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Nonhypoxic regulation and role of hypoxia-inducible factor 1 in aromatase inhibitor resistant breast cancer
Kazi, Armina A
Gilani, Rabia A
Schech, Amanda J
Chumsri, Saranya
Sabnis, Gauri
Shah, Preeti
Goloubeva, Olga
Kronsberg, Shari
Brodie, Angela H
Breast Cancer Research : BCR
Although aromatase inhibitors (AIs; for example, letrozole) are highly effective in treating estrogen receptor positive (ER+) breast cancer, a significant percentage of patients either do not respond to AIs or become resistant to them. Previous studies suggest that acquired resistance to AIs involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as human epidermal growth factor receptor-2 (HER2). However, the role of HER2, and the identity of other relevant factors that may be used as biomarkers or therapeutic targets remain unknown. This study investigated the potential role of transcription factor hypoxia inducible factor 1 (HIF-1) in acquired AI resistance, and its regulation by HER2.
In vitro studies using AI (letrozole or exemestane)-resistant and AI-sensitive cells were conducted to investigate the regulation and role of HIF-1 in AI resistance. Western blot and RT-PCR analyses were conducted to compare protein and mRNA expression, respectively, of ERα, HER2, and HIF-1α (inducible HIF-1 subunit) in AI-resistant versus AI-sensitive cells. Similar expression analyses were also done, along with chromatin immunoprecipitation (ChIP), to identify previously known HIF-1 target genes, such as breast cancer resistance protein (BCRP), that may also play a role in AI resistance. Letrozole-resistant cells were treated with inhibitors to HER2, kinase pathways, and ERα to elucidate the regulation of HIF-1 and BCRP. Lastly, cells were treated with inhibitors or inducers of HIF-1α to determine its importance.
Basal HIF-1α protein and BCRP mRNA and protein are higher in AI-resistant and HER2-transfected cells than in AI-sensitive, HER2- parental cells under nonhypoxic conditions. HIF-1α expression in AI-resistant cells is likely regulated by HER2 activated-phosphatidylinositide-3-kinase/Akt-protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, as its expression was inhibited by HER2 inhibitors and kinase pathway inhibitors. Inhibition or upregulation of HIF-1α affects breast cancer cell expression of BCRP; AI responsiveness; and expression of cancer stem cell characteristics, partially through BCRP.
One of the mechanisms of AI resistance may be through regulation of nonhypoxic HIF-1 target genes, such as BCRP, implicated in chemoresistance. Thus, HIF-1 should be explored further for its potential as a biomarker of and therapeutic target.
Related Articles
NEGATIVE REGULATION OF HIF-1α BY AN FBW7-MEDIATED DEGRADATION PATHWAY DURING HYPOXIA
Cassavaugh, Jessica M.
Hale, Sarah A.
Wellman, Theresa L.
Howe, Alan K.
Wong, Cheung
Lounsbury, Karen M.
Journal of cellular biochemistry
Hypoxia inducible factor-1α (HIF-1α) stimulates expression of genes associated with angiogenesis and is associated with poor outcomes in ovarian and other cancers. In normoxia, HIF-1α is ubiquitinated and degraded through the E3 ubiquitin ligase, von Hippel-Lindau; however, little is known about the regulation of HIF-1α in hypoxic conditions. FBW7 is an E3 ubiquitin ligase that recognizes proteins phosphorylated by glycogen synthase kinase 3β (GSK3β) and targets them for destruction. This study used an ovarian cancer cell model to test the hypothesis that HIF-1α phosphorylation by GSK3β in hypoxia leads to interaction with FBW7 and ubiquitin-dependent degradation. Expression of constitutively active GSK3β reduced HIF-1α protein and transcriptional activity and increased ubiquitination of HIF-1α in hypoxia, whereas pharmacologic inhibition of GSK3 or expression of siGSK3β promoted HIF-1α stabilization and activity. A mechanism through FBW7 was supported by the observed decrease in HIF-1α stabilization when FBW7 was overexpressed and both the elevation of HIF-1α levels and decrease in ubiquitinated HIF-1α when FBW7 was suppressed. Furthermore, HIF-1α associated with FBW7γ by co-immunoprecipitation, and the interaction was weakened by inhibition of GSK3 or mutation of GSK3β phosphorylation sites. The relevance of this pathway to angiogenic signaling was supported by the finding that endothelial cell tube maturation was increased by conditioned media from hypoxic SK-OV-3 cell lines expressing suppressed GSK3β or FBW7. These data introduce a new mechanism for regulation of HIF-1α during hypoxia that utilizes phosphorylation to target HIF-1α for ubiquitin-dependent degradation through FBW7 and may identify new targets in the regulation of angiogenesis.
angiogenesis; AKT; ubiquitination; transcription factor; ovarian cancer; endothelial cells
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Endothelin-1 Inhibits Prolyl Hydroxylase Domain 2 to Activate Hypoxia-Inducible Factor-1α in Melanoma Cells
Spinella, Francesca
Rosanò, Laura
Del Duca, Martina
Di Castro, Valeriana
Nicotra, Maria Rita
Natali, Pier Giorgio
Bagnato, Anna
PLoS ONE
The endothelin B receptor (ETBR) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1α is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation.
Principal Findings
Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ETBR, enhance the expression and activity of HIF-1α and HIF-2α that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-α stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1α oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ETBR markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKT-mammalian target of rapamycin (mTOR) pathway is required for ETBR-mediated PHD2 inhibition, HIF-1α, HIF-2α, and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1α, ETBR expression is associated with low PHD2 levels. In melanoma xenografts, ETBR blockade by ETBR antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1α, and HIF-2α expression, and an increase in PHD2 levels.
In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1α stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ETBR may represent a potential therapeutic treatment of melanoma by impairing HIF-1α stability.
Related Articles
Nicotine Induces Hypoxia-Inducible Factor-1α Expression in Human Lung Cancer Cells via Nicotinic Acetylcholine Receptor – Mediated Signaling Pathways
Zhang, Qunzhou
Tang, Xudong
Zhang, Zuo-Feng
Velikina, Rita
Shi, Shihong
Le, Anh D.
Clinical cancer research : an official journal of the American Association for Cancer Research
Nicotine, the major component in cigarette smoke, can promote tumor growth and angiogenesis in various cancers, including lung cancer. Hypoxia-inducible factor-1α (HIF-1α) is overexpressed in human lung cancers, particularly in non – small cell lung cancers (NSCLC), and is closely associated with an advanced tumor grade, increased angiogenesis, and resistance to chemotherapy and radiotherapy. The purpose of this study was to investigate the effects of nicotine on the expression of HIF-1α and its downstream target gene, vascular endothelial growth factor (VEGF), in human lung cancer cells.
Experimental Design
Human NSCLC cell lines A549 and H157 were treated with nicotine and examined for expression of HIF-1α and VEGF using Western blot or ELISA. Loss of HIF-1α function using specific small interfering RNA was used to determine whether HIF-1α is directly involved in nicotine-induced tumor angiogenic activities, including VEGF expression, cancer cell migration, and invasion.
Nicotine increased HIF-1α and VEGF expression in NSCLC cells. Pharmacologically blocking nicotinic acetylcholine receptor – mediated signaling cascades, including the Ca2+/calmodulin, c-Src, protein kinase C, phosphatidylinositol 3-kinase, mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2, and the mammalian target of rapamycin pathways, significantly attenuated nicotine-induced up-regulation of HIF-1α protein. Functionally, nicotine potently stimulated in vitro tumor angiogenesis by promoting tumor cell migration and invasion. These proangiogenic and invasive effects were partially abrogated by treatment with small interfering RNA specific for HIF-1α.
These findings identify novel mechanisms by which nicotine promotes tumor angiogenesis and metastasis and provide further evidences that HIF-1αis a potential anticancer target in nicotine-associated lung cancer.
Related Articles
Expression of hypoxia-inducible factor-1α and cell cycle proteins in invasive breast cancer are estrogen receptor related
Bos, Reinhard
van Diest, Paul J
van der Groep, Petra
Shvarts, Avi
Greijer, Astrid E
van der Wall, Elsken
Breast Cancer Research
The transcription factor hypoxia-inducible factor-1 (HIF-1) is a key regulator of the cellular response to hypoxia. Previous studies showed that concentrations of its subunit HIF-1α, as a surrogate for HIF-1 activity, are increased during breast carcinogenesis and can independently predict prognosis in breast cancer. During carcinogenesis, the cell cycle is progressively deregulated, and proliferation rate is a strong prognostic factor in breast cancer. In this study we undertook a detailed evaluation of the relationships between HIF-1α and cell cycle-associated proteins.
In a representative estrogen receptor (ER) group of 150 breast cancers, the expression of HIF-1α, vascular endothelial growth factor, the ER, HER-2/neu, Ki-67, cyclin A, cyclin D1, p21, p53, and Bcl-2 was investigated by immunohistochemistry.
High concentrations (5% or more) of HIF-1α were associated with increased proliferation as shown by positive correlations with Ki-67 (P < 0.001) and the late S–G2-phase protein cyclin A (P < 0.001), but not with the G1-phase protein cyclin D1. High HIF-1α concentrations were also strongly associated with p53 positivity (P < 0.001) and loss of Bcl-2 expression (P = 0.013). No association was found between p21 and HIF-1α (P = 0.105) in the whole group of patients. However, the subgroup of ER-positive cancers was characterized by a strong positive association between HIF-1α and p21 (P = 0.023), and HIF-1α lacked any relation with proliferation.
