios (ORs) and associated 95 % confidence intervals (CIs) where luminal A patients served as the reference group. Obese premenopausal women [body mass index (BMI) ≥30 kg/m(2)] had an 82 % (95 % CI 1.32-2.51) increased risk of TN breast cancer compared to women whose BMI <25 kg/m(2), and those in the highest weight quartile (quartiles were categorized based on the distribution among luminal A patients) had a 79 % (95 % CI 1.23-2.64) increased risk of TN disease compared to those in the lowest quartile. Among postmenopausal women obesity was associated with reduced risks of both TN (OR = 0.74, 95 % CI 0.54-1.00) and H2E (OR = 0.47, 95 % CI 0.32-0.69) cancers. Our results suggest obesity has divergent impacts on risk of aggressive subtypes of breast cancer in premenopausal versus postmenopausal women, which may contribute to the higher incidence rates of TN cancers observed among younger African American and Hispanic women. PMID:27220749
Genetic variants in the HER2 gene: Influence on HER2 overexpression and loss of heterozygosity in breast cancer.
Cresti, Nicola; Lee, Joanne; Rourke, Emma; Televantou, Despina; Jamieson, David; Verrill, Mark; Boddy, Alan V
Human epidermal growth factor receptor 2 (HER2) overexpression in breast cancer is an indicator of poor prognosis and is the pre-requisite for treatment with the agents targeting this member of the epidermal growth factor receptor family. In order to determine the influence of these common single-nucleotide polymorphisms (SNPs) in the HER2 gene, genomic DNA was obtained from 361 patients with breast cancer, aged between 29 and 82 years. Samples of tumour tissue were obtained from 241 (66%) patients and material for extraction of DNA is isolated from surrounding normal tissue by laser capture microdissection. Genotyping was performed using the Taqman fluorogenic 5' nuclease assay. Of the 360 patients with definitive determination of HER2 status, 49% were positive. The Ile655Val SNP had no influence on the frequency of HER2 expression. However, the proline allele of the Ala1170Pro SNP was associated with a higher frequency of HER2 overexpression (56% versus 43%, p = 0.015). Where the germline genotype was homozygous, the tumour genotype was identical in every case and for both SNPs. In HER2-positive tumours, heterozygosity was maintained in only 15% and 18% of the Ile655Val and Ala1170Pro SNPs, respectively. This was lower than in the HER2-negative tumours (46% and 43%, respectively). Normal breast tissue (n = 23) retained the germline genotype in all but one case. The underlying link between the Ala1170Pro SNP and HER2 positivity is not known, nor is the significance of HER2 overexpression and loss of heterozygosity in breast cancer. However, these results illustrate the complexity of HER2 genotype and overexpression in this disease. PMID:26773371
EGFR over-expression and activation in high HER2, ER negative breast cancer cell line induces trastuzumab resistance.
Dua, Rajiv; Zhang, Jianhuan; Nhonthachit, Phets; Penuel, Elicia; Petropoulos, Chris; Parry, Gordon
HER2 is gene amplified or over-expressed in 20-25% of breast cancers resulting in elevated HER2 activation. Trastuzumab (Herceptin), a humanized monoclonal antibody, targets activated HER2 and is clinically effective in HER2-over-expressing breast cancers. However, despite prolonged survival, treated breast cancer patients develop resistance. Resistance to trastuzumab occurs upon inactivation of HER2 regulatory proteins or upon up-regulation of alternative receptors. In particular, elevated levels of EGFR, present in estrogen receptor (ER) positive, trastuzumab-resistant BT-474 xenografts caused, a trastuzumab-resistant phenotype (Ritter et al. Clin Cancer Res 13:4909-4919, 2007). However, the role of EGFR in acquired trastuzumab resistance in ER negative cell models is not well defined. In this study, SKBR3 cell line clones expressing EGFR were generated to examine the role of EGFR over-expression on trastuzumab sensitivity in an, ER-negative breast carcinoma cell line. A stable clone, SKBR3/EGFR (clone 4) expressing moderate levels of EGFR remained sensitive to trastuzumab, whereas a stable clone, SKBR3/EGFR (clone 5) expressing high levels of EGFR, became resistant to trastuzumab. Depletion of EGFR by EGFR small-interfering RNAs in the SKBR3/EGFR (clone 5) reversed trastuzumab resistance. However, the SKBR3/EGFR (clone 5) cell line remained sensitive to lapatinib, an EGFR/HER2 inhibitor. Biochemical analysis using co-immunoprecipitation and proximity-based quantitative VeraTag assays demonstrated that high levels of EGFR phosphorylation, EGFR/EGFR homo-dimerization, and EGFR/HER2 hetero-dimerization were present in the trastuzumab-resistant cells. We conclude that EGFR over-expression can mediate trastuzumab resistance in both ER positive and ER negative cells and hypothesize that a threshold level of EGFR, in the absence of autocrine ligand production, is required to induce the resistant phenotype. PMID:19859802
Trastuzumab-targeted gene delivery to Her2-overexpressing breast cancer cells.
Mann, K; Kullberg, M
We describe a novel gene delivery system that specifically targets human epidermal growth factor receptor 2 (Her2)-overexpressing breast cancer cells. The targeting complexes consist of a PEGylated polylysine core that is bound to DNA molecules coding for either green fluorescent protein or shrimp luciferase. The complex is disulfide linked to the monoclonal antibody trastuzumab and to a pore-forming protein, Listeriolysin O (LLO). Trastuzumab is responsible for specific targeting of Her2 receptors and uptake of the gene delivery complex into endosomes of recipient cells, whereas LLO ensures that the DNA molecules are capable of transit from the endosomes into the cytoplasm. Omission of either trastuzumab or LLO from the nanocomplexes results in minimal gene product in targeted cells. Treatment of isogeneic MCF7 and MCF7/Her18 cell lines, differing only in number of Her2 receptors, with the complete gene delivery system results in a 30-fold greater expression of luciferase activity in the Her2-overexpressing MCF7/Her18 cells. Our nanocomplexes are small (150-250 nm), stable to storage, nontoxic and generic in make-up such that any plasmid DNA or antibody specific for cell-surface receptors can be coupled to the PEGylated polylysine core. PMID:27199219
Trastuzumab-targeted gene delivery to Her2-overexpressing breast cancer cells
Mann, K; Kullberg, M
We describe a novel gene delivery system that specifically targets human epidermal growth factor receptor 2 (Her2)-overexpressing breast cancer cells. The targeting complexes consist of a PEGylated polylysine core that is bound to DNA molecules coding for either green fluorescent protein or shrimp luciferase. The complex is disulfide linked to the monoclonal antibody trastuzumab and to a pore-forming protein, Listeriolysin O (LLO). Trastuzumab is responsible for specific targeting of Her2 receptors and uptake of the gene delivery complex into endosomes of recipient cells, whereas LLO ensures that the DNA molecules are capable of transit from the endosomes into the cytoplasm. Omission of either trastuzumab or LLO from the nanocomplexes results in minimal gene product in targeted cells. Treatment of isogeneic MCF7 and MCF7/Her18 cell lines, differing only in number of Her2 receptors, with the complete gene delivery system results in a 30-fold greater expression of luciferase activity in the Her2-overexpressing MCF7/Her18 cells. Our nanocomplexes are small (150–250 nm), stable to storage, nontoxic and generic in make-up such that any plasmid DNA or antibody specific for cell-surface receptors can be coupled to the PEGylated polylysine core. PMID:27199219
Amplification and protein over-expression of the neu/HER-2/c-erbB-2 protooncogene in human breast carcinomas: relationship to loss of gene sequences on chromosome 17, family history and prognosis.
Børresen, A. L.; Ottestad, L.; Gaustad, A.; Andersen, T. I.; Heikkilä, R.; Jahnsen, T.; Tveit, K. M.; Nesland, J. M.
c-erbB-2 gene amplification and protein over-expression were investigated in 89 primary tumours and 24 metastases from Norwegian breast cancer patients. Amplification occurred in 22.5% of the primary tumours and 50% of the metastases. The amplification was negatively correlated to the oestrogen receptor (ER) content in both the primary tumours and the metastases. No significant differences between amplified and non-amplified tumours were observed with regard to node status, clinical stage, tumour size or menopausal status, although correlations of borderline significance were found between node status, clinical stage and high degree of gene amplification. All the amplified tumours were of the invasive ductal type. Follow-up data of patients observed for more than 1 year showed a significantly higher recurrence rate in the c-erbB-2 amplified group. Allele loss of chromosome 17p and of 7q was seen in 55% and 48% of the tumours respectively. No significant correlation was found between these losses and clinico-histological parameters. More than 50% of the tumours with a loss of 17q sequences had an amplification of c-erbB-2 which is located on 17q12-21, indicating that only one of the chromosomes may be involved in the amplification of the c-erbB-2. A trend towards a correlation between loss of 17q and high degree of amplification were found. No correlation was found between positive family history of breast cancer and c-erbB-2 gene amplification, nor loss of 17p or 17q sequences. Our data support the hypothesis that amplification correlates with aggressive tumour behaviour, and thus may be used as a prognostic factor in breast carcinomas. The allele losses on 17p and 17q points to tumour suppressor gene or genes on this chromosome, although not as predisposing genes in families. Images Figure 2 Figure 3 Figure 4 PMID:1977466
Synthesis of folate- pegylated polyester nanoparticles encapsulating ixabepilone for targeting folate receptor overexpressing breast cancer cells.
Siafaka, P; Betsiou, M; Tsolou, A; Angelou, E; Agianian, B; Koffa, M; Chaitidou, S; Karavas, E; Avgoustakis, K; Bikiaris, D
The aim of this study was the preparation of novel polyester nanoparticles based on folic acid (FA)-functionalized poly(ethylene glycol)-poly(propylene succinate) (PEG-PPSu) copolymer and loaded with the new anticancer drug ixabepilone (IXA). These nanoparticles may serve as a more selective (targeted) treatment of breast cancer tumors overexpressing the folate receptor. The synthesized materials were characterized by (1)H-NMR, FTIR, XRD and DSC. The nanoparticles were prepared by a double emulsification and solvent evaporation method and characterized with regard to their morphology by scanning electron microscopy, drug loading with HPLC-UV and size by dynamic light scattering. An average size of 195 nm and satisfactory drug loading efficiency (3.5%) were observed. XRD data indicated that IXA was incorporated into nanoparticles in amorphous form. The nanoparticles exhibited sustained drug release properties in vitro. Based on in vitro cytotoxicity studies, the blank FA-PEG-PPSu nanoparticles were found to be non-toxic to the cells. Fluorescent nanoparticles were prepared by conjugating Rhodanine B to PEG-PPSu, and live cell, fluorescence, confocal microscopy was applied in order to demonstrate the ability of FA-PEG-PPSu nanoparticles to enter into human breast cancer cells expressing the folate receptor. PMID:26543021
PGC-1β regulates HER2-overexpressing breast cancer cells proliferation by metabolic and redox pathways.
Victorino, Vanessa Jacob; Barroso, W A; Assunção, A K M; Cury, V; Jeremias, I C; Petroni, R; Chausse, B; Ariga, S K; Herrera, A C S A; Panis, C; Lima, T M; Souza, H P
Breast cancer is a prevalent neoplastic disease among women worldwide which treatments still present several side effects and resistance. Considering that cancer cells present derangements in their energetic homeostasis, and that peroxisome proliferator-activated receptor- gamma coactivator 1 (PGC-1) is crucial for cellular metabolism and redox signaling, the main objective of this study was to investigate whether there is a relationship between PGC-1 expression, the proliferation of breast cancer cells and the mechanisms involved. We initially assessed PGC-1β expression in complementary DNA (cDNA) from breast tumor of patients bearing luminal A, luminal B, and HER2-overexpressed and triple negative tumors. Our data showed that PGC-1β expression is increased in patients bearing HER2-overexpressing tumors as compared to others subtypes. Using quantitative PCR and immunoblotting, we showed that breast cancer cells with HER2-amplification (SKBR-3) have greater expression of PGC-1β as compared to a non-tumorous breast cell (MCF-10A) and higher proliferation rate. PGC-1β expression was knocked down with short interfering RNA in HER2-overexpressing cells, and cells decreased proliferation. In these PGC-1β-inhibited cells, we found increased citrate synthase activity and no marked changes in mitochondrial respiration. Glycolytic pathway was decreased, characterized by lower intracellular lactate levels. In addition, after PGC-1β knockdown, SKBR-3 cells showed increased reactive oxygen species production, no changes in antioxidant activity, and decreased expression of ERRα, a modulator of metabolism. In conclusion, we show an association of HER2-overexpression and PGC-1β. PGC-1β knockdown impairs HER2-overexpressing cells proliferation acting on ERRα signaling, metabolism, and redox balance. PMID:26602383
The Cooperation between hMena Overexpression and HER2 Signalling in Breast Cancer
Di Modugno, Francesca; Mottolese, Marcella; DeMonte, Lucia; Trono, Paola; Balsamo, Michele; Conidi, Andrea; Melucci, Elisa; Terrenato, Irene; Belleudi, Francesca; Torrisi, Maria Rosaria; Alessio, Massimo; Santoni, Angela; Nisticò, Paola
hMena and the epithelial specific isoform hMena11a are actin cytoskeleton regulatory proteins belonging to the Ena/VASP family. EGF treatment of breast cancer cell lines upregulates hMena/hMena11a expression and phosphorylates hMena11a, suggesting cross-talk between the ErbB receptor family and hMena/hMena11a in breast cancer. The aim of this study was to determine whether the hMena/hMena11a overexpression cooperates with HER-2 signalling, thereby affecting the HER2 mitogenic activity in breast cancer. In a cohort of breast cancer tissue samples a significant correlation among hMena, HER2 overexpression, the proliferation index (high Ki67), and phosphorylated MAPK and AKT was found and among the molecular subtypes the highest frequency of hMena overexpressing tumors was found in the HER2 subtype. From a clinical viewpoint, concomitant overexpression of HER2 and hMena identifies a subgroup of breast cancer patients showing the worst prognosis, indicating that hMena overexpression adds prognostic information to HER2 overexpressing tumors. To identify a functional link between HER2 and hMena, we show here that HER2 transfection in MCF7 cells increased hMena/hMena11a expression and hMena11a phosphorylation. On the other hand, hMena/hMena11a knock-down reduced HER3, AKT and p44/42 MAPK phosphorylation and inhibited the EGF and NRG1-dependent HER2 phosphorylation and cell proliferation. Of functional significance, hMena/hMena11a knock-down reduced the mitogenic activity of EGF and NRG1. Collectively these data provide new insights into the relevance of hMena and hMena11a as downstream effectors of the ErbB receptor family which may represent a novel prognostic indicator in breast cancer progression, helping to stratify patients. PMID:21209853
Kif18A is involved in human breast carcinogenesis.
Zhang, Chunpeng; Zhu, Changjun; Chen, Hongyan; Li, Linwei; Guo, Liping; Jiang, Wei; Lu, Shih Hsin
Microtubule (MT) kinesin motor proteins orchestrate various cellular processes (e.g. mitosis, motility and organelle transportation) and have been implicated in human carcinogenesis. Kif18A, a plus-end directed MT depolymerase kinesin, regulates MT dynamics, chromosome congression and cell division. In this study, we report that Kif18A is overexpressed in human breast cancers and Kif18A overexpression is associated with tumor grade, metastasis and poor survival. Functional analyses reveal that ectopic overexpression of Kif18A results in cell multinucleation, whereas ablation of Kif18A expression significantly inhibits the proliferative capability of breast cancer cells in vitro and in vivo. Inhibition of Kif18A not only affects the critical mitotic function of Kif18A but also decreases cancer cell migration by stabilizing MTs at leading edges and ultimately induces anoikis of cells with inactivation of the phosphatidylinositol 3-kinase-Akt signaling pathway. Together, our results indicate that Kif18A is involved in human breast carcinogenesis and may serve as a potential therapeutic target for human breast cancer. PMID:20595236
Application of intrathecal trastuzumab (Herceptintrade mark) for treatment of meningeal carcinomatosis in HER2-overexpressing metastatic breast cancer.
Stemmler, H J; Schmitt, M; Harbeck, N; Willems, A; Bernhard, H; Lässig, D; Schoenberg, S; Heinemann, V
Leptomeningeal carcinomatosis represents a rare manifestation of metastatic breast cancer (MBC). A 39-year-old female presenting with HER2-overexpressing MBC and suffering from meningeal carcinomatosis was treated with the humanized antibody trastuzumab directed to HER2 by intrathecal administration. The patient was diagnosed with HER2-overexpressing stage III breast cancer in December 2003. In August 2004, the patient developed a singular intracerebral metastasis which was resected by neurosurgery followed by whole-brain radiotherapy. Since MRI and cerebrospinal fluid (CSF) analyses indicated meningeal carcinomatosis, the patient was commenced on trastuzumab (6 mg/kg q3w) and capecitabine (2.500 mg/m2 d1-14, q3w). Prompted by clinical deterioration, 5 repeated doses of intrathecal methotrexate (15 mg/dose) were administered, yet without clinical improvement. There is initial evidence that trastuzumab does not reach an adequate concentration in CSF after intravenous application. Nevertheless, infiltration of trastuzumab into CSF is facilitated under conditions of an impaired blood-brain barrier, as it is known for meningeal carcinomatosis. For patients with leptomeningeal disease, intrathecal application of trastuzumab may provide an interesting therapeutical approach for patients with HER2 overexpressing metastatic breast cancer. Therefore, an Ommaya reservoir for intrathecal treatment with trastuzumab was placed surgically and intrathecal therapy was begun with escalating doses of trastuzumab (5-20 mg), which proved to be effective and well tolerated by the patient. Within 2 weeks after treatment, the patients' condition improved significantly and cell counts in CSF obtained from the Ommaya reservoir remained low for 11 months after first diagnosis of meningeal carcinomatosis when clinical symptoms and MRI indicated progression of meningeal and cerebral disease. PMID:16596213
MACROD2 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers
Mohseni, Morassa; Cidado, Justin; Croessmann, Sarah; Cravero, Karen; Cimino-Mathews, Ashley; Wong, Hong Yuen; Scharpf, Rob; Zabransky, Daniel J.; Abukhdeir, Abde M.; Garay, Joseph P.; Wang, Grace M.; Beaver, Julia A.; Cochran, Rory L.; Blair, Brian G.; Rosen, D. Marc; Erlanger, Bracha; Argani, Pedram; Hurley, Paula J.; Lauring, Josh; Park, Ben Ho
Tamoxifen is effective for treating estrogen receptor-alpha (ER) positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. Here we describe a previously unidentified gene, MACROD2 that confers tamoxifen resistance and estrogen independent growth. We found MACROD2 is amplified and overexpressed in metastatic tamoxifen-resistant tumors. Transgene overexpression of MACROD2 in breast cancer cell lines results in tamoxifen resistance, whereas RNAi-mediated gene knock down reverses this phenotype. MACROD2 overexpression also leads to estrogen independent growth in xenograft assays. Mechanistically, MACROD2 increases p300 binding to estrogen response elements in a subset of ER regulated genes. Primary breast cancers and matched metastases demonstrate MACROD2 expression can change with disease evolution, and increased expression and amplification of MACROD2 in primary tumors is associated with worse overall survival. These studies establish MACROD2 as a key mediator of estrogen independent growth and tamoxifen resistance, as well as a potential novel target for diagnostics and therapy. PMID:25422431
CHL1 is involved in human breast tumorigenesis and progression
He, Li-Hong; Ma, Qin; Shi, Ye-Hui; Ge, Jie; Zhao, Hong-Meng; Li, Shu-Fen; Tong, Zhong-Sheng
Highlights: •CHL1 is down-regulation in breast cancer tissues. •Down-regulation of CHL1 is related to high grade. •Overexpression of CHL1 inhibits breast cancer cell proliferation and invasion in vitro. •CHL1 deficiency induces breast cancer cell proliferation and invasion both in vitro and in vivo. -- Abstract: Neural cell adhesion molecules (CAM) play important roles in the development and regeneration of the nervous system. The L1 family of CAMs is comprised of L1, Close Homolog of L1 (CHL1, L1CAM2), NrCAM, and Neurofascin, which are structurally related trans-membrane proteins in vertebrates. Although the L1CAM has been demonstrated play important role in carcinogenesis and progression, the function of CHL1 in human breast cancer is limited. Here, we found that CHL1 is down-regulated in human breast cancer and related to lower grade. Furthermore, overexpression of CHL1 suppresses proliferation and invasion in MDA-MB-231 cells and knockdown of CHL1 expression results in increased proliferation and invasion in MCF7 cells in vitro. Finally, CHL1 deficiency promotes tumor formation in vivo. Our results may provide a strategy for blocking breast carcinogenesis and progression.
Impact of cyclooxygenase-2 over-expression on the prognosis of breast cancer patients
Güler, Sertaç Ata; Uğurlu, Mustafa Ümit; Kaya, Handan; Şen, Semiha; Nazlı, Yasemin; Güllüoğlu, Bahadır M.
Objective: The aim of this present study was to assess the impact of COX-2 over-expression on breast cancer survival. Material and Methods: Non-metastatic invasive breast cancer patients who received adequate loco-regional and systemic treatments were evaluated. Patients' demographic, clinical, pathologic, and treatment-related and survival data were retrieved from their hospital files. COX-2, estrogen/progesterone receptor (ER/PR), HER-2/neu expression and Ki67 index of the tumors were determined immunohistochemically. As the primary objective, COX-2 positive and negative patients were compared in terms of overall (OS), disease-free (DFS) and breast cancer-specific survival (BCSS). Secondary objectives were to assess the independent prognostic factors for survival. In addition, the correlation of COX-2 expression with conventional prognostic and predictive factors of breast cancer was assessed. Results: Two hundred and seventeen patients who underwent adequate breast cancer treatment between November 2004 and December 2013 were included in the study. The median follow-up was 37 months (range: 5–107). Eighty-one (37%) patients were COX-2 positive. OS, DFS, and BCSS were similar in COX-2 positive and negative patients. Ki67 index and age were significantly correlated with COX-2 expression (r=−0.116; p=0.02; r=0.159; p=0.02). PR expression was found to be the only independent factor for predicting OS, tumor size and molecular subtype classification were found to be the only independent factors for predicting DFS, and PR expression was found to be the only independent factor for predicting BCSS. Conclusion: Among the independent predictive and prognostic factors of breast cancer, COX-2 over-expression was only correlated with Ki67 index and age. PMID:27436928
Geminin Overexpression Promotes Imatinib Sensitive Breast Cancer: A Novel Treatment Approach for Aggressive Breast Cancers, Including a Subset of Triple Negative
Blanchard, Zannel; Mullins, Nicole; Ellipeddi, Pavani; Lage, Janice M.; McKinney, Shawn; El-Etriby, Rana; Zhang, Xu; Isokpehi, Raphael; Hernandez, Brenda; ElShamy, Wael M.
Breast cancer is the second leading cause of cancer-related deaths in women. Triple negative breast cancer (TNBC) is an aggressive subtype that affects 10–25% mostly African American women. TNBC has the poorest prognosis of all subtypes with rapid progression leading to mortality in younger patients. So far, there is no targeted treatment for TNBC. To that end, here we show that c-Abl is one of several tyrosine kinases that phosphorylate and activate geminin's ability to promote TNBC. Analysis of >800 breast tumor samples showed that geminin is overexpressed in ∼50% of all tumors. Although c-Abl is overexpressed in ∼90% of all tumors, it is only nuclear in geminin overexpressing tumors. In geminin-negative tumors, c-Abl is only cytoplasmic. Inhibiting c-Abl expression or activity (using imatinib or nilotinib) prevented geminin Y150 phosphorylation, inactivated the protein, and most importantly converted overexpressed geminin from an oncogene to an apoptosis inducer. In pre-clinical orthotopic breast tumor models, geminin-overexpressing cells developed aneuploid and invasive tumors, which were suppressed when c-Abl expression was blocked. Moreover, established geminin overexpressing orthotopic tumors regressed when treated with imatinib or nilotinib. Our studies support imatinib/nilotonib as a novel treatment option for patients with aggressive breast cancer (including a subset of TNBCs)-overexpressing geminin and nuclear c-Abl. PMID:24789045
relA over-expression reduces tumorigenicity and activates apoptosis in human cancer cells
Ricca, A; Biroccio, A; Trisciuoglio, D; Cippitelli, M; Zupi, G; Bufalo, D Del
We previously demonstrated that bcl-2 over-expression increases the malignant behaviour of the MCF7 ADR human breast cancer cell line and enhances nuclear factor-kappa B (NF-k B) transcriptional activity. Here, we investigated the direct effect of increased NF-k B activity on the tumorigenicity of MCF7 ADR cells by over-expressing the NF-k B subunit relA/p65. Surprisingly, our results demonstrated that over-expression of relA determines a considerable reduction of the tumorigenic ability in nude mice as indicated by the tumour take and the median time of tumour appearance. In vitro studies also evidenced a reduced cell proliferation and the activation of the apoptotic programme after relA over-expression. Apoptosis was associated with the production of reactive oxygen species, and the cleavage of the specific substrate Poly-ADP-ribose-polymerrase. Our data indicate that there is no general role for NF-k B in the regulation of apoptosis and tumorigenicity. In fact, even though inhibiting NF-k B activity has been reported to be lethal to tumour cells, our findings clearly suggest that an over-induction of nuclear NF-k B activity may produce the same effect. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11747334
t-Darpp overexpression in HER2-positive breast cancer confers a survival advantage in lapatinib
Christenson, Jessica L.; Denny, Erin C.; Kane, Susan E.
Drug resistance is a major barrier to successful cancer treatment. For patients with HER2-positive breast cancer who initially respond to therapy, the majority develop resistance within one year of treatment. Patient outcomes could improve significantly if we can find and exploit common mechanisms of acquired resistance to different targeted therapies. Overexpression of t-Darpp, a truncated form of the dual kinase/phosphatase inhibitor Darpp-32, has been linked to acquired resistance to trastuzumab, a front-line therapy for HER2-positive breast cancer. Darpp-32 reverses t-Darpp's effect on trastuzumab resistance. In this study, we examined whether t-Darpp could be involved in resistance to lapatinib, another HER2-targeted therapeutic. Lapatinib-resistant SKBR3 cells (SK/LapR) showed a marked change in the Darpp-32:t-Darpp ratio toward a predominance of t-Darpp. Overexpression of t-Darpp alone was not sufficient to confer lapatinib resistance, but cells that overexpress t-Darpp partially mimicked the molecular resistance phenotype observed in SK/LapR cells exposed to lapatinib. SK/LapR cells failed to down-regulate Survivin and failed to induce BIM accumulation in response to lapatinib; cells overexpressing t-Darpp exhibited only the failed BIM accumulation. t-Darpp knock-down reversed this phenotype. Using a fluorescence-based co-culture system, we found that cells overexpressing t-Darpp formed colonies in lapatinib within 3–4 weeks, whereas parental cells in the same co-culture did not. Overall, t-Darpp appears to mediate a survival advantage in lapatinib, possibly linked to failed lapatinib-induced BIM accumulation. t-Darpp might also be relevant to acquired resistance to other cancer drugs that rely on BIM accumulation to induce apoptosis. PMID:26430732
MiR-506 Over-Expression Inhibits Proliferation and Metastasis of Breast Cancer Cells
Yu, Fei; Lv, Mingli; Li, Dan; Cai, Haidong; Ma, Lishui; Luo, Qiong; Yuan, Xueyu; Lv, Zhongwei
Background This study aimed to investigate the relationship between miR-506 and proliferation and migration of breast cancer cells. Material/Methods MiR-506 mimics, inhibitor, and negative control (NC) were transfected into MDA-MB-231 breast cancer cells. Cell proliferation, cell counting, colony formation assay, and Transwell assay were applied to evaluate the proliferation and migration of breast cancer cells. Data are shown as mean ± standard deviation and the experiment was performed 3 times. Statistical analyses were performed with SPSS version 10.0. Results At 1 day after transfection, cell proliferation detected by CCK-8 assay was significantly promoted in miR-506 inhibitor when compared with the miR-506 mimics group and the NC group (P<0.05). At 3 days or 5 days after transfection, cell proliferation was markedly inhibited in the miR-506 mimics group, and miR-506 inhibitor was still significantly promoted. Cell counting with a hemocytometer showed similar results to cell proliferation. Colony formation assay showed that the number of colonies in the miR-506 mimics group was significantly smaller than that in the miR-506 inhibitor group and NC group. Transwell assay revealed that the number of migrated cells in miR-506 mimics was markedly smaller than that in the miR-506 inhibitor group and NC group. Conclusions MiR-506 over-expression significantly inhibits the proliferation, colony formation, and migration of breast cancer cells. miR-506 over-expression may thus be able to improve the malignant phenotype of breast cancer cells. PMID:26059632
Cooperation between Dmp1 Loss and Cyclin D1 Overexpression in Breast Cancer
Zhu, Sinan; Mott, Ryan T.; Fry, Elizabeth A.; Taneja, Pankaj; Kulik, George; Sui, Guangchao; Inoue, Kazushi
Cyclin D1 is a component of the core cell-cycle machinery and is frequently overexpressed in breast cancer. It physically interacts with the tumor suppressor Dmp1 that attenuates the oncogenic signals from Ras and HER2 by inducing Arf/p53-dependent cell-cycle arrest. Currently, the biological significance of Dmp1–cyclin D1 interplay in breast cancer has not been determined. Here, we show that cyclin D1 bound to Dmp1 to activate both Arf and Ink4a promoters and, consequently, induced apoptosis or G2/M cell-cycle delay in normal cells to protect them from neoplastic transformation. The cyclin D1–induced Ink4a/Arf gene expression was dependent on Dmp1 because the induction was not detected in Dmp1-deficient or DMP1-depleted cells. Arf/Ink4a expression was increased in pre-malignant mammary glands from Dmp1+/+;MMTV-cyclin D1 and Dmp1+/+;MMTV-D1T286A mice but significantly down-regulated in those from Dmp1-deficient mice. Selective Dmp1 deletion was found in 21% of the MMTV-D1 and D1T286A mammary carcinomas, and the Dmp1 heterozygous status significantly accelerated mouse mammary tumorigenesis with reduced apoptosis and increased metastasis. Overall, our study reveals a pivotal role of combined Dmp1 loss and cyclin D1 overexpression in breast cancer. PMID:23938323
[Overexpression of IL-8 promotes migration of BT549 breast cancer cells].
Deng, Fang; Wang, Jing; Fan, Mengtian; Guo, Yangliu; Li, Ya; Shi, Qiong
Objective To construct a recombinant adenovirus vector containing IL-8 gene and observe its effect on the proliferation, cell cycle and migration of BT549 breast cancer cells. Methods IL-8 gene was amplified by PCR using the cDNA from 143B bone sarcoma cells and inserted into shuttle plasmid pAdTrack-TO4. The recombinant shuttle plasmid pAdTrack-TO4-IL-8 was digested by PmeI and then transformed to AdEasier competent cells. The obtained recombinant adenovirus plasmid pAdIL-8 was digested by PacI, and then transfected to HEK293 cells for package and amplification by Lipofectamine(TM) 2000. The titer was tested by dilution assay. The expression of IL-8 mRNA and protein in BT549 cells was detected by reverse transcription PCR and ELISA, respectively. Effect of IL-8 overexpression on proliferation, cell cycle and migration in BT549 cells was respectively investigated by MTT assay, flow cytometry and wound-healing test. Results PCR and DNA sequence analysis verified the recombinant shuttle plasmid pAdTrack-TO4-IL-8. Restriction enzymes PacI confirmed the recombinant adenovirus plasmid pAdIL-8. IL-8 was overexpressed in BT549 cells after AdIL-8 infection. Overexpression of IL-8 promoted BT549 cell migration and arrested the cell cycle in the S phase, but it made no significant difference in the proliferation of BT549 cells. Conclusion IL-8 overexpression can promote migration of BT549 breast cancer cells. PMID:27126933
Downregulation of GLUT4 contributes to effective intervention of estrogen receptor-negative/HER2-overexpressing early stage breast disease progression by lapatinib.
Acharya, Sunil; Xu, Jia; Wang, Xiao; Jain, Shalini; Wang, Hai; Zhang, Qingling; Chang, Chia-Chi; Bower, Joseph; Arun, Banu; Seewaldt, Victoria; Yu, Dihua
Tamoxifen and aromatase inhibitors (AIs) have shown efficacy in prevention of estrogen receptor-positive (ER+) breast cancer; however, there exists no proven prevention strategy for estrogen receptor-negative (ER-) breast cancer. Up to 40% of ER- breast cancers have human epidermal growth factor receptor 2 overexpression (HER2+), suggesting HER2 signaling might be a good target for chemoprevention for certain ER- breast cancers. Here, we tested the feasibility of the HER2-targeting agent lapatinib in prevention and/or early intervention of an ER-/HER2+ early-stage breast disease model. We found that lapatinib treatment forestalled the progression of atypical ductal hyperplasia (ADH)-like acini to ductal carcinoma in situ (DCIS)-like acini in ER-/HER2+ human mammary epithelial cells (HMECs) in 3D culture. Mechanistically, we found that inhibition of HER2/Akt signaling by lapatinib led to downregulation of GLUT4 and a reduced glucose uptake in HER2-overexpressing cells, resulting in decreased proliferation and increased apoptosis of these cells in 3D culture. Additionally, our data suggest that HER2-driven glycolytic metabolic dysregulation in ER-/HER2+ HMECs might promote early-stage breast disease progression, which can be reversed by lapatinib treatment. Furthermore, low-dose lapatinib treatment, starting at the early stages of mammary grand transformation in the MMTV-neu* mouse model, significantly delayed mammary tumor initiation and progression, extended tumor-free survival, which corresponded to effective inhibition of HER2/Akt signaling and downregulation of GLUT4 in vivo. Taken together, our results indicate that lapatinib, through its inhibition of key signaling pathways and tumor-promoting metabolic events, is a promising agent for the prevention/early intervention of ER-/HER2+ breast cancer progression. PMID:27293993
Downregulation of GLUT4 contributes to effective intervention of estrogen receptor-negative/HER2-overexpressing early stage breast disease progression by lapatinib
Acharya, Sunil; Xu, Jia; Wang, Xiao; Jain, Shalini; Wang, Hai; Zhang, Qingling; Chang, Chia-Chi; Bower, Joseph; Arun, Banu; Seewaldt, Victoria; Yu, Dihua
Tamoxifen and aromatase inhibitors (AIs) have shown efficacy in prevention of estrogen receptor-positive (ER+) breast cancer; however, there exists no proven prevention strategy for estrogen receptor-negative (ER-) breast cancer. Up to 40% of ER- breast cancers have human epidermal growth factor receptor 2 overexpression (HER2+), suggesting HER2 signaling might be a good target for chemoprevention for certain ER- breast cancers. Here, we tested the feasibility of the HER2-targeting agent lapatinib in prevention and/or early intervention of an ER-/HER2+ early-stage breast disease model. We found that lapatinib treatment forestalled the progression of atypical ductal hyperplasia (ADH)-like acini to ductal carcinoma in situ (DCIS)-like acini in ER-/HER2+ human mammary epithelial cells (HMECs) in 3D culture. Mechanistically, we found that inhibition of HER2/Akt signaling by lapatinib led to downregulation of GLUT4 and a reduced glucose uptake in HER2-overexpressing cells, resulting in decreased proliferation and increased apoptosis of these cells in 3D culture. Additionally, our data suggest that HER2-driven glycolytic metabolic dysregulation in ER-/HER2+ HMECs might promote early-stage breast disease progression, which can be reversed by lapatinib treatment. Furthermore, low-dose lapatinib treatment, starting at the early stages of mammary grand transformation in the MMTV-neu* mouse model, significantly delayed mammary tumor initiation and progression, extended tumor-free survival, which corresponded to effective inhibition of HER2/Akt signaling and downregulation of GLUT4 in vivo. Taken together, our results indicate that lapatinib, through its inhibition of key signaling pathways and tumor-promoting metabolic events, is a promising agent for the prevention/early intervention of ER-/HER2+ breast cancer progression. PMID:27293993
hMENA(11a) contributes to HER3-mediated resistance to PI3K inhibitors in HER2-overexpressing breast cancer cells.