HIF-1α overexpression is associated with increased proliferation, which might explain the adverse prognostic impact of increased concentrations of HIF-1α in invasive breast cancer. In ER-positive tumors, HIF-1α is associated with p21 but not against proliferation. This shows the importance of further functional analysis to unravel the role of HIF-1 in late cell cycle progression, and the link between HIF-1, p21, and ER.
Bcl-2; breast cancer; estrogen receptor; hypoxia-inducible factor-1α; p53
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Latent Kaposi's Sarcoma-Associated Herpesvirus Infection of Endothelial Cells Activates Hypoxia-Induced Factors▿
Carroll, Patrick A.
Kenerson, Heidi L.
Yeung, Raymond S.
Lagunoff, Michael
Journal of Virology
Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) is the etiological agent of Kaposi's sarcoma, a highly vascularized, endothelial-derived tumor. A direct role for KSHV-mediated induction of angiogenesis has been proposed based upon the nature of the neoplasia and various KSHV gene overexpression and infection model systems. We have found that KSHV infection of endothelial cells induces mRNA of hypoxia-induced factor 1α (HIF1α) and HIF2α, two homologous alpha subunits of the heterodimeric transcription factor HIF. HIF is a master regulator of both developmental and pathological angiogenesis, composed of an oxygen-sensitive alpha subunit and a constitutively expressed beta subunit. HIF is classically activated posttranscriptionally with hypoxia, leading to increased protein stability of HIF1α and/or HIF2α. However, we demonstrate that both alpha subunits are up-regulated at the transcript level by KSHV infection. The transcriptional activation of HIF leads to a functional increase in HIF activity under normoxic conditions, as demonstrated by both luciferase reporter assay and the increased expression of vascular endothelial growth factor receptor 1 (VEGFR1), an HIF-responsive gene. KSHV infection synergizes with hypoxia mimics and induces higher expression levels of HIF1α and HIF2α protein, and HIF1α is increased in a significant proportion of the latently infected endothelial cells. Src family kinases are required for the activation of HIF and the downstream gene VEGFR1 by KSHV. We also show that KS lesions, in vivo, express elevated levels of HIF1α and HIF2α proteins. Thus, KSHV stimulates the HIF pathway via transcriptional up-regulation of both HIF alphas, and this activation may play a role in KS formation, localization, and progression.
Related Articles
Retinoblastoma Binding Protein 2 (RBP2) Promotes HIF-1α–VEGF-Induced Angiogenesis of Non-Small Cell Lung Cancer via the Akt Pathway
Qi, Lei
Zhu, Feng
Li, Shu-hai
Si, Li-bo
Hu, Li-kuan
Tian, Hui
PLoS ONE
Pathological angiogenesis plays an essential role in tumor aggressiveness and leads to unfavorable prognosis. The aim of this study is to detect the potential role of Retinoblastoma binding protein 2 (RBP2) in the tumor angiogenesis of non-small cell lung cancer (NSCLC).
Immunohistochemical staining was used to detect the expression of RBP2, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and CD34. Two pairs of siRNA sequences and pcDNA3-HA-RBP2 were used to down-regulate and up-regulate RBP2 expression in H1975 and SK-MES-1 cells. An endothelial cell tube formation assay, VEGF enzyme-linked immunosorbent assay, real-time PCR and western blotting were performed to detect the potential mechanisms mediated by RBP2 in tumor angiogenesis.
Of the 102 stage I NSCLC specimens analyzed, high RBP2 protein expression is closely associated with tumor size (P = 0.030), high HIF-1α expression (P = 0.028), high VEGF expression (P = 0.048), increased tumor angiogenesis (P = 0.033) and poor prognosis (P = 0.037); high MVD was associated with high HIF-1α expression (P = 0.034), high VEGF expression (P = 0.001) and poor prognosis (P = 0.040). Multivariate analysis indicated that RBP2 had an independent influence on the survival of patients with stage I NSCLC (P = 0.044). By modulating the expression of RBP2, our findings suggested that RBP2 protein depletion decreased HUVECs tube formation by down-regulating VEGF in a conditioned medium. RBP2 stimulated the up-regulation of VEGF, which was dependent on HIF-1α, and activated the HIF-1α via phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Moreover, VEGF increased the activation of Akt regulated by RBP2.
The RBP2 protein may stimulate HIF-1α expression via the activation of the PI3K/Akt signaling pathway under normoxia and then stimulate VEGF expression. These findings indicate that RBP2 may play a critical role in tumor angiogenesis and serve as an attractive therapeutic target against tumor aggressiveness for early-stage NSCLC patients.
Related Articles
TNFα induces HIF-1α expression through activation of IKKβ
Kuo, Hsu-Ping
Lee, Dung-Fang
Xia, Weiya
Wei, Yongkun
Hung, Mien-Chie
Biochemical and biophysical research communications
The transcription factor hypoxia-inducible factor 1α (HIF-1α) is regulated by oxygen availability as well as various inflammatory mediators, including tumor necrosis factor α (TNFα). Early work suggested that the phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways are involved in TNFα-mediated HIF-1α accumulation and activation under normoxic conditions. Here, we provide evidence showing that IκBkinase β (IKKβ) is required for HIF-1α regulation by TNFα. We found that TNFα enhances HIF-1α protein expression in various breast cancer cell lines under either normoxic or hypoxia-mimicking conditions, but has little effect on the HIF-1α mRNA level. Increased HIF-1α expression was found in IKKβ stable clones and transient transfectants, and depletion of IKKβ consistently reduced the amount of HIF-1α protein. Treatment of cells with the IKKβ inhibitor Bay 11-7082 reduced the TNFα-induced HIF-1α expression, suggesting that IKKβ is required in this signaling pathway. Decreased expression of vascular endothelial growth factor (VEGF), a direct target of HIF-1α, was shown in IKKβ-knockout mouse embryonic fibroblast cells. We further demonstrated a positive correlation between IKKβ and VEGF expression in primary human breast cancer specimens. Our findings indicate that TNFα-induced HIF-1α accumulation is IKKβ dependent, and may enable further understanding of the HIF-1α regulation by inflammatory signals.
Tumor necrosis factor α; Hypoxia-inducible factor 1α; IκBkinase β; Vascular endothelial growth factor; Breast cancer
Related Articles
Physical and Functional Interactions between Runx2 and HIF-1α Induce Vascular Endothelial Growth Factor Gene Expression
Kwon, Tae-Geon
Zhao, Xiang
Yang, Qian
Li, Yan
Ge, Chunxi
Zhao, Guisheng
Franceschi, Renny T.
Journal of cellular biochemistry
Angiogenesis and bone formation are intimately related processes. Hypoxia during early bone development stabilizes hypoxia-inducible factor-1α (HIF-1α) and increases angiogenic signals including vascular endothelial growth factor (VEGF). Furthermore, stabilization of HIF-1α by genetic or chemical means stimulates bone formation. On the other hand, deficiency of Runx2, a key osteogenic transcription factor, prevents vascular invasion of bone and VEGF expression. This study explores the possibility that HIF-1α and Runx2 interact to activate angiogenic signals. Runx2 over-expression in mesenchymal cells increased VEGF mRNA and protein under both normoxic and hypoxic conditions. In normoxia, Runx2 also dramatically increased HIF-1α protein. In all cases, the Runx2 response was inhibited by siRNA-mediated suppression of HIF-1α and completely blocked by the HIF-1α inhibitor, echinomycin. Similarly, treatment of preosteoblast cells with Runx2 siRNA reduced VEGF mRNA in normoxia or hypoxia. However, Runx2 is not essential for the HIF-1α response since VEGF is induced by hypoxia even in Runx2-null cells. Endogenous Runx2 and HIF-1α were colocalized to the nuclei of MC3T3-E1 preosteoblast cells. Moreover, HIF-1α and Runx2 physically interact using sites within the Runx2 RUNT domain. Chromatin immunoprecipitation also provided evidence for colocalization of Runx2 and HIF-1α on the VEGF promoter. In addition, Runx2 stimulated HIF-1α-dependent activation of an HRE-luciferase reporter gene without requiring a separate Runx2-binding enhancer. These studies indicate that Runx2 functions together with HIF-1α to stimulate angiogenic gene expression in bone cells and may in part explain the known requirement for Runx2 in bone vascularization.
Osteoblast; vascularization; angiogenesis; transcriptional factors; hypoxia
Related Articles
Mutation of von Hippel–Lindau Tumour Suppressor and Human Cardiopulmonary Physiology
Smith, Thomas G
Brooks, Jerome T
Balanos, George M
Lappin, Terence R
Layton, D. Mark
Leedham, Dawn L
Liu, Chun
Maxwell, Patrick H
McMullin, Mary F
McNamara, Christopher J
Percy, Melanie J
Pugh, Christopher W
Ratcliffe, Peter J
Talbot, Nick P
Treacy, Marilyn
Robbins, Peter A
PLoS Medicine
The von Hippel–Lindau tumour suppressor protein–hypoxia-inducible factor (VHL–HIF) pathway has attracted widespread medical interest as a transcriptional system controlling cellular responses to hypoxia, yet insights into its role in systemic human physiology remain limited. Chuvash polycythaemia has recently been defined as a new form of VHL-associated disease, distinct from the classical VHL-associated inherited cancer syndrome, in which germline homozygosity for a hypomorphic VHL allele causes a generalised abnormality in VHL–HIF signalling. Affected individuals thus provide a unique opportunity to explore the integrative physiology of this signalling pathway. This study investigated patients with Chuvash polycythaemia in order to analyse the role of the VHL–HIF pathway in systemic human cardiopulmonary physiology.
Methods and Findings
Twelve participants, three with Chuvash polycythaemia and nine controls, were studied at baseline and during hypoxia. Participants breathed through a mouthpiece, and pulmonary ventilation was measured while pulmonary vascular tone was assessed echocardiographically. Individuals with Chuvash polycythaemia were found to have striking abnormalities in respiratory and pulmonary vascular regulation. Basal ventilation and pulmonary vascular tone were elevated, and ventilatory, pulmonary vasoconstrictive, and heart rate responses to acute hypoxia were greatly increased.
The features observed in this small group of patients with Chuvash polycythaemia are highly characteristic of those associated with acclimatisation to the hypoxia of high altitude. More generally, the phenotype associated with Chuvash polycythaemia demonstrates that VHL plays a major role in the underlying calibration and homeostasis of the respiratory and cardiovascular systems, most likely through its central role in the regulation of HIF.
Editors' Summary
Human cells (like those of other multicellular animals) use oxygen to provide the energy needed for daily life. Having not enough oxygen is a problem, but having too much is also dangerous because it damages proteins, DNA, and other large molecules that keep cells functioning. Consequently, the physiological systems—including the heart, lungs, and circulation—work together to balance oxygen supply and demand throughout the body. When oxygen is limiting (a condition called hypoxia), as happens at high altitudes, the cellular oxygen supply is maintained by increasing the heart rate, increasing the speed and depth of breathing (hyperventilation), constricting the blood vessels in the lung (pulmonary vasoconstriction), and increasing the number of oxygen-carrying cells in the blood. All these physiological changes increase the amount of oxygen that can be absorbed from the air, but how they are regulated is poorly understood. By contrast, researchers know quite a bit about how individual cells respond to hypoxia. When oxygen is limited, a protein called hypoxia-inducible factor (or HIF) activates a number of target proteins that help the cell get enough oxygen (for example, proteins that stimulate the growth of new blood vessels). When there is plenty of oxygen, another protein, called von Hippel–Lindau tumor suppressor (abbreviated VHL), rapidly destroys HIF. Recently, researchers discovered that a genetic condition called Chuvash polycythaemia, characterised by the overproduction of red blood cells, is caused by a specific defect in VHL that reduces its ability to destroy HIF. As a result, the expression of certain HIF target proteins is increased even when oxygen levels are normal.
Why Was This Study Done?
Chuvash polycythaemia is very rare, and so far little is known about how this genetic abnormality affects the physiology and long-term health of patients. By studying heart and lung function in patients with Chuvash polycythaemia, the researchers involved in this study hoped to discover more about the health consequences of the condition and to find out whether the VHL–HIF system controls systemic responses to hypoxia as well as cellular responses.
What Did the Researchers Do and Find?
The researchers recruited and studied three patients with Chuvash polycythaemia, and, as controls for the comparison, several normal individuals and patients with an unrelated form of polycythaemia. They then measured how the lungs and hearts of these people reacted to mild hypoxia (similar to that experienced on commercial air flights) and moderate hypoxia (equiv alent to being on the top of an Alpine peak). They found that patients with Chuvash polycythaemia naturally breathe slightly quicker and deeper than normal individuals, and that their breathing rate increased dramatically and abnormally when oxygen was reduced. They also found that at normal oxygen levels the pulmonary blood vessels of these patients were more constricted than those of control individuals, and that they reacted more extremely to hypoxia. Similarly, the normal heart rate of the patients was slightly higher than that of the controls and increased much more in response to mild hypoxia.
What Do These Findings Mean?
The physiological differences measured by the researchers between Chuvash polycythaemia patients and control individuals are similar to the adaptations seen in people traveling to high altitudes where oxygen is limited. Thus, the VHL–HIF proteins may regulate the response to different oxygen concentrations both in individual cells and at the systemic level, although more physiological studies are needed to confirm this. Because the pulmonary blood vessels of patients with Chuvash polycythaemia are always abnormally constricted, and even more so when oxygen is limited, these people should avoid living at high altitude and should minimise air travel, suggest the researchers. The increased blood pressure in their lungs (pulmonary hypertension) could conceivably cause heart failure under such circumstances. Finally, this study has implications for the development of drugs directed at the VHL–HIF system. Agents are currently being designed to promote the development of new blood vessels after strokes or heart attacks by preventing the destruction of HIF, but based on the findings here such agents might have undesirable physiological affects. Conversely, HIF inhibitors (which act as anti-cancer reagents by increasing hypoxia in the centre of tumors and so inhibiting their growth) might be useful in the treatment of pulmonary hypertension.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030290.
• Online Mendelian Inheritance in Man page on Chuvash polycythaemia
• Information from the VHL Family Alliance on von Hippel–Lindau disease, including information on Chuvash polycythaemia
• Wikipedia page on polycythaemia and von Hippel–Lindau disease (note: Wikipedia is a free online encyclopaedia that anyone can edit)
Physiological study of patients with Chuvash polycythemia (caused by mutation of VHL) reveals characteristics similar to those associated with acclimatization to the hypoxia of high altitude.
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Rapid non-genomic signalling by 17β-oestradiol through c-Src involves mTOR-dependent expression of HIF-1α in breast cancer cells
Sudhagar, S
Sathya, S
Lakshmi, B S
British Journal of Cancer
Hypoxia-inducible factor 1 (HIF1) has been implicated in regulating many of the genes responsible for angiogenesis, erythropoiesis, glucose metabolism and cancer pathogenesis. In this study, we demonstrate that exposure of human breast cancer lines to 17β-oestradiol (E2) rapidly induced the expression of HIF-1α, the regulated subunit of HIF1, in normoxic condition. Hypoxia-inducible factor-1α is normally degraded in normoxia through ubiquitination-mediated proteolysis, whereas hypoxia modulates HIF-1α level by inhibiting ubiquitination-mediated degradation.
Oestradiol-induced accumulation of HIF-1α in breast cancer lines was detected by western blot analysis and its promoter activity was measured by HIF1 reporter assay. Molecular signalling of oestradiol-mediated HIF-1α expression was studied using specific pharmacological inhibitors and small interference RNA by co-immunoprecipitation and western blotting analysis.
Oestradiol has been observed to rapidly activate the nongenomic signalling cascade leading to HIF-1α protein synthesis. The results define a signalling pathway in breast cancer cells whereby oestradiol induces a rapid protein–protein interaction of ERα-c-Src-PI3K, resulting in the activation of PI3K/AKT pathway leading to mammalian target of rapamycin (mTOR) phosphorylation. The mTOR then stimulates translation by phosphorylating p70 S6 kinase and 4EB-P1, modulating HIF-1α protein synthesis. Oestradiol-stimulated HIF-1α activity was inhibited by either siRNA or pharmacological inhibitors to ERα, c-Src, PI3K and mTOR, providing a mechanism for the modulation of HIF-1α protein synthesis.
These results show oestradiol-induced expression of HIF-1α, downstream of the ERα/c-Src/PI3K/AKT/mTOR pathway in human breast cancer cells.
oestradiol; HIF-1α; c-Src; mTOR; breast cancer
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Epstein-Barr Virus Latent Membrane Protein 1 Induces Synthesis of Hypoxia-Inducible Factor 1α
Wakisaka, Naohiro
Kondo, Satoru
Yoshizaki, Tomokazu
Murono, Shigeyuki
Furukawa, Mitsuru
Pagano, Joseph S.
Molecular and Cellular Biology
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1α and HIF-1β that is the central regulator of responses to hypoxia. The specific binding of HIF-1 to the hypoxia-responsive element (HRE) induces the transcription of genes that respond to hypoxic conditions, including vascular endothelial growth factor (VEGF). Here we report that expression of HIF-1α is increased in diverse Epstein-Barr virus (EBV)-infected type II and III cell lines, which express EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein, as well as other latency proteins, but not in the parental EBV-negative cell lines. We show first that transfection of an LMP1 expression plasmid into Ad-AH cells, an EBV-negative nasopharyngeal epithelial cell line, induces synthesis of HIF-1α protein without increasing its stability or mRNA level. The mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 markedly reduces induction of HIF-1α by LMP1. Catalase, an H2O2 scavenger, strongly suppresses LMP1-induced production of H2O2, which results in a decrease in the expression of HIF-1α induced by LMP1. Inhibition of the NF-κB, c-jun N-terminal kinase, p38 MAPK, and phosphatidylinositol 3-kinase pathways did not affect HIF-1α expression. Moreover, LMP1 induces HIF-1 DNA binding activity and upregulates HRE and VEGF promoter transcriptional activity. Finally, LMP1 increases the appearance of VEGF protein in extracellular fluids; induction of VEGF is suppressed by PD98059 or catalase. These results suggest that LMP1 increases HIF-1 activity through induction of HIF-1α protein expression, which is controlled by p42/p44 MAPK activity and H2O2. The ability of EBV, and specifically its major oncoprotein, LMP1, to induce HIF-1α along with other invasiveness and angiogenic factors reported previously discloses additional oncogenic properties of this tumor virus.
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Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer
Khong, Tak L
Thairu, Ngayu
Larsen, Helene
Dawson, Peter M
Kiriakidis, Serafim
Paleolog, Ewa M
BMC Cancer
Colorectal cancer (CRC) is characterised by hypoxia, which activates gene transcription through hypoxia-inducible factors (HIF), as well as by expression of epidermal growth factor (EGF) and EGF receptors, targeting of which has been demonstrated to provide therapeutic benefit in CRC. Although EGF has been demonstrated to induce expression of angiogenic mediators, potential interactions in CRC between EGF-mediated signalling and the hypoxia/HIF pathway remain uncharacterised.