Trono, P; Di Modugno, F; Circo, R; Spada, S; Di Benedetto, A; Melchionna, R; Palermo, B; Matteoni, S; Soddu, S; Mottolese, M; De Maria, R; Nisticò, P
Human Mena (hMENA), an actin regulatory protein of the ENA/VASP family, cooperates with ErbB receptor family signaling in breast cancer. It is overexpressed in high-risk preneoplastic lesions and in primary breast tumors where it correlates with HER2 overexpression and an activated status of AKT and MAPK. The concomitant overexpression of hMENA and HER2 in breast cancer patients is indicative of a worse prognosis. hMENA is expressed along with alternatively expressed isoforms, hMENA(11a) and hMENAΔv6 with opposite functions. A novel role for the epithelial-associated hMENA(11a) isoform in sustaining HER3 activation and pro-survival pathways in HER2-overexpressing breast cancer cells has been identified by reverse phase protein array and validated in vivo in a series of breast cancer tissues. As HER3 activation is crucial in mechanisms of cell resistance to PI3K inhibitors, we explored whether hMENA(11a) is involved in these resistance mechanisms. The specific hMENA(11a) depletion switched off the HER3-related pathway activated by PI3K inhibitors and impaired the nuclear accumulation of HER3 transcription factor FOXO3a induced by PI3K inhibitors, whereas PI3K inhibitors activated hMENA(11a) phosphorylation and affected its localization. At the functional level, we found that hMENA(11a) sustains cell proliferation and survival in response to PI3K inhibitor treatment, whereas hMENA(11a) silencing increases molecules involved in cancer cell apoptosis. As shown in three-dimensional cultures, hMENA(11a) contributes to resistance to PI3K inhibition because its depletion drastically reduced cell viability upon treatment with PI3K inhibitor BEZ235. Altogether, these results indicate that hMENA(11a) in HER2-overexpressing breast cancer cells sustains HER3/AKT axis activation and contributes to HER3-mediated resistance mechanisms to PI3K inhibitors. Thus, hMENA(11a) expression can be proposed as a marker of HER3 activation and resistance to PI3K inhibition therapies, to
Hepatic steatosis in transgenic mice overexpressing human histone deacetylase 1
Wang, Ai-Guo; Seo, Sang-Beom; Moon, Hyung-Bae; Shin, Hye-Jun; Kim, Dong Hoon; Kim, Jin-Man; Lee, Tae-Hoon; Kwon, Ho Jeong; Yu, Dae-Yeul . E-mail: firstname.lastname@example.com; Lee, Dong-Seok . E-mail: email@example.com
It is generally thought that histone deacetylases (HDACs) play important roles in the transcriptional regulation of genes. However, little information is available concerning the specific functions of individual HDACs in disease states. In this study, two transgenic mice lines were established which harbored the human HDAC1 gene. Overexpressed HDAC1 was detected in the nuclei of transgenic liver cells, and HDAC1 enzymatic activity was significantly higher in the transgenic mice than in control littermates. The HDAC1 transgenic mice exhibited a high incidence of hepatic steatosis and nuclear pleomorphism. Molecular studies showed that HDAC1 may contribute to nuclear pleomorphism through the p53/p21 signaling pathway.
Ethanol Enhances the Interaction of Breast Cancer Cells Over-Expressing ErbB2 With Fibronectin
Xu, Mei; Bower, Kimberly A.; Chen, Gang; Shi, Xianglin; Dong, Zheng; Ke, Zunji; Luo, Jia
Background Ethanol is a tumor promoter and may enhance the metastasis of breast cancer. However, the underlying cellular / molecular mechanisms remain unknown. Amplification of ErbB2 or HER2, a receptor tyrosine kinase of the ErbB family, is found in 20 to 30% of patients with breast cancer. We have previously demonstrated that the effect of ethanol on the migration / invasion of breast cancer cells positively correlated with the expression levels of ErbB2. Adhesion to the extracellular matrix (ECM) is an important initial step for cancer cell invasion and metastasis. In this study, we investigated the effects of ethanol on the adhesion of MCF7 breast cancer cells over-expressing ErbB2 (MCF7ErbB2) to human plasma fibronectin. Methods To test the hypothesis that ethanol may enhance the attachment of human breast cancer cells to fibronectin, an important component of the ECM, we evaluated the effect of ethanol on the expression of focal adhesions, cell attachment, and ErbB2 signaling in cultured MCF7ErbB2 cells. Results Exposure to ethanol drastically enhanced the adhesion of MCFErbB2 cells to fibronectin and increased the expression of focal adhesions. Ethanol induced phosphorylation of ErbB2 at Tyr1248, FAK at Tyr861, and cSrc at Try216. Ethanol promoted the interaction among ErbB2, FAK, and cSrc, and the formation of a focal complex. AG825, a selective ErbB2 inhibitor, attenuated the ethanol-induced phosphorylation of ErbB2 and its association with FAK. Furthermore, AG825 blocked ethanol-promoted cell / fibronectin adhesion as well as the expression of focal adhesions. Conclusions Our results suggest that ethanol enhances the adhesion of breast cancer cells to fibronectin in an ErbB2-dependent manner, and the FAK pathway plays an important role in ethanol-induced formation of a focal complex. PMID:20201928
Statins affect ETS1-overexpressing triple-negative breast cancer cells by restoring DUSP4 deficiency.
Jung, Hae Hyun; Lee, Soo-Hyeon; Kim, Ji-Yeon; Ahn, Jin Seok; Park, Yeon Hee; Im, Young-Hyuck
We investigated the molecular mechanisms underlying statin-induced growth suppression of triple-negative breast cancer (TNBC) that overexpress the transcription factor ets proto-oncogene 1(ets-1) and downregulate dual specific protein phosphatase 4(dusp4) expression. We examined the gene expression of BC cell lines using the nCounter expression assay, MTT viability assay, cell proliferation assay and Western blot to evaluate the effects of simvastatin. Finally, we performed cell viability testing in TNBC cell line-transfected DUSP4. We demonstrated that ETS1 mRNA and protein were overexpressed in TNBC cells compared with other BC cell lines (P = <0.001) and DUSP4 mRNA was downregulated (P = <0.001). MTT viability assay showed that simvastatin had significant antitumor activity (P = 0.002 in 0.1 μM). In addition, simvastatin could restore dusp4 deficiency and suppress ets-1 expression in TNBC. Lastly, we found that si-DUSP4 RNA transfection overcame the antitumor activity of statins. MAPK pathway inhibitor, U0126 and PI3KCA inhibitor LY294002 also decreased levels of ets-1, phosphor-ERK and phosphor-AKT on Western blot assay. Accordingly, our study indicates that simvastatin potentially affects the activity of transcriptional factors such as ets-1 and dusp4 through the MAPK pathway. In conclusion, statins might be potential candidates for TNBC therapy reducing ets-1 expression via overexpression of dusp4. PMID:27604655
Statins affect ETS1-overexpressing triple-negative breast cancer cells by restoring DUSP4 deficiency
Jung, Hae Hyun; Lee, Soo-Hyeon; Kim, Ji-Yeon; Ahn, Jin Seok; Park, Yeon Hee; Im, Young-Hyuck
We investigated the molecular mechanisms underlying statin-induced growth suppression of triple-negative breast cancer (TNBC) that overexpress the transcription factor ets proto-oncogene 1(ets-1) and downregulate dual specific protein phosphatase 4(dusp4) expression. We examined the gene expression of BC cell lines using the nCounter expression assay, MTT viability assay, cell proliferation assay and Western blot to evaluate the effects of simvastatin. Finally, we performed cell viability testing in TNBC cell line-transfected DUSP4. We demonstrated that ETS1 mRNA and protein were overexpressed in TNBC cells compared with other BC cell lines (P = <0.001) and DUSP4 mRNA was downregulated (P = <0.001). MTT viability assay showed that simvastatin had significant antitumor activity (P = 0.002 in 0.1 μM). In addition, simvastatin could restore dusp4 deficiency and suppress ets-1 expression in TNBC. Lastly, we found that si-DUSP4 RNA transfection overcame the antitumor activity of statins. MAPK pathway inhibitor, U0126 and PI3KCA inhibitor LY294002 also decreased levels of ets-1, phosphor-ERK and phosphor-AKT on Western blot assay. Accordingly, our study indicates that simvastatin potentially affects the activity of transcriptional factors such as ets-1 and dusp4 through the MAPK pathway. In conclusion, statins might be potential candidates for TNBC therapy reducing ets-1 expression via overexpression of dusp4. PMID:27604655
Enhanced exposure of phosphatidylserine in human gastric carcinoma cells overexpressing the half-size ABC transporter BCRP (ABCG2).
Woehlecke, Holger; Pohl, Antje; Alder-Baerens, Nele; Lage, Hermann; Herrmann, Andreas
Members of the ABC (ATP-binding cassette) transporter super-family are emerging to be involved in lipid transport. In the present study, we studied the organization of phospholipids in the plasma membrane of EPG85-257 human gastric carcinoma cells overexpressing BCRP (breast cancer resistance protein, ABCG-2), a half-size transporter belonging to the ABCG subfamily. A significantly increased plasma membrane association of the PS (phosphatidylserine)-binding probe FITC-Annexin V in comparison with control cells was observed. Treatment of BCRP -overexpressing cells with the inhibitor Tryprostatin A decreased FITC-Annexin V binding almost to the control level. This suggests an enhanced exposure of PS in BCRP -overexpressing cells, which is dependent on functional BCRP. A role of BCRP in the transverse distribution of lipids in the plasma membrane is supported by the increased outward transport of the lipid analogue C6- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PS in BCRP -overexpressing EPG85-257 cells and MCF-7 human breast cancer cells. As shown for BCRP -overexpressing EPG85-257 cells, enhanced outward redistribution of the lipid analogue is inhibited by Tryprostatin A as well as by reduction of BCRP expression on transfection with an anti- BCRP -ribozyme. We also observed an enhanced outward transport of C6- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phosphatidylcholine in BCRP -overexpressing EPG85-257 cells, suggesting that the influence of BCRP on transverse lipid organization is not highly specific. PMID:12946267
High prevalence of HER-2/neu overexpression in female breast cancer among an Iraqi population exposed to depleted uranium
AL-Dujaily, Esraa A.; Al-Janabi, Asad A.; Pierscionek, Tomasz
Background This study aimed to estimate the rate of HER-2/neu (c-erbB2) immunohistochemical overexpression in different histological types of breast cancer found in the middle Euphrates region of Iraq, a region that was exposed to high levels of depleted uranium. HER-2/neu (c-erbB2) overexpression was correlated with common clinicopathological parameters such as age, grade, stage, tumor size and lymph node involvement to determine if any particular biomarker for exposure to depleted uranium could be found in the tumor samples from this region. Materials and Methods The present investigation was performed over a period starting from September 2007 to June 2008. Formalin-fixed, paraffin-embedded blocks from 90 patients with breast cancer were included in this study. A group of 25 patients with benign breast lesions (fibroadenoma) was included as a comparative group, and 20 breast tissue sections were used as controls. Labeled streptavidin-biotin (LSAB) complex method was employed for immunohistochemical detection of HER-2/neu. Results HER-2/neu immuno-expression was positive in 67.8% of breast cancer, while it was negative in all benign breast lesions (fibroadenoma) (P < 0.05). HER-2/neu immunostaining was significantly associated with histological type and recurrence of breast cancer (P < 0.05). It was positively correlated with tumor grade, but this finding was not significant (P > 0.05). Conclusion Based upon the findings of this study, it can be concluded that HER-2/neu overexpression plays an important role in the pathogenesis of breast cancer and is associated with a worse prognosis. The findings indicate that in regions exposed to high levels of depleted uranium, HER-2/neu overexpression is high, but its correlation with age, grade, stage, tumor size, and lymph node involvement is similar to studies that have been conducted on populations not exposed to depleted uranium. PMID:19008567
Adoptive transfer of autologous, HER2-specific, cytotoxic T lymphocytes for the treatment of HER2-overexpressing breast cancer.
Bernhard, Helga; Neudorfer, Julia; Gebhard, Kerstin; Conrad, Heinke; Hermann, Christine; Nährig, Jörg; Fend, Falko; Weber, Wolfgang; Busch, Dirk H; Peschel, Christian
The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated antigen by immunotherapeutical approaches based on HER2-directed monoclonal antibodies and cancer vaccines. We describe the adoptive transfer of autologous HER2-specific T-lymphocyte clones to a patient with metastatic HER2-overexpressing breast cancer. The HLA/multimer-based monitoring of the transferred T lymphocytes revealed that the T cells rapidly disappeared from the peripheral blood. The imaging studies indicated that the T cells accumulated in the bone marrow (BM) and migrated to the liver, but were unable to penetrate into the solid metastases. The disseminated tumor cells in the BM disappeared after the completion of adoptive T-cell therapy. This study suggests the therapeutic potential for HER2-specific T cells for eliminating disseminated HER2-positive tumor cells and proposes the combination of T cell-based therapies with strategies targeting the tumor stroma to improve T-cell infiltration into solid tumors. PMID:17646988
Focal adhesion kinase overexpression and its impact on human osteosarcoma
Chen, Yong; Yang, Aizhen; Chen, Hui; Zhang, Jian; Wu, Sujia; Shi, Xin; Wang, Chen; Sun, Xiaoliang
Focal adhesion kinase (FAK) has been implicated in tumorigenesis in various malignancies. We sought to examine the expression patterns of FAK and the activated form, phosphorylated FAK (pFAK), in human osteosarcoma and to investigate the correlation of FAK expression with clinicopathologic parameters and prognosis. In addition, the functional consequence of manipulating the FAK protein level was investigated in human osteosarcoma cell lines. Immunohistochemical staining was used to detect FAK and pFAK in pathologic archived materials from 113 patients with primary osteosarcoma. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognoses. The role of FAK in the cytological behavior of MG63 and 143B human osteosarcoma cell lines was studied via FAK protein knock down with siRNA. Cell proliferation, migration, invasiveness and apoptosis were assessed using the CCK8, Transwell and Annexin V/PI staining methods. Both FAK and pFAK were overexpressed in osteosarcoma. There were significant differences in overall survival between the FAK-/pFAK- and FAK+/pFAK- groups (P = 0.016), the FAK+/pFAK- and FAK+/pFAK+ groups (P = 0.012) and the FAK-/pFAK- and FAK+/pFAK+ groups (P < 0.001). There were similar differences in metastasis-free survival between groups. The Cox proportional hazards analysis showed that the FAK expression profile was an independent indicator of both overall and metastasis-free survival. siRNA-based knockdown of FAK not only dramatically reduced the migration and invasion of MG63 and 143B cells, but also had a distinct effect on osteosarcoma cell proliferation and apoptosis. These results collectively suggest that FAK overexpression and phosphorylation might predict more aggressive biologic behavior in osteosarcoma and may be an independent predictor of poor prognosis. PMID:26393679
Frequent methylation of the KLOTHO gene and overexpression of the FGFR4 receptor in invasive ductal carcinoma of the breast.
Dallol, Ashraf; Buhmeida, Abdelbaset; Merdad, Adnan; Al-Maghrabi, Jaudah; Gari, Mamdooh A; Abu-Elmagd, Muhammad M; Elaimi, Aisha; Assidi, Mourad; Chaudhary, Adeel G; Abuzenadah, Adel M; Nedjadi, Taoufik; Ermiah, Eramah; Alkhayyat, Shadi S; Al-Qahtani, Mohammed H
Invasive ductal carcinoma of the breast is the most common cancer affecting women worldwide. The marked heterogeneity of breast cancer is matched only with the heterogeneity in its associated or causative factors. Breast cancer in Saudi Arabia is apparently an early onset with many of the affected females diagnosed before they reach the age of 50 years. One possible rationale underlying this observation is that consanguinity, which is widely spread in the Saudi community, is causing the accumulation of yet undetermined cancer susceptibility mutations. Another factor could be the accumulation of epigenetic aberrations caused by the shift toward a Western-like lifestyle in the past two decades. In order to shed some light into the molecular mechanisms underlying breast cancer in the Saudi community, we identified KLOTHO (KL) as a tumor-specific methylated gene using genome-wide methylation analysis of primary breast tumors utilizing the MBD-seq approach. KL methylation was frequent as it was detected in 55.3 % of breast cancer cases from Saudi Arabia (n = 179) using MethyLight assay. Furthermore, KL is downregulated in breast tumors with its expression induced following treatment with 5-azacytidine. The involvement of KL in breast cancer led us to investigate its relationship in the context of breast cancer, with one of the protagonists of its function, fibroblast growth factor receptor 4 (FGFR4). Overexpression of FGFR4 in breast cancer is frequent in our cohort and this overexpression is associated with poor overall survival. Interestingly, FGFR4 expression is higher in the absence of KL methylation and lower when KL is methylated and presumably silenced, which is suggestive of an intricate relationship between the two factors. In conclusion, our findings further implicate "metabolic" genes or pathways in breast cancer that are disrupted by epigenetic mechanisms and could provide new avenues for understanding this disease in a new context. PMID:26152288
Signaling Pathway of GP88 (Progranulin) in Breast Cancer Cells: Upregulation and Phosphorylation of c-myc by GP88/Progranulin in Her2-Overexpressing Breast Cancer Cells
Kim, Wes E.; Yue, Binbin; Serrero, Ginette
Her2 is a receptor tyrosine kinase overexpressed in 25% of breast tumors. We have shown that the 88 kDa autocrine growth and survival factor GP88 (progranulin) stimulated Her2 phosphorylation and proliferation and conferred Herceptin resistance in Her2-overexpressing cells. Herein, we report that GP88 stimulates c-myc phosphorylation and upregulates c-myc levels in Her2-overexpressing cells. c-myc phosphorylation and upregulation by GP88 were not observed in non-Her2-overexpressing breast cancer cells. c-myc activation was inhibited upon treatment with ERK, PI3 kinase, and c-src pathway inhibitors, U0126, LY294002, and PP2. GP88 also stimulated c-src phosphorylation, a known upstream regulator of c-myc. Thus, we describe here a signaling pathway for GP88 in Her2-overexpressing cells, with GP88 stimulating Src phosphorylation, followed by phosphorylation and upregulation of c-myc. These data would suggest that targeting GP88 could provide a novel treatment approach in breast cancer. PMID:27168723
The SH2 domain protein GRB-7 is co-amplified, overexpressed and in a tight complex with HER2 in breast cancer.
Stein, D; Wu, J; Fuqua, S A; Roonprapunt, C; Yajnik, V; D'Eustachio, P; Moskow, J J; Buchberg, A M; Osborne, C K; Margolis, B
SH2 domain proteins are important components of the signal transduction pathways activated by growth factor receptor tyrosine kinases. We have been cloning SH2 domain proteins by bacterial expression cloning using the tyrosine phosphorylated C-terminus of the epidermal growth factor receptor as a probe. One of these newly cloned SH2 domain proteins, GRB-7, was mapped on mouse chromosome 11 to a region which also contains the tyrosine kinase receptor, HER2/erbB-2. The analogous chromosomal locus in man is often amplified in human breast cancer leading to overexpression of HER2. We find that GRB-7 is amplified in concert with HER2 in several breast cancer cell lines and that GRB-7 is overexpressed in both cell lines and breast tumors. GRB-7, through its SH2 domain, binds tightly to HER2 such that a large fraction of the tyrosine phosphorylated HER2 in SKBR-3 cells is bound to GRB-7. GRB-7 can also bind tyrosine phosphorylated SHC, albeit at a lower affinity than GRB2 binds SHC. We also find that GRB-7 has a strong similarity over > 300 amino acids to a newly identified gene in Caenorhabditis elegans. This region of similarity, which lies outside the SH2 domain, also contains a pleckstrin homology domain. The presence of evolutionarily conserved domains indicates that GRB-7 is likely to perform a basic signaling function. The fact that GRB-7 and HER2 are both overexpressed and bound tightly together suggests that this basic signaling pathway is greatly amplified in certain breast cancers. PMID:7907978
Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells.
Fang, Huafeng; Yue, Xuan; Li, Xiaoxu; Taylor, John-Stephen
We have recently shown that an MCF-7 tumor can be imaged in a mouse by PET with 64Cu-labeled Peptide nucleic acids (PNAs) tethered to the permeation peptide Lys4 that recognize the uniquely overexpressed and very abundant upstream of N-ras or N-ras related gene (unr mRNA) expressed in these cells. Herein we describe how the high affinity antisense PNAs to the unr mRNA were identified and characterized. First, antisense binding sites on the unr mRNA were mapped by an reverse transcriptase random oligonucleotide library (RT-ROL) method that we have improved, and by a serial analysis of antisense binding sites (SAABS) method that we have developed which is similar to another recently described method. The relative binding affinities of oligodeoxynucleotides (ODNs) complementary to the antisense binding sites were then qualitatively ranked by a new Dynabead-based dot blot assay. Dissociation constants for a subset of the ODNs were determined by a new Dynabead-based solution assay and were found to be 300 pM for the best binders in 1 M salt. PNAs corresponding to the ODNs with the highest affinities were synthesized with an N-terminal CysTyr and C-terminal Lys4 sequence. Dissociation constants of these hybrid PNAs were determined by the Dynabead-based solution assay to be about 10 pM for the highest affinity binders. PMID:16314303
Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris
NASA Technical Reports Server (NTRS)
Karr, Laurel; Malone, Christine, C.; Rose, M. Franklin (Technical Monitor)
The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.
Human cancers overexpress genes that are specific to a variety of normal human tissues
Lotem, Joseph; Netanely, Dvir; Domany, Eytan; Sachs, Leo
We have analyzed gene expression data from three different kinds of samples: normal human tissues, human cancer cell lines, and leukemic cells from lymphoid and myeloid leukemia pediatric patients. We have searched for genes that are overexpressed in human cancer and also show specific patterns of tissue-dependent expression in normal tissues. Using the expression data of the normal tissues, we identified 4,346 genes with a high variability of expression and clustered these genes according to their relative expression level. Of 91 stable clusters obtained, 24 clusters included genes preferentially expressed either only in hematopoietic tissues or in hematopoietic and one to two other tissues; 28 clusters included genes preferentially expressed in various nonhematopoietic tissues such as neuronal, testis, liver, kidney, muscle, lung, pancreas, and placenta. Analysis of the expression levels of these two groups of genes in the human cancer cell lines and leukemias identified genes that were highly expressed in cancer cells but not in their normal counterparts and, thus, were overexpressed in the cancers. The different cancer cell lines and leukemias varied in the number and identity of these overexpressed genes. The results indicate that many genes that are overexpressed in human cancer cells are specific to a variety of normal tissues, including normal tissues other than those from which the cancer originated. It is suggested that this general property of cancer cells plays a major role in determining the behavior of the cancers, including their metastatic potential. PMID:16339305
Claudin-20 promotes an aggressive phenotype in human breast cancer cells
Martin, Tracey A; Lane, Jane; Ozupek, Hulya; Jiang, Wen G
Claudin-20 is a member of the Claudin family of transmembrane proteins located in the tight junction (TJ) of cells of epithelial origin. Due to the increasing evidence supporting the role of TJ proteins in preventing tumor cell metastatic behavior, this study sought to evaluate the distribution of Claudin-20 in human breast cancer and the effect of Claudin-20 overexpression in human breast cancer cells. Q-PCR data from breast cancer primary tumors (n = 114) and matched background tissue (n = 30) showed that high claudin-20 expression was correlated with poor survival of patients with breast cancer (p = 0.022). Following transformation of the breast cancer cell lines MDA-MB-231 and MCF7 with a Claudin-20 expression construct functional assays were performed to ascertain changes in cell behavior. Claudin-20 transformed cells showed significantly increased invasion (p < 0.005) and were significantly less adhesive than wild type cells (p < 0.05). There was no effect on growth (either in vitro or in vivo) for either cell line. Overexpression of Claudin-20 resulted in reduced transepithelial resistance (induced by the motogen HGF at 25 ng/ml, p = 0.0007). Interestingly, this was not mirrored by paracellular permeability, as overexpression of Claudin-20 caused a decrease in permeability. The introduction of Claudin-20 into human breast cancer cells resulted in breast cancer cells with an aggressive phenotype and reduced trans-epithelial resistance. There was no corresponding decrease in paracellular permeability, indicating that this Claudin has a differential function in epithelial TJ. This provides further insight into the importance of correctly functioning TJ in preventing the progression of human breast cancer. PMID:24665404
Induction of breast cancer in wild type p53 cells by BRCA1-IRIS overexpression.
Elshamy, Wael M
Cells ability to evade cell death and to proliferate post geno-/cell-toxic stresses, likely leads to formation of cancer. Activation of p38MAPK and p53 following these stresses help protect cells against cancer development by initiating apoptosis. The duration of p38MAPK and p53 activation is regulated by the WIP1 phosphatase. BRCA1-IRIS triggers WIP1 expression in p53-dependent and -independent manner. BRCA1-IRIS triggers the expression and cytoplasmic localization of the mRNA stabilization and translation inducer, HuR that binds p53 and PPM1D mRNA. Hence, BRCA1-IRIS overexpression inactivates p38MAPK and/or p53 by upregulating WIP1 expression. BRCA1-IRIS abrogation of the homeostatic balance maintained by p38MAPK-p53-WIP1 pathway suppressed cell death induced by a lethal dose of UVC, high dosages of etoposide or H2O2, and allowed cells to survive and proliferate post geno-/cell-toxic stresses. This mechanism represents a new link between geno-/cell-toxic stress and aggressive breast cancer formation in p53 wild-type cells. PMID:20845286
[Binding capability of lidamycin apoprotein to human breast cancer detected by tissue microarrays].
Cai, Lin; Gao, Rui-Juan; Guo, Xiao-Zhong; Li, Yi; Zhen, Yong-Su
This study is to investigate the binding capability of lidamycin apoprotein (LDP), an enediyne-associated apoprotein of the chromoprotein antitumor antibiotic family, to human breast cancer and normal tissues, the correlation of LDP binding capability to human breast cancer tissues and the expression of tumor therapeutic targets such as VEGF and HER2. In this study, the binding capability of LDP to human breast cancer tissues was detected with tissue microarray. The correlation study of LDP binding capability to human breast tumor tissues and relevant therapeutic targets was performed on breast cancer tissue microarrays. Immunocytochemical examination was used to detect the binding capability of LDP to human breast carcinoma MCF-7 cells. As a result, tissue microarray showed that LDP staining of 73.2% (30/41) of breast cancer tissues was positive, whereas that of 48.3% (15/31) of the adjacent normal breast specimens was positive. The difference between the tumor and normal samples was significant (Chi2 = 4.63, P < 0.05). LDP immunoreactivity in breast cancer correlated significantly with the overexpression of VEGF and HER2 (P < 0.001 and < 0.01, r = 0.389 and 0.287, respectively). Determined with confocal immunofluorescent analysis, LDP showed the binding capability to mammary carcinoma MCF-7 cells. It is demonstrated that LDP can bind to human breast cancer tissues and there is significant difference between the breast cancer tissues and the corresponding normal tissues. Notably, the binding reactivity shows positive correlation with the expression of VEGF and HER2 in breast carcinoma tissues. The results imply that LDP may have a potential use as targeting drug carrier in the research and development of new anticancer therapeutics. This study may provide reference for drug combination of LDM and other therapeutic agents. PMID:20931759
Plasma membrane calcium-ATPase 2 and 4 in human breast cancer cell lines
Lee, Won Jae; Roberts-Thomson, Sarah J.; Monteith, Gregory R. . E-mail: email@example.com
There is evidence to suggest that plasma membrane Ca{sup 2+}-ATPase (PMCA) isoforms are important mediators sssof mammary gland physiology. PMCA2 in particular is upregulated extensively during lactation. Expression of other isoforms such as PMCA4 may influence mammary gland epithelial cell proliferation and aberrant regulation of PMCA isoform expression may lead or contribute to mammary gland pathophysiology in the form of breast cancers. To explore whether PMCA2 and PMCA4 expression may be deregulated in breast cancer, we compared mRNA expression of these PMCA isoforms in tumorigenic and non-tumorigenic human breast epithelial cell lines using real time RT-PCR. PMCA2 mRNA has a higher level of expression in some breast cancer cell lines and is overexpressed more than 100-fold in ZR-75-1 cells, compared to non-tumorigenic 184B5 cells. Although differences in PMCA4 mRNA levels were observed between breast cell lines, they were not of the magnitude observed for PMCA2. We conclude that PMCA2 mRNA can be highly overexpressed in some breast cancer cells. The significance of PMCA2 overexpression on tumorigenicity and its possible correlation with other properties such as invasiveness requires further study.
Protection of normal human reconstructed epidermis from UV by catalase overexpression.
Rezvani, H R; Cario-André, M; Pain, C; Ged, C; deVerneuil, H; Taïeb, A
Reactive oxygen species (ROS) generated by ultraviolet (UV) irradiation are counterbalanced by endogenous antioxidant systems. To test the hypothesis of a novel photoprotective approach, we irradiated epidermis reconstructed with normal human keratinocytes overexpressing sustainably lentivirus-mediated catalase (CAT), copper/zinc superoxide dismutase (CuZnSOD) or manganese superoxide dismutase (MnSOD) enzymes. We found that following UVB irradiation there was a marked decrease in sunburn cell formation, caspase-3 activation and p53 accumulation in human reconstructed epidermis overexpressing CAT. Moreover, UVA-induced hypertrophy and DNA oxidation (8-oxodeoxyguanosine) were decreased by CAT overexpression. These effects were not achieved by overexpression of CuZnSOD or MnSOD. In conclusion, vector-mediated CAT overexpression could be a promising photoprotective tool against deleterious effects of UV irradiation such skin cancer especially in monogenic/polygenic photosensitive disorders characterized by ROS accumulation. PMID:17053817
Reovirus oncolysis of human breast cancer.
Norman, Kara L; Coffey, Matthew C; Hirasawa, Kensuke; Demetrick, Douglas J; Nishikawa, Sandra G; DiFrancesco, Lisa M; Strong, James E; Lee, Patrick W K
We have previously shown that human reovirus replication is restricted to cells with an activated Ras pathway, and that reovirus could be used as an effective oncolytic agent against human glioblastoma xenografts. This study examines in more detail the feasibility of reovirus as a therapeutic for breast cancer, a subset of cancer in which direct activating mutations in the ras proto-oncogene are rare, and yet where unregulated stimulation of Ras signaling pathways is important in the pathogenesis of the disease. We demonstrate herein the efficient lysis of breast tumor-derived cell lines by the virus, whereas normal breast cells resist infection in vitro. In vivo studies of reovirus breast cancer therapy reveal that viral administration could cause tumor regression in an MDA-MB-435S mammary fat pad model in severe combined immunodeficient mice. Reovirus could also effect regression of tumors remote from the injection site in an MDA-MB-468 bilateral tumor model, raising the possibility of systemic therapy of breast cancer by the oncolytic agent. Finally, the ability of reovirus to act against primary breast tumor samples not propagated as cell lines was evaluated; we found that reovirus could indeed replicate in ex vivo surgical specimens. Overall, reovirus shows promise as a potential breast cancer therapeutic. PMID:11916487
PI3K inhibition results in enhanced HER signaling and acquired ERK dependency in HER2-overexpressing breast cancer
Serra, V; Scaltriti, M; Prudkin, L; Eichhorn, P J A; Ibrahim, Y H; Chandarlapaty, S; Markman, B; Rodriguez, O; Guzman, M; Rodriguez, S; Gili, M; Russillo, M; Parra, J L; Singh, S; Arribas, J; Rosen, N; Baselga, J
There is a strong rationale to therapeutically target the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway in breast cancer since it is highly deregulated in this disease and it also mediates resistance to anti-HER2 therapies. However, initial studies with rapalogs, allosteric inhibitors of mTORC1, have resulted in limited clinical efficacy probably due to the release of a negative regulatory feedback loop that triggers AKT and ERK signaling. Since activation of AKT occurs via PI3K, we decided to explore whether PI3K inhibitors prevent the activation of these compensatory pathways. Using HER2-overexpressing breast cancer cells as a model, we observed that PI3K inhibitors abolished AKT activation. However, PI3K inhibition resulted in a compensatory activation of the ERK signaling pathway. This enhanced ERK signaling occurred as a result of activation of HER family receptors as evidenced by induction of HER receptors dimerization and phosphorylation, increased expression of HER3 and binding of adaptor molecules to HER2 and HER3. The activation of ERK was prevented with either MEK inhibitors or anti-HER2 monoclonal antibodies and tyrosine kinase inhibitors. Combined administration of PI3K inhibitors with either HER2 or MEK inhibitors resulted in decreased proliferation, enhanced cell death and superior anti-tumor activity compared with single agent PI3K inhibitors. Our findings indicate that PI3K inhibition in HER2-overexpressing breast cancer activates a new compensatory pathway that results in ERK dependency. Combined anti-MEK or anti-HER2 therapy with PI3K inhibitors may be required in order to achieve optimal efficacy in HER2-overexpressing breast cancer. This approach warrants clinical evaluation. PMID:21278786
Overexpression of angiotensin II type 1 receptor in breast cancer cells induces epithelial-mesenchymal transition and promotes tumor growth and angiogenesis.
Oh, Eunhye; Kim, Ji Young; Cho, Youngkwan; An, Hyunsook; Lee, Nahyun; Jo, Hunho; Ban, Changill; Seo, Jae Hong
The angiotensin II type I receptor (AGTR1) has been implicated in diverse aspects of human disease, from the regulation of blood pressure and cardiovascular homeostasis to cancer progression. We sought to investigate the role of AGTR1 in cell proliferation, epithelial-mesenchymal transition (EMT), migration, invasion, angiogenesis and tumor growth in the breast cancer cell line MCF7. Stable overexpression of AGTR1 was associated with accelerated cell proliferation, concomitant with increased expression of survival factors including poly(ADP-ribose) polymerase (PARP) and X-linked inhibitor of apoptosis (XIAP), as well as extracellular signal-regulated kinase (ERK) activation. AGTR1-overexpressing MCF7 cells were more aggressive than their parent line, with significantly increased activity in migration and invasion assays. These observations were associated with changes in EMT markers, including reduced E-cadherin expression and increased p-Smad3, Smad4 and Snail levels. Treatment with the AGTR1 antagonist losartan attenuated these effects. AGTR1 overexpression also accelerated tumor growth and increased Ki-67 expression in a xenograft model. This was associated with increased tumor angiogenesis, as evidenced by a significant increase in microvessels in the intratumoral and peritumoral areas, and enhanced tumor invasion, with the latter response associated with increased EMT marker expression and matrix metallopeptidase 9 (MMP-9) upregulation. In vivo administration of losartan significantly reduced both tumor growth and angiogenesis. Our findings suggest that AGTR1 plays a significant role in tumor aggressiveness, and its inhibition may have therapeutic implications. PMID:26975580
Epigenetic Effects of Human Breast Milk
Verduci, Elvira; Banderali, Giuseppe; Barberi, Salvatore; Radaelli, Giovanni; Lops, Alessandra; Betti, Federica; Riva, Enrica; Giovannini, Marcello
A current aim of nutrigenetics is to personalize nutritional practices according to genetic variations that influence the way of digestion and metabolism of nutrients introduced with the diet. Nutritional epigenetics concerns knowledge about the effects of nutrients on gene expression. Nutrition in early life or in critical periods of development, may have a role in modulating gene expression, and, therefore, have later effects on health. Human breast milk is well-known for its ability in preventing several acute and chronic diseases. Indeed, breastfed children may have lower risk of neonatal necrotizing enterocolitis, infectious diseases, and also of non-communicable diseases, such as obesity and related-disorders. Beneficial effects of human breast milk on health may be associated in part with its peculiar components, possible also via epigenetic processes. This paper discusses about presumed epigenetic effects of human breast milk and components. While evidence suggests that a direct relationship may exist of some components of human breast milk with epigenetic changes, the mechanisms involved are still unclear. Studies have to be conducted to clarify the actual role of human breast milk on genetic expression, in particular when linked to the risk of non-communicable diseases, to potentially benefit the infant's health and his later life. PMID:24763114
Efficacy and safety of trastuzumab combined with chemotherapy for first-line treatment and beyond progression of HER2-overexpressing advanced breast cancer
Shao, Bin; Yan,, Yin; Song, Guohong; Liu, Xiaoran; Wang, Jing; Liang, Xu
Objective To observe the efficacy and safety of trastuzumab combined with chemotherapy in patients with human epidermal growth factor receptor 2 (HER2)-overexpressing advanced breast cancer. Methods A total of 90 patients with HER2-overexpressing advanced breast cancer were enrolled in this study. All patients were diagnosed with ductal invasive breast cancer by pathological analysis, and were aged between 31–73 years with a median of 51 years. HER2-positivity was defined as 3(+) staining in immunochemistry or amplification of fluorescence in situ hybridization (FISH, ratio ≥2.0). Trastuzumab was administered in combination with chemotherapy as first-line treatment and beyond progression as a secondline, third-line, and above treatment in 90, 34, 14, and 6 patients, respectively. The chemotherapy regimen was given according to normal clinical practice. The response rate was evaluated every two cycles, and the primary endpoints were progression-free survival (PFS) and overall survival (OS). Survival curves were estimated by using Kaplan-Meier graphs and were compared by using log-rank test statistics. Multivariate analysis was done using Cox's proportional hazards regression model, and the level of significance was P<0.05. Results All 90 patients received at least one dose of trastuzumab, and efficacy could be evaluated in 85 patients. The median follow-up was 50 months. In total, 72 (80.00%) patients had visceral metastasis, and 43 (47.78%) patients had progressed after one or more extensive chemotherapy regimens for metastatic diseases. The median PFS for first-line trastuzumab was 10 months (range, 2–59 months), and the median OS after metastasis or initially local advanced disease was 22 months (range, 2–116 months). Conclusions Trastuzumab combined with chemotherapy was active and well-tolerated as a first-line treatment and even beyond progression in HER2-overexpressing advanced breast cancer as a second-line or third-line treatment. However, its
Engineering targeted chromosomal amplifications in human breast epithelial cells.
Springer, Simeon; Yi, Kyung H; Park, Jeenah; Rajpurohit, Anandita; Price, Amanda J; Lauring, Josh
Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including "sawtooth" patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes. PMID:26099605
Cystathionine: A novel oncometabolite in human breast cancer.
Sen, Suvajit; Kawahara, Brain; Mahata, Sushil K; Tsai, Rebecca; Yoon, Alexander; Hwang, Lin; Hu-Moore, Kayla; Villanueva, Carissa; Vajihuddin, Abdulqadir; Parameshwar, Pooja; You, Michelle; Bhaskar, Divya Lakshmi; Gomez, Omar; Faull, Kym F; Farias-Eisner, Robin; Chaudhuri, Gautam
In this study, we have identified cystathionine (CTH), a sulfur containing metabolite, to be selectively enriched in human breast cancer (HBC) tissues (∼50-100 pmoles/mg protein) compared with undetectable levels in normal breast tissues. The accumulation of CTH, specifically in HBC, was attributed to the overexpression of cystathionine beta synthase (CBS), its synthesizing enzyme, and the undetectable levels of its downstream metabolizing enzyme, cystathionine gamma lyase (CGL). Interestingly both CBS and CGL could not be detected in normal breast tissues. We further observed that CTH protected HBC cells against excess reactive oxygen species (ROS) and chemotherapeutic drug-induced apoptosis. Moreover, CTH promoted both mitochondrial and endoplasmic reticulum homeostasis in HBC cells. As both the mitochondria and the endoplasmic reticulum are key organelles regulating the onset of apoptosis, we reasoned that endogenous CTH could be contributing towards increasing the apoptotic threshold in HBC cells. An increased apoptotic threshold is a hallmark of all cancer types, including HBC, and is primarily responsible for drug resistance. Hence this study unravels one of the possible pathways that may contribute towards drug resistance in HBC. PMID:27311614
Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers
Smalley, Matthew J; Iravani, Marjan; Leao, Maria; Grigoriadis, Anita; Kendrick, Howard; Dexter, Tim; Fenwick, Kerry; Regan, Joseph L; Britt, Kara; McDonald, Sarah; Lord, Christopher J; MacKay, Alan; Ashworth, Alan
-quantitative RT-PCR analysis, data mining and quantitative real-time RT-PCR approaches, which showed that RGS2 was expressed in the majority of solid breast cancers at much higher levels than in normal human mammary cells. Conclusion Molecular analysis of prospectively isolated mammary epithelial cells identified RGS2 as a modulator of oxytocin receptor signalling, which is highly expressed in the myoepithelial cells. The RGS2 gene, but not the oxytocin receptor, was also shown to be over-expressed in the majority of breast cancers, identifying the product of this gene, or the pathway(s) it regulates, as potentially significant therapeutic targets. PMID:18067675
Co-transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice
Wege, Anja K; Schmidt, Marcus; Ueberham, Elke; Ponnath, Marvin; Ortmann, Olaf; Brockhoff, Gero; Lehmann, Jörg
Humanized tumor mice (HTM) were generated by the co-transplantation of human hematopoietic stem cells and human breast cancer cells overexpressing HER2 into neonatal NOD-scid IL2Rγnull (NSG) mice. These mice are characterized by the development of a human immune system in combination with human breast cancer growth. Due to concurrent transplantation into newborn mice, transfer of MHC-mismatched tumor cells resulted in solid coexistence and immune cell activation (CD4+ T cells, natural killer cells, and myeloid cells), but without evidence for rejection. Histological staining of the spleen of HTM revealed co-localization of human antigen-presenting cells together with human T and B cells allowing MHC-dependent interaction, and thereby the generation of T cell-dependent antibody production. Here, we investigated the capability of these mice to generate human tumor-specific antibodies and correlated immunoglobulin titers with tumor outgrowth. We found detectable IgM and also IgG amounts in the serum of HTM, which apparently controlled tumor development when IgG serum concentrations were above 10 µg/ml. Western blot analyses revealed that the tumor-specific antibodies generated in HTM did not recognize HER2/neu antigens, but different, possibly relevant antigens for breast cancer therapy. In conclusion, HTM offer a novel approach to generate complete human monoclonal antibodies that do not require further genetic manipulation (e. g., humanization) for a potential application in humans. In addition, efficacy and safety of the generated antibodies can be tested in the same mouse model under human-like conditions. This might be of particular interest for cancer subtypes with no currently available antibody therapy. PMID:24870377
Nondestructive testing of the human breast
NASA Astrophysics Data System (ADS)
Cockburn, William
The utilization of thermal imaging in the evaluation of the human breast has been for the past two decades a highly effective form of screening for breast cancer and other breast disease. The procedure however, is not without controversy and a continuing debate concerning the competitive paradox with mammography as the gold standard in breast cancer screening/detection still exists. This paper and its accompanying oral presentation at Thermosense XXI will provide a brief historic overview of breast thermal imaging and will explore the authors concepts of the paradigm shift which needs to occur in order for breast thermal imaging to gain acceptance in the scientific, medical, and public communities. Early thermal imaging equipment sold for medical application were based on liquid crystal detector plates, or electronic low band infrared detectors. While the final output of these devices was quite colorful and impressive, they lacked the quantification necessary to accurately measure temperature from a medical perspective, and as such, many false positive findings and papers were produced which damaged the early credibility of the procedure. The author has previously suggested appropriate changes in both technology and in utilization protocol for correction of errors which have hindered the advancement and indeed, the further development and implementation of this most beneficial quantitative diagnostic tool.
Aromatase overexpression in dysfunctional adipose tissue links obesity to postmenopausal breast cancer.
Wang, Xuyi; Simpson, Evan R; Brown, Kristy A
The number of breast cancer cases has increased in the last a few decades and this is believed to be associated with the increased prevalence of obesity worldwide. The risk of breast cancer increases with age beyond menopause and the relationship between obesity and the risk of breast cancer in postmenopausal women is well established. The majority of postmenopausal breast cancers are estrogen receptor (ER) positive and estrogens produced in the adipose tissue promotes tumor formation. Obesity results in the secretion of inflammatory factors that stimulate the expression of the aromatase enzyme, which converts androgens into estrogens in the adipose tissue. Evidence demonstrating a link between obesity and breast cancer has led to the investigation of metabolic pathways as novel regulators of estrogen production, including pathways that can be targeted to inhibit aromatase specifically within the breast. This review aims to present some of the key findings in this regard. PMID:26209254
MUC4 Overexpression Augments Cell Migration and Metastasis through EGFR Family Proteins in Triple Negative Breast Cancer Cells
Mukhopadhyay, Partha; Lakshmanan, Imayavaramban; Ponnusamy, Moorthy P.; Chakraborty, Subhankar; Jain, Maneesh; Pai, Priya; Smith, Lynette M.; Lele, Subodh M.; Batra, Surinder K.
Introduction Current studies indicate that triple negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and an early pattern of metastasis. Emerging evidence suggests that MUC4 mucin is associated with metastasis of various cancers, including breast cancer. However, the functional role of MUC4 remains unclear in breast cancers, especially in TNBCs. Method In the present study, we investigated the functional and mechanistic roles of MUC4 in potentiating pathogenic signals including EGFR family proteins to promote TNBC aggressiveness using in vitro and in vivo studies. Further, we studied the expression of MUC4 in invasive TNBC tissue and normal breast tissue by immunostaining. Results MUC4 promotes proliferation, anchorage-dependent and-independent growth of TNBC cells, augments TNBC cell migratory and invasive potential in vitro, and enhances tumorigenicity and metastasis in vivo. In addition, our studies demonstrated that MUC4 up-regulates the EGFR family of proteins, and augments downstream Erk1/2, PKC-γ, and FAK mediated oncogenic signaling. Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition. We also demonstrated in this study, for the first time, that knockdown of MUC4 was associated with reduced expression of EGFR and ErbB3 (EGFR family proteins) in TNBC cells, suggesting that MUC4 uses an alternative to ErbB2 mechanism to promote aggressiveness. We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue. Conclusions MUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo. Overall, our findings suggest that MUC4 mucin promotes invasive activities of TNBC cells by altering the expression of EGFR, ErbB2, and ErbB3 molecules and their downstream signaling. PMID
Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer.
Duffy, Supipi; Fam, Hok Khim; Wang, Yi Kan; Styles, Erin B; Kim, Jung-Hyun; Ang, J Sidney; Singh, Tejomayee; Larionov, Vladimir; Shah, Sohrab P; Andrews, Brenda; Boerkoel, Cornelius F; Hieter, Philip
Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1 Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors. PMID:27551064
Human SPF45, a splicing factor, has limited expression in normal tissues, is overexpressed in many tumors, and can confer a multidrug-resistant phenotype to cells.
Sampath, Janardhan; Long, Pandy R; Shepard, Robert L; Xia, Xiaoling; Devanarayan, Viswanath; Sandusky, George E; Perry, William L; Dantzig, Anne H; Williamson, Mark; Rolfe, Mark; Moore, Robert E
Our effort to identify novel drug-resistant genes in cyclophosphamide-resistant EMT6 mouse mammary tumors led us to the identification of SPF45. Simultaneously, other groups identified SPF45 as a component of the spliceosome that is involved in alternative splicing. We isolated the human homologue and examined the normal human tissue expression, tumor expression, and the phenotype caused by overexpression of human SPF45. Our analyses revealed that SPF45 is expressed in many, but not all, normal tissues tested with predominant expression in normal ductal epithelial cells of the breast, liver, pancreas, and prostate. Our analyses using tissue microarrays and sausages of tumors indicated that SPF45 is highly expressed in numerous carcinomas including bladder, breast, colon, lung, ovarian, pancreatic, and prostate. Interestingly, this study revealed that overexpression of SPF45 in HeLa, a cervical carcinoma cell line, resulted in drug resistance to doxorubicin and vincristine, two chemotherapeutic drugs commonly used in cancer. To our knowledge, this is the first study showing tumor overexpression of an alternate splicing factor resulting in drug resistance. PMID:14578179
miR-342 overexpression results in a synthetic lethal phenotype in BRCA1-mutant HCC1937 breast cancer cells
Crippa, Elisabetta; Folini, Marco; Pennati, Marzia; Zaffaroni, Nadia; Pierotti, Marco A.; Gariboldi, Manuela
Expression of miR-342 has been strongly correlated with estrogen receptor (ER) status in breast cancer, where it is highest in ER-positive and lowest in triple-negative tumors. We investigated the effects of miR-342 transfection in the triple-negative breast cancer cell lines MDA-MB-231 and HCC1937, the latter carrying a germ-line BRCA1 mutation. Reconstitution of miR-342 led to caspase-dependent induction of apoptosis only in HCC1937 cells, while overexpression of wild-type BRCA1 in HCC1937 cells counteracted miR-342-mediated induction of apoptosis, suggesting that miR-342 overexpression and the lack of functional BRCA1 result in a synthetic lethal phenotype. Moreover, siRNA-mediated depletion of BRCA1 in MDA-MB-231 cells expressing the wild-type protein led to apoptosis upon transfection with miR-342. Using an in silico approach and a luciferase reporter system, we identified and functionally validated the Baculoviral IAP repeat-containing 6 gene (BIRC6), which encodes the anti-apoptotic factor Apollon/BRUCE, as a target of miR-342. In our model, BIRC6 likely acts as a determinant of the miRNA-dependent induction of apoptosis in BRCA1-mutant HCC1937 cells. Together, our findings suggest a tumor-suppressive function of miR-342 that could be exploited in the treatment of a subset of BRCA1-mutant hereditary breast cancers. PMID:26919240
Intrathecal trastuzumab (Herceptin) and methotrexate for meningeal carcinomatosis in HER2-overexpressing metastatic breast cancer: a case report.
Stemmler, Hans-Joachim; Mengele, Karin; Schmitt, Manfred; Harbeck, Nadia; Laessig, Dorit; Herrmann, Karin A; Schaffer, Pamela; Heinemann, Volker
Leptomeningeal carcinomatosis represents a rare manifestation of metastatic breast cancer (MBC). We herewith report on a patient suffering from HER2 overexpressing MBC who received intrathecal methotrexate and trastuzumab for meningeal carcinomatosis. A 48-year-old woman was diagnosed with breast cancer in December 2002. Following surgery, six cycles of adjuvant FE100C plus irradiation and, subsequently for 1 year, trastuzumab were given. As a result of disseminated metastatic spread in October 2005, the patient received whole-brain radiotherapy for symptomatic central nervous system involvement, and was put on several trastuzumab-based combination regimens (capecitabine, vinorelbine, paclitaxel). In June 2006, the patient developed clinical signs of terminal cone involvement with overflow incontinence and paraparesis of the legs. Immediate radiation led to partial relief from clinical symptoms. Subsequently, the patient was put on the tyrosine kinase inhibitor lapatinib and capecitabine (August to October 2007), but on November 6th the patient suffered again from overflow incontinence and weakness of the legs. Failing to respond to lapatinib, the patient received gemcitabine/cisplatin and, additionally, was recommenced on intravenous trastuzumab. Owing to progressive leptomeningeal disease, the patient received repeated doses of intrathecal methotrexate and trastuzumab. Within 2 weeks and four intrathecal treatments, cerebrospinal fluid cytology showed the absence of tumor cells. Moreover, a striking clinical improvement with resolution of the paraparesis of the legs and overflow incontinence was observed. This case report gives details regarding the clinical course of a breast cancer patient who received intrathecal trastuzumab and methotrexate via lumbar puncture for meningeal carcinomatosis of HER2-overexpressing MBC. PMID:18690096
COPS5 amplification and overexpression confers tamoxifen-resistance in ERα-positive breast cancer by degradation of NCoR
Lu, Renquan; Hu, Xiaobo; Zhou, Junmei; Sun, Jiajun; Zhu, Alan Z.; Xu, Xiaofeng; Zheng, Hui; Gao, Xiang; Wang, Xian; Jin, Hongchuan; Zhu, Ping; Guo, Lin
Oestrogen receptor α (ERα) antagonists are used in endocrine therapies for ERα-positive (ERα+) breast cancer patients. Unfortunately the clinical benefit is limited due to intrinsic and acquired drug resistance. Here using integrated genomic and functional studies, we report that amplification and/or overexpression of COPS5 (CSN5/JAB1) confers resistance to tamoxifen. Amplification and overexpression of COPS5, a catalytic subunit of the COP9 complex, is present in about 9% of the ERα+ primary breast cancer and more frequently (86.7%, 26/30) in tamoxifen-refractory tumours. Overexpression of COPS5, through its isopeptidase activity, leads to ubiquitination and proteasome-mediated degradation of NCoR, a key corepressor for ERα and tamoxifen-mediated suppression of ERα target genes. Importantly, COPS5 overexpression causes tamoxifen-resistance in preclinical breast cancer models in vitro and in vivo. We also demonstrate that genetic inhibition of the isopeptidase activity of COPS5 is sufficient to re-sensitize the resistant breast cancer cells to tamoxifen-treatment, offering a potential therapeutic approach for endocrine-resistant breast cancer patients. PMID:27375289
Drug Efflux Transporters Are Overexpressed in Short-Term Tamoxifen-Induced MCF7 Breast Cancer Cells.
Krisnamurti, Desak Gede Budi; Louisa, Melva; Anggraeni, Erlia; Wanandi, Septelia Inawati
Tamoxifen is the first line drug used in the treatment of estrogen receptor-positive (ER+) breast cancer. The development of multidrug resistance (MDR) to tamoxifen remains a major challenge in the treatment of cancer. One of the mechanisms related to MDR is decrease of drug influx via overexpression of drug efflux transporters such as P-glycoprotein (P-gp/MDR1), multidrug resistance associated protein (MRP), or BCRP (breast cancer resistance protein). We aimed to investigate whether the sensitivity of tamoxifen to the cells is maintained through the short period and whether the expressions of several drug efflux transporters have been upregulated. We exposed MCF7 breast cancer cells with tamoxifen 1 μM for 10 passages (MCF7 (T)). The result showed that MCF7 began to lose their sensitivity to tamoxifen from the second passage. MCF7 (T) also showed a significant increase in all transporters examined compared with MCF7 parent cells. The result also showed a significant increase of CC50 in MCF7 (T) compared to that in MCF7 (97.54 μM and 3.04 μM, resp.). In conclusion, we suggest that the expression of several drug efflux transporters such as P-glycoprotein, MRP2, and BCRP might be used and further studied as a marker in the development of tamoxifen resistance. PMID:26981116
Drug Efflux Transporters Are Overexpressed in Short-Term Tamoxifen-Induced MCF7 Breast Cancer Cells
Krisnamurti, Desak Gede Budi; Louisa, Melva; Anggraeni, Erlia; Wanandi, Septelia Inawati
Tamoxifen is the first line drug used in the treatment of estrogen receptor-positive (ER+) breast cancer. The development of multidrug resistance (MDR) to tamoxifen remains a major challenge in the treatment of cancer. One of the mechanisms related to MDR is decrease of drug influx via overexpression of drug efflux transporters such as P-glycoprotein (P-gp/MDR1), multidrug resistance associated protein (MRP), or BCRP (breast cancer resistance protein). We aimed to investigate whether the sensitivity of tamoxifen to the cells is maintained through the short period and whether the expressions of several drug efflux transporters have been upregulated. We exposed MCF7 breast cancer cells with tamoxifen 1 μM for 10 passages (MCF7 (T)). The result showed that MCF7 began to lose their sensitivity to tamoxifen from the second passage. MCF7 (T) also showed a significant increase in all transporters examined compared with MCF7 parent cells. The result also showed a significant increase of CC50 in MCF7 (T) compared to that in MCF7 (97.54 μM and 3.04 μM, resp.). In conclusion, we suggest that the expression of several drug efflux transporters such as P-glycoprotein, MRP2, and BCRP might be used and further studied as a marker in the development of tamoxifen resistance. PMID:26981116
Pomolic acid inhibits metastasis of HER2 overexpressing breast cancer cells through inactivation of the ERK pathway.
Kim, Buyun; Kim, Yu Chul; Park, Byoungduck
Expression of the CXC chemokine receptor-4 (CXCR4), a G protein-coupled receptor, and HER2, a receptor tyrosine kinase, strongly correlates with tumor progression and metastatic potential of breast cancer cells. We report the identification of pomolic acid (PA) as a novel regulator of HER2 and CXCR4 expression. We found that PA downregulated the expression of HER2 and CXCR4 in SKBR3 cells in a dose- and time-dependent manner. When investigated for the molecular mechanism(s), it was found that the downregulation of HER2 and CXCR4 was not due to proteolytic degradation but rather to transcriptional regulation as indicated by downregulation of mRNA expression. Moreover, we show that PA inhibits phosphorylation of ERK and reduces NF-κB activation. Suppression of CXCR4 expression by PA correlated with the inhibition of CXCL12-induced invasion of HER2-overexpressing breast cancer cells. Overall, our results demonstrate for the first time that PA is a novel inhibitor of HER2 and CXCR4 expression via kinase pathways and may play a critical role in determining the metastatic potential of breast cancer cells. PMID:27277173
Excretion of drugs in human breast milk
Welch, R.M.; Findlay, J.W.
The present report briefly discusses some of the morphological, physiological, and compositional aspects of animal and human breast milk and how these characteristics might be important for the accumulation of drugs and foreign compounds. In addition, a study is described confirming the presence of caffeine, codeine, morphine, phenacetin, acetaminophen, and salicylic acid in the breast milk of a lactating mother following oral administration of a combination analgesic containing aspirin, phenacetin, caffeine, and codeine. Although the study is limited to one subject, it has provided critically needed data on the rates of appearance in, and elimination of these drugs from, breast milk. A similar amount of information is presented on phenacetin, also a component of the analgesic mixture, which has not been previously reported to enter human milk. The distribution of these drugs between the slightly more acidic breast milk and the relatively neutral plasma is consistent with their weakly basic, acidic, or relatively neutral properties. In general, the study shows that codeine and morphine milk concentrations are higher than, salicylic acid milk levels are much lower than, and phenacetin, caffeine, and acetaminophen milk concentrations are relatively similar to their respective plasma levels. It is projected, from estimated steady-state milk concentrations of the drugs and their metabolites studied, that very low percentages of the therapeutic dosages (less than 0.7%) would be excreted in mother's milk, too low an amount to be clinically significant to the infant.
Chemical Biomarkers of Human Breast Milk Pollution
Massart, Francesco; Gherarducci, Giulia; Marchi, Benedetta; Saggese, Giuseppe
Human milk is, without question, the best source of nutrition for infants containing the optimal balance of fats, carbohydrates and proteins for developing babies. Breastfeeding provides a range of benefits for growth, immunity and development building a powerful bond between mother and her child. Recognition of the manifold benefits of breast milk has led to the adoption of breast-feeding policies by numerous health and professional organizations such as the World Health Organization and American Academy of Pediatrics. In industrially developed as well as in developing nations, human milk contamination by toxic chemicals such as heavy metals, dioxins and organohalogen compounds, however, is widespread and is the consequence of decades of inadequately controlled pollution. Through breastfeeding, the mother may transfer to the suckling infant potentially toxic chemicals to which the mother has previously been exposed. In the present review, environmental exposure, acquisition and current levels of old and emerging classes of breast milk pollutants are systematically presented. Although scientific evidences indicated that the advantages of breast-feeding outweigh any risks from contaminants, it is important to identify contaminant trends, to locate disproportionately exposed populations, and to take public health measures to improve chemical BM pollution as possible. PMID:19578503
Overexpression of centromere protein H is significantly associated with breast cancer progression and overall patient survival.
Liao, Wen-Ting; Feng, Yan; Li, Men-Lin; Liu, Guang-Lin; Li, Man-Zhi; Zeng, Mu-Sheng; Song, Li-Bing
Breast cancer is one of the leading causes of cancer death worldwide. This study aimed to analyze the expression of centromere protein H (CENP-H) in breast cancer and to correlate it with clinicopathologic data, including patient survival. Using reverse transcription-polymerase chain reaction and Western blotting to detect the expression of CENP-H in normal mammary epithelial cells, immortalized mammary epithelial cell lines, and breast cancer cell lines, we observed that the mRNA and protein levels of CENP-H were higher in breast cancer cell lines and in immortalized mammary epithelial cells than in normal mammary epithelial cells. We next examined CENP-H expression in 307 paraffin-embedded archived samples of clinicopathologically characterized breast cancer using immunohistochemistry, and detected high CENP-H expression in 134 (43.6%) samples. Statistical analysis showed that CENP-H expression was related with clinical stage (P = 0.001), T classification (P = 0.032), N classification (P = 0.018), and Ki-67 (P < 0.001). Patients with high CENP-H expression had short overall survival. Multivariate analysis showed that CENP-H expression was an independent prognostic indicator for patient survival. Our results suggest that CENP-H protein is a valuable marker of breast cancer progression and prognosis. PMID:21880184
Overexpression of HE4 (human epididymis protein 4) enhances proliferation, invasion and metastasis of ovarian cancer
Wang, Huimin; Tan, Mingzi; Schwab, Carlton L.; Deng, Lu; Gao, Jian; Hao, Yingying; Li, Xiao; Gao, Song; Liu, Juanjuan; Lin, Bei
Overexpression of Human epididymis protein 4 (HE4) related with a role in ovarian cancer tumorigenesis while little is known about the molecular mechanism alteration by HE4 up regulation. Here we reported that overexpressed HE4 promoted ovarian cancer cells proliferation, invasion and metastasis. Furthermore, human whole genome gene expression profile microarrays revealed that 231 differentially expressed genes (DEGs) were altered in response to HE4, in which MAPK signaling, ECM receptor, cell cycle, steroid biosynthesis pathways were involved. The findings suggested that overexpressed HE4 played an important role in ovarian cancer progression and metastasis and that HE4 has the potential to serve as a novel therapeutic target for ovarian cancer. PMID:26575020
Rab23 is overexpressed in human bladder cancer and promotes cancer cell proliferation and invasion.
Jiang, Yuanjun; Han, Yushuang; Sun, Chaonan; Han, Chuyang; Han, Ning; Zhi, Weiwei; Qiao, Qiao
Rab23 overexpression has been implicated in several human cancers. However, its expression pattern and biological roles in human bladder cancer have not been elucidated. In this study, we examined Rab23 expression in 93 bladder cancer specimens and analyzed its correlation with clinicopathological parameters. We found that Rab23 was overexpressed in 45 of 93 (48.3 %) cancer specimens. Significant association was found between Rab23 overexpression and tumor invasion depth (p = 0.0027). Rab23 overexpression also negatively correlated with FGFR3 protein expression (p = 0.021). We found that Rab23 expression was lower in normal bladder transitional cell line SV-HUC-1 than in bladder cancer cell lines BIU-87, 5637, and T24. We knocked down Rab23 expression in T24 cancer cells and transfected a Rab23 plasmid in the BIU-87 cell line. Rab23 depletion inhibited cell growth rate and invasion, while its overexpression resulted in increased cell growth and invasion. In addition, we demonstrated that Rab23 depletion decreased and its transfection upregulated expression of cyclin E, c-myc, and MMP-9. Furthermore, we showed that Rab23 knockdown inhibited NF-κB signaling and its overexpression upregulated NF-κB signaling. BAY 11-7082 (NF-κB inhibitor) partly inhibited the effect of Rab23 on cyclin E and MMP-9 expression. In conclusion, the present study demonstrated that Rab23 overexpression facilitates malignant cell growth and invasion in bladder cancer through the NF-κB pathway. PMID:26715272
Integrin α6/Akt/Erk signaling is essential for human breast cancer resistance to radiotherapy.
Hu, Ting; Zhou, Rui; Zhao, Yanxia; Wu, Gang
Integrin α6 (ITGA6), a transmembrane glycoprotein adhesion receptor protein, is widely upregulated in many types of tumors and promotes migration and invasion in cancer cells. However, the role that the ITGA6-associated signaling network plays in radiosensitivity in breast cancer has not been described. The expression of ITGA6 was examined in human breast cancer and normal breast cell lines using western blot analysis. We also explored the role of ITGA6 in the regulation of radiation sensitivity in breast cancer using the colony formation assays, cell cycle analyses, apoptosis assays and immunofluorescence analyses. The results showed that the protein and mRNA expression levels of ITGA6 was higher in breast cancer cells than in normal cells. ITGA6 protectived responses to radiotherapy in breast cancer cells by altering cell apoptosis, DNA damage repair and cell-cycle regulation. Furthermore, ITGA6 enhanced radiation resistance via PI3K/Akt and MEK/Erk signaling. In addition, overexpressing ITGA6 promoted radiation resistance in cells, and this effect was neutralized by the PI3K inhibitor LY294002 and MEK inhibitor U0126. Taken together, these findings indicate that ITGA6 might be involved in a mechanism that underlies radiation resistance and that ITGA6 could be a potential target for therapies aimed at overcoming radiation resistance in breast cancer. PMID:27624978
The significance of Brf1 overexpression in human hepatocellular carcinoma
Shi, Ganggang; Huang, Yi; Zhang, Yanmei; Levy, Daniel; Zhong, Shuping
Brf1 (TFIIB-related factor 1) plays a crucial role in cell transformation and tumorigenesis. However, the significance of Brf1 expression in human HCC (hepatocellular carcinoma) cases remains to be addressed. In this study, biopsies of human HCC, liver tumor samples of mice and cell lines of normal and tumor liver were utilized to determine the alteration of Brf1 expression using cytological and molecular biological approaches. Brf1 expression is increased in human HCC cases, which is correlated with shorter survival times. Levels of Brf1 and Pol III (RNA polymerase III-dependent) gene transcription in HCC patients with alcohol consumption are higher than the cases of non-HCC with or without alcohol intake. Induction of Brf1 and Pol III genes by ethanol in hepatoma cells is higher than in non-tumor cells. Ethanol increases the rate of cell transformation. Repression of Brf1 inhibits alcohol-promoted cell transformation. Alcohol consumption enhances Brf1 expression to promote cell transformation. These studies demonstrate that Brf1 is a new biomarker of HCC. PMID:26701855
Defining the cellular precursors to human breast cancer
Keller, Patricia J.; Arendt, Lisa M.; Skibinski, Adam; Logvinenko, Tanya; Klebba, Ina; Dong, Shumin; Smith, Avi E.; Prat, Aleix; Perou, Charles M.; Gilmore, Hannah; Schnitt, Stuart; Naber, Stephen P.; Garlick, Jonathan A.; Kuperwasser, Charlotte
Human breast cancers are broadly classified based on their gene-expression profiles into luminal- and basal-type tumors. These two major tumor subtypes express markers corresponding to the major differentiation states of epithelial cells in the breast: luminal (EpCAM+) and basal/myoepithelial (CD10+). However, there are also rare types of breast cancers, such as metaplastic carcinomas, where tumor cells exhibit features of alternate cell types that no longer resemble breast epithelium. Until now, it has been difficult to identify the cell type(s) in the human breast that gives rise to these various forms of breast cancer. Here we report that transformation of EpCAM+ epithelial cells results in the formation of common forms of human breast cancer, including estrogen receptor-positive and estrogen receptor-negative tumors with luminal and basal-like characteristics, respectively, whereas transformation of CD10+ cells results in the development of rare metaplastic tumors reminiscent of the claudin-low subtype. We also demonstrate the existence of CD10+ breast cells with metaplastic traits that can give rise to skin and epidermal tissues. Furthermore, we show that the development of metaplastic breast cancer is attributable, in part, to the transformation of these metaplastic breast epithelial cells. These findings identify normal cellular precursors to human breast cancers and reveal the existence of a population of cells with epidermal progenitor activity within adult human breast tissues. PMID:21940501
Immunoreactive opioid peptides in human breast cancer.
Scopsi, L.; Balslev, E.; Brünner, N.; Poulsen, H. S.; Andersen, J.; Rank, F.; Larsson, L. I.
Opioid peptides have a variety of actions on inter alia pituitary hormone secretion and the immune system. Release of endogenous opioids has been found to stimulate growth of experimental breast cancers and opiate receptor blockers have reduced the growth of chemically induced rat breast tumors. Opioid peptides may therefore play a role in human breast cancer. Invasive ductal carcinomas from 61 premenopausal women were immunocytochemically analyzed for the presence of opioid peptide immunoreactivity. Positive staining was unambiguously identified in 34 of the tumors (56%). In addition, a medullary carcinoma was positive. In a smaller series of tumors, opioid peptide immunoreactive cells were detected in both primary tumors and metastases. Positive tumor cells were usually few and scattered. Therefore, underestimates of their true frequency of occurrence are likely to have occurred, making accurate correlations with clinical behavior and estrogen receptor status difficult. No correlations with estrogen receptors were established for the unambiguously opioid peptide-positive tumors. Many of the positive tumors also stained with antibodies to gamma-endorphin and alpha-melanocyte-stimulating hormone, suggesting the presence of proopiomelanocortin-derived peptides in them. However, peptides derived from other opioid precursors also may be present in breast cancer. Images Figure 1 PMID:2464945
Molecular Portrait of the Normal Human Breast Tissue and Its Influence on Breast Carcinogenesis.
Margan, Madalin Marius; Jitariu, Andreea Adriana; Cimpean, Anca Maria; Nica, Cristian; Raica, Marius
Normal human breast tissue consists of epithelial and nonepithelial cells with different molecular profiles and differentiation grades. This molecular heterogeneity is known to yield abnormal clones that may contribute to the development of breast carcinomas. Stem cells that are found in developing and mature breast tissue are either positive or negative for cytokeratin 19 depending on their subtype. These cells are able to generate carcinogenesis along with mature cells. However, scientific data remains controversial regarding the monoclonal or polyclonal origin of breast carcinomas. The majority of breast carcinomas originate from epithelial cells that normally express BRCA1. The consecutive loss of the BRCA1 gene leads to various abnormalities in epithelial cells. Normal breast epithelial cells also express hypoxia inducible factor (HIF) 1α and HIF-2α that are associated with a high metastatic rate and a poor prognosis for malignant lesions. The nuclear expression of estrogen receptor (ER) and progesterone receptor (PR) in normal human breast tissue is maintained in malignant tissue as well. Several controversies regarding the ability of ER and PR status to predict breast cancer outcome remain. Both ER and PR act as modulators of cell activity in normal human breast tissue. Ki-67 positivity is strongly correlated with tumor grade although its specific role in applied therapy requires further studies. Human epidermal growth factor receptor 2 (HER2) oncoprotein is less expressed in normal human breast specimens but is highly expressed in certain malignant lesions of the breast. Unlike HER2, epidermal growth factor receptor expression is similar in both normal and malignant tissues. Molecular heterogeneity is not only found in breast carcinomas but also in normal breast tissue. Therefore, the molecular mapping of normal human breast tissue might represent a key research area to fully elucidate the mechanisms of breast carcinogenesis. PMID:27382385
Molecular Portrait of the Normal Human Breast Tissue and Its Influence on Breast Carcinogenesis
Margan, Madalin Marius; Jitariu, Andreea Adriana; Nica, Cristian; Raica, Marius
Normal human breast tissue consists of epithelial and nonepithelial cells with different molecular profiles and differentiation grades. This molecular heterogeneity is known to yield abnormal clones that may contribute to the development of breast carcinomas. Stem cells that are found in developing and mature breast tissue are either positive or negative for cytokeratin 19 depending on their subtype. These cells are able to generate carcinogenesis along with mature cells. However, scientific data remains controversial regarding the monoclonal or polyclonal origin of breast carcinomas. The majority of breast carcinomas originate from epithelial cells that normally express BRCA1. The consecutive loss of the BRCA1 gene leads to various abnormalities in epithelial cells. Normal breast epithelial cells also express hypoxia inducible factor (HIF) 1α and HIF-2α that are associated with a high metastatic rate and a poor prognosis for malignant lesions. The nuclear expression of estrogen receptor (ER) and progesterone receptor (PR) in normal human breast tissue is maintained in malignant tissue as well. Several controversies regarding the ability of ER and PR status to predict breast cancer outcome remain. Both ER and PR act as modulators of cell activity in normal human breast tissue. Ki-67 positivity is strongly correlated with tumor grade although its specific role in applied therapy requires further studies. Human epidermal growth factor receptor 2 (HER2) oncoprotein is less expressed in normal human breast specimens but is highly expressed in certain malignant lesions of the breast. Unlike HER2, epidermal growth factor receptor expression is similar in both normal and malignant tissues. Molecular heterogeneity is not only found in breast carcinomas but also in normal breast tissue. Therefore, the molecular mapping of normal human breast tissue might represent a key research area to fully elucidate the mechanisms of breast carcinogenesis. PMID:27382385
Different apoptotic effects of saxifragifolin C in human breast cancer cells.