PCR-based profiling was applied to identify angiogenic genes in Caco-2 CRC cells regulated by hypoxia, the hypoxia mimetic dimethyloxallylglycine (DMOG) and/or EGF. Western blotting was used to determine the role of HIF-1alpha, HIF-2alpha and MAPK cell signalling in mediating the angiogenic responses.
We identified a total of 9 angiogenic genes, including angiopoietin-like (ANGPTL) 4, ephrin (EFNA) 3, transforming growth factor (TGF) β1 and vascular endothelial growth factor (VEGF), to be upregulated in a HIF dependent manner in Caco-2 CRC cells in response to both hypoxia and the hypoxia mimetic dimethyloxallylglycine (DMOG). Stimulation with EGF resulted in EGFR tyrosine autophosphorylation, activation of p42/p44 MAP kinases and stabilisation of HIF-1α and HIF-2α proteins. However, expression of 84 angiogenic genes remained unchanged in response to EGF alone. Crucially, addition of DMOG in combination with EGF significantly increased expression of a further 11 genes (in addition to the 9 genes upregulated in response to either DMOG alone or hypoxia alone). These additional genes included chemokines (CCL-11/eotaxin-1 and interleukin-8), collagen type IV α3 chain, integrin β3 chain, TGFα and VEGF receptor KDR.
These findings suggest that although EGFR phosphorylation activates the MAP kinase signalling and promotes HIF stabilisation in CRC, this alone is not sufficient to induce angiogenic gene expression. In contrast, HIF activation downstream of hypoxia/DMOG drives expression of genes such as ANGPTL4, EFNA3, TGFβ1 and VEGF. Finally, HIF activation synergises with EGF-mediated signalling to additionally induce a unique sub-group of candidate angiogenic genes. Our data highlight the complex interrelationship between tumour hypoxia, EGF and angiogenesis in the pathogenesis of CRC.
Colorectal cancer; Angiogenesis; Hypoxia; EGF
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Stathmin Regulates Hypoxia-Inducible Factor-1α Expression through the Mammalian Target of Rapamycin Pathway in Ovarian Clear Cell Adenocarcinoma
Tamura, Kazuhiro
Yoshie, Mikihiro
Miyajima, Eri
Kano, Mika
Tachikawa, Eiichi
ISRN Pharmacology
Stathmin, a microtubule-destabilizing phosphoprotein, is highly expressed in ovarian cancer, but the pathophysiological significance of this protein in ovarian carcinoma cells remains poorly understood. This study reports the involvement of stathmin in the mTOR/HIF-1α/VEGF pathway in ovarian clear cell adenocarcinoma (CCA) during hypoxia. HIF-1α protein and VEGF mRNA levels were markedly elevated in RMG-1 cells, a CCA cell line, cultured under hypoxic conditions. Rapamycin, an inhibitor of mTOR complex 1, reduced the level of HIF-1α and blocked phosphorylation of ribosomal protein S6 kinase 1 (S6K), a transcriptional regulator of mTOR, demonstrating that hypoxia activates mTOR/S6K/HIF-1α signaling in CCA. Furthermore, stathmin knockdown inhibited hypoxia-induced HIF-1α and VEGF expression and S6K phosphorylation. The silencing of stathmin expression also reduced Akt phosphorylation, a critical event in the mTOR/HIF-1α/VEGF signaling pathway. By contrast, stathmin overexpression upregulated hypoxia-induced HIF-1α and VEGF expression in OVCAR-3 cells, another CCA cell line. In addition, suppression of Akt activation by wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, decreased HIF-1α and VEGF expression. These results illustrate that regulation of HIF-1α through the PI3K/Akt/mTOR pathway is controlled by stathmin in CCA. Our findings point to a new mechanism of stathmin regulation during ovarian cancer.
Related Articles
Hypoxia Promotes Uveal Melanoma Invasion through Enhanced Notch and MAPK Activation
Asnaghi, Laura
Lin, Michael H.
Lim, Kah Suan
Lim, Kah Jing
Tripathy, Arushi
Wendeborn, Murilo
Merbs, Shannath L.
Handa, James T.
Sodhi, Akrit
Bar, Eli E.
Eberhart, Charles G.
PLoS ONE
The transcriptional response promoted by hypoxia-inducible factors has been associated with metastatic spread of uveal melanoma. We found expression of hypoxia-inducible factor 1α (HIF-1α) protein in well-vascularized tumor regions as well as in four cell lines grown in normoxia, thus this pathway may be important even in well-oxygenated uveal melanoma cells. HIF-1α protein accumulation in normoxia was inhibited by rapamycin. As expected, hypoxia (1% pO2) further induced HIF-1α protein levels along with its target genes VEGF and LOX. Growth in hypoxia significantly increased cellular invasion of all 5 uveal melanoma lines tested, as did the introduction of an oxygen-insensitive HIF-1α mutant into Mel285 cells with low HIF-1α baseline levels. In contrast, HIF-1α knockdown using shRNA significantly decreased growth in hypoxia, and reduced by more than 50% tumor invasion in four lines with high HIF-1α baseline levels. Pharmacologic blockade of HIF-1α protein expression using digoxin dramatically suppressed cellular invasion both in normoxia and in hypoxia. We found that Notch pathway components, including Jag1-2 ligands, Hes1-Hey1 targets and the intracellular domain of Notch1, were increased in hypoxia, as well as the phosphorylation levels of Erk1-2 and Akt. Pharmacologic and genetic inhibition of Notch largely blocked the hypoxic induction of invasion as did the pharmacologic suppression of Erk1-2 activity. In addition, the increase in Erk1-2 and Akt phosphorylation by hypoxia was partially reduced by inhibiting Notch signaling. Our findings support the functional importance of HIF-1α signaling in promoting the invasive capacity of uveal melanoma cells in both hypoxia and normoxia, and suggest that pharmacologically targeting HIF-1α pathway directly or through blockade of Notch or Erk1-2 pathways can slow tumor spread.
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Regulation of Hypoxia-Inducible Factor 1α Expression and Function by the Mammalian Target of Rapamycin
Hudson, Christine C.
Liu, Mei
Chiang, Gary G.
Otterness, Diane M.
Loomis, Dawn C.
Kaper, Fiona
Giaccia, Amato J.
Abraham, Robert T.
Molecular and Cellular Biology
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor containing an inducibly expressed HIF-1α subunit and a constititutively expressed HIF-1β subunit. Under hypoxic conditions, the HIF-1α subunit accumulates due to a decrease in the rate of proteolytic degradation, and the resulting HIF-1α-HIF-1β heterodimers undergo posttranslational modifications that promote transactivation. Recent studies suggest that amplified signaling through phosphoinositide 3-kinase, and its downstream target, mTOR, enhances HIF-1-dependent gene expression in certain cell types. In the present study, we have explored further the linkage between mTOR and HIF-1 in PC-3 prostate cancer cells treated with hypoxia or the hypoxia mimetic agent, CoCl2. Pretreatment of PC-3 cells with the mTOR inhibitor, rapamycin, inhibited both the accumulation of HIF-1α and HIF-1-dependent transcription induced by hypoxia or CoCl2. Transfection of these cells with wild-type mTOR enhanced HIF-1 activation by hypoxia or CoCl2, while expression of a rapamycin-resistant mTOR mutant rendered both HIF-1α stabilization and HIF-1 transactivating function refractory to inhibition by rapamycin. Studies with GAL4-HIF-1α fusion proteins pinpointed the oxygen-dependent degradation domain as a critical target for the rapamycin-sensitive, mTOR-dependent signaling pathway leading to HIF-1α stabilization by CoCl2. These studies position mTOR as an upstream activator of HIF-1 function in cancer cells and suggest that the antitumor activity of rapamycin is mediated, in part, through the inhibition of cellular responses to hypoxic stress.
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HIF-1α stimulates aromatase expression driven by prostaglandin E2 in breast adipose stroma
Samarajeewa, Nirukshi U
Yang, Fangyuan
Docanto, Maria M
Sakurai, Minako
McNamara, Keely M
Sasano, Hironobu
Fox, Stephen B
Simpson, Evan R
Brown, Kristy A
Breast Cancer Research : BCR
The majority of postmenopausal breast cancers are estrogen-dependent. Tumor-derived factors, such as prostaglandin E2 (PGE2), stimulate CREB1 binding to cAMP response elements (CREs) on aromatase promoter II (PII), leading to the increased expression of aromatase and biosynthesis of estrogens within human breast adipose stromal cells (ASCs). Hypoxia inducible factor-1α (HIF-1α), a key mediator of cellular adaptation to low oxygen levels, is emerging as a novel prognostic marker in breast cancer. We have identified the presence of a consensus HIF-1α binding motif overlapping with the proximal CRE of aromatase PII. However, the regulation of aromatase expression by HIF-1α in breast cancer has not been characterized. This study aimed to characterize the role of HIF-1α in the activation of aromatase PII.
HIF-1α expression and localization were examined in human breast ASCs using quantitative PCR (QPCR), Western blotting, immunofluorescence and high content screening. QPCR and tritiated water-release assays were performed to assess the effect of HIF-1α on aromatase expression and activity. Reporter assays and chromatin immunoprecipitation (ChIP) were performed to assess the effect of HIF-1α on PII activity and binding. Treatments included PGE2 or DMOG ((dimethyloxalglycine), HIF-1α stabilizer). Double immunohistochemistry for HIF-1α and aromatase was performed on tissues obtained from breast cancer and cancer-free patients.