Kim, Kyung-Ho; Kim, Ji-Yun; Kwak, Jong-Hwan; Kim, Byung Oh; Pyo, Suhkneung
Breast cancer is currently the most common form of cancer affecting women. Recent studies have reported that triterpenoid saponins isolated from Androsace umbellata exhibit anti-proliferative effects in several types of cancer cells. However, the cytotoxic effect of saxifragifolin C (Saxi C) on breast cancer cells remains unclear. The purpose of this study is to evaluate the in vitro anti-tumor activity of Saxi C in human breast cancer cells. Our data indicated that MDA-MB-231 cells were more sensitive than MCF-7 cells to Saxi C treatment. In addition, Saxi C inhibited cell survival through the induction of reactive oxygen species and the caspase-dependent pathway in the MDA-MB-231 cells, whereas MCF-7 cells treated with Saxi C underwent the apoptotic cell death in a caspase-independent manner. Although Saxi C treatment resulted in the induction of activation of MAPKs in both types of human breast cancer cells, p38 MAPK and JNK, but not ERK1/2, appeared to be involved in Saxi C-induced apoptosis. Moreover, ERα-overexpressing MDA-MB-231 cells remained alive, whereas the survival of shERα-transfected MCF-7 cells decreased. Taken together, Saxi C induced apoptosis in MCF-7 cells and MDA-MB-231 cells via different regulatory mechanisms, and ERα status might be essential for regulating Saxi C-induced apoptosis in breast cancer cells. Thus, Saxi C is a potential chemotherapeutic agent in breast cancer. PMID:26965415
Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase
NASA Technical Reports Server (NTRS)
Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand
Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 220.127.116.11) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For
Overexpression of sonic hedgehog in the triple negative breast cancer: clinicopathological characteristics of high burden breast cancer patients from Bangladesh
Noman, A. S.; Uddin, M.; Rahman, M. Z.; Nayeem, M. J.; Alam, S. S.; Khatun, Z.; Wahiduzzaman, M.; Sultana, A.; Rahman, M. L.; Ali, M. Y.; Barua, D.; Ahmed, I.; Islam, M. S.; Aboussekhra, A.; Yeger, H.; Farhat, W. A.; Islam, S. S.
Dysregulation of Hedgehog (Hh) signaling pathway has been documented in mammary gland development and breast cancer (BC) progression. Despite the remarkable progress in therapeutic interventions, BC related mortality in Bangladesh increased in the last decade. Triple negative breast cancer (TNBC) still presents a critical therapeutic challenge. Thus effective targeted therapy is urgently needed. In this study, we report the clinicopathological characteristics and prognosis of BC patients from Bangladesh. Routine immunohistochemical analysis and high throughput RNA-Seq data from the TCGA library were used to analyze the expression pattern and association of high and low level of Shh expression in a collection of BC patients with a long-term follow-up. High levels of Shh were observed in a subset of BC tumors with poor prognostic pathological features. Higher level of Shh expression correlated with a significantly poorer overall survival of patients compared with patients whose tumors expressed a low level of Shh. These data support the contention that Shh could be a novel biomarker for breast cancer that is involved in mediating the aggressive phenotype of BC. We propose that BC patients exhibiting a higher level of Shh expression, representing a subset of BC patients, would be amenable to Shh targeted therapy. PMID:26727947
Overexpression of sonic hedgehog in the triple negative breast cancer: clinicopathological characteristics of high burden breast cancer patients from Bangladesh.
Noman, A S; Uddin, M; Rahman, M Z; Nayeem, M J; Alam, S S; Khatun, Z; Wahiduzzaman, M; Sultana, A; Rahman, M L; Ali, M Y; Barua, D; Ahmed, I; Islam, M S; Aboussekhra, A; Yeger, H; Farhat, W A; Islam, S S
Dysregulation of Hedgehog (Hh) signaling pathway has been documented in mammary gland development and breast cancer (BC) progression. Despite the remarkable progress in therapeutic interventions, BC related mortality in Bangladesh increased in the last decade. Triple negative breast cancer (TNBC) still presents a critical therapeutic challenge. Thus effective targeted therapy is urgently needed. In this study, we report the clinicopathological characteristics and prognosis of BC patients from Bangladesh. Routine immunohistochemical analysis and high throughput RNA-Seq data from the TCGA library were used to analyze the expression pattern and association of high and low level of Shh expression in a collection of BC patients with a long-term follow-up. High levels of Shh were observed in a subset of BC tumors with poor prognostic pathological features. Higher level of Shh expression correlated with a significantly poorer overall survival of patients compared with patients whose tumors expressed a low level of Shh. These data support the contention that Shh could be a novel biomarker for breast cancer that is involved in mediating the aggressive phenotype of BC. We propose that BC patients exhibiting a higher level of Shh expression, representing a subset of BC patients, would be amenable to Shh targeted therapy. PMID:26727947
Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression
Lu, Li; Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin; Song, Wen-Hui; Yan, Ba-Yi; Yang, Gui-Jiao; Li, Ang; Yang, Wu-Lin
Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5.
Protein expression and amplification of AIB1 in human urothelial carcinoma of the bladder and overexpression of AIB1 is a new independent prognostic marker of patient survival.
Luo, Jun-Hang; Xie, Dan; Liu, Meng-Zhong; Chen, Wei; Liu, Yong-Dong; Wu, Guo-Qing; Kung, Hsiang-Fu; Zeng, Yi-Xin; Guan, Xin-Yuan
AIB1, a candidate oncogene in human breast cancer, is frequently amplified and overexpressed in several types of human cancers, but the status of AIB1 amplification and expression in urothelial carcinoma of the bladder (UC) and its clinical/prognostic significance is unclear. In our study, the methods of immunohistochemistry and fluorescence in situ hybridization were utilized to examine protein expression and amplification of AIB1 in 163 primary UCs and in 30 samples of normal bladder mucosa. Overexpression of AIB1 and amplification of AIB1 was found in 32.5 and 7.0% of UCs, respectively. In univariate survival analysis of the UC cohorts, a highly significant association of overexpression of AIB1 with shortened patient survival (mean: 45.6 months vs. 59.0 months, p < 0.001, log rank test) was demonstrated. In different subsets of UC patients, overexpression of AIB1 was also a prognostic indicator in grade 1 (p = 0.007), grade 2 (p = 0.010) and grade 3 (p = 0.015) tumor patients, and in pTa (p = 0.025), pT2-4 (p = 0.004), pN0 (p < 0.001) and pT2-4/pN0 (p = 0.040) tumor patients. Importantly, AIB1 expression (p < 0.001) together with pT and pN status (p < 0.05) provided significant independent prognostic parameters in multivariate analysis. In addition, a significant correlation (p < 0.05) of overexpression of AIB1 with an increased UC labeling index of Ki-67 (a cell proliferation marker) was observed in these UCs. Thus, these findings provide evidence that an overexpression of AIB1, as detected by immunohistochemistry, is an independent molecular marker for poor prognosis (shortened survival time) of patients with UC. PMID:18246597
A novel nanoparticle formulation overcomes multiple types of membrane efflux pumps in human breast cancer cells.
Prasad, Preethy; Cheng, Ji; Shuhendler, Adam; Rauth, Andrew M; Wu, Xiao Yu
Multidrug resistance (MDR) in cancer cells can involve overexpression of different types of membrane drug efflux pumps and other drug resistance mechanisms. Hence, inhibition of one resistance mechanism may not be therapeutically effective. Previously we demonstrated a new polymer lipid hybrid nanoparticle (PLN) system was able to circumvent drug resistance of P-glycoprotein (P-gp) overexpressing breast cancer cells. The objectives of the present study were 2-fold: (1) to evaluate the ability of the PLN system to overcome two other membrane efflux pumps-multidrug resistance protein 1 (MRP1+) and breast cancer resistance protein (BCRP+) overexpressed on human breast cancer cell lines MCF7 VP (MRP1+) and MCF7 MX (BCRP+); and (2) to evaluate possible synergistic effects of doxorubicin (Dox)-mitomycin C (MMC) in these cell lines. These objectives were accomplished by measuring in vitro cellular uptake, intracellular trafficking, and cytotoxicity (using a clonogenic assay and median effect analysis), of Dox, MMC, or Dox-MMC co-loaded PLN. Treatment of MDR cells with PLN encapsulating single anticancer agents significantly enhanced cell kill compared to free Dox or MMC solutions. Dox-MMC co-loaded PLN were 20-30-folds more effective in killing MDR cells than free drugs. Co-encapsulated Dox-MMC was more effective in killing MDR cells than single agent-encapsulated PLN. Microscopic images showed perinuclear localization of fluorescently labelled PLN in all cell lines. These results are consistent with our previous results for P-gp overexpressing breast cancer cells suggesting the PLN system can overcome multiple types of membrane efflux pumps increasing the cytotoxicity of Dox-MMC at significantly lower doses than free drugs. PMID:25786718
Scanning electrochemical microscopy of living cells: different redox activities of nonmetastatic and metastatic human breast cells.
Liu, B; Rotenberg, S A; Mirkin, M V
Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Calpha), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators. PMID:10963658
Scanning electrochemical microscopy of living cells: Different redox activities of nonmetastatic and metastatic human breast cells
Liu, Biao; Rotenberg, Susan A.; Mirkin, Michael V.
Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Cα), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators. PMID:10963658
Brachyury, a vaccine target, is overexpressed in triple-negative breast cancer.
Hamilton, Duane H; Roselli, Mario; Ferroni, Patrizia; Costarelli, Leopoldo; Cavaliere, Francesco; Taffuri, Mariateresa; Palena, Claudia; Guadagni, Fiorella
Patients diagnosed with triple-negative breast cancer (TNBC) have a high rate of tumor metastasis and a poor prognosis. The treatment option for these patients is currently chemotherapy, which results in very low response rates. Strategies that exploit the immune system for the treatment of cancer have now shown the ability to improve survival in several tumor types. Identifying potential targets for immune therapeutic interventions is an important step in developing novel treatments for TNBC. In this study, in silico analysis of publicly available datasets and immunohistochemical analysis of primary and metastatic tumor biopsies from TNBC patients were conducted to evaluate the expression of the transcription factor brachyury, which is a driver of tumor metastasis and resistance and a target for cancer vaccine approaches. Analysis of breast cancer datasets demonstrated a predominant expression of brachyury mRNA in TNBC and in basal vs luminal or HER2 molecular breast cancer subtypes. At the protein level, variable levels of brachyury expression were detected both in primary and metastatic TNBC lesions. A strong association was observed between nuclear brachyury protein expression and the stage of disease, with nuclear brachyury being more predominant in metastatic vs primary tumors. Survival analysis also demonstrated an association between high levels of brachyury in the primary tumor and poor prognosis. Two brachyury-targeting cancer vaccines are currently undergoing clinical evaluation; the data presented here provide rationale for using brachyury-targeting immunotherapy approaches for the treatment of TNBC. PMID:27580659
Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase
Brizio, Carmen; Galluccio, Michele; Wait, Robin; Torchetti, Enza Maria; Bafunno, Valeria; Accardi, Rosita; Gianazza, Elisabetta; Indiveri, Cesare; Barile, Maria . E-mail: firstname.lastname@example.com
FAD synthetase (FADS) (EC 220.127.116.11) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl{sub 2}, as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 {+-} 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K {sub M} value for FMN of 1.5 {+-} 0.3 {mu}M. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast.
Aquaporin-5: A Marker Protein for Proliferation and Migration of Human Breast Cancer Cells
Jung, Hyun Jun; Park, Ji-Young; Jeon, Hyo-Sung; Kwon, Tae-Hwan
Aquaporin (AQP) is a family of transmembrane proteins for water transport. Recent studies revealed that AQPs are likely to play a role in tumor progression and invasion. We aimed to examine the potential role of AQP5 in the progression of human breast cancer cells. Expression of AQP5 mRNA and protein was seen in human breast cancer cell line (both MCF7 and MDA-MB-231) by RT-PCR and immunoblotting analysis. Immunoperoxidase labeling of AQP5 was observed at ductal epithelial cells of human breast tissues. In benign tumor, AQP5 labeling was mainly seen at the apical domains of ductal epithelial cells. In contrast, in invasive ductal carcinoma, prominent AQP5 labeling was associated with cancer cells, whereas some ducts were unlabeled and apical polarity of AQP5 in ducts was lost. Cell proliferation (BrdU incorporation assay) and migration of MCF7 cells were significantly attenuated by lentivirus-mediated AQP5-shRNA transduction. Hyperosmotic stress induced by sorbitol treatment (100 mM, 24 h) reduced AQP5 expression in MCF7 cells, which was also associated with a significant reduction in cell proliferation and migration. Taken together, prominent AQP5 expression in breast cancer cells with the loss of polarity of ductal epithelial cells was seen during the progression of breast carcinoma. shRNA- or hyperosmotic stress-induced reduction in AQP5 expression of MCF7 cells was associated with significantly reduced cell proliferation and migration. In conclusion, AQP5 overexpression is likely to play a role in cell growth and metastasis of human breast cancer and could be a novel target for anti-breast cancer treatment. PMID:22145049
Human Breast Progenitor Cell Numbers Are Regulated by WNT and TBX3
Arendt, Lisa M.; St. Laurent, Jessica; Wronski, Ania; Caballero, Silvia; Lyle, Stephen R.; Naber, Stephen P.; Kuperwasser, Charlotte
Background Although human breast development is mediated by hormonal and non-hormonal means, the mechanisms that regulate breast progenitor cell activity remain to be clarified. This limited understanding of breast progenitor cells has been due in part to the lack of appropriate model systems to detect and characterize their properties. Methods To examine the effects of WNT signaling and TBX3 expression on progenitor activity in the breast, primary human mammary epithelial cells (MEC) were isolated from reduction mammoplasty tissues and transduced with lentivirus to overexpress WNT1 or TBX3 or reduce expression of their cognate receptors using shRNA. Changes in progenitor activity were quantified using characterized assays. We identified WNT family members expressed by cell populations within the epithelium and assessed alterations in expression of WNT family ligands by MECs in response to TBX3 overexpression and treatment with estrogen and progesterone. Results Growth of MECs on collagen gels resulted in the formation of distinct luminal acinar and basal ductal colonies. Overexpression of TBX3 in MECs resulted in increased ductal colonies, while shTBX3 expression diminished both colony types. Increased WNT1 expression led to enhanced acinar colony formation, shLRP6 decreased both types of colonies. Estrogen stimulated the formation of acinar colonies in control MEC, but not shLRP6 MEC. Formation of ductal colonies was enhanced in response to progesterone. However, while shLRP6 decreased MEC responsiveness to progesterone, shTBX3 expression did not alter this response. Conclusions We identified two phenotypically distinguishable lineage-committed progenitor cells that contribute to different structural elements and are regulated via hormonal and non-hormonal mechanisms. WNT signaling regulates both types of progenitor activity. Progesterone favors the expansion of ductal progenitor cells, while estrogen stimulates the expansion of acinar progenitor cells. Paracrine
Methylation status and overexpression of COX-2 in Tunisian patients with ductal invasive breast carcinoma.
Karray-Chouayekh, Sondes; Trifa, Fatma; Khabir, Abdelmajid; Boujelbene, Noureddine; Sellami-Boudawara, Tahia; Daoud, Jamel; Frikha, Mounir; Gargouri, Ali; Mokdad-Gargouri, Raja
Inflammation and hormonal signalling induce the cyclooxygenase-2 (COX-2) expression in solid tumours including breast cancer, which in turn affects cell proliferation, apoptosis and metastasis. The aim of this study was to investigate the expression of COX-2 and its association with clinical parameters, patient's survival, hormones receptors (oestrogen, progesterone), ERBB2 and TP53 expression in 83 cases of infiltrating ductal breast carcinomas. Moreover, the methylation status at the CpG islands of the COX-2 gene promoter was also explored in 70 specimens. We showed that tumours exhibiting moderate to intense COX-2 immunostaining were significantly more frequent in patients over 45 years old (p = 0.027). Moreover, a high level of COX-2 expression correlated with a shorter survival time (p log-rank = 0.04) and was an independent prognostic factor (p = 0.022; HR 6.4; 95% CI = 1.3-31.4). On the other hand, hypermethylation of the COX-2 gene promoter was observed in 27% of cases and strongly associated with smaller tumours (<5 cm, p = 0.011). Furthermore, patients with methylated COX-2 pattern have a better 4-year disease-free survival (p = 0.022) as well as a prolonged overall survival (p log-rank test = 0.034). In conclusion, we showed that high COX-2 expression was associated with reduced survival and was an independent prognostic factor. However, hypermethylation of the COX-2 promoter correlated with a better overall survival in Tunisian patients with breast carcinoma. PMID:21153458
Upregulation of GRIM-19 inhibits the growth and invasion of human breast cancer cells.
Zhang, Wei; Du, Ye; Jiang, Tong; Geng, Wei; Yuan, Jiuli; Zhang, Duo
Gene associated with retinoid-interferon (IFN)-induced mortality 19 (GRIM-19), a novel IFN-β/retinoic acid-inducible gene product, has been identified as a potential tumor suppressor, which is associated with the inhibition of tumor growth. GRIM-19 has been demonstrated to be downregulated in the ovarian tissue of patients with breast cancer, however, its role in breast cancer remains to be fully elucidated. In the present study, a recombinant eukaryotic expression plasmid carrying GRIM-19 was constructed and then transfected into the MCF7 human breast cancer cell line to examine its effects on breast cancer cell growth, migration and invasion using several in vitro approaches. The results demonstrated that upregulation GRIM-19 in the MCF7 cells significantly inhibited cell proliferation, colony formation, migration and invasion, and induced cell apoptosis. Additionally, upregulation of GRIM-19 also suppressed the secretion of urokinase-type plasminogen activator (u-PA), matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF). It was also demonstrated that the activation of signal transducer and activator of transcription 3 (STAT3) was downregulated by the expression of GRIM-19. These results revealed that overexpression of the GRIM-19 gene may be an effective approach to control the growth and invasion of human breast cancer cells. PMID:25955394
High DRC Levels Are Associated with Let-7b Overexpression in Women with Breast Cancer
Encarnación, Jarline; Ortiz, Carmen; Vergne, Ralphdy; Vargas, Wanda; Coppola, Domenico; Matta, Jaime L.
Nucleotide Excision Repair (NER) is a critical pathway involved in breast cancer (BC). We have previously published that a low DNA repair capacity (DRC) is associated with a higher risk of BC in Puerto Rican women. Let-7b belongs to a miRNA family with tumor suppressor activity that targets oncogenes. We isolated miRNAs from plasma of 153 Puerto Rican women with and without BC. DRC was measured in lymphocytes by means of a host cell reactivation assay. These women were divided into four groups according to their DRC level: High (>3.8%) and low (<3.8%). The four groups consisted of BC patients with high (n = 35) and low (n = 43) DRC and controls with high (n = 39) and low (n = 36) DRC. Epidemiologic data were collected at initial BC diagnosis and almost five years after diagnosis. A significant difference in Let-7b expression was found in BC patients with high DRC versus the remaining groups (p < 0.001). Thus, our data reveal a possible role of Let-7b on DRC during breast carcinogenesis. Our study is innovative because it provides the first evidence that Let-7b may play role in DRC regulation (through the NER repair pathway) in BC. PMID:27271599
High DRC Levels Are Associated with Let-7b Overexpression in Women with Breast Cancer.
Encarnación, Jarline; Ortiz, Carmen; Vergne, Ralphdy; Vargas, Wanda; Coppola, Domenico; Matta, Jaime L
Nucleotide Excision Repair (NER) is a critical pathway involved in breast cancer (BC). We have previously published that a low DNA repair capacity (DRC) is associated with a higher risk of BC in Puerto Rican women. Let-7b belongs to a miRNA family with tumor suppressor activity that targets oncogenes. We isolated miRNAs from plasma of 153 Puerto Rican women with and without BC. DRC was measured in lymphocytes by means of a host cell reactivation assay. These women were divided into four groups according to their DRC level: High (>3.8%) and low (<3.8%). The four groups consisted of BC patients with high (n = 35) and low (n = 43) DRC and controls with high (n = 39) and low (n = 36) DRC. Epidemiologic data were collected at initial BC diagnosis and almost five years after diagnosis. A significant difference in Let-7b expression was found in BC patients with high DRC versus the remaining groups (p < 0.001). Thus, our data reveal a possible role of Let-7b on DRC during breast carcinogenesis. Our study is innovative because it provides the first evidence that Let-7b may play role in DRC regulation (through the NER repair pathway) in BC. PMID:27271599
Overexpression of caspase 7 is ERα dependent to affect proliferation and cell growth in breast cancer cells by targeting p21Cip
Chaudhary, S; Madhukrishna, B; Adhya, A K; Keshari, S; Mishra, S K
Caspase 7 (CASP7) expression has important function during cell cycle progression and cell growth in certain cancer cells and is also involved in the development and differentiation of dental tissues. However, the function of CASP7 in breast cancer cells is unclear. The aim of this study was to analyze the expression of CASP7 in breast carcinoma patients and determine the role of CASP7 in regulating tumorigenicity in breast cancer cells. In this study, we show that the CASP7 expression is high in breast carcinoma tissues compared with normal counterpart. The ectopic expression of CASP7 is significantly associated with ERα expression status and persistently elevated in different stages of the breast tumor grades. High level of CASP7 expression showed better prognosis in breast cancer patients with systemic endocrine therapy as observed from Kaplan–Meier analysis. S3 and S4, estrogen responsive element (ERE) in the CASP7 promoter, is important for estrogen-ERα-mediated CASP7 overexpression. Increased recruitment of p300, acetylated H3 and pol II in the ERE region of CASP7 promoter is observed after hormone stimulation. Ectopic expression of CASP7 in breast cancer cells results in cell growth and proliferation inhibition via p21Cip reduction, whereas small interfering RNA (siRNA) mediated reduction of CASP7 rescued p21Cip levels. We also show that pro- and active forms of CASP7 is located in the nucleus apart from cytoplasmic region of breast cancer cells. The proliferation and growth of breast cancer cells is significantly reduced by broad-spectrum peptide inhibitors and siRNA of CASP7. Taken together, our findings show that CASP7 is aberrantly expressed in breast cancer and contributes to cell growth and proliferation by downregulating p21Cip protein, suggesting that targeting CASP7-positive breast cancer could be one of the potential therapeutic strategies. PMID:27089142
Humoral immunity to human breast cancer: antigen definition and quantitative analysis of mRNA expression.
Scanlan, M J; Gout, I; Gordon, C M; Williamson, B; Stockert, E; Gure, A O; Jäger, D; Chen, Y T; Mackay, A; O'Hare, M J; Old, L J
The ability of the immune system to recognize structurally altered, amplified or aberrantly expressed proteins can be used to identify molecules of etiologic relevance to cancer and to define targets for cancer immunotherapy. In the current study, ninety-four distinct antigens reactive with serum IgG from breast cancer patients were identified by immunoscreening breast cancer-derived cDNA expression libraries (SEREX). A serological profile was generated for each antigen on the basis of reactivity with allogeneic sera from normal individuals and cancer patients, and mRNA expression profiles for coding sequences were assembled based upon the tissue distribution of expressed sequence tags, Northern blots and real-time RT-PCR. Forty antigens reacted exclusively with sera from cancer patients. These included well-characterized tumor antigens, e.g. MAGE-3, MAGE-6, NY-ESO-1, Her2neu and p53, as well as newly-defined breast cancer antigens, e.g. kinesin 2, TATA element modulatory factor 1, tumor protein D52 and MAGE D, and novel gene products, e.g. NY-BR-62, NY-BR-75, NY-BR-85, and NY-BR-96. With regard to expression profiles, two of the novel gene products, NY-BR-62 and NY-BR-85, were characterized by a high level of testicular mRNA expression, and were overexpressed in 60% and 90% of breast cancers, respectively. In addition, mRNA encoding tumor protein D52 was overexpressed in 60% of breast cancer specimens, while transcripts encoding SNT-1 signal adaptor protein were downregulated in 70% of these cases. This study adds to the growing list of breast cancer antigens defined by SEREX and to the ultimate objective of identifying the complete repertoire of immunogenic gene products in human cancer (the cancer immunome). PMID:12747765
The Polycomb group protein RING1B is overexpressed in ductal breast carcinoma and is required to sustain FAK steady state levels in breast cancer epithelial cells
Bosch, Almudena; Panoutsopoulou, Konstantina; Corominas, Josep Maria; Gimeno, Ramón; Moreno-Bueno, Gema; Martín-Caballero, Juan; Morales, Saleta; Lobato, Tania; Martínez-Romero, Carles; Farias, Eduardo F.; Mayol, Xavier; Cano, Amparo; Hernández-Muáoz, Inmaculada
In early stages of metastasis malignant cells must acquire phenotypic changes to enhance their migratory behavior and their ability to breach the matrix surrounding tumors and blood vessel walls. Epigenetic regulation of gene expression allows the acquisition of these features that, once tumoral cells have escape from the primary tumor, can be reverted. Here we report that the expression of the Polycomb epigenetic repressor Ring1B is enhanced in tumoral cells that invade the stroma in human ductal breast carcinoma and its expression is coincident with that of Fak in these tumors. Ring1B knockdown in breast cancer cell lines revealed that Ring1B is required to sustain Fak expression in basal conditions as well as in Tgfβ-treated cells. Functionally, endogenous Ring1B is required for cell migration and invasion in vitro and for in vivo invasion of the mammary fat pad by tumoral cells. Finally we identify p63 as a target of Ring1B to regulate Fak expression: Ring1B depletion results in enhanced p63 expression, which in turns represses Fak expression. Importantly, Fak downregulation upon Ring1B depletion is dependent on p63 expression. Our findings provide new insights in the biology of the breast carcinoma and open new avenues for breast cancer prognosis and therapy. PMID:24742605
Geminin overexpression-dependent recruitment and crosstalk with mesenchymal stem cells enhance aggressiveness in triple negative breast cancers
Ananthula, Suryatheja; Sinha, Abhilasha; Gassim, Mohamed El; Batth, Simran; Marshall, Gailen D.; Gardner, Lauren H.; Shimizu, Yoshiko; ElShamy, Wael M.
Resident mesenchymal stem cells (MSCs) promote cancer progression. However, pathways and mechanisms involved in recruiting MSCs into breast tumors remain largely undefined. Here we show that geminin-dependent acetylation releases HMGB1 from the chromatin to the cytoplasm and extracellular space. Extracellular acetylated HMGB1 (Ac-HMGB1) promotes geminin overexpressing (GemOE) cells survival by binding to RAGE and activating NF-κB signaling. Extracellular Ac-HMGB1 also triggers expression and activation of RAGE in the non-expressing MSCs. RAGE activation induces expression of CXCR4 in MSCs and directional migration towards SDF1 (aka CXCL12)-expressing GemOE cells in vitro and in vivo. These effects augmented by the necrotic and hypoxic environment in GemOE tumors, especially within their cores. Reciprocal interactions between newly recruited MSCs and GemOE tumor cells elevate tumor-initiating (TIC), basal and epithelial-to-mesenchymal transition (EMT) traits and enhance aggressiveness in vitro and in vivo in GemOE tumor cells. Indeed, faster, larger and more aggressive tumors develop when GemOE cells are co-injected with MSCs in orthotopic breast tumor model. Concurrently, inhibiting c-Abl (and thus geminin function), RAGE or CXCR4 prevented MSCs recruitment to GemOE cells in vitro and in vivo, and decreased the TIC, basal and EMT phenotypes in these tumor cells. Accordingly, we propose that GemOE tumor cells present within tumor cores represent metastatic precursors, and suppressing the GemOE→HMGB1/RAGE→SDF1/CXCR4 signaling circuit could be a valid target for therapies to inhibit GemOE tumors and their metastases. PMID:26989079
Increased expression of CYP4Z1 promotes tumor angiogenesis and growth in human breast cancer
Yu, Wei; Chai, Hongyan; Li, Ying; Zhao, Haixia; Xie, Xianfei; Zheng, Hao; Wang, Chenlong; Wang, Xue; Yang, Guifang; Cai, Xiaojun; Falck, John R.; Yang, Jing
Cytochrome P450 (CYP) 4Z1, a novel CYP4 family member, is over-expressed in human mammary carcinoma and associated with high-grade tumors and poor prognosis. However, the precise role of CYP4Z1 in tumor progression is unknown. Here, we demonstrate that CYP4Z1 overexpression promotes tumor angiogenesis and growth in breast cancer. Stable expression of CYP4Z1 in T47D and BT-474 human breast cancer cells significantly increased mRNA expression and production of vascular endothelial growth factor (VEGF)-A, and decreased mRNA levels and secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), without affecting cell proliferation and anchorage-independent cell growth in vitro. Notably, the conditioned medium from CYP4Z1-expressing cells enhanced proliferation, migration and tube formation of human umbilical vein endothelial cells, and promoted angiogenesis in the zebrafish embryo and chorioallantoic membrane of the chick embryo. In addition, there were lower levels of myristic acid and lauric acid, and higher contents of 20-hydroxyeicosatetraenoic acid (20-HETE) in CYP4Z1-expressing T47D cells compared with vector control. CYP4Z1 overexpression significantly increased tumor weight and microvessel density by 2.6-fold and 1.9-fold in human tumor xenograft models, respectively. Moreover, CYP4Z1 transfection increased the phosphorylation of ERK1/2 and PI3K/Akt, while PI3K or ERK inhibitors and siRNA silencing reversed CYP4Z1-mediated changes in VEGF-A and TIMP-2 expression. Conversely, HET0016, an inhibitor of the CYP4 family, potently inhibited the tumor-induced angiogenesis with associated changes in the intracellular levels of myristic acid, lauric acid and 20-HETE. Collectively, these data suggest that increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation. -- Highlights: ► CYP4Z1 overexpression promotes human breast cancer growth and angiogenesis. ► The pro-angiogenic effects of CYP4Z1 have
Overexpression of Snail induces epithelial–mesenchymal transition and a cancer stem cell–like phenotype in human colorectal cancer cells
Fan, Fan; Samuel, Shaija; Evans, Kurt W; Lu, Jia; Xia, Ling; Zhou, Yunfei; Sceusi, Eric; Tozzi, Federico; Ye, Xiang-Cang; Mani, Sendurai A; Ellis, Lee M
Epithelial–mesenchymal transition (EMT) is a critical process providing tumor cells with the ability to migrate and escape from the primary tumor and metastasize to distant sites. Recently, EMT was shown to be associated with the cancer stem cell (CSC) phenotype in breast cancer. Snail is a transcription factor that mediates EMT in a number of tumor types, including colorectal cancer (CRC). Our study was done to determine the role of Snail in mediating EMT and CSC function in CRC. Human CRC specimens were stained for Snail expression, and human CRC cell lines were transduced with a retroviral Snail construct or vector control. Cell proliferation and chemosensitivity to oxaliplatin of the infected cells were determined by the MTT (colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Migration and invasion were determined in vitro using modified Boyden chamber assays. EMT and putative CSC markers were analyzed using Western blotting. Intravenous injection of tumor cells was done to evaluate their metastatic potential in mice. Snail was overexpressed in human CRC surgical specimens. This overexpression induced EMT and a CSC-like phenotype in human CRC cells and enhanced cell migration and invasion (P < 0.002 vs. control). Snail overexpression also led to an increase in metastasis formation in vivo (P < 0.002 vs. control). Furthermore, the Snail-overexpressing CRC cells were more chemoresistant to oxaliplatin than control cells. Increased Snail expression induces EMT and the CSC-like phenotype in CRC cells, which enhance cancer cell invasion and chemoresistance. Thus, Snail is a potential therapeutic target in metastatic CRC. PMID:23342249
Overexpressed human heme Oxygenase-1 decreases adipogenesis in pigs and porcine adipose-derived stem cells.
Park, Eun Jung; Koo, Ok Jae; Lee, Byeong Chun
Adipose-derived mesenchymal stem cells (ADSC) are multipotent, which means they are able to differentiate into several lineages in vivo and in vitro under proper conditions. This indicates it is possible to determine the direction of differentiation of ADSC by controlling the microenvironment. Heme oxygenase 1 (HO-1), a type of antioxidant enzyme, attenuates adipogenicity and obesity. We produced transgenic pigs overexpressing human HO-1 (hHO-1-Tg), and found that these animals have little fatty tissue when autopsied. To determine whether overexpressed human HO-1 suppresses adipogenesis in pigs, we analyzed body weight increases of hHO-1-Tg pigs and wild type (WT) pigs of the same strain, and induced adipogenic differentiation of ADSC derived from WT and hHO-1-Tg pigs. The hHO-1-Tg pigs had lower body weights than WT pigs from 16 weeks of age until they died. In addition, hHO-1-Tg ADSC showed reduced adipogenic differentiation and expression of adipogenic molecular markers such as PPARγ and C/EBPα compared to WT ADSC. These results suggest that HO-1 overexpression reduces adipogenesis both in vivo and in vitro, which could support identification of therapeutic targets of obesity and related metabolic diseases. PMID:26471299
Docosahexaenoic Acid Modulates a HER2-Associated Lipogenic Phenotype, Induces Apoptosis, and Increases Trastuzumab Action in HER2-Overexpressing Breast Carcinoma Cells
Ravacci, Graziela Rosa; Brentani, Maria Mitzi; Tortelli, Tharcisio Citrângulo; Torrinhas, Raquel Suzana M. M.; Santos, Jéssica Reis; Logullo, Angela Flávia; Waitzberg, Dan Linetzky
In breast cancer, lipid metabolic alterations have been recognized as potential oncogenic stimuli that may promote malignancy. To investigate whether the oncogenic nature of lipogenesis closely depends on the overexpression of HER2 protooncogene, the normal breast cell line, HB4a, was transfected with HER2 cDNA to obtain HER2-overexpressing HB4aC5.2 cells. Both cell lines were treated with trastuzumab and docosahexaenoic acid. HER2 overexpression was accompanied by an increase in the expression of lipogenic genes involved in uptake (CD36), transport (FABP4), and storage (DGAT) of exogenous fatty acids (FA), as well as increased activation of "de novo" FA synthesis (FASN). We further investigate whether this lipogenesis reprogramming might be regulated by mTOR/PPARγ pathway. Inhibition of the mTORC1 pathway markers, p70S6 K1, SREBP1, and LIPIN1, as well as an increase in DEPTOR expression (the main inhibitor of the mTOR) was detected in HB4aC5.2. Based on these results, a PPARγ selective antagonist, GW9662, was used to treat both cells lines, and the lipogenic genes remained overexpressed in the HB4aC5.2 but not HB4a cells. DHA treatment inhibited all lipogenic genes (except for FABP4) in both cell lines yet only induced death in the HB4aC5.2 cells, mainly when associated with trastuzumab. Neither trastuzumab nor GW9662 alone was able to induce cell death. In conclusion, oncogenic transformation of breast cells by HER2 overexpression may require a reprogramming of lipogenic genetic that is independent of mTORC1 pathway and PPARγ activity. This reprogramming was inhibited by DHA. PMID:26640797
Nucleolin overexpression in breast cancer cell sub-populations with different stem-like phenotype enables targeted intracellular delivery of synergistic drug combination.
Fonseca, Nuno A; Rodrigues, Ana S; Rodrigues-Santos, Paulo; Alves, Vera; Gregório, Ana C; Valério-Fernandes, Ângela; Gomes-da-Silva, Lígia C; Rosa, Manuel Santos; Moura, Vera; Ramalho-Santos, João; Simões, Sérgio; Moreira, João Nuno
Breast cancer stem cells (CSC) are thought responsible for tumor growth and relapse, metastization and active evasion to standard chemotherapy. The recognition that CSC may originate from non-stem cancer cells (non-SCC) through plastic epithelial-to-mesenchymal transition turned these into relevant cell targets. Of crucial importance for successful therapeutic intervention is the identification of surface receptors overexpressed in both CSC and non-SCC. Cell surface nucleolin has been described as overexpressed in cancer cells as well as a tumor angiogenic marker. Herein we have addressed the questions on whether nucleolin was a common receptor among breast CSC and non-SCC and whether it could be exploited for targeting purposes. Liposomes functionalized with the nucleolin-binding F3 peptide, targeted simultaneously, nucleolin-overexpressing putative breast CSC and non-SCC, which was paralleled by OCT4 and NANOG mRNA levels in cells from triple negative breast cancer (TNBC) origin. In murine embryonic stem cells, both nucleolin mRNA levels and F3 peptide-targeted liposomes cellular association were dependent on the stemness status. An in vivo tumorigenic assay suggested that surface nucleolin overexpression per se, could be associated with the identification of highly tumorigenic TNBC cells. This proposed link between nucleolin expression and the stem-like phenotype in TNBC, enabled 100% cell death mediated by F3 peptide-targeted synergistic drug combination, suggesting the potential to abrogate the plasticity and adaptability associated with CSC and non-SCC. Ultimately, nucleolin-specific therapeutic tools capable of simultaneous debulk multiple cellular compartments of the tumor microenvironment may pave the way towards a specific treatment for TNBC patient care. PMID:26283155
Efficacy and Safety of HER2-Targeted Agents for Breast Cancer with HER2-Overexpression: A Network Meta-Analysis
Yu, Qiuyan; Zhu, Zhenli; Liu, Yan; Zhang, Jun; Li, Ke
Background Clinical trials of human epidermal growth factor receptor 2 (HER2)-targeted agents added to standard treatment have been efficacious for HER2-positive (HER2+) advanced breast cancer. To our knowledge, no meta-analysis has evaluated HER2-targeted therapy including trastuzumab emtansine (T-DM1) and pertuzumab for HER2-positive breast caner and ranked the targeted treatments. We performed a network meta-analysis of both direct and indirect comparisons to evaluate the effect of adding HER2-targeted agents to standard treatment and examined side effects. Methods We performed a Bayesian-framework network meta-analysis of randomized controlled trials to compare 6 HER2-targeted treatment regimens and 1 naïve standard treatment (NST, without any-targeted drugs) in targeted treatment of HER2+ breast cancer in adults. These treatment regimens were T-DM1, LC (lapatinib), HC (trastuzumab), PEC (pertuzumab), LHC (lapatinib and trastuzumab), and PEHC (pertuzumab and trastuzumab). The main outcomes were overall survival and response rates. We also examined side effects of rash, LVEF (left ventricular ejection fraction), fatigue, and gastrointestinal disorders, and performed subgroup analysis for the different treatment regimens in metastatic or advanced breast cancer. Results We identified 25 articles of 21 trials, with data for 11,276 participants. T-DM1 and PEHC were more efficient drug regimens with regard to overall survival as compared with LHC, LC, HC and PEC. The incidence of treatment-related rash occurs more frequently in the patients who received LC treatment regimen than PEHC and T-DM1 and HC. In subgroup analysis, T-DM1 was associated with increased overall survival as compared with LC and HC. PEHC was associated with increased overall response as compared with LC, HC, and NST. Conclusions Overall, the regimen of T-DM1 as well as pertuzumab in combination with trastuzumab and docetaxel is efficacious with fewer side effects as compared with other regimens
Over-expression of miR-675 in formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer patients
Zhai, Ling-Ling; Wang, Peng; Zhou, Ling-Yu; Yin, Jia-Yu; Tang, Qin; Zhang, Tin-Juan; Wang, Yu-Xin; Yang, Dong-Qin; Lin, Jiang; Deng, Zhao-Qun
Background: Dysregulation of miR-675 has been found in a variety of solid tumors. MiR-675 has been suggested as having both oncogenic and tumor suppression properties in cancer. However, there is no evidence whether miR-675 is involved in breast cancer. The objective of this study was to evaluate the expression status of miR-675 and its clinical relevance in breast cancer patients. Methods: The expression level of miR-675 was detected in 100 breast cancer patients and 38 cancer-free controls using real-time quantitative PCR. The clinicopathological characteristics of miR-675 in breast cancer were also investigated. All statistical analyses were performed using SPSS 20.0. Results: The study showed that miR-675 was significantly up-regulated in breast cancer patients compared with controls (P < 0.01). There was no significant difference in age, lymph nodes stage, ER status and PR status between patients with and without miR-675 over-expression (P > 0.05). The frequency of miR-675 over-expression was higher in the patients of histological grade I-II than in others (50% versus 9%, P = 0.011). The expression level of miR-675 had a high correlation with miR-24/93/98/378 in breast cancer patients. Conclusions: Taken together, our study demonstrated that miR-675 in formalin-fixed paraffin-embedded (FFPE) tissues might serve as a good source for biomarker discovery and breast cancer validation. PMID:26379923
Screening of HER2 Overexpressed Breast Cancer Subtype In Vivo by the Validation of High-Performance, Long-Term, and Noninvasive Fluorescence Tracer.
Ding, Jie; Zhou, Ying; Li, Jingjing; Jiang, Liping; He, Zhiwei; Zhu, Jun-Jie
The high-performance and noninvasive screening of heterogeneous tumor subtypes in vivo is particularly desirable for the diagnosis and symptomatic treatment of cancer. Therefore, we report a near-infrared (NIR) fluorescence tracer "smartly identified HER2" (SI-HER2) for rapid, accurate, and highly specific screening of HER2 overexpressed breast cancer. An antibody against HER2 protein receptor, EP1045Y, was conjugated with NIR emitting CdSeTe/CdS/ZnS QDs via polyhistidine-driven self-assembly approach. The further adsorption of black hole quencher 3 on antibody enabled a "turn on" fluorescence response of the fluorescence tracer to HER2 protein receptor. Aside from the capability of differentiating the HER2 overexpressed MCF-7 cells from its counterparts, the fluorescence tracer can also accurately and rapidly identify the HER2 overexpressed breast tumor subtype in two tumors-bearing mouse model, providing a platform for the investigation of advanced pathways to distinguish the different breast cancer subtypes. PMID:26598802
Human antimicrobial protein hCAP18/LL-37 promotes a metastatic phenotype in breast cancer
Weber, Günther; Chamorro, Clara Ibel; Granath, Fredrik; Liljegren, Annelie; Zreika, Sami; Saidak, Zuzana; Sandstedt, Bengt; Rotstein, Samuel; Mentaverri, Romuald; Sánchez, Fabio; Pivarcsi, Andor; Ståhle, Mona
Introduction Human cathelicidin antimicrobial protein, hCAP18, and its C-terminal peptide LL-37 is a multifunctional protein. In addition to being important in antimicrobial defense, it induces chemotaxis, stimulates angiogenesis and promotes tissue repair. We previously showed that human breast cancer cells express high amounts of hCAP18, and hypothesised that hCAP18/LL-37 may be involved in tumour progression. Methods hCAP18 mRNA was quantified in 109 primary breast cancers and compared with clinical findings and ERBB2 mRNA expression. Effects of exogenous LL-37 and transgenic overexpression of hCAP18 on ErbB2 signalling were investigated by immunoblotting using extracts from breast cancer cell lines ZR75-1 and derivatives of MCF7. We further analysed the impact of hCAP18/LL-37 on the morphology of breast cancer cells grown in soft agar, on cell migration and on tumour development in severe combined immunodeficiency (SCID) mice. Results The expression of hCAP18 correlated closely with that of ERBB2 and with the presence of lymph node metastases in oestrogen receptor-positive tumours. hCAP18/LL-37 amplified Heregulin-induced mitogen-activated protein kinase (MAPK) signalling through ErbB2, identifying a functional association between hCAP18/LL-37 and ErbB2 in breast cancer. Treatment with LL-37 peptide significantly stimulated the migration of breast cancer cells and their colonies acquired a dispersed morphology indicative of increased metastatic potential. A truncated version of LL-37 competitively inhibited LL-37 induced MAPK phosphorylation and significantly reduced the number of altered cancer cell colonies induced by LL-37 as well as suppressed their migration. Transgenic overexpression of hCAP18 in a low malignant breast cancer cell line promoted the development of metastases in SCID mice, and analysis of hCAP18 transgenic tumours showed enhanced activation of MAPK signalling. Conclusions Our results provide evidence that hCAP18/LL-37 contributes to breast
Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT
Xu, Hanwen; Pirisi, Lucia; Creek, Kim E.
Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial–mesenchymal transition (EMT). Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistance to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes. - Highlights: • Six1 expression increases during HPV16-mediated transformation. • Six1 overexpression causes differentiation resistance in HPV16-immortalized cells. • Six1 overexpression in HPV16-immortalized keratinocytes activates MAPK. • Activation of MAPK promotes EMT and differentiation resistance. • Six1 overexpression reduces Smad-dependent TGF-β signaling.
Overexpression of human β-defensin 2 promotes growth and invasion during esophageal carcinogenesis
Shi, Ni; Jin, Feng; Zhang, Xiaoli; Clinton, Steven K.; Pan, Zui; Chen, Tong
Human β-defensin 2 (HBD-2) is an antimicrobial peptide produced by mucosal surfaces in response to microbial exposure or inflammatory cytokines. Although HBD-2 is expressed in the esophagus in response to stress and infectious agents, little is known regarding its expression and functional role in esophageal carcinogenesis. In the current investigation, normal esophagus and N-nitrosomethylbenzylamine (NMBA)-induced precancerous and papillomatous lesions of the rat esophagus were characterized for HBD-2 encoding gene Defb4 and protein. HBD-2 was found to be overexpressed in esophagi of rats treated with NMBA compared to animals in control group. Results of Real-time PCR, Western blot and immunohistochemistry demonstrated a positive correlation between the overexpression of HBD-2 and the progression of rat squamous cell carcinogenesis (SCC) in the esophagus. We also observed that HBD-2 is overexpressed in tumor tissues removed from patients with esophageal SCC. Moreover, Defb4 silencing in vitro suppresses the tumor cell proliferation, mobility and invasion in esophageal SCC cell line KYSE-150. The results from this study provide experimental evidence that HBD-2 may play an oncogenic role in the initiation and progression of esophageal SCC and thus serves as a target for chemopreventive and therapeutic interventions. PMID:25226614
Overexpression of human β-defensin 2 promotes growth and invasion during esophageal carcinogenesis.
Shi, Ni; Jin, Feng; Zhang, Xiaoli; Clinton, Steven K; Pan, Zui; Chen, Tong
Human β-defensin 2 (HBD-2) is an antimicrobial peptide produced by mucosal surfaces in response to microbial exposure or inflammatory cytokines. Although HBD-2 is expressed in the esophagus in response to stress and infectious agents, little is known regarding its expression and functional role in esophageal carcinogenesis. In the current investigation, normal esophagus and N-nitrosomethylbenzylamine (NMBA)-induced precancerous and papillomatous lesions of the rat esophagus were characterized for HBD-2 encoding gene Defb4 and protein. HBD-2 was found to be overexpressed in esophagi of rats treated with NMBA compared to animals in control group. Results of Real-time PCR, Western blot and immunohistochemistry demonstrated a positive correlation between the overexpression of HBD-2 and the progression of rat squamous cell carcinogenesis (SCC) in the esophagus. We also observed that HBD-2 is overexpressed in tumor tissues removed from patients with esophageal SCC. Moreover, Defb4 silencing in vitro suppresses the tumor cell proliferation, mobility and invasion in esophageal SCC cell line KYSE-150. The results from this study provide experimental evidence that HBD-2 may play an oncogenic role in the initiation and progression of esophageal SCC and thus serves as a target for chemopreventive and therapeutic interventions. PMID:25226614
Durable Clinical Benefit of Pertuzumab in a Young Patient with BRCA2 Mutation and HER2-Overexpressing Breast Cancer Involving the Brain
Koumarianou, Anna; Kontopoulou, Christina; Kouloulias, Vassilis; Tsionou, Christina
Patients with HER2-positive breast cancer and brain metastases have limited treatment options, and, as a result of their poor performance status and worse prognosis, they are underrepresented in clinical trials. Not surprisingly, these patients may not be fit enough to receive any active treatment and are offered supportive therapy. BRCA2 mutations are reported to be rarely associated with HER2-overexpressing advanced breast cancer and even more rarely with brain metastases at diagnosis. We report on a BRCA2-positive breast cancer patient with metastatic disease in multiple sites, including the brain, and poor performance status who exhibited an extraordinary clinical and imaging response to the novel anti-HER2 therapy pertuzumab after multiple lines of therapy including anti-HER2 targeting. To our knowledge, the clinicopathologic and therapeutic characteristics of this patient point to a unique case and an urgent need for further investigation of pertuzumab in patients with brain metastases. PMID:27195161
Overexpression of S100A4 in human cancer cell lines resistant to methotrexate
Background Methotrexate is a chemotherapeutic drug that is used in therapy of a wide variety of cancers. The efficiency of treatment with this drug is compromised by the appearance of resistance. Combination treatments of MTX with other drugs that could modulate the expression of genes involved in MTX resistance would be an adequate strategy to prevent the development of this resistance. Methods The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. A global comparison of all the studied cell lines was performed in order to find out differentially expressed genes in the majority of the MTX-resistant cells. S100A4 mRNA and protein levels were determined by RT-Real-Time PCR and Western blot, respectively. Functional validations of S100A4 were performed either by transfection of an expression vector for S100A4 or a siRNA against S100A4. Transfection of an expression vector encoding for β-catenin was used to inquire for the possible transcriptional regulation of S100A4 through the Wnt pathway. Results S100A4 is overexpressed in five out of the seven MTX-resistant cell lines studied. Ectopic overexpression of this gene in HT29 sensitive cells augmented both the intracellular and extracellular S100A4 protein levels and caused desensitization toward MTX. siRNA against S100A4 decreased the levels of this protein and caused a chemosensitization in combined treatments with MTX. β-catenin overexpression experiments support a possible involvement of the Wnt signaling pathway in S100A4 transcriptional regulation in HT29 cells. Conclusions S100A4 is overexpressed in many MTX-resistant cells. S100A4 overexpression decreases the sensitivity of HT29 colon cancer human cells to MTX, whereas its knockdown causes chemosensitization toward MTX. Both approaches highlight a role for S100A4 in MTX resistance. PMID:20515499
[Study of Her-2/neu oncogene in relation to prognosis of human breast cancer].
Chen, R S
A follow-up study of 143 cases of human breast cancer for over 5 years proved that Her-2/neu oncogene overexpression is much more common in the high risk group (patients died within 5 years) in comparison with the low risk group (patients survived over 5 years). The difference between these 2 groups was statistically significant. The Her-2/neu oncogene positive rate in infiltrative ductal carcinoma was 33.3%, the lower the differentiation, the higher the positive rate. Histological typing is also related to the positive rate, comedocarcinoma (intraductal carcinoma) expresses the highest positive rate while lobular carcinoma the lowest. Selection of fixation fluid and the mastering of diagnostic criteria are also important. In the author's opinion, only membrane staining in monoclonal antibody C-erbB-2 can be recognized as truly positive. In conclusion, Her-2/neu oncogene expression can be used as a supplemental marker when considering prognosis in breast cancer. PMID:7909501
In vitro comparative models for canine and human breast cancers
During the past four decades, an increased number of similarities between canine mammary tumors and human breast cancer have been reported: molecular, histological, morphological, clinical and epidemiological, which lead to comparative oncological studies. One of the most important goals in human and veterinary oncology is to discover potential molecular biomarkers that could detect breast cancer in an early stage and to develop new effective therapies. Recently, cancer cell lines have successfully been used as an in vitro model to study the biology of cancer, to investigate molecular pathways and to test the efficiency of anticancer drugs. Moreover, establishment of an experimental animal model for the study of human breast cancer will improve testing potential anti-cancer therapies and the discovery of effective therapeutic schemes suitable for human clinical trials. In this review, we collected data from previous studies that strengthen the value of canine mammary cancer cell lines as an in vitro model for the study of human breast cancer. PMID:27004024
Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency.
Tsai, Steven C; Chang, David F; Hong, Chang-Mu; Xia, Ping; Senadheera, Dinithi; Trump, Lisa; Mishra, Suparna; Lutzko, Carolyn
Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. The level of octamer-binding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC. PMID:24627557
Overexpression of CD99 Increases the Migration and Invasiveness of Human Malignant Glioma Cells.
Seol, Ho Jun; Chang, Jong Hee; Yamamoto, Junkoh; Romagnuolo, Rocco; Suh, Youngchul; Weeks, Adrienne; Agnihotri, Sameer; Smith, Christian A; Rutka, James T
The malignant glioma is the most common primary human brain tumor, and its migration and invasiveness away from the primary tumor mass are considered a leading cause of tumor recurrence and treatment failure. Recently, gene expression profiling revealed that the transmembrane glycoprotein CD99 is more highly expressed in malignant glioma than in normal brain. Although its function is not completely understood, CD99 is implicated in cell adhesion and migration in a variety of different cell types. CD99 has wild-type and splice variant isoforms. Previous studies have shown that wild-type CD99 may be an oncosuppressor in some tumors, distinct from the role of the splice variant isoform. In this study, our data reveal that only wild-type CD99 is expressed in human glioma cells and tissues. Using a tissue microarray, we validated that gliomas demonstrate higher expression of CD99 compared with nonneoplastic brain. To assess the role of CD99 in glioma migration and invasion, we inhibited CD99 expression by siRNA and demonstrated decreased glioma migration and invasion. In contrast, when CD99 was overexpressed in glioma cells, we observed enhancement of cell migration and invasiveness. An orthotopic brain tumor model demonstrates that CD99 overexpression significantly increases invasiveness and decreases survival rate. Interestingly, Rac activity was decreased and Rho activity was increased in CD99 overexpressing glioma cells, and the proportion of amoeboid cells to mesenchymal cells was significantly increased. Taken together, our findings suggest that CD99 may play an important role in the migration and invasion of human gliomas independent of Akt, ERK, or JNK signaling pathways. Moreover, CD99 might be involved in amoeboid-mesenchymal transition in glioma migration. CD99 may be an important future target to inhibit migration and invasion, especially in CD99-expressing gliomas. PMID:23486730
Method to Screen Multidrug Transport Inhibitors Using Yeast Overexpressing a Human MDR Transporter.
Fiorini, Laura; Mus-Veteau, Isabelle
Multidrug resistance has appeared to mitigate the efficiency of anticancer drugs and the possibility of successful cancer chemotherapy. The Hedgehog receptor Patched is a multidrug transporter expressed in several cancers and as such it represents a new target to circumvent chemotherapy resistance. In this chapter, we describe the screening test developed to identify molecules able to inhibit the drug efflux activity of Patched. This screening test uses yeast overexpressing functional human Patched that have been shown to resist to chemotherapeutic agents. This test can be adapted to other MDR transporters. PMID:27485344
Is human cytomegalovirus associated with breast cancer progression?
Background It has been hypothesized that human cytomegalovirus (HCMV) may be associated with breast cancer progression. However, the role of HCMV infection in breast cancer remains controversial. We aimed to assess whether HCMV genes (UL122 and UL83) could be detected in breast carcinomas and reinvestigated their possible association with breast cancer progression. DNA from paraffin-embedded tissues was analyzed by real-time PCR. We investigated 20 fibroadenomas and 27 primary breast carcinomas (stages II, III, and IV). Findings Two carcinomas were positive for HCMV, one was positive for two TaqMan viral detection probes, and one was positive for a sole TaqMan viral detection probe (UL83), whereas the remainder of the samples was negative. Conclusions Samples studied showed no association between HCMV infection and breast cancer progression. PMID:23557440
PRKAR1A is overexpressed and represents a possible therapeutic target in human cholangiocarcinoma.
Loilome, Watcharin; Juntana, Sirinun; Namwat, Nisana; Bhudhisawasdi, Vajarabhongsa; Puapairoj, Anucha; Sripa, Banchob; Miwa, Masanao; Saya, Hideyuki; Riggins, Gregory J; Yongvanit, Puangrat
The protein kinase A regulatory subunit 1 alpha (PRKAR1A/PKAI) pathway is overexpressed in varieties of tumors and cancer cell lines including cholangiocarcinoma (CCA), although its role in CCA growth modulation is unclear. In our study, we evaluated the effect of PRKAR1A/PKAI targeting on CCA cell proliferation. Real-time PCR demonstrated an increased mRNA expression of PRKAR1A/PKAI, whereas protein kinase A regulatory subunit 2 beta (PRKAR2B/PKAII) was downregulated in human CCA tissues and CCA cell lines. Immunohistochemistry of human CCA tissues revealed increased PRKAR1A with decreased PRKAR2B protein expression. Moreover, CCA cell lines showed abundantly expressed PRKAR1A, while lacking PRKAR2B expression. Silencing PRKAR1A expression induced growth inhibition and apoptosis of CCA cells, with an associated decrease in mitogen-activated protein kinases, PI3K/Akt, JAK/STAT and Wnt/β-catenin pathway signaling. The inhibition of PKA using a PKA inhibitor and cAMP analogs also led to a significant cell growth inhibition. In conclusion, our study reports the overexpression as well as molecular mechanisms by which PRKAR1A/PKA regulates human CCA cell growth. Importantly, abrogation of gene expression caused significant CCA cell growth inhibition, oncogenic signaling and coupled apoptosis induction, suggesting PRKAR1A's potential as a drug target for CCA therapy. PMID:20824711
Cyclopamine and jervine induce COX-2 overexpression in human erythroleukemia cells but only cyclopamine has a pro-apoptotic effect
Ghezali, Lamia; Leger, David Yannick; Limami, Youness; Cook-Moreau, Jeanne; Beneytout, Jean-Louis; Liagre, Bertrand
Erythroleukemia is generally associated with a very poor response and survival to current available therapeutic agents. Cyclooxygenase-2 (COX-2) has been described to play a crucial role in the proliferation and differentiation of leukemia cells, this enzyme seems to play an important role in chemoresistance in different cancer types. Previously, we demonstrated that diosgenin, a plant steroid, induced apoptosis in HEL cells with concomitant COX-2 overexpression. In this study, we investigated the antiproliferative and apoptotic effects of cyclopamine and jervine, two steroidal alkaloids with similar structures, on HEL and TF1a human erythroleukemia cell lines and, for the first time, their effect on COX-2 expression. Cyclopamine, but not jervine, inhibited cell proliferation and induced apoptosis in these cells. Both compounds induced COX-2 overexpression which was responsible for apoptosis resistance. In jervine-treated cells, COX-2 overexpression was NF-κB dependent. Inhibition of NF-κB reduced COX-2 overexpression and induced apoptosis. In addition, cyclopamine induced apoptosis and COX-2 overexpression via PKC activation. Inhibition of the PKC pathway reduced both apoptosis and COX-2 overexpression in both cell lines. Furthermore, we demonstrated that the p38/COX-2 pathway was involved in resistance to cyclopamine-induced apoptosis since p38 inhibition reduced COX-2 overexpression and increased apoptosis in both cell lines. - Highlights: ► Cyclopamine alone but not jervine induces apoptosis in human erythroleukemia cells. ► Cyclopamine and jervine induce COX-2 overexpression. ► COX-2 overexpression is implicated in resistance to cyclopamine-induced apoptosis. ► Apoptotic potential of jervine is restrained by NF-κB pathway activation. ► PKC is involved in cyclopamine-induced apoptosis and COX-2 overexpression.
Human breast milk provides better antioxidant capacity than infant formula.
Oveisi, Mohammad Reza; Sadeghi, Naficeh; Jannat, Behrooz; Hajimahmoodi, Mannan; Behfar, Abd-Ol-Azim; Jannat, Forouzandeh; Mokhtarinasab, Fariba
Human milk contains all of the constituents that are required for the optimal growth and development of a neonate. It supports the development of brain, immune, and physiological systems. This study aimed to consider the significance of breast milk in preventing oxidative stress by comparing total antioxidant capacity (TAC) in breast and formula milk for premature infants, demonstrating the relationship between TAC in breast milk and postnatal age in days. The Ferric reducing antioxidant power assay (FRAP) method was used to spectophotometrically measure of TAC in breast and formula milk. One hundred and fourty (n = 140) lactating mothers agreed to participate in the study. TAC was also measured in two brands of formula milk (n = 80). The Range of TAC in human breast milk was 234.27-1442.31 μM and in two formula was 160.04-630.92 μM. The average TAC was significantly higher in breast milk (642.94 ± 241.23 μM) compared to formula milk (280.986 ± 100.34 μM) p < 0.0001. The TAC of breast milk was increased with some nutritional parameter such as increased consumption of cheese, vegetables, fruits, bread and nuts. Infants' height at the birthday was directly correlated with antioxidant capacity of breast milk, whilst a reversed correlation was observed between TAC in breast milk and infant age. Based on our results, it is concluded that the TAC of breast milk is varied and affected by nutrition. It is alo observed that TAC is significantly higher in breast milk than formula, which means that breast milk provides better antioxidant potency than infant formula. PMID:24381611
Plasma Membrane Proteomics of Human Breast Cancer Cell Lines Identifies Potential Targets for Breast Cancer Diagnosis and Treatment
Ziegler, Yvonne S.; Moresco, James J.; Tu, Patricia G.; Yates, John R.; Nardulli, Ann M.
The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease. PMID:25029196
Expression of the glioma-associated oncogene homolog (GLI) 1 in human breast cancer is associated with unfavourable overall survival
Background The transcription factor GLI1, a member of the GLI subfamily of Krüppel-like zinc finger proteins is involved in signal transduction within the hedgehog pathway. Aberrant hedgehog signalling has been implicated in the development of different human tumour entities such as colon and lung cancer and increased GLI1 expression has been found in these tumour entities as well. In this study we questioned whether GLI1 expression might also be important in human breast cancer development. Furthermore we correlated GLI1 expression with histopathological and clinical data to evaluate whether GLI1 could represent a new prognostic marker in breast cancer treatment. Methods Applying semiquantitative realtime PCR analysis and immunohistochemistry (IHC) GLI1 expression was analysed in human invasive breast carcinomas (n = 229) in comparison to normal human breast tissues (n = 58). GLI1 mRNA expression was furthermore analysed in a set of normal (n = 3) and tumourous breast cell lines (n = 8). IHC data were statistically interpreted using SPSS version 14.0. Results Initial analysis of GLI1 mRNA expression in a small cohort of (n = 5) human matched normal and tumourous breast tissues showed first tendency towards GLI1 overexpression in human breast cancers. However only a small sample number was included into these analyses and values for GLI1 overexpression were statistically not significant (P = 0.251, two-tailed Mann-Whitney U-test). On protein level, nuclear GLI1 expression in breast cancer cells was clearly more abundant than in normal breast epithelial cells (P = 0.008, two-tailed Mann-Whitney U-test) and increased expression of GLI1 protein in breast tumours significantly correlated with unfavourable overall survival (P = 0.019), but also with higher tumour stage (P < 0.001) and an increased number of tumour-positive axillar lymph nodes (P = 0.027). Interestingly, a highly significant correlation was found between GLI1 expression and the expression of SHH, a
Overexpression of potassium channel ether à go-go in human osteosarcoma.
Wu, J; Wu, X; Lian, K; Lin, B; Guo, L; Ding, Z
Human ether à go-go (hEAG) potassium channels are primarily expressed in brain but also frequently overexpressed in solid tumors, which could indicate their potential value for cancer diagnosis and therapy. hEAG1, one member of the hEAG subfamily, has been shown to play a role in neoplastic process. Here we report the expression of hEAG1 in human osteosarcoma detected by a new polyclonal antibody. The full-length hEAG1 cDNA was cloned from human osteosarcoma cell line MG63 by RT-PCR and expressed in Escherichia coli as His tagged protein. The 6His-hEAG1F protein was purified by nickel agarose and used as the antigen to immunize rabbits following standard protocols. The obtained antiserum could detect hEAG1 exogenously expressed in HEK 293 cells. Furthermore, the polyclonal antibody was used to evaluate hEAG1 expression in 42 human osteosarcoma specimens and 19 osteochondromas specimens by immunohistochemistry. hEAG1 was expressed in 71.4% (30/42) osteosarcoma, and 15.8% (3/19) osteochondromas. Moreover, statistical analysis revealed that hEAG1 expression was not dependent on age, sex, site, histology, grade and type in the osteosarcoma specimens. Our data provide evidence that hEAG1 is overexpressed in human osteosarcoma and the hEAG1 polyclonal antibody offers a good tool for further characterization of the oncogenic function of hEAG1 in osteosarcoma. PMID:22248279
Overexpression, purification and preliminary crystallographic analysis of human M-ficolin fibrinogen-like domain
Tanio, Michikazu; Kondo, Shin; Sugio, Shigetoshi; Kohno, Toshiyuki
Human M-ficolin fibrinogen-like domain has been overexpressed in P. pastoris, purified and crystallized. Diffraction data have been collected to 1.9 Å. Ficolins, which are comprised of a collagen-like domain and a fibrinogen-like domain, are a kind of pattern-recognition molecule for pathogens in the innate immunity system. To investigate the molecular mechanism of the discrimination between self and non-self by ficolins, human M-ficolin fibrinogen-like domain (FD1), which contains the ligand-binding site, was overexpressed in Pichia pastoris, purified and crystallized using the vapour-diffusion method at 293 K. The crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 55.16, b = 117.45, c = 55.19 Å, β = 99.88°, and contain three molecules per asymmetric unit. An X-ray data set was collected to 1.9 Å resolution using synchrotron radiation at beamline BL24XU at the SPring-8 facility in Japan.
Human Neural Stem Cells Overexpressing Choline Acetyltransferase Restore Unconditioned Fear in Rats with Amygdala Injury
Shin, Kyungha; Cha, Yeseul; Kim, Kwang Sei; Choi, Ehn-Kyoung; Choi, Youngjin; Guo, Haiyu; Ban, Young-Hwan; Kim, Jong-Choon; Park, Dongsun; Kim, Yun-Bae
Amygdala is involved in the fear memory that recognizes certain environmental cues predicting threatening events. Manipulation of neurotransmission within the amygdala affects the expression of conditioned and unconditioned emotional memories such as fear freezing behaviour. We previously demonstrated that F3.ChAT human neural stem cells (NSCs) overexpressing choline acetyltransferase (ChAT) improve cognitive function of Alzheimer's disease model rats with hippocampal or cholinergic nerve injuries by increasing acetylcholine (ACh) level. In the present study, we examined the effect of F3.ChAT cells on the deficit of unconditioned fear freezing. Rats given N-methyl-d-aspartate (NMDA) in their amygdala 2 weeks prior to cat odor exposure displayed very short resting (freezing) time compared to normal animals. NMDA induced neuronal degeneration in the amygdala, leading to a decreased ACh concentration in cerebrospinal fluid. However, intracerebroventricular transplantation of F3.ChAT cells attenuated amygdala lesions 4 weeks after transplantation. The transplanted cells were found in the NMDA-injury sites and produced ChAT protein. In addition, F3.ChAT-receiving rats recuperated freezing time staying remote from the cat odor source, according to the recovery of brain ACh concentration. The results indicate that human NSCs overexpressing ChAT may facilitate retrieval of unconditioned fear memory by increasing ACh level. PMID:27087745
Human Neural Stem Cells Overexpressing Choline Acetyltransferase Restore Unconditioned Fear in Rats with Amygdala Injury.
Shin, Kyungha; Cha, Yeseul; Kim, Kwang Sei; Choi, Ehn-Kyoung; Choi, Youngjin; Guo, Haiyu; Ban, Young-Hwan; Kim, Jong-Choon; Park, Dongsun; Kim, Yun-Bae
Amygdala is involved in the fear memory that recognizes certain environmental cues predicting threatening events. Manipulation of neurotransmission within the amygdala affects the expression of conditioned and unconditioned emotional memories such as fear freezing behaviour. We previously demonstrated that F3.ChAT human neural stem cells (NSCs) overexpressing choline acetyltransferase (ChAT) improve cognitive function of Alzheimer's disease model rats with hippocampal or cholinergic nerve injuries by increasing acetylcholine (ACh) level. In the present study, we examined the effect of F3.ChAT cells on the deficit of unconditioned fear freezing. Rats given N-methyl-d-aspartate (NMDA) in their amygdala 2 weeks prior to cat odor exposure displayed very short resting (freezing) time compared to normal animals. NMDA induced neuronal degeneration in the amygdala, leading to a decreased ACh concentration in cerebrospinal fluid. However, intracerebroventricular transplantation of F3.ChAT cells attenuated amygdala lesions 4 weeks after transplantation. The transplanted cells were found in the NMDA-injury sites and produced ChAT protein. In addition, F3.ChAT-receiving rats recuperated freezing time staying remote from the cat odor source, according to the recovery of brain ACh concentration. The results indicate that human NSCs overexpressing ChAT may facilitate retrieval of unconditioned fear memory by increasing ACh level. PMID:27087745
Peroxisome proliferator-activated receptor gamma overexpression suppresses proliferation of human colon cancer cells
Tsukahara, Tamotsu; Haniu, Hisao
Highlights: Black-Right-Pointing-Pointer We examined the correlation between PPAR{gamma} expression and cell proliferation. Black-Right-Pointing-Pointer PPAR{gamma} overexpression reduces cell viability. Black-Right-Pointing-Pointer We show the synergistic effect of cell growth inhibition by a PPAR{gamma} agonist. -- Abstract: Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) plays an important role in the differentiation of intestinal cells and tissues. Our previous reports indicate that PPAR{gamma} is expressed at considerable levels in human colon cancer cells. This suggests that PPAR{gamma} expression may be an important factor for cell growth regulation in colon cancer. In this study, we investigated PPAR{gamma} expression in 4 human colon cancer cell lines, HT-29, LOVO, DLD-1, and Caco-2. Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that the relative levels of PPAR{gamma} mRNA and protein in these cells were in the order HT-29 > LOVO > Caco-2 > DLD-1. We also found that PPAR{gamma} overexpression promoted cell growth inhibition in PPAR{gamma} lower-expressing cell lines (Caco-2 and DLD-1), but not in higher-expressing cells (HT-29 and LOVO). We observed a correlation between the level of PPAR{gamma} expression and the cells' sensitivity for proliferation.
Dysregulation of autophagy in human follicular lymphoma is independent of overexpression of BCL-2
McCarthy, Aine; Marzec, Jacek; Clear, Andrew; Petty, Robert D.; Coutinho, Rita; Matthews, Janet; Wilson, Andrew; Iqbal, Sameena; Calaminici, Maria; Gribben, John G.; Jia, Li
Overexpression of the anti-apoptotic protein BCL-2 is characteristic of human follicular lymphoma (FL) and some cases of diffuse large B cell lymphoma (DLBCL). We aimed to determine autophagy status in primary FL and DLBCL samples and the BCL-2+/BCL-2− lymphoma cell lines using both autophagy PCR array and tissue microarray (TMA). A greater number of autophagy machinery genes were up-regulated in the BCL-2+ Su-DHL4 cell line compared with BCL-2− Su-DHL8 cells, at both the basal level and in response to autophagic stress. The autophagy-related gene expression profiles were determined in purified and unpurified malignant human lymph node biopsies. Seven autophagy machinery genes were up-regulated in purified FL B-cells compared with reactive B-cells. Only 2 autophagy machinery genes were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. Expression of autophagy substrates p62 and LC3 were determined by TMAs. FL samples showed significantly decreased levels of both p62 and LC3 compared with reactive and DLBCL, indicative of an increased autophagy activity in FL. In summary, these results demonstrate that FL showed increased basal autophagy activity, regardless of overexpression of BCL-2 in this disease. PMID:25362242
Transgenic mice with overexpression of mutated human optineurin(E50K) in the retina.
Meng, Qingfeng; Xiao, Zheng; Yuan, Huiping; Xue, Fei; Zhu, Yuanmao; Zhou, Xinrong; Yang, Binbin; Sun, Jingbo; Meng, Bo; Sun, Xian; Cheng, Fang
In the present work, Site-directed mutagenesis to insert the Glu50Lys amino acid substitution was achieved by PCR using plasmid pBluescript-OPTN. Mutated human OPTN(E50K) gene-driven mouse c-kit promoter was constructed and confirmed by endonuclease digestion and sequence analysis. Transgenic mice were generated via the microinjection method. PCR and DNA dot blot were used to screen the positive transgenic mice. RT-PCR analyzed the RNA level and location of mutated human OPTN(E50K) mRNA expression in transgenic mice. Western blot and immunohistochemistry were used to detect the level and location of mutated human OPTN(E50K) expression in transgenic mice. A transgenic mouse model with overexpression of mutated human OPTN(E50K) in retina was successfully established. The transgene was integrated and transmitted into the chromosome of transgenic mice. Mutated human OPTN(E50K) gene was controlled by c-kit promoter and expressed in the retina in mice. Mutated human OPTN(E50K) in transgenic mice was higher than that of wild type C57BL/6J mice. Our studies had provided a new transgenic model for investigating the molecular properties of mutated human OPTN(E50K). PMID:21681420
Promotion of breast cancer cells MDA-MB-231 invasion by di(2-ethylhexyl)phthalate through matrix metalloproteinase-2/-9 overexpression.