Results indicate that PGE2 increases HIF-1α transcript and protein expression, nuclear localization and binding to aromatase PII in human breast ASCs. Results also demonstrate that HIF-1α significantly increases PII activity, and aromatase transcript expression and activity, in the presence of DMOG and/or PGE2, and that HIF-1α and CREB1 act co-operatively on PII. There is a significant increase in HIF-1α positive ASCs in breast cancer patients compared to cancer-free women, and a positive association between HIF-1α and aromatase expression.
This study is the first to identify HIF-1α as a modulator of PII-driven aromatase expression in human breast tumor-associated stroma and provides a novel mechanism for estrogen regulation in obesity-related, post-menopausal breast cancer. Together with our on-going studies on the role of AMP-activated protein kinase (AMPK) in the regulation of breast aromatase, this work provides another link between disregulated metabolism and breast cancer.
Related Articles
1, 9-Pyrazoloanthrones Downregulate HIF-1α and Sensitize Cancer Cells to Cetuximab-Mediated Anti-EGFR Therapy
Lu, Yang
Li, Xinqun
Lu, Haiquan
Fan, Zhen
PLoS ONE
Cetuximab, a monoclonal antibody that blocks the epidermal growth factor receptor (EGFR), is currently approved for the treatment of several types of solid tumors. We previously showed that cetuximab can inhibit hypoxia-inducible factor-1 alpha (HIF-1α) protein synthesis by inhibiting the activation of EGFR downstream signaling pathways including Erk, Akt, and mTOR. 1, 9-pyrazoloanthrone (1, 9 PA) is an anthrapyrazolone compound best known as SP600125 that specifically inhibits c-jun N-terminal kinase (JNK). Here, we report 1, 9 PA can downregulate HIF-1α independently of its inhibition of JNK. This downregulatory effect was abolished when the oxygen-dependent domain (ODD) of HIF-1α (HIF-1α-ΔODD, the domain responsible for HIF-1α degradation) was experimentally deleted or when the activity of HIF-1α prolyl hydroxylase (PHD) or the 26S proteasomal complex was inhibited, indicating that the 1, 9 PA downregulates HIF-1α by promoting PHD-dependent HIF-1α degradation. We found that the combination of 1, 9 PA and cetuximab worked synergistically to induce apoptosis in cancer cells in which cetuximab or 1, 9 PA alone had no or only weak apoptotic activity. This synergistic effect was substantially decreased in cancer cells transfected with HIF-1α-ΔODD, indicating that downregulation of HIF-1α was the mechanism of this synergistic effect. More importantly, 1, 9 PA can downregulate HIF-1α in cancer cells that are insensitive to cetuximab-induced inhibition of HIF-1α expression due to overexpression of oncogenic Ras (RasG12V). Our findings suggest that 1, 9 PA is a lead compound of a novel class of drugs that may be used to enhance the response of cancer cells to cetuximab through a complementary effect on the downregulation of HIF-1α.
Related Articles
Hypoxia-Inducible Factor-2α Is an Essential Catabolic Regulator of Inflammatory Rheumatoid Arthritis
Ryu, Je-Hwang
Chae, Chang-Suk
Kwak, Ji-Sun
Oh, Hwanhee
Shin, Youngnim
Huh, Yun Hyun
Lee, Choong-Gu
Park, Yong-Wook
Chun, Churl-Hong
Kim, Young-Myeong
Im, Sin-Hyeog
Chun, Jang-Soo
PLoS Biology
Hypoxia-inducible factor-2α (HIF-2α) is sufficient to cause experimental rheumatoid arthritis and acts to regulate the functions of fibroblast-like cells from tissue surrounding joints, independent of HIF-1α.
Rheumatoid arthritis (RA) is a systemic autoimmune disorder that manifests as chronic inflammation and joint tissue destruction. However, the etiology and pathogenesis of RA have not been fully elucidated. Here, we explored the role of the hypoxia-inducible factors (HIFs), HIF-1α (encoded by HIF1A) and HIF-2α (encoded by EPAS1). HIF-2α was markedly up-regulated in the intimal lining of RA synovium, whereas HIF-1α was detected in a few cells in the sublining and deep layer of RA synovium. Overexpression of HIF-2α in joint tissues caused an RA-like phenotype, whereas HIF-1α did not affect joint architecture. Moreover, a HIF-2α deficiency in mice blunted the development of experimental RA. HIF-2α was expressed mainly in fibroblast-like synoviocytes (FLS) of RA synovium and regulated their proliferation, expression of RANKL (receptor activator of nuclear factor–κB ligand) and various catabolic factors, and osteoclastogenic potential. Moreover, HIF-2α–dependent up-regulation of interleukin (IL)-6 in FLS stimulated differentiation of TH17 cells—crucial effectors of RA pathogenesis. Additionally, in the absence of IL-6 (Il6−/− mice), overexpression of HIF-2α in joint tissues did not cause an RA phenotype. Thus, our results collectively suggest that HIF-2α plays a pivotal role in the pathogenesis of RA by regulating FLS functions, independent of HIF-1α.
Author Summary
Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by chronic inflammation in joint tissues leading to destruction of cartilage and bone. Despite some therapeutic advances, the etiology of RA pathogenesis is not yet clear, and effective treatment of RA remains a significant, unmet medical need. Hypoxia is a prominent feature of inflamed tissue within RA-affected joints, and earlier work has implicated limited involvement of hypoxia-inducible factor (HIF)-1 α. We explored the role of a second HIF family member, HIF-2α, in RA pathogenesis. We showed that HIF-2α is markedly increased in the tissue lining the RA-affected joints. Notably and in contrast to HIF-1α, when overexpressed in normal mouse joint tissues, HIF-2α is sufficient to cause RA-like symptoms. Conversely, an HIF-2α deficiency blocks the development of experimental arthritis in mice. We discovered further that HIF-2α regulates RA pathogenesis by modulating various RA-associated functions of joint-specific fibroblast-like cells, including proliferation, expression of cytokines, chemokines, and matrix-degrading enzymes, and bone-remodeling potential. HIF-2α also increases the ability of these cells to promote interleukin-6–dependent differentiation of TH17 cells, a known effector of RA pathogenesis. We thus show that HIF-1α and HIF-2α have distinct roles and act via different mechanisms in RA pathogenesis.
Related Articles
IFN-α Induces Transcription of Hypoxia-Inducible Factor-1α to Inhibit Proliferation of Human Endothelial Cells1
Gerber, Scott A.
Pober, Jordan S.
Journal of immunology (Baltimore, Md. : 1950)
Expression of hypoxia-inducible factor (HIF)-1α, a transcription factor subunit increased by protein stabilization in response to hypoxia, is increased in human endothelial cells (ECs) by IFN-α under normoxic conditions. IFN-α increases HIF-1α transcript levels within 2 h by up to 50% and doubles HIF-1α protein expression. Based on pharmacological inhibition studies, the increase in HIF-1α mRNA involves new transcription, is independent of new protein synthesis, and requires JAK signaling. Protein knockdown by small interfering RNA confirms the involvement of JAK1 and TYK2, as well of IFN-stimulated gene factor 3 (ISGF3). IFN-γ does not significantly induce HIF-1α mRNA, but increases the magnitude and duration of the IFN-α effect. IFN-α-induced HIF-1α protein translocates to the nucleus and can bind to hypoxia response elements in DNA. However, IFN-α treatment fails to induce transcription of several prototypic HIF-responsive genes (VEGF-A, PPARγ, and prostacyclin synthase) due to an insufficient increase in HIF-1α protein levels. Although certain other HIF-responsive genes (PHD3 and VEGF-C) are induced following IFN-α and/or IFN-γ treatment, these responses are not inhibited by siRNA knockdown of HIF-1α. Additionally, IFN-α induction of ISGF3-dependent genes involved in innate immunity (viperin, OAS2, and CXCL10) are also unaffected by knockdown of HIF-1α. Interestingly, knockdown of HIF-1α significantly reduces the capacity of IFN-α to inhibit endothelial cell proliferation. We conclude that IFN-α induces the transcription of HIF-1α in human endothelial cells though a JAK-ISGF3 pathway under normoxic conditions, and that this response contributes to the antiproliferative activity of this cytokine.
Related Articles
HIF-1α: A key survival factor for serum-deprived prostate cancer cells
Thomas, Rusha
Kim, Myoung H.
The Prostate
Hypoxia-inducible factor-1α (HIF-1α) is commonly overexpressed in prostate cancer (PCa) cells. As PCa cells are known to survive serum deprivation, we investigated the effect of prolonged serum deprivation on HIF-1α expression, and the function of HIF-1α in regulating the survival of normoxic serum-deprived PCa cells.
HIF-1α protein was assessed by immunoblots. Cell viability and proliferation were assessed by trypan blue assay and flow cytometric analysis. Transcriptional activity was assessed by luciferase reporter assay and RT-PCR. HIF-1α expression was suppressed with siRNA. Activities of HIF-1α–target genes were inhibited with neutralizing antibody.