Zhang, Shuya; Ma, Jiehua; Fu, Ziyi; Zhang, Zhilei; Cao, Jian; Huang, Lei; Li, Wenqu; Xu, Pengfei; Cao, Xin
Di(2-ethylhexyl)phthalate (DEHP) is an estrogenic chemical that is widely used in polyvinyl products. We aimed to determine the mechanisms behind the effects of DEHP on ERα-negative breast cancer cells MDA-MB-231 invasion and matrix metalloproteinases-2/-9 (MMP-2/-9) up-regulation in this study. Transwell assay indicated that DEHP exposure (>50 μg/ml) significantly enhanced the invasion ability of MDA-MB-231 cells. Quantitative real-time PCR (qPCR) and western blotting revealed that MMP-2/-9 is overexpressed in mRNA and protein levels after DEHP treatment. Gelatin zymography consistently demonstrated that DEHP exposure also enhances the activity of MMP-2/-9. Immunofluorescence assay showed that DEHP could accelerate NF-kappaB (NF-κB) subunits-p65 translocation into the nucleus, which is confirmed by western blotting assay, suggesting that the ratio of nuclear/cytosolic level of p65 was significantly increased. Furthermore, the invasion and MMP-2/-9 overexpression of MDA-MB-231 cells after DEHP-treated were reversed by the NF-κB chemical inhibitor JSH-23 via drug inhibition assay. This study suggested that DEHP could promote ERα-negative breast cancer cells MDA-MB-231 invasion through activating NF-κB and MMP-2/-9 overexpression. PMID:26850096
Nodal signaling promotes a tumorigenic phenotype in human breast cancer.
Kirsammer, Gina; Strizzi, Luigi; Margaryan, Naira V; Gilgur, Alina; Hyser, Matthew; Atkinson, Janis; Kirschmann, Dawn A; Seftor, Elisabeth A; Hendrix, Mary J C
The Ras-ERK pathway is deregulated in approximately a third of human cancers, particularly those of epithelial origin. In aggressive, triple-negative, basal-like breast cancers, most tumors display increased MEK and ERK phosphorylation and exhibit a gene expression profile characteristic of Kras or EGFR mutant tumors; however, Ras family genetic mutations are uncommon in triple-negative breast cancer and EGFR mutations account for only a subset of these tumors. Therefore, the upstream events that activate MAPK signaling and promote tumor aggression in triple-negative breast cancers remain poorly defined. We have previously shown that a secreted TGF-β family signaling ligand, Nodal, is expressed in breast cancer in correlation with disease progression. Here we highlight key findings demonstrating that Nodal is required in aggressive human breast cancer cells to activate ERK signaling and downstream tumorigenic phenotypes both in vitro and in vivo. Experimental knockdown of Nodal signaling downregulates ERK activity, resulting in loss of c-myc, upregulation of p27, G1 cell cycle arrest, increased apoptosis and decreased tumorigenicity. The data suggest that ERK activation by Nodal signaling regulates c-myc and p27 proteins post-translationally and that this cascade is essential for aggressive breast tumor behavior in vivo. As the MAPK pathway is an important target for treating triple-negative breast cancers, upstream Nodal signaling may represent a promising target for breast cancer diagnosis and combined therapies aimed at blocking ERK pathway activation. PMID:25073112
Nodal signaling promotes a tumorigenic phenotype in human breast cancer
Kirsammer, Gina; Strizzi, Luigi; Margaryan, Naira V.; Gilgur, Alina; Hyser, Matthew; Atkinson, Janis; Kirschmann, Dawn A.; Seftor, Elisabeth A.; Hendrix, Mary J.C.
The Ras-ERK pathway is deregulated in approximately a third of human cancers, particularly those of epithelial origin. In aggressive, triple-negative, basal-like breast cancers, most tumors display increased MEK and ERK phosphorylation and exhibit a gene expression profile characteristic of Kras or EGFR mutant tumors; however, Ras family genetic mutations are uncommon in triple-negative breast cancer and EGFR mutations account for only a subset of these tumors. Therefore, the upstream events that activate MAPK signaling and promote tumor aggression in triple-negative breast cancers remain poorly defined. We have previously shown that a secreted TGF-β family signaling ligand, Nodal, is expressed in breast cancer in correlation with disease progression. Here we highlight key findings demonstrating that Nodal is required in aggressive human breast cancer cells to activate ERK signaling and downstream tumorigenic phenotypes both in vitro and in vivo. Experimental knockdown of Nodal signaling downregulates ERK activity, resulting in loss of c-myc, upregulation of p27, G1 cell cycle arrest, increased apoptosis and decreased tumorigenicity. The data suggest that ERK activation by Nodal signaling regulates c-myc and p27 proteins post-translationally and that this cascade is essential for aggressive breast tumor behavior in vivo. As the MAPK pathway is an important target for treating triple-negative breast cancers, upstream Nodal signaling may represent a promising target for breast cancer diagnosis and combined therapies aimed at blocking ERK pathway activation. PMID:25073112
Overexpression of Dishevelled-2 contributes to proliferation and migration of human esophageal squamous cell carcinoma.
Zhou, Guoren; Ye, Jinjun; Sun, Lei; Zhang, Zhi; Feng, Jifeng
Dishevelled-2 (Dvl2) was associated with tumor cell proliferation and migration. We aimed to examine the mechanism of Dvl2 in esophageal squamous cell carcinoma (ESCC). Dvl2 was overexpressed in human ESCC tissues and cell lines ECA109 and TE1 cells. CCK-8 and colony formation assay was performed to evaluate the proliferation in ECA109 cells transfected with Dvl2-shRNA. Wound-healing assay and transwell assay were used to examine the activities of migration and invasion in Dvl2-silenced ESCC cells. Knockdown of Dvl2 significantly reduced ECA109 cell proliferation and migration. Moreover, we demonstrated that the proliferation and migration ability of Dvl2 might through the activation of Wnt pathway by targeting the Cyclin D1 and MMP-9. We came to the conclusion that the proliferation and migration effects of Dvl2 might contribute to malignant development of human ESCC. PMID:27083564
Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane
ErbB2 is known to upregulate glycolysis in breast cancer, however, the precise mechanisms remain unclear. In the present study, ErbB2 upregulated Hexokinase II (HK II) activity by increasing the binding of HK II to the mitochondrial outer membrane. Dysregulated glucose metabolism in high ErbB2-expressing breast cancer cells induces susceptibility to glucose starvation and glycolysis inhibition. Additionally, HK II has a tendency to dissociate from the mitochondria outer membrane in ErbB2-overexpressing cells following treatment with the HK II inhibitor, 3-BrPA. Furthermore, 3-BrPA treatment results in decreased mitochondria membrane potential and release of cytochrome c into cytoplasm in ErbB2-overexpressing cells, leading to activation of the mitochondrial apoptotic signaling pathway. In summary, the results demonstrate a novel mechanism for ErbB2-activated glycolysis and reveal that 3-BrPA is effective in reducing ErbB2-positive breast cancer cell viability by targeting HK II in vitro and in vivo. PMID:26893781
Bovine Leukemia Virus DNA in Human Breast Tissue
Shen, Hua Min; Jensen, Hanne M.; Choi, K. Yeon; Sun, Dejun; Nuovo, Gerard
Bovine leukemia virus (BLV), a deltaretrovirus, causes B-cell leukemia/lymphoma in cattle and is prevalent in herds globally. A previous finding of antibodies against BLV in humans led us to examine the possibility of human infection with BLV. We focused on breast tissue because, in cattle, BLV DNA and protein have been found to be more abundant in mammary epithelium than in lymphocytes. In human breast tissue specimens, we identified BLV DNA by using nested liquid-phase PCR and DNA sequencing. Variations from the bovine reference sequence were infrequent and limited to base substitutions. In situ PCR and immunohistochemical testing localized BLV to the secretory epithelium of the breast. Our finding of BLV in human tissues indicates a risk for the acquisition and proliferation of this virus in humans. Further research is needed to determine whether BLV may play a direct role in human disease. PMID:24750974
Transient overexpression of TGFBR3 induces apoptosis in human nasopharyngeal carcinoma CNE-2Z cells
Zheng, Fangfang; He, Kaiwen; Li, Xin; Zhao, Dan; Sun, Fei; Zhang, Yu; Nie, Dan; Li, Xingda; Chu, Wenfeng; Sun, Yan; Lu, Yanjie
NPC (nasopharyngeal carcinoma) is a common malignancy in southern China without defined aetiology. Recent studies have shown that TGFBR3 (transforming growth factor type III receptor, also known as betaglycan), exhibits anticancer activities. This study was to investigate the effects of TGFBR3 on NPC growth and the mechanisms for its actions. Effects of TGFBR3 overexpression on cell viability and apoptosis were measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], AO/EB (acridine orange/ethidium bromide) staining and electron microscopy in human NPC CNE-2Z cells. The expression of apoptosis-related proteins, p-Bad, Bad, XIAP (X-linked inhibitor of apoptosis), AIF (apoptosis-inducing factor), Bax and Bcl-2, was determined by Western blot or immunofluorescence analysis. Caspase 3 activity was measured by caspase 3 activity kit and [Ca2+]i (intracellular Ca2+ concentration) was detected by confocal microscopy. Transfection of TGFBR3 containing plasmid DNA at concentrations of 0.5 and 1 μg/ml reduced viability and induced apoptosis in CNE-2Z in concentration- and time-dependent manners. Forced expression of TGFBR3 up-regulated pro-apoptotic Bad and Bax protein, and down-regulated anti-apoptotic p-Bad, Bcl-2 and XIAP protein. Furthermore, transient overexpression of TGFBR3 also enhanced caspase 3 activity, increased [Ca2+]i and facilitated AIF redistribution from the mitochondria to the nucleus in CNE-2Z cells, which is independent of the caspase 3 pathway. These events were associated with TGFBR3-regulated multiple targets involved in CNE-2Z proliferation. Therefore transient overexpression of TGFBR3 may be a novel strategy for NPC prevention and therapy. PMID:23387308
Characterization of the 5'-regulatory regions of the rat and human apelin genes and regulation of breast apelin by USF.
Wang, Guiyun; Qi, Xiang; Wei, Wei; Englander, Ella W; Greeley, George H
Apelin, a peptide widely expressed in the body, is the endogenous ligand for the APJ receptor. To investigate how the apelin gene is regulated transcriptionally, we cloned and characterized approximately 3000 and approximately 4000 bp 5'-upstream fragments of the rat and human apelin genes. Putative CAAT-like box, but not TATA-box sites were identified. The rat (-207/-1 bp) and human (-100/+74 bp) core promoter sequences contain putative binding sites for upstream stimulatory factor (USF)-1/-2. Mutagenesis and overexpression assays showed that USF up-regulates basal and inducible apelin transcription. EMSA and supershift experiments indicated binding of USF-1/-2 to the rat (-114/-109 bp) and human (-84/-79 bp) apelin promoters. ChIP experiments show that USF is recruited to the putative USF binding site in the human apelin promoter in cultured breast cells. In concert with increased breast apelin expression during pregnancy and lactation in rats, EMSAs demonstrate an elevated binding of pregnant and lactating rat breast nuclear proteins to a consensus USF oligonucleotide. In vivo ChIP assays verified increased USF binding to the apelin promoter in breast of lactating rats. Together, our findings show that USF exerts a stimulatory role in regulation of breast apelin expression during pregnancy and lactation. PMID:17060400
Clinical impact of human breast milk metabolomics.
Cesare Marincola, Flaminia; Dessì, Angelica; Corbu, Sara; Reali, Alessandra; Fanos, Vassilios
Metabolomics is a research field concerned with the analysis of metabolome, the complete set of metabolites in a given cell, tissue, or biological sample. Being able to provide a molecular snapshot of biological systems, metabolomics has emerged as a functional methodology in a wide range of research areas such as toxicology, pharmacology, food technology, nutrition, microbial biotechnology, systems biology, and plant biotechnology. In this review, we emphasize the applications of metabolomics in investigating the human breast milk (HBM) metabolome. HBM is the recommended source of nutrition for infants since it contains the optimal balance of nutrients for developing babies, and it provides a range of benefits for growth, immunity, and development. The molecular mechanisms beyond the inter- and intra-variability of HBM that make its composition unique are yet to be well-characterized. Although still in its infancy, the study of HBM metabolome has already proven itself to be of great value in providing insights into this biochemical variability in relation to mother phenotype, diet, disease, and lifestyle. The results of these investigations lay the foundation for further developments useful to identify normal and aberrant biochemical changes as well as to develop strategies to promote healthy infant feeding practices. PMID:25689794
No evidence for TSLP pathway activity in human breast cancer.
Ghirelli, Cristina; Sadacca, Benjamin; Reyal, Fabien; Zollinger, Raphaël; Michea, Paula; Sirven, Philémon; Pattarini, Lucia; Martínez-Cingolani, Carolina; Guillot-Delost, Maude; Nicolas, André; Scholer-Dahirel, Alix; Soumelis, Vassili
Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that primes dendritic cells for Th2 induction. It has been implicated in different types of allergic diseases. Recent work suggested that TSLP could play an important role in the tumor microenvironment and influence tumor progression, in particular in breast cancer. In this study we systematically assessed the production of TSLP at the mRNA and protein levels in several human breast cancer cell lines, large-scale public transcriptomics data sets, and primary human breast tumors. We found that TSLP production was marginal, and concerned less than 10% of the tumors, with very low mRNA and protein levels. In most cases TSLP was undetectable and found to be expressed at lower levels in breast cancer as compared to normal breast tissue. Last, we could not detect any functional TSLP receptor (TSLPR) expression neither on hematopoietic cells nor on stromal cells within the primary tumor microenvironment. We conclude that TSLP-TSLPR pathway activity is not significantly detected within human breast cancer. Taken together, these observations do not support TSLP targeting in breast cancer. PMID:27622057
Parkin deletion causes cerebral and systemic amyloidosis in human mutated tau over-expressing mice.
Rodríguez-Navarro, Jose A; Gómez, Ana; Rodal, Izaskun; Perucho, Juan; Martinez, Armando; Furió, Vicente; Ampuero, Israel; Casarejos, María J; Solano, Rosa M; de Yébenes, Justo García; Mena, Maria A
Deposition of proteins leading to amyloid takes place in some neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. Mutations of tau and parkin proteins produce neurofibrillary abnormalities without deposition of amyloid. Here we report that mature, parkin null, over-expressing human mutated tau (PK(-/-)/Tau(VLW)) mice have altered behaviour and dopamine neurotransmission, tau pathology in brain and amyloid deposition in brain and peripheral organs. PK(-/-)/Tau(VLW) mice have abnormal behaviour and severe drop out of dopamine neurons in the ventral midbrain, up to 70%, at 12 months and abundant phosphorylated tau positive neuritic plaques, neuro-fibrillary tangles, astrogliosis, microgliosis and plaques of murine beta-amyloid in the hippocampus. PK(-/-)/Tau(VLW) mice have organomegaly of the liver, spleen and kidneys. The electron microscopy of the liver confirmed the presence of a fibrillary protein deposits with amyloid characteristics. There is also accumulation of mouse tau in hepatocytes. These mice have lower levels of CHIP-HSP70, involved in the proteosomal degradation of tau, increased oxidative stress, measured as depletion of glutathione which, added to lack of parkin, could trigger tau accumulation and amyloidogenesis. This model is the first that demonstrates beta-amyloid deposits caused by over-expression of tau and without modification of the amyloid precursor protein, presenilins or secretases. PK(-/-)/Tau(VLW) mice provide a link between the two proteins more important for the pathogenesis of Alzheimer disease. PMID:18640988
Overexpression of Recombinant Human Beta Interferon (rhINF-β) in Periplasmic Space of Escherichia coli
Morowvat, Mohammad Hossein; Babaeipour, Valiollah; Rajabi-Memari, Hamid; Vahidi, Hossein; Maghsoudi, Nader
Human Interferon β (INF-β) is a member of cytokines family which different studies have shown its immunomodulatory and antiviral activities. In this study an expression vector was designed and constructed for expression of human INF-β-1b either in shake flasks or bench top bioreactor. The designed vector was constructed based upon pET-25b(+) with T7 promoter. Recombinant human beta interferon (rhINF-β) was codon optimized and overexpressed as a soluble, N-terminal pelB fusion protein and secreted into the periplasmic space of Escherichia coli BL21 (DE3). The sugar, Isopropyl-β-D-thiogalactopyranoside (IPTG) was used as a chemical inducer for rhINF-β production in the shake flasks and bench top bioreactor. Timing of beta interferon expression was controlled by using the T7 promoter. The rhINF-β protein was extracted from periplasmic space by osmotic shock treatment and the expression of the beta interferon encoding gene in random selected transformants, was confirmed by western and dot blot methods. The maximum of product formation achieved at the OD600nm = 3.42 was found to be 35 % of the total protein content of the strain which translates to 0.32 g L-1. The constructed vector could efficiently overexpress the rhINF-β into the periplasmic space of E. coli. The obtained yield of the produced rhINF-β was more than previous reports. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable to express other recombinant proteins. PMID:24711841
Overexpression of Recombinant Human Beta Interferon (rhINF-β) in Periplasmic Space of Escherichia coli.
Morowvat, Mohammad Hossein; Babaeipour, Valiollah; Rajabi-Memari, Hamid; Vahidi, Hossein; Maghsoudi, Nader
Human Interferon β (INF-β) is a member of cytokines family which different studies have shown its immunomodulatory and antiviral activities. In this study an expression vector was designed and constructed for expression of human INF-β-1b either in shake flasks or bench top bioreactor. The designed vector was constructed based upon pET-25b(+) with T7 promoter. Recombinant human beta interferon (rhINF-β) was codon optimized and overexpressed as a soluble, N-terminal pelB fusion protein and secreted into the periplasmic space of Escherichia coli BL21 (DE3). The sugar, Isopropyl-β-D-thiogalactopyranoside (IPTG) was used as a chemical inducer for rhINF-β production in the shake flasks and bench top bioreactor. Timing of beta interferon expression was controlled by using the T7 promoter. The rhINF-β protein was extracted from periplasmic space by osmotic shock treatment and the expression of the beta interferon encoding gene in random selected transformants, was confirmed by western and dot blot methods. The maximum of product formation achieved at the OD600nm = 3.42 was found to be 35 % of the total protein content of the strain which translates to 0.32 g L-1. The constructed vector could efficiently overexpress the rhINF-β into the periplasmic space of E. coli. The obtained yield of the produced rhINF-β was more than previous reports. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable to express other recombinant proteins. PMID:24711841
Human papillomavirus and breast cancer in Iran: a meta- analysis
Haghshenas, Mohammad Reza; Mousavi, Tahoora; Moosazadeh, Mahmood; Afshari, Mahdi
Objective(s): This study aims to investigate the relationship between human papillomavirus (HPV) and breast cancer using meta- analysis. Materials and Methods: Relevant studies were identified reviewing the national and international databases. We also increased the search sensitivity by investigating the references as well as interview with research centers and experts. Finally, quality assessment and implementation of inclusion/exclusion criteria determined the eligible articles for meta-analysis. Based on the heterogeneity observed among the results of the primary studies, random effects model was used to estimate the pooled prevalence of HPV infection and also pooled odds ratio between HPV and developing breast cancer using Stata SE V. 11 software. Results: This meta- analysis included 11 primary studies investigating the HPV infection prevalence among 1539 Iranian women. Pooled prevalence (95% confidence interval) of HPV infection among Iranian women with breast cancer was estimated as of 23.6% (6.7- 40.5), while, the odds ratio (95% confidence interval) between HPV infection and developing breast cancer was estimated as of 5.7% (0.7- 46.8). Conclusion: This meta- analysis showed a high prevalence of HPV infection among women with breast cancer. We also found that the odds of developing breast cancer among women with breast cancer was more than that of women without breast cancer. PMID:27114791
Targeted Vaccination against Human α-Lactalbumin for Immunotherapy and Primary Immunoprevention of Triple Negative Breast Cancer.
Tuohy, Vincent K; Jaini, Ritika; Johnson, Justin M; Loya, Matthew G; Wilk, Dennis; Downs-Kelly, Erinn; Mazumder, Suparna
We have proposed that safe and effective protection against the development of adult onset cancers may be achieved by vaccination against tissue-specific self-proteins that are "retired" from expression at immunogenic levels in normal tissues as we age, but are overexpressed in emerging tumors. α-Lactalbumin is an example of a "retired" self-protein because its expression in normal tissues is confined exclusively to the breast during late pregnancy and lactation, but is also expressed in the vast majority of human triple negative breast cancers (TNBC)-the most aggressive and lethal form of breast cancer and the predominant form that occurs in women at high genetic risk including those with mutated BRCA1 genes. In anticipation of upcoming clinical trials, here we provide preclinical data indicating that α-lactalbumin has the potential as a vaccine target for inducing safe and effective primary immunoprevention as well as immunotherapy against TNBC. PMID:27322324
Targeted Vaccination against Human α-Lactalbumin for Immunotherapy and Primary Immunoprevention of Triple Negative Breast Cancer
Tuohy, Vincent K.; Jaini, Ritika; Johnson, Justin M.; Loya, Matthew G.; Wilk, Dennis; Downs-Kelly, Erinn; Mazumder, Suparna
We have proposed that safe and effective protection against the development of adult onset cancers may be achieved by vaccination against tissue-specific self-proteins that are "retired" from expression at immunogenic levels in normal tissues as we age, but are overexpressed in emerging tumors. α-Lactalbumin is an example of a "retired" self-protein because its expression in normal tissues is confined exclusively to the breast during late pregnancy and lactation, but is also expressed in the vast majority of human triple negative breast cancers (TNBC)—the most aggressive and lethal form of breast cancer and the predominant form that occurs in women at high genetic risk including those with mutated BRCA1 genes. In anticipation of upcoming clinical trials, here we provide preclinical data indicating that α-lactalbumin has the potential as a vaccine target for inducing safe and effective primary immunoprevention as well as immunotherapy against TNBC. PMID:27322324
The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase
Watanabe, H.; Grubb, J.H.; Sly, W.S. )
The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functional receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.
Diosgenin, a plant steroid, induces apoptosis in human rheumatoid arthritis synoviocytes with cyclooxygenase-2 overexpression
Liagre, Bertrand; Vergne-Salle, Pascale; Corbiere, Cecile; Charissoux, Jean L; Beneytout, Jean L
In the present study, we have shown for the first time that a plant steroid, diosgenin, causes an inhibition of the growth of fibroblast-like synoviocytes from human rheumatoid arthritis, with apoptosis induction associated with cyclooxygenase-2 (COX-2) up-regulation. Celecoxib, a selective COX-2 inhibitor, provoked a large decrease in diosgenin-induced apoptosis even in the presence of exogenous prostaglandin E2, whereas interleukin-1β, a COX-2 inducer, strongly increased diosgenin-induced apoptosis of these synoviocytes. These findings suggest that the proapoptotic effect of diosgenin is associated with overexpression of COX-2 correlated with overproduction of endogenous prostaglandin E2. We also observed a loss of mitochondrial membrane potential, caspase-3 activation, and DNA fragmentation after diosgenin treatment. PMID:15225373
Rad: A member of the Ras family overexpressed in muscle of type II diabetic humans
Reynet, C.; Kahn, C.R. )
To identify the gene or genes associated with insulin resistance in Type II (non-insulin-dependent) diabetes mellitus, subtraction libraries were prepared from skeletal muscle of normal and diabetic humans and screened with subtracted probes. Only one clone out of 4000 was selectively overexpressed in Type II diabetic muscle as compared to muscle of non-diabetic or Type I diabetic individuals. This clone encoded a new 290 kilodalton member of the Ras-guanosine triphosphatase superfamily and was termed Rad (Ras associated with diabetes). Messenger ribonucleic acid of Rad was expressed primarily in skeletal and cardiac muscle and was increased an average of 8.6-fold in the muscle of Type II diabetics as compared to normal individuals.
Overexpression of Long Non-Coding RNA HOTAIR Promotes Tumor Growth and Metastasis in Human Osteosarcoma
Wang, Bo; Su, Yun; Yang, Qun; Lv, Decheng; Zhang, Weiguo; Tang, Kai; Wang, Hong; Zhang, Rui; Liu, Yang
Human osteosarcoma usually presented a high tendency to metastatic spread and caused poor outcomes, however, the underlying mechanism was still largely unknown. In the present study, using a series of in vitro experiments and an animal model, we investigated the roles of HOX antisense intergenic RNA (HOTAIR) during the proliferation and invasion of osteosarcoma. According with our results, HOTAIR was commonly overexpressed in osteosarcoma, which significantly correlated with advanced tumor stage, highly histological grade and poor prognosis. In vitro and in vivo experiments demonstrated that knockdown of HOTAIR could notably suppress cellular proliferation, inhibit invasion and decrease the secretion of MMP2 and MMP9 in osteosarcoma. Collectively, our results suggested that HOTAIR might be a potent therapeutic target for osteosarcoma. PMID:25728753
Function of RasGRP3 in the formation and progression of human breast cancer
Introduction Ras guanine nucleotide exchange factors (RasGEFs) mediate the activation of the Ras signaling pathway that is over activated in many human cancers. The RasGRP3, an activator of H-Ras and R-Ras protein exerts oncogenic effects and the overexpression of the protein is observed in numerous malignant cancer types. Here, we investigated the putative alteration of expression and potential function of RasGRP3 in the formation and progression of human breast cancer. Methods The RasGRP3 and phosphoRasGRP3 expressions were examined in human invasive ductal adenocarcinoma derived samples and cell lines (BT-474, JIMT-1, MCF7, SK-BR-3, MDA-MB-453, T-47D) both in mRNA (Q-PCR) and protein (Western blot; immunohistochemistry) levels. To explore the biological function of the protein, RasGRP3 knockdown cultures were established. To assess the role of RasGRP3 in the viability of cells, annexin-V/PI staining and MitoProbe™ DilC1 (5) assay were performed. To clarify the function of the protein in cell proliferation and in the development of chemotherapeutic resistance, CyQuant assay was performed. To observe the RasGRP3 function in tumor formation, the Severe combined immunodeficiency (SCID) mouse model was used. To investigate the role of the protein in Ras-related signaling Q-PCR and Western blot experiments were performed. Results RasGRP3 expression was elevated in human breast tumor tissue samples as well as in multiple human breast cancer cell lines. Down-regulation of RasGRP3 expression in breast cancer cells decreased cell proliferation, induced apoptosis in MCF7 cells, and sensitized T-47D cells to the action of drugs Tamoxifen and trastuzumab (Herceptin). Gene silencing of RasGRP3 reduced tumor formation in mouse xenografts as well. Inhibition of RasGRP3 expression also reduced Akt, ERK1/2 and estrogen receptor alpha phosphorylation downstream from IGF-I insulin like growth factor-I (IGF-I) or epidermal growth factor (EGF) stimulation confirming the functional
Comprehensive molecular portraits of human breast tumors
Summary We analyzed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, mRNA arrays, microRNA sequencing and reverse phase protein arrays. Our ability to integrate information across platforms provided key insights into previously-defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity. Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at > 10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the Luminal A subtype. We identified two novel protein expression-defined subgroups, possibly contributed by stromal/microenvironmental elements, and integrated analyses identified specific signaling pathways dominant in each molecular subtype including a HER2/p-HER2/HER1/p-HER1 signature within the HER2-Enriched expression subtype. Comparison of Basal-like breast tumors with high-grade Serous Ovarian tumors showed many molecular commonalities, suggesting a related etiology and similar therapeutic opportunities. The biologic finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biologic subtypes of breast cancer. PMID:23000897
Human leukocyte antigen-G overexpression predicts poor clinical outcomes in low-grade gliomas.
Fan, Xing; Wang, Yinyan; Zhang, Chuanbao; Liu, Xing; Qian, Zenghui; Jiang, Tao
Overexpression of human leukocyte antigen-G (HLA-G), a non-classical major histocompatibility complex class-I molecule associated with immunosuppression, has been reported in various human malignancies. In the present study, we examined the role of HLA-G in gliomas. Clinical characteristics, mRNA expression microarrays and follow-up data pertaining to 293 patients with histologically confirmed gliomas were analyzed. The expression levels of HLA-G were compared between different grades of gliomas and correlated with progression-free survival (PFS) and overall survival (OS) to evaluate its prognostic value. We found that HLA-G was overexpressed in gliomas as compared to that in normal brain tissue samples (-1.288±0.265). The highest expression levels were in glioblastomas (GBMs), anaplastic gliomas (AGs) and low-grade gliomas (LGGs), in that order (0.328±0.778, 0.176±0.881, -0.388±0.686, respectively). Significant inter-group differences were observed between low-grade and high-grade glioma tissues (p<0.001 and p<0.001, t-test, AGs and GBMs, respectively). More astrocytoma patients exhibited increased HLA-G expression as compared to other LGG patients (p=0.004, Chi-square test). Significant differences were observed with respect to PFS and OS (p=0.009 and 0.032, log-rank test, for PFS and OS, respectively) between the high- and low-expression subgroups in patients with LGGs. On Cox regression analysis, overexpression of HLA-G appeared to be an independent predictor of clinical outcomes (p=0.007 and 0.026, for PFS and OS, respectively). Our results suggest that HLA-G expression may serve as a potential biomarker for predicting aggressive tumor grades of gliomas and for histological subtype of LGGs. Elevated HLA-G expression could serve as an independent predictor of poor clinical outcomes in patients with low-grade gliomas. PMID:27138095
Induction of cell proliferation, clonogenicity and cell accumulation in S phase as a consequence of human UBE2Q1 overexpression
Fahmidehkar, Mohammad Ali; Shafiee, Sayed Mohammad; Eftekhar, Ebrahim; Mahbudi, Laleh; Seghatoleslam, Atefeh
Ubiquitination is an important cellular mechanism with a pivotal role in the degradation of abnormal or short-lived proteins and the regulation of cell cycle and cell growth. The ubiquitin-proteasome pathway is altered in multiple types of human malignancies, including colorectal cancer (CRC). The alteration in the expression of the novel human gene ubiquitin-conjugating enzyme E2 Q1 (UBE2Q1), as a putative member of the E2 ubiquitin-conjugating enzyme family, has been reported in several malignancies, including carcinoma of the breast, hepatocellular and colorectal cancer, and pediatric acute lymphoblastic leukemia. In the present study, the effect of UBE2Q1 overexpression on cell growth, clonogenicity, motility and cell cycle was investigated in a CRC cell line. The UBE2Q1 gene was cloned in the pCMV6-AN-GFP expression vector. A series of stable transfectants of SW1116 cells overexpressing UBE2Q1 protein were established and confirmed by fluorescence microscopy and western blotting. Using these cells, MTT assay was performed to evaluate cell growth and proliferation, while crystal violet staining was used for clonogenicity assay. Cell cycle analysis was also performed to survey the ratio of cells accumulated in different phases of the cell cycle upon transfection. The motility of these cells was also studied using wound healing assay. UBE2Q1 transfectants exhibited a faster growth in cell culture, increased colony formation capacity and enhanced motility compared with control non-transfected cells and cells transfected with empty vector (mock-transfected cells). UBE2Q1 overexpression also resulted in a significant decrease in the number of cells accumulated in the G0/G1 phase of the cell cycle. The present findings suggest that UBE2Q1 may function as an oncogene that induces proliferation of cancer cells, and could be a novel diagnostic tool and a potential therapeutic target for CRC. PMID:27602158
Cdx2 Polymorphism Affects the Activities of Vitamin D Receptor in Human Breast Cancer Cell Lines and Human Breast Carcinomas
Di Benedetto, Anna; Korita, Etleva; Goeman, Frauke; Sacconi, Andrea; Biagioni, Francesca; Blandino, Giovanni; Strano, Sabrina; Muti, Paola; Mottolese, Marcella; Falvo, Elisabetta
Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). It regulates the action of hormone responsive genes and is involved in cell cycle regulation, differentiation and apoptosis. VDR is a critical component of the vitamin D pathway and different common single nucleotide polymorphisms have been identified. Cdx2 VDR polymorphism can play an important role in breast cancer, modulating the activity of VDR. The objective of this study is to assess the relationship between the Cdx2 VDR polymorphism and the activities of VDR in human breast cancer cell lines and carcinomas breast patients. Cdx2 VDR polymorphism and antiproliferative effects of vitamin D treatment were investigated in a panel of estrogen receptor-positive (MCF7 and T-47D) and estrogen receptor-negative (MDA-MB-231, SUM 159PT, SK-BR-3, BT549, MDA-MB-468, HCC1143, BT20 and HCC1954) human breast cancer cell lines. Furthermore, the potential relationship among Cdx2 VDR polymorphism and a number of biomarkers used in clinical management of breast cancer was assessed in an ad hoc set of breast cancer cases. Vitamin D treatment efficacy was found to be strongly dependent on the Cdx2 VDR status in ER-negative breast cancer cell lines tested. In our series of breast cancer cases, the results indicated that patients with variant homozygote AA were associated with bio-pathological characteristics typical of more aggressive tumours, such as ER negative, HER2 positive and G3. Our results may suggest a potential effect of Cdx2 VDR polymorphism on the efficacy of vitamin D treatment in aggressive breast cancer cells (estrogen receptor negative). These results suggest that Cdx2 polymorphism may be a potential biomarker for vitamin D treatment in breast cancer, independently of the VDR receptor expression. PMID:25849303
Cdx2 polymorphism affects the activities of vitamin D receptor in human breast cancer cell lines and human breast carcinomas.
Pulito, Claudio; Terrenato, Irene; Di Benedetto, Anna; Korita, Etleva; Goeman, Frauke; Sacconi, Andrea; Biagioni, Francesca; Blandino, Giovanni; Strano, Sabrina; Muti, Paola; Mottolese, Marcella; Falvo, Elisabetta
Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). It regulates the action of hormone responsive genes and is involved in cell cycle regulation, differentiation and apoptosis. VDR is a critical component of the vitamin D pathway and different common single nucleotide polymorphisms have been identified. Cdx2 VDR polymorphism can play an important role in breast cancer, modulating the activity of VDR. The objective of this study is to assess the relationship between the Cdx2 VDR polymorphism and the activities of VDR in human breast cancer cell lines and carcinomas breast patients. Cdx2 VDR polymorphism and antiproliferative effects of vitamin D treatment were investigated in a panel of estrogen receptor-positive (MCF7 and T-47D) and estrogen receptor-negative (MDA-MB-231, SUM 159PT, SK-BR-3, BT549, MDA-MB-468, HCC1143, BT20 and HCC1954) human breast cancer cell lines. Furthermore, the potential relationship among Cdx2 VDR polymorphism and a number of biomarkers used in clinical management of breast cancer was assessed in an ad hoc set of breast cancer cases. Vitamin D treatment efficacy was found to be strongly dependent on the Cdx2 VDR status in ER-negative breast cancer cell lines tested. In our series of breast cancer cases, the results indicated that patients with variant homozygote AA were associated with bio-pathological characteristics typical of more aggressive tumours, such as ER negative, HER2 positive and G3. Our results may suggest a potential effect of Cdx2 VDR polymorphism on the efficacy of vitamin D treatment in aggressive breast cancer cells (estrogen receptor negative). These results suggest that Cdx2 polymorphism may be a potential biomarker for vitamin D treatment in breast cancer, independently of the VDR receptor expression. PMID:25849303
Human wings apart-like gene is specifically overexpressed in cervical cancer
Cervical cancer is the third most commonly diagnosed cancer in women. The human wings apart-like (hWAPL) gene, which is 30,793 bp long and located on 10q23.2., is a human homologue of the WAPL gene in Drosophila melanogaster. hWAPL has the characteristics of an oncogene in uterine cervical cancer. The present study investigated the expression of the hWAPL gene in tissues, including 9 common cancers, consisting of cervical, gastric and lung cancers, liver, bladder, esophageal, endometrial, renal and rectal carcinomas, cervical intraepithelial neoplasia (CIN) and benign squamous epithelia. The immunohistochemical analysis was conducted using paraffin-embedded tissues obtained from 413 patients, consisting of 27 benign squamous epithelial tissue samples, and 47 cervical cancer, 30 cervical intraepithelial neoplasia (CIN)I, 33 CINII, 38 CINIII, 29 gastric cancer, 28 liver carcinoma, 26 bladder carcinoma, 35 esophageal carcinoma, 25 endometrial, 26 renal carcinoma, 36 rectal carcinoma and 33 lung cancer tissues. The expression of hWAPL mRNA was evaluated by reverse transcription-quantitative polymerase chain reaction in 8 benign squamous epithelia and 11 cervical cancer tissues. Compared to benign squamous epithelia and the 8 other cancers, hWAPL protein was significantly increased in cervical cancer (P<0.001). The expression of the hWAPL protein in cervical cancer and CINIII tissues was markedly increased compared to the expression in CINI and CINII tissues (P<0.001). Despite the significant difference in the staining scores (P<0.001), no significant difference was observed in the percentage of tissues expressing hWAPL (P=0.102) between cervical cancer and CINIII. The hWAPL gene may therefore be specifically overexpressed in cervical cancer. The overexpression of hWAPL may play an important role in occurrence and development of cervical cancer. PMID:27347120
Development of realistic physical breast phantoms matched to virtual breast phantoms based on human subject data
Kiarashi, Nooshin; Nolte, Adam C.; Sturgeon, Gregory M.; Ghate, Sujata V.; Segars, William P.; Nolte, Loren W.; Samei, Ehsan; and others
Purpose: Physical phantoms are essential for the development, optimization, and evaluation of x-ray breast imaging systems. Recognizing the major effect of anatomy on image quality and clinical performance, such phantoms should ideally reflect the three-dimensional structure of the human breast. Currently, there is no commercially available three-dimensional physical breast phantom that is anthropomorphic. The authors present the development of a new suite of physical breast phantoms based on human data. Methods: The phantoms were designed to match the extended cardiac-torso virtual breast phantoms that were based on dedicated breast computed tomography images of human subjects. The phantoms were fabricated by high-resolution multimaterial additive manufacturing (3D printing) technology. The glandular equivalency of the photopolymer materials was measured relative to breast tissue-equivalent plastic materials. Based on the current state-of-the-art in the technology and available materials, two variations were fabricated. The first was a dual-material phantom, the Doublet. Fibroglandular tissue and skin were represented by the most radiographically dense material available; adipose tissue was represented by the least radiographically dense material. The second variation, the Singlet, was fabricated with a single material to represent fibroglandular tissue and skin. It was subsequently filled with adipose-equivalent materials including oil, beeswax, and permanent urethane-based polymer. Simulated microcalcification clusters were further included in the phantoms via crushed eggshells. The phantoms were imaged and characterized visually and quantitatively. Results: The mammographic projections and tomosynthesis reconstructed images of the fabricated phantoms yielded realistic breast background. The mammograms of the phantoms demonstrated close correlation with simulated mammographic projection images of the corresponding virtual phantoms. Furthermore, power
Therapeutic targeting of erbB3 with MM-121/SAR256212 enhances antitumor activity of paclitaxel against erbB2-overexpressing breast cancer
Introduction Elevated expression of erbB3 rendered erbB2-overexpressing breast cancer cells resistant to paclitaxel via PI-3 K/Akt-dependent upregulation of Survivin. It is unclear whether an erbB3-targeted therapy may abrogate erbB2-mediated paclitaxel resistance in breast cancer. Here, we study the antitumor activity of an anti-erbB3 antibody MM-121/SAR256212 in combination with paclitaxel against erbB2-overexpressing breast cancer. Methods Cell growth assays were used to determine cell viability. Cells undergoing apoptosis were quantified by a specific apoptotic ELISA. Western blot analyses were performed to assess the protein expression and activation. Lentiviral vector containing shRNA was used to specifically knockdown Survivin. Tumor xenografts were established by inoculation of BT474-HR20 cells into nude mice. The tumor-bearing mice were treated with paclitaxel and/or MM-121/SAR256212 to determine whether the antibody (Ab) enhances paclitaxel's antitumor activity. Immunohistochemistry was carried out to study the combinatorial effects on tumor cell proliferation and induction of apoptosis in vivo. Results MM-121 significantly facilitated paclitaxel-mediated anti-proliferative/anti-survival effects on SKBR3 cells transfected with a control vector or erbB3 cDNA. It specifically downregulated Survivin associated with inactivation of erbB2, erbB3, and Akt. MM-121 enhances paclitaxel-induced poly(ADP-ribose) polymerase (PARP) cleavage, activation of caspase-8 and -3, and apoptosis in both paclitaxel-sensitive and -resistant cells. Specific knockdown of Survivin in the trastuzumab-resistant BT474-HR20 cells dramatically enhanced paclitaxel-induced apoptosis, suggesting that increased Survivin caused a cross-resistance to paclitaxel. Furthermore, the studies using a tumor xenograft model-established from BT474-HR20 cells revealed that either MM-121 (10 mg/kg) or low-dose (7.5 mg/kg) paclitaxel had no effect on tumor growth, their combinations significantly
Over-expression, purification, and characterization of recombinant human arylamine N-acetyltransferase 1.
Wang, Haiqing; Vath, Gregory M; Kawamura, Akane; Bates, Caleb A; Sim, Edith; Hanna, Patrick E; Wagner, Carston R
Human arylamine N-acetyltransferase 1 (NAT1) has been overexpressed in E. coli as a mutant dihydrofolic acid reductase (DHFR) fusion protein with a thrombin sensitive linker. An initial DEAE anion-exchange chromatography resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin, and human rNAT1 was purified with a second DEAE column. A total of 8 mg of human rNAT1 from 2 1 of cell culture was purified to homogeneity with this methodology. Arylamine substrate specificities were determined for human rNATI and hamster rNAT2. With both NATs, the second order rate constants (k(cat)/ Kmb) for p-aminobenzoic acid (PABA) and 2-aminofluorene (2-AF) were several thousand-fold higher than those for procainamide (PA), consistent with the expected substrate specificities of the enzymes. However, p-aminosalicylic acid (PAS), previously reported to be a human NAT1 and hamster NAT2 selective substrate, exhibits 20-fold higher specificity for hamster rNAT2 (k(cat)/Kmb 3410 microM(-1) s(-1)) than for human rNAT1 (k(cat)/Kmb 169.4 microM(-1) s(-1)). p-aminobenzoyl-glutamic acid (pABglu) was acetylated 10-fold more efficiently by human rNAT1 than by hamster rNAT2. Inhibition studies of human rNAT1 and hamster rNAT2 revealed that folic acid and methotrexate (MTX) are competitive inhibitors of both the unacetylated and acetylated forms of the enzymes, with K(I) values in 50 - 300 micro range. Dihydrofolic acid (DHF) was a much poorer inhibitor of human rNAT1 than of hamster rNAT2. The combined results demonstrate that human rNAT1 and hamster rNAT2 have similar but distinct kinetic properties with certain substrates, and suggest that folic acid, at least in the non-polyglutamate form, may not have an effect on human NAT1 activity in vivo. PMID:16003948
Human HMGA2 protein overexpressed in mice induces precursor T-cell lymphoblastic leukemia
Efanov, A; Zanesi, N; Coppola, V; Nuovo, G; Bolon, B; Wernicle-Jameson, D; Lagana, A; Hansjuerg, A; Pichiorri, F; Croce, C M
T-cell acute lymphoblastic leukemia (T-ALL) is a neoplasia of thymocytes characterized by the rapid accumulation of the precursors of T lymphocytes. HMGA2 (high-mobility group AT-hook 2) gene expression is extremely low in normal adult tissues, but it is overexpressed in many tumors. To identify the biological function of HMGA2, we generated transgenic mice carrying the human HMGA2 gene under control of the VH promoter/Eμ enhancer. Approximately 90% of Eμ-HMGA2 transgenic mice became visibly sick between 4 and 8 months due to the onset and progression of a T-ALL-like disease. Characteristic features included severe alopecia (30% of mice); enlarged lymph nodes and spleen; and profound immunological abnormalities (altered cytokine levels, hypoimmunoglobulinemia) leading to reduced immune responsiveness. Immunophenotyping showed accumulation of CD5+CD4+, CD5+CD8+ or CD5+CD8+CD4+ T-cell populations in the spleens and bone marrow of sick animals. These findings show that HMGA2-driven leukemia in mice closely resembles spontaneous human T-ALL, indicating that HMGA2 transgenic mice should serve as an important model for investigating basic mechanisms and potential new therapies of relevance to human T-ALL. PMID:25014774
G protein-coupled receptors GPR4 and TDAG8 are oncogenic and overexpressed in human cancers.
Sin, Wun Chey; Zhang, Yaoping; Zhong, Wendy; Adhikarakunnathu, Sree; Powers, Scott; Hoey, Tim; An, Songzhu; Yang, Jianxin
The GPR4 subfamily consists of four G protein-coupled receptors that share significant sequence homology. In addition to GPR4, this subfamily includes OGR1, TDAG8 and G2A. G2A has previously been shown to be a potent transforming oncogene for murine 3T3 cells. Here we show that GPR4 also malignantly transforms NIH3T3 cells and that TDAG8 malignantly transforms the normal mammary epithelial cell line NMuMG. Overexpression of GPR4 or TDAG8 in HEK293 cells led to transcriptional activation from SRE- and CRE-driven promoters, independent of exogenously added ligand. TDAG8 and GPR4 are also overexpressed in a range of human cancer tissues. Our results suggest that GPR4 and TDAG8 overexpression in human tumors plays a role in driving or maintaining tumor formation. PMID:15221007
Evaluation of a quantitative RT-PCR assay to detect HER2 mRNA overexpression for diagnosis and selection of trastuzumab therapy in breast cancer tissue samples.
Wang, Hye-Young; Kim, Sunghyun; Park, Sangjung; Kim, Seungil; Jung, Dongju; Park, Kwang Hwa; Lee, Hyeyoung
Breast cancer patients who have a positive result for HER2 overexpression are commonly treated with Herceptin, a HER2-targeted therapy. In the present study, the BrightGen HER2 RT-qDx (Syantra, Calgary, Canada), which is based on a one-tube nested RT-qPCR method that detects HER2 mRNA overexpression, was clinically evaluated in a total of 237 formalin-fixed paraffin-embedded (FFPE) tissue samples from breast cancer patients. Among the 38 HER2 positive samples, which were determined via IHC/FISH methods, 13 samples out of 16 (81.3%) that were IHC2+/FISH+ and 22 samples out of 22 (100%) that were IHC3+ have been decided positive for HER2 expression via the RT-qPCR method. The true positivity and false positivity results for the RT-qPCR were 92% (35/38) and 2% (1/65), respectively. The concordance between RT-qPCR and IHC results and RT-qPCR and IHC/FISH was 87.2% and 92.1%, respectively. Conclusively, the BrightGen HER2 RT-qDx may be a reliable and convenient method that can supplement traditional IHC and FISH methods for efficient use of trastuzumab. PMID:25236569
Development of octreotide-conjugated polymeric prodrug of bufalin for targeted delivery to somatostatin receptor 2 overexpressing breast cancer in vitro and in vivo
Liu, Tao; Jia, Tingting; Yuan, Xia; Liu, Cheng; Sun, Jian; Ni, Zhenhua; Xu, Jian; Wang, Xuhui; Yuan, Yi
Background Development of polymeric prodrugs of small molecular anticancer drugs has become one of the most promising strategies to overcome the intrinsic shortcomings of small molecular anticancer drugs and improve their anticancer performance. Materials and methods In the current work, we fabricated a novel octreotide (Oct)-modified esterase-sensitive tumor-targeting polymeric prodrug of bufalin (BUF) and explored its anticancer performance against somatostatin receptor 2 overexpressing breast cancer. Results The obtained tumor-targeting polymeric prodrug of BUF, P(oligo[ethylene glycol] monomethyl ether methacrylate [OEGMA]-co-BUF-co-Oct), showed a nanosize dimension and controlled drug release features in the presence of esterase. It was demonstrated by in vitro experiment that P(OEGMA-co-BUF-co-Oct) showed enhanced cytotoxicity, cellular uptake, and apoptosis in comparison with those of free BUF. In vivo experiment further revealed the improved accumulation of drugs in tumor tissues and enhanced anticancer performance of P(OEGMA-co-BUF-co-Oct). Conclusion Taken together, this study indicated that polymeric prodrug of BUF holds promising potential toward the treatment of somatostatin receptor 2 overexpressing breast cancer. PMID:27284243
808 nm-excited upconversion nanoprobes with low heating effect for targeted magnetic resonance imaging and high-efficacy photodynamic therapy in HER2-overexpressed breast cancer.
Zeng, Leyong; Pan, Yuanwei; Zou, Ruifen; Zhang, Jinchao; Tian, Ying; Teng, Zhaogang; Wang, Shouju; Ren, Wenzhi; Xiao, Xueshan; Zhang, Jichao; Zhang, Lili; Li, Aiguo; Lu, Guangming; Wu, Aiguo
To avoid the overheating effect of excitation light and improve the efficacy of photodynamic therapy (PDT) of upconversion nanoplatform, a novel nanoprobe based on 808 nm-excited upconversion nanocomposites (T-UCNPs@Ce6@mSiO2) with low heating effect and deep penetration has been successfully constructed for targeted upconversion luminescence, magnetic resonance imaging (MRI) and high-efficacy PDT in HER2-overexpressed breast cancer. In this nanocomposite, photosensitizers (Ce6) were covalently conjugated inside of mesoporous silica to enhance the PDT efficacy by shortening the distance of fluorescence resonance energy transfer and to decrease the cytotoxicity by preventing the undesired leakage of Ce6. Compared with UCNPs@mSiO2@Ce6, UCNPs@Ce6@mSiO2 greatly promoted the singlet oxygen generation and amplified the PDT efficacy under the excitation of 808 nm laser. Importantly, the designed nanoprobe can greatly improve the uptake of HER2-positive cells and tumors by modifying the site-specific peptide, and the in vivo experiments showed excellent MRI and PDT via intravenous injection by modeling MDA-MB-435 tumor-bearing nude mice. Our strategy may provide an effective solution for overcoming the heating effect and improving the PDT efficacy of upconversion nanoprobes, and has potential application in visualized theranostics of HER2-overexpressed breast cancer. PMID:27376560
Positional cloning of ZNF217 and NABC1: genes amplified at 20q13.2 and overexpressed in breast carcinoma.
Collins, C; Rommens, J M; Kowbel, D; Godfrey, T; Tanner, M; Hwang, S I; Polikoff, D; Nonet, G; Cochran, J; Myambo, K; Jay, K E; Froula, J; Cloutier, T; Kuo, W L; Yaswen, P; Dairkee, S; Giovanola, J; Hutchinson, G B; Isola, J; Kallioniemi, O P; Palazzolo, M; Martin, C; Ericsson, C; Pinkel, D; Albertson, D; Li, W B; Gray, J W
We report here the molecular cloning of an approximately 1-Mb region of recurrent amplification at 20q13.2 in breast cancer and other tumors and the delineation of a 260-kb common region of amplification. Analysis of the 1-Mb region produced evidence for five genes, ZNF217, ZNF218, and NABC1, PIC1L (PIC1-like), CYP24, and a pseudogene CRP (Cyclophillin Related Pseudogene). ZNF217 and NABC1 emerged as strong candidate oncogenes and were characterized in detail. NABC1 is predicted to encode a 585-aa protein of unknown function and is overexpressed in most but not all breast cancer cell lines in which it was amplified. ZNF217 is centrally located in the 260-kb common region of amplification, transcribed in multiple normal tissues, and overexpressed in all cell lines and tumors in which it is amplified and in two in which it is not. ZNF217 is predicted to encode alternately spliced, Kruppel-like transcription factors of 1,062 and 1,108 aa, each having a DNA-binding domain (eight C2H2 zinc fingers) and a proline-rich transcription activation domain. PMID:9671742
The Oncogenic Potential of Human Cytomegalovirus and Breast Cancer
Herbein, Georges; Kumar, Amit
Breast cancer is the leading causes of cancer-related death among women. The vast majority of breast cancers are carcinomas that originate from cells lining the milk-forming ducts of the mammary gland. Numerous articles indicate that breast tumors exhibit diverse phenotypes depending on their distinct physiopathological signatures, clinical courses, and therapeutic possibilities. The human cytomegalovirus (HCMV) is a multifaceted highly host specific betaherpesvirus that is regarded as asymptomatic or mildly pathogenic virus in immunocompetent host. HCMV may cause serious in utero infections as well as acute and chronic complications in immunocompromised individual. The involvement of HCMV in late inflammatory complications underscores its possible role in inflammatory diseases and cancer. HCMV targets a variety of cell types in vivo, including macrophages, epithelial cells, endothelial cells, fibroblasts, stromal cells, neuronal cells, smooth muscle cells, and hepatocytes. HCMV can be detected in the milk after delivery and thereby HCMV could spread to adjacent mammary epithelial cells. HCMV also infects macrophages and induces an atypical M1/M2 phenotype, close to the tumor-associated macrophage phenotype, which is associated with the release of cytokines involved in cancer initiation or promotion and breast cancer of poor prognosis. HCMV antigens and DNA have been detected in tissue biopsies of breast cancers and elevation in serum HCMV IgG antibody levels has been reported to precede the development of breast cancer in some women. In this review, we will discuss the potential role of HCMV in the initiation and progression of breast cancer. PMID:25202681
Evidence for altered ion transport in Saccharomyces cerevisiae overexpressing human MDR 1 protein.
Fritz, F; Howard, E M; Hoffman, M M; Roepe, P D
Recently [Hoffman, M. M., and Roepe, P. D. (1997) Biochemistry 36, 11153-11168] we presented evidence for a novel Na+- and Cl--dependent H+ transport process in LR73/hu MDR 1 CHO transfectants that likely explains pHi, volume, and membrane potential changes in eukaryotic cells overexpressing the hu MDR 1 protein. To further explore this process, we have overexpressed human MDR 1 protein in yeast strain 9.3 following a combination of approaches used previously [Kuchler, K., and Thorner, J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2302-2306; Ruetz, S., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11588-11592]. Thus, a truncated hu MDR 1 cDNA was cloned behind a tandem array of sterile 6 (Ste6) and alchohol dehydrogenase (Adh) promoters to create the yeast expression vector pFF1. Valinomycin resistance of intact cells and Western blot analysis with purified yeast plasma membranes confirmed the overexpression of full length, functional, and properly localized hu MDR 1 protein in independently isolated 9.3/pFF1 colonies. Interestingly, relative valinomycin resistance and growth of the 9.3/hu MDR 1 strains are found to strongly depend on the ionic composition of the growth medium. Atomic absorption reveals significant differences in intracellular K+ for 9.3/hu MDR 1 versus control yeast. Transport assays using [3H]tetraphenylphosphonium ([3H]TPP+) reveal perturbations in membrane potential for 9.3/hu MDR 1 yeast that are stimulated by KCl and alkaline pHex. ATPase activity of purified plasma membrane fractions from yeast strains and LR73/hu MDR 1 CHO transfectants constructed previously [Hoffman, M. M., et al. (1996) J. Gen. Physiol. 108, 295-313] was compared. MDR 1 ATPase activity exhibits a higher pH optimum and different salt dependencies, relative to yeast H+ ATPase. Inside-out plasma membrane vesicles (ISOV) fabricated from 9.3/hu MDR 1 and control strains were analyzed for formation of H+ gradients +/- verapamil. Similar pharmacologic profiles are found for
Expression of K+ channels in normal and cancerous human breast.
Brevet, Marie; Ahidouch, Ahmed; Sevestre, Henri; Merviel, Philippe; El Hiani, Yassine; Robbe, Micheline; Ouadid-Ahidouch, Halima
Potassium (K+) channels contribute to the regulation of cell proliferation and apoptosis and are also involved in tumor generation and malignant growth. Using immunohistochemical analysis, we investigated the expression of four K+ channels GIRK1 (G-Protein Inwardly Rectifying Potassium Channel 1), Ca2+-activated K channel (K Ca 1.1), voltage activated K+ channels (KV 1.1 and KV 1.3) and of the anti-apoptotic protein Bcl2 in normal and cancerous breast tissues and compared their expression with clinicopathological data. GIRK1 was overexpressed in carcinomatous tissues. In contrast, K V 1.1 and K V 1.3 were less expressed in cancerous tissue. The expression of Bcl-2 was similar in both tissues. As to the clinicopathological data, a correlation between K Ca 1.1 channel and estrogen receptor (ER) expression was observed. GIRK1 was overexpressed in breast carcinoma suggesting its involvement in proliferation and oncogenesis and its possible use as a putative pharmaceutical target. The correlation between K Ca 1.1 channel and ER suggests the involvement of this channel in proliferation. The loss of expression of the two channels K V 1.1 and K V 1.3 may correspond to their role in apoptosis. PMID:18498071
MicroRNA-106b targets FUT6 to promote cell migration, invasion, and proliferation in human breast cancer.
Li, Nana; Liu, Yuejian; Miao, Yuan; Zhao, Lifen; Zhou, Huimin; Jia, Li
It is demonstrated that the maladjustment of microRNA (miRNA) plays significant roles in the occurrence and development of tumors. MicroRNA-106b-5p (miR-106b), a carcinogenic miRNA, is identified as a dysregulated miRNA in human breast cancer. In this article, the expression levels of miR-106b were discovered to be particularly higher in breast cancer tissues than that in the corresponding adjacent tissues. Accordingly, miR-106b was higher expressed in the breast cancer cell lines compared with that in the normal breast cell lines. Moreover, according to the data previously reported, increased expression of miR-106b was significantly associated with advanced clinical stages and poor prognosis in breast cancer. Fucosyltransferase 6 (FUT6), a member of the fucosyltransferase (FUT) family, was found to have a reduced expression in tissues or cells with higher level of miR-106b in breast cancer. Additionally, down-regulation of miR-106b increased the expression of FUT6 and resulted in an obvious decrease of cell migration, invasion, and proliferation in MDA-MB-231 cells. Furthermore, over-expressed FUT6 reversed the impacts of up-regulated miR-106b on cell migration, invasion, and proliferation in MCF-7 cells, indicating that FUT6 might be directly targeted by miR-106b and serve as therapeutic targets for breast cancer. In brief, our results strongly showed that the low expression of FUT6 regulated by miR-106b contributed to cell migration, invasion, and proliferation in human breast cancer. © 2016 IUBMB Life, 68(9):764-775, 2016. PMID:27519168
Profilin-1 overexpression in MDA-MB-231 breast cancer cells is associated with alterations in proteomics biomarkers of cell proliferation, survival, and motility as revealed by global proteomics analyses.
Coumans, Joëlle V F; Gau, David; Poljak, Anne; Wasinger, Valerie; Roy, Partha; Moens, Pierre D J
Despite early screening programs and new therapeutic strategies, metastatic breast cancer is still the leading cause of cancer death in women in industrialized countries and regions. There is a need for novel biomarkers of susceptibility, progression, and therapeutic response. Global analyses or systems science approaches with omics technologies offer concrete ways forward in biomarker discovery for breast cancer. Previous studies have shown that expression of profilin-1 (PFN1), a ubiquitously expressed actin-binding protein, is downregulated in invasive and metastatic breast cancer. It has also been reported that PFN1 overexpression can suppress tumorigenic ability and motility/invasiveness of breast cancer cells. To obtain insights into the underlying molecular mechanisms of how elevating PFN1 level induces these phenotypic changes in breast cancer cells, we investigated the alteration in global protein expression profiles of breast cancer cells upon stable overexpression of PFN1 by a combination of three different proteome analysis methods (2-DE, iTRAQ, label-free). Using MDA-MB-231 as a model breast cancer cell line, we provide evidence that PFN1 overexpression is associated with alterations in the expression of proteins that have been functionally linked to cell proliferation (FKPB1A, HDGF, MIF, PRDX1, TXNRD1, LGALS1, STMN1, LASP1, S100A11, S100A6), survival (HSPE1, HSPB1, HSPD1, HSPA5 and PPIA, YWHAZ, CFL1, NME1) and motility (CFL1, CORO1B, PFN2, PLS3, FLNA, FLNB, NME2, ARHGDIB). In view of the pleotropic effects of PFN1 overexpression in breast cancer cells as suggested by these new findings, we propose that PFN1-induced phenotypic changes in cancer cells involve multiple mechanisms. Our data reported here might also offer innovative strategies for identification and validation of novel therapeutic targets and companion diagnostics for persons with, or susceptibility to, breast cancer. PMID:25454514
Overexpression of SATB1 Is Associated with Biologic Behavior in Human Renal Cell Carcinoma
Liu, Lian; Zeng, Fuqing; Xing, Shi'an; Wu, Xiaofei; Chen, Xuepan; Zhu, Zhaohui
Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be aberrantly expressed in various cancers and correlated with the malignant behavior of cancer cells. However, the function of SATB1 in RCC remains unclear. With the combination of immunohistochemistry, western blotting, immunofluorescence, qRT-PCR, and cell proliferation, migration and invasion assays, we found that levels of SATB1 mRNA and protein were dramatically increased in human ccRCC tissues (P<0.001 for both), and upregulation of SATB1 was significantly associated with depth of invasion (P<0.001), lymph node status (P = 0.001) and TNM stage (P = 0.009). SATB1 knockdown inhibited the proliferation, migration and invasion of 786-O cells, whereas SATB1 overexpression promoted the growth and aggressive phenotype of ACHN cells in vitro. Furthermore, SATB1 expression was positively correlated with ZEB2 expression (P = 0.013), and inversely linked to levels of SATB2 and E-cadherin (P = 0.005 and P<0.001, respectively) in ccRCC tissues. Our data provide a basis for the concept that overexpression of SATB1 may play a critical role in the acquisition of an aggressive phenotype for RCC cells through EMT, providing new insights into the significance of SATB1 in invasion and metastasis of ccRCC, which may contribute to fully elucidating the exact mechanism of development and progression of RCC. PMID:24835085
Pancreatic islet differentiation of human embryonic stem cells by microRNA overexpression.
Lahmy, Reyhaneh; Soleimani, Masoud; Sanati, Mohammad H; Behmanesh, Mehrdad; Kouhkan, Fatemeh; Mobarra, Naser
Development of stem cell-based therapies for the treatment of type 1 diabetes would provide a renewable supply of human β-cells. Human embryonic stem cells (ESCs) are considered to be one of the stem cell populations with sufficient proliferative capacity to achieve this goal. Currently, differentiation protocols directing ESCs toward a pancreatic fate employ a variety of expensive cytokines and inhibitors. With the known significance of microRNAs in islet development, we present a novel and cost-effective strategy in which miR-375 overexpression promotes pancreatic endocrine differentiation in hESCs in the absence of any extrinsic factors. miR-375 has been shown to be a key regulator of pancreatic development and function in zebrafish, mouse and human. In this study, hESCs were transduced with lentiviral vectors containing human miR-375 precursor and aggregated to form human embryoid bodies (hEBs) for up to 21 days. Morphological assessment, immunocytochemistry and DTZ staining confirmed that miR-375-induced hEBs have similar characteristics to those of mature islets. In addition, the dynamic expression profile of endodermal marker Foxa2 and endocrine-specific genes, including HNF4α, Pdx1, Pax6, Nkx6.1, Glut2 and insulin, were detected by quantitative real-time PCR. Finally, insulin release upon glucose stimulation was detected in our differentiated clusters. The data presented here demonstrate the feasibility of using microRNAs to direct differentiation into the pancreatic lineage. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23897763
Luteinizing hormone/human chorionic gonadotropin receptors in breast cancer.
Meduri, G; Charnaux, N; Loosfelt, H; Jolivet, A; Spyratos, F; Brailly, S; Milgrom, E
Recent studies have suggested that human choriogonadotropin (hCG), in addition to its function in regulating steroidogenesis, may also play a role as a growth factor. Immunocytochemistry using two different monoclonal antibodies (LHR29 and LHR1055) raised against the human luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor allowed us to detect this receptor in breast cancer cell lines (T47D, MCF7, and ZR75) in individual cancer biopsies and in benign breast lesions. The receptor was also present in epithelial cells of normal human and sow breast. In the latter, its concentration increased after ovulation. The presence of LH/hCG receptor mRNA was confirmed by reverse transcription-PCR using primers extending over exons 2-4, 5-11, and 9-11. The proportion of LH/hCG-receptor positive cells and the intensity of the immunolabeling varied in individual biopsies, but there was no obvious correlation with the histological type of the cancer. These results are compatible with previous studies suggesting that during pregnancy, hCG is involved in the differentiation of breast glandular epithelium and that this hormone may play an inhibitory role in mammary carcinogenesis and in the growth of breast tumors. PMID:9041186
MicroRNA Regulation of Human Breast Cancer Stem Cells
Shimono, Yohei; Mukohyama, Junko; Nakamura, Shun-ichi; Minami, Hironobu
MicroRNAs (miRNAs) are involved in virtually all biological processes, including stem cell maintenance, differentiation, and development. The dysregulation of miRNAs is associated with many human diseases including cancer. We have identified a set of miRNAs differentially expressed between human breast cancer stem cells (CSCs) and non-tumorigenic cancer cells. In addition, these miRNAs are similarly upregulated or downregulated in normal mammary stem/progenitor cells. In this review, we mainly describe the miRNAs that are dysregulated in human breast CSCs directly isolated from clinical specimens. The miRNAs and their clusters, such as the miR-200 clusters, miR-183 cluster, miR-221-222 cluster, let-7, miR-142 and miR-214, target the genes and pathways important for stem cell maintenance, such as the self-renewal gene BMI1, apoptosis, Wnt signaling, Notch signaling, and epithelial-to-mesenchymal transition. In addition, the current evidence shows that metastatic breast CSCs acquire a phenotype that is different from the CSCs in a primary site. Thus, clarifying the miRNA regulation of the metastatic breast CSCs will further advance our understanding of the roles of human breast CSCs in tumor progression. PMID:26712794
GSK-3 inhibition overcomes chemoresistance in human breast cancer.
Ugolkov, Andrey; Gaisina, Irina; Zhang, Jin-San; Billadeau, Daniel D; White, Kevin; Kozikowski, Alan; Jain, Sarika; Cristofanilli, Massimo; Giles, Francis; O'Halloran, Thomas; Cryns, Vincent L; Mazar, Andrew P
Glycogen Synthase Kinase-3β (GSK-3β), a serine/threonine protein kinase, is an emerging therapeutic target in the treatment of human breast cancer. In this study, we demonstrate that the pharmacological inhibition of GSK-3 by two novel small molecule GSK-3 inhibitors, 9-ING-41 and 9-ING-87, reduced the viability of breast cancer cells but had little effect on non-tumorigenic cell growth. Moreover, treatment with 9-ING-41 enhanced the antitumor effect of irinotecan (CPT-11) against breast cancer cells in vitro. We next established two patient-derived xenograft tumor models (BC-1 and BC-2) from metastatic pleural effusions obtained from patients with progressive, chemorefractory breast cancer and demonstrated that 9-ING-41 also potentiated the effect of the chemotherapeutic drug CPT-11 in vivo, leading to regression of established BC-1 and BC-2 tumors in mice. Our results suggest that the inhibition of GSK-3 is a promising therapeutic approach to overcome chemoresistance in human breast cancer, and identify the GSK-3 inhibitor 9-ING-41 as a candidate targeted agent for metastatic breast cancer therapy. PMID:27424289
Overexpression of ubiquitin carboxyl terminal hydrolase-L1 enhances multidrug resistance and invasion/metastasis in breast cancer by activating the MAPK/Erk signaling pathway.
Wang, Wenjuan; Zou, Liping; Zhou, Danmei; Zhou, Zhongwen; Tang, Feng; Xu, Zude; Liu, Xiuping
Multidrug resistant (MDR) cancer cells overexpressing P-glycoprotein (P-gp) exhibit enhanced invasive/metastatic ability as compared with the sensitive cells. We aimed to clarify the mechanism underlying this observation and found that during the development of drug resistance to adriamycin in MCF7 cells, the elevated expression of UCH-L1 coincides with the up-regulation of MDR1, CD147, MMP2, and MMP9 as well as increased cellular migration/invasion. Overexpression of UCH-L1 in MCF7 cells up-regulated MDR1, CD147, MMP2, and MMP9, which conferred MDR and promoted migration/invasion. On the other hand, silencing of UCH-L1 in MCF7/Adr cells led to the opposite effect. Immunohistochemistry in 203 breast cancer samples revealed that UCH-L1 expression is positively correlated with P-gp, CD147, MMP2, and MMP9 expression and standard tumor spread indicators. Kaplan-Meier survival analysis indicated a correlation between UCH-L1 expression and shorter recurrent and survival times. Moreover, UCH-L1-overexpressing clones treated with U0126 (an Erk1/2-specific inhibitor) significantly decreased the expression of MDR1, CD147, MMP2, and MMP9. These data indicate that UCH-L1 may assume a dual role, because it had intrinsic stimulatory effects on tumor migration/invasion and increased MDR. © 2015 Wiley Periodicals, Inc. PMID:26293643
Analysis of human breast tissues with Raman microspectroscopy
NASA Astrophysics Data System (ADS)
Liu, Gang; Zhang, Lin; Liu, Jianhong; Yu, Fan; Sun, Shizhong
Raman microspectroscopy was used to study normal, benign and malignant human breast tissues. The Raman spectrum of normal breast tissue recorded with 514.5 nm line of Ar + laser excitation contains features attributed to carotenoids and lipids. The CH II bending mode near 1447 cm -1 in normal tissue shifts up to 1454 cm -1 in diseased tissues (benign and malignant). The band near 1660 cm -1 in normal tissue is narrow and sharp; whereas the band is broaden in the diseased tissues. In the region of C-H stretching mode, the 2902-/2860-cm -1 intensity ratio shows differences among normal, benign and malignant breast tissues. The ratio is the smallest in carcinoma tissue. The observed spectra differences may be used to probe breast lesion. The results show that Raman spectroscopic technique may have clinical applications.
Overexpression of molecular chaperons GRP78 and GRP94 in CD44hi/CD24lo breast cancer stem cells
Nami, Babak; Ghasemi-Dizgah, Armin; Vaseghi, Akbar
Introduction: Breast cancer stem cell with CD44hi/CD24lo phonotype is described having stem cell properties and represented as the main driving factor in breast cancer initiation, growth, metastasis and low response to anti-cancer agents. Glucoseregulated proteins (GRPs) are heat shock protein family chaperons that are charged with regulation of protein machinery and modulation of endoplasmic reticulum homeostasis whose important roles in stem cell development and invasion of various cancers have been demonstrated. Here, we investigated the expression levels of GRP78 and GRP94 in CD44hi/CD24lo phenotype breast cancer stem cells (BCSCs). Methods: MCF7, T-47D and MDA-MB-231 breast cancer cell lines were used. CD44hi/CD24lo phenotype cell population were analyzed and sorted by fluorescence-activated cell sorting (FACS). Transcriptional and translational expression of GRP78 and GRP94 were investigated by western blotting and quantitative real time PCR. Results: Results showed different proportion of CD44hi/CD24lo phenotype cell population in their original bulk cells. The ranking of the cell lines in terms of CD44hi/CD24lo phenotype cell population was as MCF7
Prodigiosin down-regulates survivin to facilitate paclitaxel sensitization in human breast carcinoma cell lines
Ho, T.-F.; Peng, Y.-T.; Chuang, S.-M.; Lin, S.-C.; Feng, B.-L.; Lu, C.-H.; Yu, W.-J.; Chang, J.-S. Chang, C.-C.
Prodigiosin is a bacterial metabolite with potent anticancer activity, which is attributed to its proapoptotic effect selectively active in malignant cells. Still, the molecular mechanisms whereby prodigiosin induces apoptosis remain largely unknown. In particular, the role of survivin, a vital inhibitor of apoptosis, in prodigiosin-induced apoptosis has never been addressed before and hence was the primary goal of this study. Our results showed that prodigiosin dose-dependently induced down-regulation of survivin in multiple breast carcinoma cell lines, including MCF-7, T-47D and MDA-MB-231. This down-regulation is mainly regulated at the level of transcription, as prodigiosin reduced the levels of both survivin mRNA and survivin promoter activity but failed to rescue survivin expression when proteasome-mediated degradation is abolished. Importantly, overexpression of survivin rendered cells more resistant to prodigiosin, indicating an essential role of survivin down-regulation in prodigiosin-induced apoptosis. In addition, we found that prodigiosin synergistically enhanced cell death induced by paclitaxel, a chemotherapy drug known to up-regulate survivin that in turn confers its own resistance. This paclitaxel sensitization effect of prodigiosin is ascribed to the lowering of survivin expression, because prodigiosin was shown to counteract survivin induction by paclitaxel and, notably, the sensitization effect was severely abrogated in cells that overexpress survivin. Taken together, our results argue that down-regulation of survivin is an integral component mediating prodigiosin-induced apoptosis in human breast cancer cells, and further suggest the potential of prodigiosin to sensitize anticancer drugs, including paclitaxel, in the treatment of breast cancer.