Prolonged serum deprivation is a potent inducer of HIF-1α in PC-3 and LNCaP PCa cells, despite normal oxygen conditions. In contrast, cells grown in the presence of serum did not show HIF-1α protein accumulation. Moreover, HIF-1α protein increase during serum deprivation correlated with increased cell survival, while suppression of HIF-1α expression significantly decreased PCa cell viability. Our results further demonstrate that HIF-1α protein increase is due to increased HIF-1α protein synthesis. First, there was no significant increase in HIF-1α mRNA. Secondly, cycloheximide, a protein synthesis inhibitor, prevented HIF-1α protein increase in serum-deprived PCa cells. Moreover, the expression of HIF-1α-target genes, VEGF and IGF-2, was concomitantly increased in serum-deprived PCa cells, while suppression of HIF-1α expression significantly inhibited their induction. Furthermore, inhibition of IGF-2 activity resulted in a significant decline in PCa cell survival.
PCa cells counteract the stress of prolonged serum deprivation by upregulating HIF-1α protein which increases IGF-2 expression to promote cell survival.
HIF-1α; IGF-2; survival; serum deprivation; prostate cancer
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Bcl-2 Regulates HIF-1α Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90
Trisciuoglio, Daniela
Gabellini, Chiara
Desideri, Marianna
Ziparo, Elio
Zupi, Gabriella
Del Bufalo, Donatella
PLoS ONE
Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1α, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis.
Methodology/Principal Findings
By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1α protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1α protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1α protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1α stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1α degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1α protein. We also showed that bcl-2, HIF-1α and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1α protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1α protein during hypoxia, and in particular the isoform HSP90β is the main player in this phenomenon.
We identified the stabilization of HIF-1α protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the β isoform of molecular chaperone HSP90.
Related Articles
Induction of HIF-1α Expression by Intermittent Hypoxia: Involvement of NADPH Oxidase, Ca2+ Signaling, Prolyl Hydroxylases, and mTOR
Yuan, Guoxiang
Nanduri, Jayasri
Khan, Shakil
Semenza, Gregg L.
Prabhakar, Nanduri R.
Journal of cellular physiology
Sleep-disordered breathing with recurrent apnea (periodic cessation of breathing) results in chronic intermittent hypoxia (IH), which leads to cardiovascular and respiratory pathology. Molecular mechanisms underlying IH-evoked cardio-respiratory co-morbidities have not been delineated. Mice with heterozygous deficiency of hypoxia-inducible factor 1α (HIF-1α) do not develop cardio-respiratory responses to chronic IH. HIF-1α protein expression and HIF-1 transcriptional activity are induced by IH in PC12 cells. In the present study, we investigated the signaling pathways associated with IH-evoked HIF-1α accumulation. PC12 cells were exposed to aerobic conditions (20% O2) or 60 cycles of IH (30 sec at 1.5% O2 followed by 5 min at 20% O2). Our results show that IH-induced HIF-1α accumulation is due to increased generation of ROS by NADPH oxidase. We further demonstrate that ROS-dependent Ca2+ signaling pathways involving phospholipase Cγ and protein kinase C activation are required for IH-evoked HIF-1α accumulation. IH leads to activation of mTOR and S6 kinase and rapamycin partially inhibited IH-induced HIF-1α accumulation. IH also decreased hydroxylation of HIF-1α protein and anti-oxidants as well as inhibitors of Ca+2 signaling prevented this response. Thus, both increased mTOR-dependent HIF-1α synthesis and decreased hydroxylase-dependent HIF-1α degradation contribute to IH-evoked HIF-1α accumulation. Following IH, HIF-1α and phosphorylated mTOR levels remained elevated during 90 min of re-oxygenation despite re-activation of prolyl hydroxylase. Rapamycin or cycloheximide, blocked increased HIF-1α levels during re-oxygenation indicating that mTOR-dependent protein synthesis is required for the persistent elevation of HIF-1α levels during re-oxygenation.
Intermittent hypoxia; HIF-1α; NADPH oxidase; Calcium signaling; Prolyl hydroxylases; mTOR
Related Articles
HIF-1α/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs)
De Francesco, Ernestina Marianna
Lappano, Rosamaria
Santolla, Maria Francesca
Marsico, Stefania
Caruso, Arnaldo
Maggiolini, Marcello
Breast Cancer Research : BCR
Carcinoma-associated fibroblasts (CAFs) play a pivotal role in cancer progression by contributing to invasion, metastasis and angiogenesis. Solid tumors possess a unique microenvironment characterized by local hypoxia, which induces gene expression changes and biological features leading to poor outcomes. Hypoxia Inducible Factor 1 (HIF-1) is the main transcription factor that mediates the cell response to hypoxia through different mechanisms that include the regulation of genes strongly associated with cancer aggressiveness. Among the HIF-1 target genes, the G-protein estrogen receptor (GPER) exerts a stimulatory role in diverse types of cancer cells and in CAFs.
We evaluated the regulation and function of the key angiogenic mediator vascular endothelial growth factor (VEGF) in CAFs exposed to hypoxia. Gene expression studies, Western blotting analysis and immunofluorescence experiments were performed in CAFs and breast cancer cells in the presence of cobalt chloride (CoCl2) or cultured under low oxygen tension (2% O2), in order to analyze the involvement of the HIF-1α/GPER signaling in the biological responses to hypoxia. We also explored the role of the HIF-1α/GPER transduction pathway in functional assays like tube formation in human umbilical vein endothelial cells (HUVECs) and cell migration in CAFs.
We first determined that hypoxia induces the expression of HIF-1α and GPER in CAFs, then we ascertained that the HIF-1α/GPER signaling is involved in the regulation of VEGF expression in breast cancer cells and in CAFs exposed to hypoxia. We also assessed by ChIP assay that HIF-1α and GPER are both recruited to the VEGF promoter sequence and required for VEGF promoter stimulation upon hypoxic condition. As a biological counterpart of these findings, conditioned medium from hypoxic CAFs promoted tube formation in HUVECs in a HIF-1α/GPER dependent manner. The functional cooperation between HIF-1α and GPER in CAFs was also evidenced in the hypoxia-induced cell migration, which involved a further target of the HIF-1α/GPER signaling like connective tissue growth factor (CTGF).
The present results provide novel insight into the role elicited by the HIF-1α/GPER transduction pathway in CAFs towards the hypoxia-dependent tumor angiogenesis. Our findings further extend the molecular mechanisms through which the tumor microenvironment may contribute to cancer progression.
Related Articles
Signal Transducer and Activator of Transcription 3 is required for hypoxia-inducible factor-1α RNA expression in both tumor cells and tumor-associated myeloid cells
Niu, Guilian
Briggs, Jon
Deng, Jiehui
Ma, Yihong
Lee, Heehyoung
Kortylewski, Marcin
Kujawski, Maciej
Kay, Heidi
Cress, W. Douglas
Jove, Richard
Yu, Hua
Molecular cancer research : MCR
Hypoxia-inducible factor 1 (HIF-1) is a potent tumorigenic factor. Its alpha subunit (HIF-1α), which is tightly regulated in normal tissues, is elevated in tumors due to hypoxia and overactive growth signaling pathways. Although much is known about HIF-1α regulation in cancer cells, crucial molecular targets that affect HIF-1α levels modulated by both hypoxia and oncogenic signaling pathways remain to be identified. Additionally, whether and how the tumor microenvironment contributes to HIF-1α accumulation is unclear. This study demonstrates a novel mechanism by which HIF-1α availability is regulated in both cancer cells and in myeloid cells in the tumor microenvironment. We show a requirement of Stat3 for HIF-1α RNA expression under both hypoxia and growth signaling conditions. Furthermore, tumor-derived myeloid cells express elevated levels of HIF-1α mRNA relative to their counterparts from normal tissues in a Stat3-dependent manner. Additionally, Stat3 activity in the non-transformed cells in the tumor milieu impacts HIF-1α RNA expression of the entire growing tumor. Consistent with a role of Stat3 in regulating HIF-1α RNA transcription, elevated Stat3 activity increases HIF-1α promoter activity, and Stat3 protein binds to the HIF-1α promoter in both transformed cells and in growing tumors. Taken together, these findings demonstrate a novel mode by which HIF-1α is regulated not only in cancer cells but also in the tumor associated inflammatory cells, suggesting Stat3 as an important molecular target for inhibiting the oncogenic potential of HIF-1 induced by both hypoxia and overactive growth signaling pathways prevalent in cancer.
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Making project management indispensable for business results. ® We serve practitioners and organizations with standards that describe good practices, globally ...Podio is the online work platform. Manage, share, and get your work done smarter together with tools that work like you. Free for 10 Users.This handbook is intended as a ready reference guide to the control and management tactics for the more important plant diseases in the Pacific Northwest.Why Project Based Learning (PBL)? Project Based Learning's time has come. The experience of thousands of teachers across all grade levels and subject areas, backed ...For everyone in the team Project Management Tool "Backlog" Backlog will make your daily tasks easier and more enjoyable, and lead your team to more enthusiasm for ...One of the key elements of project stakeholder management is the use of influence ("the ability to affect the actions, beliefs and attitudes of other people") to ...Latest news. Sphere Board unveils strategic plan 2015-2020. Developing capacity for Sphere in Cambodia. New Sphere training materials. Comparing the Core …Redbooth, the online collaboration platform for team and task management. Be more productive with project management tools like video chat and file sharing.Research and management of the fire ant from Texas Agricultural Experiment Station and other sources.The FreeBSD Project. FreeBSD is an advanced computer operating system used to power modern servers, desktops and embedded platforms.