Pertuzumab in human epidermal growth-factor receptor 2-positive breast cancer: clinical and economic considerations
Lamond, Nathan WD; Younis, Tallal
In the absence of specific therapy, the 15%–20% of breast cancers demonstrating human epidermal growth-factor receptor 2 (HER2) protein overexpression and/or gene amplification are characterized by a more aggressive phenotype and poorer prognosis compared to their HER2-negative counterparts. Trastuzumab (Herceptin), the first anti-HER2-targeted therapy, has been associated with improved survival outcomes in HER2-positive breast cancer. However, many patients with early stage disease continue to relapse, and metastatic disease remains incurable. In order to further improve these outcomes, several novel HER2-targeted agents have recently been developed. Pertuzumab (Perjeta), a monoclonal antibody against the HER2 dimerization domain, has also been associated with improved patient outcomes in clinical trials, and has recently been approved in combination with chemotherapy and trastuzumab for neoadjuvant therapy of early stage, HER2-positive breast cancer and first-line treatment of metastatic disease. This review briefly summarizes pertuzumab's clinical development as well as the published evidence supporting its use, and highlights some of the currently unanswered questions that will influence pertuzumab's incorporation into clinical practice. PMID:24876795
Brain metastasis in human epidermal growth factor receptor 2-positive breast cancer: from biology to treatment
Koo, Taeryool
Overexpression of human epidermal growth factor receptor 2 (HER2) is found in about 20% of breast cancer patients. With treatment using trastuzumab, an anti-HER2 monoclonal antibody, systemic control is improved. Nonetheless, the incidence of brain metastasis does not be improved, rather seems to be increased in HER2-positive breast cancer. The mainstay treatment for brain metastases is radiotherapy. According to the number of metastatic lesions and performance status of patients, radiosurgery or whole brain radiotherapy can be performed. The concurrent use of a radiosensitizer further improves intracranial control. Due to its large molecular weight, trastuzumab has a limited ability to cross the blood-brain barrier. However, small tyrosine kinase inhibitors such as lapatinib, has been noted to be a promising agent that can be used as a radiosensitizer to affect HER2-positive breast cancer. This review will outline general management of brain metastases and will focus on preclinical findings regarding the radiosensitizing effect of small molecule HER2 targeting agents. PMID:27104161
Let-7c overexpression inhibits dengue virus replication in human hepatoma Huh-7 cells.
Escalera-Cueto, Manuel; Medina-Martínez, Ingrid; del Angel, Rosa M; Berumen-Campos, Jaime; Gutiérrez-Escolano, Ana Lorena; Yocupicio-Monroy, Martha
MicroRNAs (miRNAs) constitute an important class of non-coding RNA implicated in gene expression regulation. More than 1900 miRNA molecules have been identified in humans and their modulation during viral infection and it is recognized to play a role in latency regulation or in establishing an antiviral state. The liver cells are targets during DENV infection, and alteration of liver functions contributes to severe disease. In this work the miRNAs expression profile of the human hepatoma cell line, Huh-7, infected with DENV-2 was determined using microarray and real-time PCR. Let-7c is one of the miRNAs up-regulated during DENV infection in the hepatic Huh-7 as well as in the macrophage-monocytic cell line U937-DC-SIGN. Let-7c overexpression down-regulates both DENV-2 and DENV-4 infection. Additionally, we found that the transcription factor BACH1, a let-7c target, is also down-regulated during DENV infection. In accordance with this finding, HO-1, the main responsive factor of BACH1 was found up-regulated. The up-regulation of HO-1 may contribute to the stress oxidative response in infected cells. PMID:25445350
GFAP expression and social deficits in transgenic mice overexpressing human sAPPα.
Bailey, Antoinette R; Hou, Huayan; Song, Min; Obregon, Demian F; Portis, Samantha; Barger, Steven; Shytle, Doug; Stock, Saundra; Mori, Takashi; Sanberg, Paul G; Murphy, Tanya; Tan, Jun
Autistic individuals display impaired social interactions and language, and restricted, stereotyped behaviors. Elevated levels of secreted amyloid precursor protein-alpha (sAPPα), the product of α-secretase cleavage of APP, are found in the plasma of some individuals with autism. The sAPPα protein is neurotrophic and neuroprotective and recently showed a correlation to glial differentiation in human neural stem cells (NSCs) via the IL-6 pathway. Considering evidence of gliosis in postmortem autistic brains, we hypothesized that subsets of patients with autism would exhibit elevations in CNS sAPPα and mice generated to mimic this observation would display markers suggestive of gliosis and autism-like behavior. Elevations in sAPPα levels were observed in brains of autistic patients compared to controls. Transgenic mice engineered to overexpress human sAPPα (TgsAPPα mice) displayed hypoactivity, impaired sociability, increased brain glial fibrillary acidic protein (GFAP) expression, and altered Notch1 and IL-6 levels. NSCs isolated from TgsAPPα mice, and those derived from wild-type mice treated with sAPPα, displayed suppressed β-tubulin III and elevated GFAP expression. These results suggest that elevations in brain sAPPα levels are observed in subsets of individuals with autism and TgsAPPα mice display signs suggestive of gliosis and behavioral impairment. PMID:23840007
Overexpression of ROCK1 and ROCK2 inhibits human laryngeal squamous cell carcinoma
Zhang, Junbo; He, Xue; Ma, Yueying; Liu, Yanli; Shi, Huaiyin; Guo, Weiwei; Liu, Liangfa
Rho-associated coiled-coil containing protein kinase (ROCK) over-expression has been implicated in the progression of many tumor types. The aim of this study was to explore the roles of ROCK1 and ROCK2 in human laryngeal squamous cell carcinoma (LSCC). ROCK1 and ROCK2 expression levels were examined in 50 cases of human LSCC samples by immunohistochemistry. Effects of ROCK1 and ROCK2 on LSCC cell proliferation and motility were investigated in the presence of the ROCK inhibitor Y-27632. The results showed that ROCK1 expression was positively correlated with tumor size and lymph node metastasis (P < 0.05); ROCK2 positively correlated with tumor size (P < 0.05). Inhibition of ROCK1 and ROCK2 by Y-27632 significantly inhibits proliferation, migration, and invasion of LSCC cells. Our data indicate that expression of ROCK1 and ROCK2 are closely associated with tumor growth and lymph node metastasis of LSCC. Thus, these two ROCK isoforms may be useful as molecular makers for LSCC diagnosis and may be useful therapeutic targets as well. PMID:25755711
GFAP expression and social deficits in transgenic mice overexpressing human sAPPα
Bailey, Antoinette R; Hou, Huayan; Song, Min; Obregon, Demian F; Portis, Samantha; Barger, Steven; Shytle, Doug; Stock, Saundra; Mori, Takashi; Sanberg, Paul G; Murphy, Tanya; Tan, Jun
Autistic individuals display impaired social interactions and language, and restricted, stereotyped behaviors. Elevated levels of secreted amyloid precursor protein-alpha (sAPPα), the product of α-secretase cleavage of APP, are found in the plasma of some individuals with autism. The sAPPα protein is neurotrophic and neuroprotective and recently showed a correlation to glial differentiation in human neural stem cells (NSCs) via the IL-6 pathway. Considering evidence of gliosis in postmortem autistic brains, we hypothesized that subsets of patients with autism would exhibit elevations in CNS sAPPα and mice generated to mimic this observation would display markers suggestive of gliosis and autism-like behavior. Elevations in sAPPα levels were observed in brains of autistic patients compared to controls. Transgenic mice engineered to overexpress human sAPPα (TgsAPPα mice) displayed hypoactivity, impaired sociability, increased brain glial fibrillary acidic protein (GFAP) expression, and altered Notch1 and IL-6 levels. NSCs isolated from TgsAPPα mice, and those derived from wild-type mice treated with sAPPα, displayed suppressed β-tubulin III and elevated GFAP expression. These results suggest that elevations in brain sAPPα levels are observed in subsets of individuals with autism and TgsAPPα mice display signs suggestive of gliosis and behavioral impairment. PMID:23840007
Assessment of the Selenoprotein M (SELM) Over-Expression on Human Hepatocellular Carcinoma Tissues by Immunohistochemistry
Guerriero, E.; Accardo, M.; Capone, F.; Colonna, G.; Castello, G.; Costantini, S.
Selenium is an essential trace mineral of fundamental importance to human healthy and exerts its biological function through selenoproteins. In particular, Selenoprotein M (SELM) is located in the endoplasmic reticulum and contains the common redox motif of cysteine-X-X-selenocysteine type. It attracts great attention due to its high expression in brain and its potential roles as antioxidant, neuroprotective, and cytosolic calcium regulator. Recently, our group found SELM over-expression in human hepatocellular carcinoma (HCC) cell lines. In this report some paraffin-embedded tissues from liver biopsy of patients with hepatitis C virus (HCV)-related cirrhosis and HCC were immunohistochemically stained and SELM expression scoring was evaluated. Our results evidence for the first time an increase of SELM expression in HCC liver tissues, and its gradual expression raise associated with an increased malignancy grade. Therefore, we propose to use i) SELM as putative marker for HCC as well as ii) simple immunohistochemistry technique to distinguish between the different grades of malignancy. PMID:25578973
Tandem overexpression of five human factors renders murine hepatocytes susceptible to hepatitis C virus.
Lv, L; Kang, Q; Yu, X; Gao, B; Hu, T; Ma, P; Zhang, Y; Yan, F; Xiao, J; Deng, J; Zhou, X; Xu, J
Development of mouse model of hepatitis C virus (HCV) infection has great significance in drug screening and vaccine research. The barriers of interspecies transmission of HCV are increasingly better understood. Human factors, namely low-density lipoprotein receptor (hLDLR), CD81 (hCD81), scavenger receptor class B type I (hSCARB1), occludin (hOCLN) and claudin 1 (hCLDN1) are all required for rendering mouse hepatocytes permissive to HCV. With the aim to humanize mouse hepatocytes we constructed two recombinant vectors tandemly expressing the first three and the last two HCV entry factors mentioned above, respectively. Cotransfection of mouse hepatocytes with these vectors made them permissive to HCV binding and entry. Tandem overexpression of hLDLR, hSCARB1, hCD81, hCLDN1 and hOCLN is a novel approach to tailoring mouse hepatocytes to HCV binding and entry which can be further used to establish a mouse model of HCV infection as a basis for developing antiviral drugs and vaccines. PMID:25790047
Constitutive Overexpression of Human Erythropoietin Protects the Mouse Retina against Induced But Not Inherited Retinal Degeneration
Grimm, Christian; Wenzel, Andreas; Stanescu, Dinu; Samardzija, Marijana; Hotop, Svenja; Groszer, Mathias; Naash, Muna; Gassmann, Max; Remé, Charlotte
Elevation of erythropoietin (Epo) concentrations by hypoxic preconditioning or application of recombinant human Epo (huEpo) protects the mouse retina against light-induced degeneration by inhibiting photoreceptor cell apoptosis. Because photoreceptor apoptosis is also the common path to cell loss in retinal dystrophies such as retinitis pigmentosa (RP), we tested whether high levels of huEpo would reduce apoptotic cell death in two mouse models of human RP. We combined the two respective mutant mouse lines with a transgenic line (tg6) that constitutively overexpresses huEpo mainly in neural tissues. Transgenic expression of huEpo caused constitutively high levels of Epo in the retina and protected photoreceptors against light-induced degeneration; however, the presence of high levels of huEpo did not affect the course or the extent of retinal degeneration in a light-independent (rd1) and a light-accelerated (VPP) mouse model of RP. Similarly, repetitive intraperitoneal injections of recombinant huEpo did not protect the retina in the rd1 and the VPP mouse. Lack of neuroprotection by Epo in the two models of inherited retinal degeneration was not caused by adaptational downregulation of Epo receptor. Our results suggest that apoptotic mechanisms during acute, light-induced photoreceptor cell death differ from those in genetically based retinal degeneration. Therapeutic intervention with cell death in inherited retinal degeneration may therefore require different drugs and treatments. PMID:15215287
Claudin 1 Expression Levels Affect miRNA Dynamics in Human Basal-Like Breast Cancer Cells.
Majer, Anna; Blanchard, Anne A; Medina, Sarah; Booth, Stephanie A; Myal, Yvonne
Deemed a putative tumor suppressor in breast cancer, the tight junction protein claudin 1 has now been shown to be highly expressed in the basal-like molecular subtype. Moreover, recent in vitro studies show that claudin 1 can regulate breast cancer cell motility and proliferation. Herein, we investigated whether microRNA (miRNA) dysregulation is associated with alterations in the level of claudin 1. Using next-generation sequencing (NGS), we identified seven miRNAs (miR-9-5p, miR-9-3p, let-7c, miR-127-3p, miR-99a-5p, miR-129-5p, and miR-146a-5p) that were deregulated as a consequence of claudin 1 overexpression in the MDA-MB231 human breast cancer (HBC) cell line. Most of these miRNAs have been associated with tumor suppression in a variety of cancers, including breast cancer. Moreover, through gene expression profiling analysis, we identified epithelial-mesenchymal transition-related genes, including platelet-derived growth factor receptor-beta (PDGFRB) and cadherin 1 (CDH1, E cadherin), whose downregulation correlated with claudin 1 overexpression. Collectively, we show for the first time that in HBC, claudin 1 can alter the dynamics of a number of miRNAs involved in tumor progression. Our data suggest that the dysregulated expression of these miRNAs, in conjunction with the high claudin 1 levels, could serve as a useful biomarker that identifies a subset of tumors within the poorly characterized basal-like subtype of breast cancer. Further studies are warranted to determine the role of these miRNAs in facilitating the function of claudin 1 in breast cancer. PMID:26982264
Modeling Breast Tumor Development with a Humanized Mouse Model.
Arendt, Lisa M
The tumor microenvironment plays a critical role in breast cancer growth and progression to metastasis. Here, we describe a method to examine stromal-epithelial interactions during tumor formation and progression utilizing human-derived mammary epithelial cells and breast stromal cells. This method outlines the isolation of each cell type from reduction mammoplasty tissue, the culture and genetic modification of both epithelial and stromal cells using lentiviral technology, and the method of humanizing and implantation of transformed epithelial cells into the cleared mammary fat pads of immunocompromised mice. This model system may be a useful tool to dissect signaling interactions that contribute to invasive tumor behavior and therapeutic resistance. PMID:27581027
Zeranol stimulates proliferation and aromatase activation in human breast preadipocytes.
Zhong, Saiyi; Liu, Shouchun; Chen, Suhua; Lin, Huajuan; Wang, Weimin; Qin, Xiaoming
Aromatase is a crucial enzyme for the biosynthesis of estrogens and is involved in the process of breast carcinogenesis. Concerns have been raised regarding the effects of environmental estrogens as potential regulators of aromatase expression in human breast cells. Zeranol is a non‑steroidal agent with potent estrogenic activity, which is widely used as a growth promoter for cattle in certain countries. The present study hypothesized that aromatase expression and activity may be elevated by low dose zeranol exposure, providing a source of estrogens that may stimulate cell proliferation. In the present study, primary cultured human breast preadipocytes were used as an in vitro model. The effects of zeranol on cell proliferation were measured using the MTS assay, aromatase expression levels were determined by immunocytochemical staining and reverse transcription‑polymerase chain reaction, and aromatase enzyme activity and estrogen production were analyzed using corresponding assay kits. The results demonstrated that low dose zeranol (2‑50 nM) was able to significantly promote cell proliferation, aromatase mRNA expression, aromatase activity and estrogen production in primary cultured human breast preadipocytes, thus suggesting that zeranol may act as an aromatase activator. The findings of the present study suggest that zeranol promotes breast cancer cell growth by stimulating aromatase activation and increasing estrogen biosynthesis in adipose tissue. PMID:27220457
Antiviral activity of purified human breast milk mucin.
Habte, Habtom H; Kotwal, Girish J; Lotz, Zoë E; Tyler, Marilyn G; Abrahams, Melissa; Rodriques, Jerry; Kahn, Delawir; Mall, Anwar S
Human breast milk is known to contain numerous biologically active components which protect breast fed infants against microbes, viruses, and toxins. The purpose of this study was to purify and characterize the breast milk mucin and determine its anti-poxvirus activity. In this study human milk mucin, free of contaminant protein and of sufficient quantity for further analysis, was isolated and purified by Sepharose CL-4B gel filtration and cesiumchloride density-gradient centrifugation. Based on the criteria of size and appearance of the bands and their electrophoretic mobility on sodium dodecyl sulfate polyacrylamide-gel electrophoresis, Western blotting together with the amino acid analysis, it is very likely that the human breast milk mucin is MUC1. It was shown that this breast milk mucin inhibits poxvirus activity by 100% using an inhibition assay with a viral concentration of 2.4 million plaque-forming units/ml. As the milk mucin seems to aggregate poxviruses prior to their entry into host cells, it is possible that this mucin may also inhibit other enveloped viruses such as HIV from entry into host cells. PMID:17361093
Significance of Heterogeneous Twist2 Expression in Human Breast Cancers
Mao, Yubin; Zhang, Nini; Xu, Jinfei; Ding, Zhijie; Zong, Rongrong; Liu, Zuguo
Background Twist2 (Dermo1) has been shown to mediate the epithelial-mesenchymal transition (EMT) to promote tumor invasion and even metastasis. However, the involvement of EMT in breast cancer progression is highly debated, partially due to clinical observations showing that the majority of human breast carcinoma metastases express E-cadherin and maintain their epithelial morphology. The molecular mechanism by which Twist2 participates in EMT of breast cancer in vivo remains poorly understood. Methods We examined Twist2 expression pattern in human breast carcinomas by western blot and tissue microarray, and analyzed Twist2 cellular localization by confocal microscopy, cell fractionation and other approaches. Results Twist2 expression was significantly increased in breast cancer. Cytoplasmic Twist2 positive cancer cells expressing E-cadherin on the cellular membrane were mainly located at tumor center of primary carcinomas and lymph metastases, while cancer cells with nuclear Twist2 clearly showed loss of E-cadherin and were detected at the invasive front in ductal breast carcinomas. In addition, ectopically stable-expressed Twist2 was found to localize in the cytoplasm of cancer cells. Collectively, these data indicate that upregulation of cytoplasmic Twist2 is correlated with tumor histological type and tumor metastasis in human breast cancers. Conclusion The differential cellular distribution of Twist2 may be associated with tumor progression. The cytoplasmic Twist2 in cancer cells at tumor center of primary carcinomas and lymph metastases contributes to the maintenance of epithelial cancer characteristics expressing E-cadherin in a noninvasive state, while the nuclear Twist2 at the cancer invasion front activates EMT to deprive epithelial property of neoplastic cells, thus facilitating invasion and metastasis. These findings suggest that heterogeneous expression of Twist2 in tumors may have a functional link to tumor progression. PMID:23133563
Overexpression of TGF-β1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells
Kim, Yong Il; Ryu, Jae-Sung; Yeo, Jee Eun; Choi, Yun Jin; Kim, Yong Sang; Ko, Kinarm; Koh, Yong-Gon
Highlights: • Continuous TGF-β1 overexpression in hSD-MSCs did not influence their phenotypes. • Retroviral-mediated transduction of TGFB1 in hSD-MSCs enhances cell proliferation. • TGF-β1 overexpression did not effect to adipo- or osteogenic potential of hSD-MSCs. • TGF-β1 overexpression in hSD-MSCs could stimulate and accelerate chondrogenesis. - Abstract: Transforming growth factor-beta (TGF-β) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-β up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuous TGF-β1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-β1. The results revealed that continuous overexpression of TGF-β1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-β1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-β1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs.
Cation-selective transporters are critical to the AMPK-mediated antiproliferative effects of metformin in human breast cancer cells.
Cai, Hao; Zhang, Yunhui; Han, Tianxiang Kevin; Everett, Ruth S; Thakker, Dhiren R
The antidiabetic drug metformin exerts antineoplastic effects against breast cancer and other cancers. One mechanism by which metformin is believed to exert its anticancer effect involves activation of its intracellular target, adenosine monophosphate-activated protein kinase (AMPK), which is also implicated in the antidiabetic effect of metformin. It is proposed that in cancer cells, AMPK activation leads to inhibition of the mammalian target of rapamycin (mTOR) and the downstream pS6K that regulates cell proliferation. Due to its hydrophilic and cationic nature, metformin requires cation-selective transporters to enter cells and activate AMPK. This study demonstrates that expression levels of cation-selective transporters correlate with the antiproliferative and antitumor efficacy of metformin in breast cancer. Metformin uptake and antiproliferative activity were compared between a cation-selective transporter-deficient human breast cancer cell line, BT-20, and a BT-20 cell line that was engineered to overexpress organic cation transporter 3 (OCT3), a representative of cation-selective transporters and a predominant transporter in human breast tumors. Metformin uptake was minimal in BT-20 cells, but increased by >13-fold in OCT3-BT20 cells, and its antiproliferative potency was >4-fold in OCT3-BT20 versus BT-20 cells. This increase in antiproliferative activity was associated with greater AMPK phosphorylation and decreased pS6K phosphorylation in OCT3-BT20 cells. In vitro data were corroborated by in vivo observations of significantly greater antitumor efficacy of metformin in xenograft mice bearing OCT3-overexpressing tumors versus low transporter-expressing wildtype tumors. Collectively, these findings establish a clear relationship between cation-selective transporter expression, the AMPK-mTOR-pS6K signaling cascade, and the antiproliferative activity of metformin in breast cancer. PMID:26669511
The modeling of Alzheimer's disease by the overexpression of mutant Presenilin 1 in human embryonic stem cells.
Honda, Makoto; Minami, Itsunari; Tooi, Norie; Morone, Nobuhiro; Nishioka, Hisae; Uemura, Kengo; Kinoshita, Ayae; Heuser, John E; Nakatsuji, Norio; Aiba, Kazuhiro
Cellular disease models are useful tools for Alzheimer's disease (AD) research. Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are promising materials for creating cellular models of such diseases. In the present study, we established cellular models of AD in hESCs that overexpressed the mutant Presenilin 1 (PS1) gene with the use of a site-specific gene integration system. The overexpression of PS1 did not affect the undifferentiated status or the neural differentiation ability of the hESCs. We found increases in the ratios of amyloid-β 42 (Aβ42)/Aβ40 and Aβ43/Aβ40. Furthermore, synaptic dysfunction was observed in a cellular model of AD that overexpressed mutant PS1. These results suggest that the AD phenotypes, in particular, the electrophysiological abnormality of the synapses in our AD models might be useful for AD research and drug discovery. PMID:26687948
Over-expression of human endosulfatase-1 exacerbates cadmium-induced injury to transformed human lung cells in vitro
Zhang, Huiying; Newman, Donna R.; Bonner, James C.; Sannes, Philip L.
Environmental exposure to cadmium is known to cause damage to alveolar epithelial cells of the lung, impair their capacity to repair, and result in permanent structural alterations. Cell surface heparan sulfate proteoglycans (HSPGs) can modulate cell responses to injury through their interactions with soluble effector molecules. These interactions are often sulfate specific, and the removal of sulfate groups from HS side chains could be expected to influence cellular injury, such as that caused by exposure to cadmium. The goal of this study was to define the role 6-O-sulfate plays in cellular responses to cadmium exposure in two pulmonary epithelial cancer cell lines (H292 and A549) and in normal human primary alveolar type II (hAT2) cells. Sulfate levels were modified by transduced transient over-expression of 6-O-endosulfatase (HSulf-1), a membrane-bound enzyme which specifically removes 6-O-sulfate groups from HSPG side chains. Results showed that cadmium decreased cell viability and activated apoptosis pathways at low concentrations in hAT2 cells but not in the cancer cells. HSulf-1 over-expression, on the contrary, decreased cell viability and activated apoptosis pathways in H292 and A549 cells but not in hAT2 cells. When combined with cadmium, HSulf-1 over-expression further decreased cell viability and exacerbated the activation of apoptosis pathways in the transformed cells but did not add to the toxicity in hAT2 cells. The finding that HSulf-1 sensitizes these cancer cells and intensifies the injury induced by cadmium suggests that 6-O-sulfate groups on HSPGs may play important roles in protection against certain environmental toxicants, such as heavy metals. -- Highlights: ► Primary human lung alveolar type 2 (hAT2) cells and H292 and A549 cells were used. ► Cadmium induced apoptosis in hAT2 cells but not in H292 or A549 cells. ► HSulf-1exacerbates apoptosis induced by cadmium in H292 and A549 but not hAT2 cells.
Overexpression of the β Subunit of Human Chorionic Gonadotropin Promotes the Transformation of Human Ovarian Epithelial Cells and Ovarian Tumorigenesis
Guo, Xiaoqing; Liu, Guangzhi; Schauer, Isaiah G.; Yang, Gong; Mercado-Uribe, Imelda; Yang, Fan; Zhang, Shiwu; He, Yuanli; Liu, Jinsong
Ovarian carcinoma is the most lethal gynecologic malignancy, however underlying molecular events remain elusive. Expression of human chorionic gonadotropin β subunit (β-hCG) is clinically significant for both trophoblastic and nontrophoblastic cancers; however, whether β-hCG facilitates ovarian epithelial cell tumorigenic potential remains uncharacterized. Immortalized nontumorigenic ovarian epithelial T29 and T80 cells stably overexpressing β-hCG were examined for alterations in cell cycle and apoptotic status by flow cytometry, expression of proteins regulating cell cycle and apoptosis by Western blot, proliferation status by MTT assay, anchorage-independent colony formation, and mouse tumor formation. Immunoreactivity for β-hCG was evaluated using mouse xenografts and on human normal ovarian, fallopian tube, endometrium, and ovarian carcinoma tissues. T29 and T80 cells overexpressing β-hCG demonstrated significantly increased proliferation, anchorage-independent colony formation, prosurvival Bcl-XL protein expression, G2-checkpoint progression, elevated cyclins E/D1 and Cdk 2/4/6, and decreased apoptosis. Collectively, these transformational alterations in phenotype facilitated increased xenograft tumorigenesis (P < 0.05). Furthermore, β-hCG immunoreactivity was elevated in malignant ovarian tumors, compared with normal epithelial expression in ovaries, fallopian tube, and endometrium (P < 0.001). Our data indicate that elevated β-hCG transforms ovarian surface epithelial cells, facilitating proliferation, cell cycle progression, and attenuated apoptosis to promote tumorigenesis. Our results further decipher the functional role and molecular mechanism of β-hCG in ovarian carcinoma. β-hCG may contribute to ovarian cancer etiology, which introduces a new therapeutic intervention target for ovarian cancer. PMID:21763678
The overexpression of SIRT1 inhibited osteoarthritic gene expression changes induced by interleukin-1β in human chondrocytes.
Matsushita, Takehiko; Sasaki, Hiroshi; Takayama, Koji; Ishida, Kazunari; Matsumoto, Tomoyuki; Kubo, Seiji; Matsuzaki, Tokio; Nishida, Kotaro; Kurosaka, Masahiro; Kuroda, Ryosuke
In this study, we examined the effects of overexpression of SIRT1 on IL-1β-induced gene expression changes in human chondrocytes to explore a protective role of SIRT1 in human chondrocytes. SIRT1 was overexpressed in human chondrocytes by expression plasmid under stimulation with IL-1β. SIRT1 was also inhibited by siRNA under stimulation with IL-1β. Gene expression changes were examined by real-time PCR. The interaction of SIRT1 and p65 (NF-κB) were examined by Western blotting. SIRT1, MMP-13, and ADAMTS-5 expressions in human cartilage were examined by immunohistochemistry. IL-1β stimulation significantly up-regulated MMP-1, 2, 9, and 13 and ADAMTS-5. Overexpression of SIRT1 significantly inhibited the up-regulation of those genes caused by IL-1β while the inhibition of SIRT1 further increased them. In addition, the overexpression of SIRT1 markedly reduced the IL-1β-induced acetylation of p65. SIRT1 expression was clearly detected in the non-OA cartilage while MMP-13 and ADAMTS-5 were undetectable. In contrast, in the OA cartilage, SIRT1 expression was decreased while MMP-13 and ADAMTS-5 were increased. Our observations suggested that SIRT1 can play a protective role by suppressing IL-1β-induced expressions of cartilage-degrading enzymes partially through the modulation of the NF-κB pathway. SIRT1 overexpression might be a new therapeutic approach for OA. PMID:23143889
Epidermal growth factor receptor coexpression modulates susceptibility to Herceptin in HER2/neu overexpressing breast cancer cells via specific erbB-receptor interaction and activation
Diermeier, Simone; Horvath, Gabor; Knuechel-Clarke, Ruth; Hofstaedter, Ferdinand; Szoellosi, Janos; Brockhoff, Gero . E-mail: firstname.lastname@example.com
Background: Growth factors and Herceptin specifically and differentially modulate cell proliferation of tumor cells. However, the mechanism of action on erbB-receptor level is incompletely understood. We evaluated Herceptin's capacity to modulate erbB-receptor activation and interaction on the cell surface level and thereby potentially impair cell proliferation of HER2/neu (c-erbB2) overexpressing breast cancer cells, both in the presence and absence of relevant growth factors. Methods: BT474 and SK-BR-3 breast cancer cell lines were treated with Epidermal Growth Factor (EGF), Heregulin, and with Herceptin in different combinations. Kinetics of cell proliferation were evaluated flow cytometrically based on BrdU-labeling. Fluorescence Resonance Energy Transfer, ELISAs and phosphorylation site specific Western Blotting was performed to investigate erbB-receptor interaction and activation. Results: EGF induced EGFR/EGFR and EGFR/c-erbB2 interactions correlate with stimulation of cell proliferation in BT474 cells. Both homo- and heterodimerization are considerably less pronounced in SK-BR-3 cells and heterointeraction is additionally reduced by EGF treatment, causing inhibition of cell proliferation. Heregulin stimulates cell proliferation extensively in both cell lines. Herceptin drives BT474 cells more efficiently into quiescence than it does with SK-BR-3 cells and thereby blocks cell cycle progress. In SK-BR-3 Herceptin treatment causes c-erbB2 phosphorylation of Y877 and Y1248, EGF induces Y877 and Y1112 phosphorylation. The Y1112 phosphorylation site, activated by EGF in SK-BR-3 cell, is bypassed in BT474. In addition the inhibitory capacity of Herceptin on BT474 and SK-BR-3 cell proliferation depends on the presence and absence of growth factors to a various extent. Conclusion: The growth inhibitory effect of Herceptin on c-erbB2 overexpressing breast cancer cells is considerably modulated by EGFR coexpression and consequently EGFR/c-erbB2 homo- and
Systems consequences of amplicon formation in human breast cancer
Inaki, Koichiro; Menghi, Francesca; Woo, Xing Yi; Wagner, Joel P.; Jacques, Pierre-Étienne; Lee, Yi Fang; Shreckengast, Phung Trang; Soon, Wendy WeiJia; Malhotra, Ankit; Teo, Audrey S.M.; Hillmer, Axel M.; Khng, Alexis Jiaying; Ruan, Xiaoan; Ong, Swee Hoe; Bertrand, Denis; Nagarajan, Niranjan; Karuturi, R. Krishna Murthy; Hidalgo Miranda, Alfredo
Chromosomal structural variations play an important role in determining the transcriptional landscape of human breast cancers. To assess the nature of these structural variations, we analyzed eight breast tumor samples with a focus on regions of gene amplification using mate-pair sequencing of long-insert genomic DNA with matched transcriptome profiling. We found that tandem duplications appear to be early events in tumor evolution, especially in the genesis of amplicons. In a detailed reconstruction of events on chromosome 17, we found large unpaired inversions and deletions connect a tandemly duplicated ERBB2 with neighboring 17q21.3 amplicons while simultaneously deleting the intervening BRCA1 tumor suppressor locus. This series of events appeared to be unusually common when examined in larger genomic data sets of breast cancers albeit using approaches with lesser resolution. Using siRNAs in breast cancer cell lines, we showed that the 17q21.3 amplicon harbored a significant number of weak oncogenes that appeared consistently coamplified in primary tumors. Down-regulation of BRCA1 expression augmented the cell proliferation in ERBB2-transfected human normal mammary epithelial cells. Coamplification of other functionally tested oncogenic elements in other breast tumors examined, such as RIPK2 and MYC on chromosome 8, also parallel these findings. Our analyses suggest that structural variations efficiently orchestrate the gain and loss of cancer gene cassettes that engage many oncogenic pathways simultaneously and that such oncogenic cassettes are favored during the evolution of a cancer. PMID:25186909
Systems consequences of amplicon formation in human breast cancer.
Inaki, Koichiro; Menghi, Francesca; Woo, Xing Yi; Wagner, Joel P; Jacques, Pierre-Étienne; Lee, Yi Fang; Shreckengast, Phung Trang; Soon, Wendy WeiJia; Malhotra, Ankit; Teo, Audrey S M; Hillmer, Axel M; Khng, Alexis Jiaying; Ruan, Xiaoan; Ong, Swee Hoe; Bertrand, Denis; Nagarajan, Niranjan; Karuturi, R Krishna Murthy; Miranda, Alfredo Hidalgo; Liu, Edison T
Chromosomal structural variations play an important role in determining the transcriptional landscape of human breast cancers. To assess the nature of these structural variations, we analyzed eight breast tumor samples with a focus on regions of gene amplification using mate-pair sequencing of long-insert genomic DNA with matched transcriptome profiling. We found that tandem duplications appear to be early events in tumor evolution, especially in the genesis of amplicons. In a detailed reconstruction of events on chromosome 17, we found large unpaired inversions and deletions connect a tandemly duplicated ERBB2 with neighboring 17q21.3 amplicons while simultaneously deleting the intervening BRCA1 tumor suppressor locus. This series of events appeared to be unusually common when examined in larger genomic data sets of breast cancers albeit using approaches with lesser resolution. Using siRNAs in breast cancer cell lines, we showed that the 17q21.3 amplicon harbored a significant number of weak oncogenes that appeared consistently coamplified in primary tumors. Down-regulation of BRCA1 expression augmented the cell proliferation in ERBB2-transfected human normal mammary epithelial cells. Coamplification of other functionally tested oncogenic elements in other breast tumors examined, such as RIPK2 and MYC on chromosome 8, also parallel these findings. Our analyses suggest that structural variations efficiently orchestrate the gain and loss of cancer gene cassettes that engage many oncogenic pathways simultaneously and that such oncogenic cassettes are favored during the evolution of a cancer. PMID:25186909
Detection of Volatile Metabolites of Garlic in Human Breast Milk
Scheffler, Laura; Sauermann, Yvonne; Zeh, Gina; Hauf, Katharina; Heinlein, Anja; Sharapa, Constanze; Buettner, Andrea
The odor of human breast milk after ingestion of raw garlic at food-relevant concentrations by breastfeeding mothers was investigated for the first time chemo-analytically using gas chromatography−mass spectrometry/olfactometry (GC-MS/O), as well as sensorially using a trained human sensory panel. Sensory evaluation revealed a clear garlic/cabbage-like odor that appeared in breast milk about 2.5 h after consumption of garlic. GC-MS/O analyses confirmed the occurrence of garlic-derived metabolites in breast milk, namely allyl methyl sulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO2). Of these, only AMS had a garlic-like odor whereas the other two metabolites were odorless. This demonstrates that the odor change in human milk is not related to a direct transfer of garlic odorants, as is currently believed, but rather derives from a single metabolite. The formation of these metabolites is not fully understood, but AMSO and AMSO2 are most likely formed by the oxidation of AMS in the human body. The excretion rates of these metabolites into breast milk were strongly time-dependent with large inter-individual differences. PMID:27275838
Detection of Volatile Metabolites of Garlic in Human Breast Milk.
Scheffler, Laura; Sauermann, Yvonne; Zeh, Gina; Hauf, Katharina; Heinlein, Anja; Sharapa, Constanze; Buettner, Andrea
The odor of human breast milk after ingestion of raw garlic at food-relevant concentrations by breastfeeding mothers was investigated for the first time chemo-analytically using gas chromatography-mass spectrometry/olfactometry (GC-MS/O), as well as sensorially using a trained human sensory panel. Sensory evaluation revealed a clear garlic/cabbage-like odor that appeared in breast milk about 2.5 h after consumption of garlic. GC-MS/O analyses confirmed the occurrence of garlic-derived metabolites in breast milk, namely allyl methyl sulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO₂). Of these, only AMS had a garlic-like odor whereas the other two metabolites were odorless. This demonstrates that the odor change in human milk is not related to a direct transfer of garlic odorants, as is currently believed, but rather derives from a single metabolite. The formation of these metabolites is not fully understood, but AMSO and AMSO₂ are most likely formed by the oxidation of AMS in the human body. The excretion rates of these metabolites into breast milk were strongly time-dependent with large inter-individual differences. PMID:27275838