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Rachel Kathryn Residential Home
Bridge Farm, High Street, Argoed, Blackwood NP12 0HG
Rachel Kathryn Residential Home is a large property set in its own grounds in the beautiful Sirhowy Valley and boasts excellent views. The grounds have a parking area, spacious lawns and patio barbeque areas to sit and relax whilst enjoying the views… more
Heol Islwyn, Nelson, Treharris CF46 6HG
Mill Lane, Llanfyllin SY22 5BG
Llwyn Teg has been purpose built and designed to offer a high standard of care and accommodation. A team of friendly and experienced professionals are on hand to offer 24 hour, individual based care for short or long term needs as well as providing expert… more
Abernant Road, Aberdare CF44 0PY
Barchester Bradshaw Manor Care Home
2 Chester Street, Rhyl LL18 3ER
Barchester Bradshaw Manor Care Home is a purpose built Nursing and residential home set in landscaped gardens adjacent to the main promenade in Rhyl. Bradshaw Manor also provides expert care for those ladies and gentlemen who are experiencing a dementia… more
Queens Square, The Crescent, Ebbw Vale NP23 6EF
The Rookery is an Ebbw Vale care home registered for forty-one service users, offering skilled elderly care packages tailored to individual needs. The aim of carers is to maintain quality care in an atmosphere that respects personal freedoms and is… more
Clos Llwynaalt, Alltwen, Pontardawe, Swansea SA8 3JG
Mayflower Care Home, formerly known as LLwynallt Care Home was established in 1982 and has recently undergone an extensive refurbishment to the highest standard. Mayflower Care Home can now provide accommodation for up to 43 residents (single en-suite… more
Great Oaks Park, Off Cefn Road, Rogerstone, Newport NP10 9FX
The Oaks is a residential care home situated in the village of Rogerstone which provides support for the elderly. The Home is nestled in a beautiful setting close to Newport Golf Course and the picturesque Monmouthshire and Brecon Canal and is surrounded… more
13 St Martin's Road, Caerphilly CF83 1EF
Raisdale Road, Penarth CF64 5BN
Morel Court is a thirty-six capacity Penarth facility, employing qualified carers to deliver a friendly and professional service and maintaining a living environment that is pleasing and flexible towards personal choice. Modern and en-suite bedrooms… more
Group / Provider:
MHA (Methodist Homes)
Bryn Eithin Hall Residential Care Home
30 Llanrwst Road, Off Victoria Park, Colwyn Bay LL29 7YU
Bryn Eithin Hall in Colwyn Bay is a well appointed, comfortable residence, in its own 3.5 acres of mature grounds. In addition to Full Time Care, they offer Day Care Services and Short Term Breaks for 'Respite Care,' from Highly-qualified staff, who… more
Cartrefle Residential Home (Conwy)
Betws Road, Llanrwst LL26 0HG
3 Pwllycrochan Avenue, Colwyn Bay LL29 7DA
Pembroke Lodge is a specialist dementia care home, with staff who are skilled in the challenges of memory problems and target personal fulfilment as a core part of their service philosophy. A friendly team demonstrate a consistent commitment to quality,… more
Group / Provider:
Pembroke Nursing Homes Ltd
Park Road, Garden City, Ebbw Vale NP23 8UP
Red Rose Nursing Home is an Ebbw Vale facility suitable for residential and nursing purposes, together with being highly-suited for younger people living with a disability. Staff are accustomed to looking after a range of care packages and the different… more
615 Birchgrove Road, Glais, Swansea SA7 9EN
Glais House is a unique, warm and welcoming nursing home based in Swansea. The home is situated on 4 acres of well-kept gardens, home to local wildlife and protected trees and an outdoor patios. Glais House believe that having spacious and green… more
77 Dyserth Road, Rhyl LL18 4DT
Marle Close, Pentwyn Road, Pentwyn, Cardiff CF23 7EP
Ty Enfys is a leading, purpose-built care home situated in Pentywn on the outskirts of Cardiff. The home is in the final stages of an extensive refurbishment and offers outstanding, modern facilities in addition to the highest standards of residential,… more
Barchester Awel-y-Mor Care Centre
Brynafon Road, Gorseinon, Swansea SA4 4YF
Located in Swansea, Awel-y-Mor Care Centre is home to adults of all ages requiring nursing care. Offering short stay, respite breaks, long term care and rehabilitation services, Awel Y Mor is able to cater for most needs. As well as providing care… more
Overstone Court, Dumballs Road, Cardiff Bay, Cardiff CF10 5NT
Shire Hall Care Home is just a short walk from Cardiff Bay. Facilities at the purpose-built home include a cinema, reception cafe area, and a farmhouse kitchen - designed to promote active and independent living. Shire Hall provides the highest… more
Woodlands Park, Colonel Road, Betws, Ammanford SA18 2HW
Cartref Annwyl Fan Care Home is a popular care home set in a beautiful rural location on the outskirts of Betws, Ammanford, and is easily accessible by public transport. The purpose-built home is tastefully decorated to create a warm and homely environment… more
Penylan, Cardiff CF23 5HB
Ty Draw Lodge is part of a family-run organisation that remains committed to care environments that have a non-institutional feel, as well as being located in scenic surroundings. The aim for professionals is to work in partnership with residents… more
Pen-y-Garth EMI Residential & Residential Home
Pleasant Lane, Brymbo, Wrexham LL11 5DH
Pen-Y-Garth is situated in well tended grounds of two acres and enjoys panoramic views of the Welsh Countryside and Cheshire Plains. The home is located just 4 miles from Wrexham in the village of Brymbo, and is within walking distance of shops and… more
Cherry Tree Care Home EMI/EMH Residential
High Street, Coedpoeth, Wrexham LL11 3UF
Cherry Tree is very well known within the small village of Coedpoeth as a friendly, family run residential home, registered to provide EMH residential care, specialising in dementia. Cherry Tree Care Home offers a homely and quiet environment with the… more
Magor Road, Wilcrick, Magor, Caldicot NP26 3DA
Pool Cottage is a converted cottage offering accommodation for eight individuals with learning disabilities. There are eight single bedrooms, seven are en-suite. Pool Cottage provides a large lounge on both the ground and first floors, a conservatory,… more
Group / Provider:
Consensus Support
Cadoc Place, Danescourt, Cardiff CF5 2QN
Danesbrook House is a care home offering a homely interior that is staffed by a team of expert and friendly care professionals. Carers look to understand personal requirements, to help residents enjoy personal freedoms, comfort and relaxation, while… more
Oakwood Lane, Port Talbot SA13 1DF
20 Russell Road, Rhyl LL18 3DD
Heol Lotwen, Capel Hendre, Ammanford SA18 3RP
Albert Edward Prince of Wales Court
Penylan Avenue, Porthcawl CF36 3LY
Purpose-built in 1973 and refurbished in 2000, Albert Edward Prince of Wales Court stands in extensive, well-kept grounds situated one mile from Porthcawl town centre and the Promenade. Within the grounds there are beautiful inner courtyard gardens… more
Group / Provider:
Royal Masonic Benevolent Institution Care Company
Windsor Road, Miskin, Mountain Ash CF45 3BH
Newport Road, Bedwas, Caerphilly CF83 8AA
Gorof Road, Ystradgynlais, Lower Cwmtwrch, Swansea SA9 1DS
Cwrt Enfys is an open and welcoming care home situated in the beautiful upper Swansea Valley on the outskirts of the village of Ystradgynlais. The home and its whole team are fully equipped and trained to provide excellence in relationship-centred… more
Group / Provider:
Silvercrest Care


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|Message view
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|DO NOT REPLY [Bug 47464] New: Do not include *.class files into src archives of TC 5.5
|Wed, 01 Jul 2009 22:44:51 GMT
https://issues.apache.org/bugzilla/show_bug.cgi?id=47464 Summary: Do not include *.class files into src archives of TC 5.5 Product: Tomcat 5 Version: 5.5.27 Platform: PC OS/Version: Windows XP Status: NEW Severity: normal Priority: P2 Component: Unknown AssignedTo: email@example.com ReportedBy: firstname.lastname@example.com src bundles in the distribution of TC 5.5.27 include *.class files. Those should not be there. Also, as I understand, those ones in .tar.gz archive have gone through fixcrlf filter and are unusable. I can reproduce this issue with current tc5.5.x using Eclipse IDE as the development environment. (The classes compiled by the IDE are the ones that are packed there). -- Configure bugmail: https://issues.apache.org/bugzilla/userprefs.cgi?tab=email ------- You are receiving this mail because: ------- You are the assignee for the bug. --------------------------------------------------------------------- To unsubscribe, e-mail: firstname.lastname@example.com For additional commands, e-mail: firstname.lastname@example.com
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Welcome to Pregnancy Care Center
Our kind and professional staff is focused on helping you by providing free and confidential services including the following: clinical pregnancy testing, limited obstetrical ultrasound, factual information about pregnancy, abortion, adoption and sexually transmitted infections (STIs). Remember, at the end of the day it's YOUR choice. But first, let us help you know your options and get the facts so you can make an informed decision!


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Home » Posts Tagged "discovery"
From the Community: A week of ecology in mixed media
Posted By Katie Kline on Mar 22, 2010
Video describing the challenges of male pregnancy, photo gallery of the oldest trees in the world, podcast outlining Earth's environmental tipping points and an article on adapting to the anthropocene. Here is ecological news from the third week in March.
Annual Meeting
2016 ESA Annual Meeting
August 7-12th, 2016
Fort Lauderdale, Florida.
Recent From Guest Posts
- Take the sustainability research leadership survey Jul 14, 2016
Zika: Are outbreaks in U.S. cities avoidable? Mar 9, 2016
Reflections on Flint and environmental justice Mar 2, 2016
Recent From Research and Field Notes
Asian tiger mosquito thrives in New York Jul 26, 2016
Battle at the bloodmeal lek #ESA2016 Jul 25, 2016
Recent From Policy
blogging ecologists
- Beneath the Treeline
- Better Know a Fish
- Biodiverse Perspectives
- Biogeography Bits
- Bird Ecology Study Group
- Buzz Hoot Roar
- Down With Time
- Dynamic Ecology
- Ecological Rants
- I'm a chordata, urochordata
- Ideas for Sustainability
- Jabberwocky Ecology
- Land Ecology
- On the Other Hand
- Reconciliation Ecology
- Sedges have Edges
- Small Pond Science
- The Comtemplative Mammoth
- The Echinoblog
- The EEB and Flow
- The Molecular Ecologist
- The Prairie Ecologist
- Travels in ecosystem science
- Uncommon Ground
Asian tiger mosquito thrives in New York Jul 26, 2016
Battle at the bloodmeal lek #ESA2016 Jul 25, 2016
Ecologists remap the biosphere to include humans Dec 13, 2007
Hybrids in the Arctic Mar 17, 2011
Shrubs Bred for Sparse Seeds Still Spread Oct 14, 2011
ESA wants you(r photographs) (Update) Jan 19, 2007


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Russian Black Bread (For the Bread Machine)
Total Time
Prep 10 mins
Cook 4 hrs
Got this from All Recipes.com where it was submitted by Mary. Posted for Zaar World Tour 4.
Ingredients Nutrition
- 1 1⁄2 cups water
- 2 tablespoons cider vinegar
- 2 1⁄2 cups bread flour
- 1 cup rye flour
- 1 teaspoon salt
- 2 tablespoons margarine
- 2 tablespoons dark corn syrup
- 1 tablespoon brown sugar
- 3 tablespoons unsweetened cocoa powder
- 1 teaspoon instant coffee granules
- 1 tablespoon caraway seed
- 2 teaspoons active dry yeast
- Place ingredients into the bread machine in order suggested by the manufacturer.
- Use the whole wheat, regular crust setting.
- Remove bread from pan and allow to cool on rack.
Most Helpful
Have made this twice and I really like it. I would argue that it takes about a day after baking to really develop. Though the texture is phenomenal when served warm, that deep rye flavor didn't really register for me until it cooled down. I subbed brown sugar for molasses and left out the fennel the first time with good results.
One of the nicest breads I've ever made in my bread machine. It's not as dense/firm as dark rye that you get at the store, it's actually sort of soft with a somewhat crusty outside. Very easy to slice. I made it as written with two exceptions: I added no caraway seed (not a fan) and had no dark corn syrup, so I used agave nectar. You could also use 1 T add'l water with 1 T add'l brown sugar. I used 3 T agave nectar, and it turned out perfectly. I'll be making this over and over, and may try to increase the ratio of rye to wheat, as rye flour is healthier for the diabetics in my family. By the way...you cannot taste the cocoa or the coffee in the bread, but it gives the bread a gorgeous deep color and flavor. My daughter always prefers white bread, and she loved it.
Really enjoyed this bread, the deep strong flavors are so good. Perfect with soups or stews and makes a great sandwich. Nice hearty loaf of bread! Made and reviewed for ZWT 4 - Family Picks.


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If you want a hostel with a homey-atmosphere, Travellers Oasis is the place to stay! We've been staying here for several weeks and already when checking in we felt like we were home, it's like a gigant family where everybody helps each other. The staff will help you with anything you might need or want to do, just ask them and you'll get all information you need! All facilities (there's lots of them!!!) are clean and tidy, the staff really does everything in their power to make it comfy and homey! The one thing you should watch out for tho is Mickey, he's the awesome and cute guy behind the reception desk that sings Carolas song Mickey to the pet fish (we all reckon he has a crush for the fish since he always smiles like a moron and flirts with it while singing……). And by the by, if you sing the Mickey-song to Mickey he just might take you out on a date (c'mon girls give it a go, you won't be disappointed just as long as you respect his love for the beloved fishey)!
- Official Description (provided by the hotel):
- Welcome to Travellers Oasis Backpackers Hostel in Cairns.Travellers Oasis is a charming little guest house offering cosy and clean facilities to the budget conscious traveller, in a friendly and relaxed environment.Centrally located in a quiet area, our guests can relax in our sparkling pool, or lie back in our hammocks and chill out to the music. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with HostelWorld so you can book your Travellers Oasis Backpackers reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Travellers Oasis Backpackers Hotel Cairns


text:
Selecting a wedding cake is a crucial part of planning your wedding, and it is a step that requires guidance to make the right decision. Many bakeries and confectioneries in Wisconsin Rapids do provide wedding cakes for you to order, but you need to know how to go about it to get your wedding cake ready and delivered on your big day. Before you contact a cake decorator in Wisconsin Rapids, do a bit of research and write down what is important for your cake to include. This will go a long way in helping you get the perfect wedding cake while staying within your budget. To help assist you, we have compiled the following comprehensive list of cake decorators in and around Wisconsin Rapids, Wisconsin.
If you do not see a listing in Wisconsin Rapids, simply choose from one of the popular cities in Wisconsin near you to find a wedding cake decorator in your area.
Wal-mart - Bakery- Store Infor
Wisconsin Rapids, WI 54494
Cakes By Heidi
Wisconsin Rapids, WI 54494
The Occasional Bakery Llc
Port Edwards, WI 54469
Gremler's Bakery
Wisconsin Rapids, WI 54495
Colligan's Bakery
Plover, WI 54467
Gerber A Decorated Cakes
Vesper, WI 54489
Lemke's Nancy Custom Cakes
Stevens Point, WI 54481
Earthcrust Bakery
Stevens Point, WI 54481
These days, many brides are steering clear of traditional portrait style wedding photography. If you find yourself in this same boat but don't know where to go except the traditional photographer's studio, you can find a more artistic wedding
One of the things that many couples forget to do before their wedding day is to put lots of time and thought into their wedding photographer. This is a big mistake, though. If there's any part of your wedding that you'll cherish for years to come,
Your wedding day is probably going to be a more hectic and confusing time than you think it will be. Most brides have a vision of everything going smoothly, but that rarely happens. Even if you turn over most of the duties to other people, someone


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This flatware display from Cal-Mil features three slots to help organize and separate flatware utensils for any self-service area. Made of bamboo, this unique and modern organizer is a green alternative to other flatware displays. The eco-friendly bamboo material is a modern, renewable resource that's being used for a number of commercial products because of its durability. Because of the neutral color of bamboo, this flatware storage box will complement any restaurant, hotel buffet, or catered event!
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|Standard Edition
|Full Game
|Title Name
|The Orange Box
|First-Person Shooter
|Game Modes
|Single-Player, Multi-Player
Please Note: All products sold on Flipkart are brand new and 100% genuine
Have you used this product?
Rate it now.Write a Review
Hashir Zahid
Apr 17, 2014
Best Bundle Ever!
Shivendu Mishra
Apr 14, 2014
Best deal you'll ever have
Shalu Samuel
Feb 25, 2014
Frustrating Installation!
Pawan Sawaimoon
Jan 28, 2014
The Orange Box Package
Shantanu Tushar
Jan 13, 2014
All the Half Life games and Portal!
'Top Reviews' lists the most relevant product reviews only. Show ALL instead?Expand all Reviews
Puneet Kapoor
Jan 13, 2012
The Orange BoxBefore buying this just make sure that you do have a high speed internet connection or else you wont be able to play any games.The games need to be updated before they can be played via steam.
Manan Bhatia
Jun 20, 2011
The best bundleThe best game is team fortress 2 and the others also make a superb addition. Its been 4 days and i have already sunk in 29 hours.....
Siddharth Mankad
Jun 8, 2011
FPS AwesomenessHalf Life is a superb title. And Half-Life 2 makes it even better. HL2, HL2 - Episode 1 and 2 trace the lives of our dear ol' Dr. Gordon Freeman in sci-fi adventures with an engaging storyline, a satisfying amount of zombies, aliens, guns and entertainment, not forgetting the ever present and mysterious G-man.
Arun P%2520T
Jul 12, 2012
FRUSTRATING INSTALLATION BUT AWESOME GAMESA GOOD INTERNET CONNECTION(3G or Broadband) IS A MUST..
Shantanu S
Jan 11, 2012
Pleasant SurpriseI received this package just 20 minutes ago. I won't comment in detail about Half Life 2, the two episodes, Portal 2 or TF2 because nothing need to be said. any gamer worth his/her salt knows that these are true classics. HL 2 and Episodes 1 & 2 carry forward the original half life's legacy, the sheer scale, deep story, and incredible visuals, sound, gameplay and atmospherics, though with one of the best game engines 2 date, "Source" the game looks gorgeous even now,8-9 years later when Battlefield 3, Crysis 2 are upon us. Team Fortress 2 is a worthy challenger to Counterstrike, a must play fo… (Expand)
Shri Ram
Oct 5, 2012
One of the best games, Painful installationWell i think i don't need any introduction about the infamous half life series. Thanks to flipkart for the discount, i was finally able to purchase the game! And by the way, there is no mouse pad with the box. Maybe it was a limited offer, so only few got it.
Anil Sahu
Dec 1, 2011
Lost CauseTrue it has bundled many awesome games but the DRM is a lost cause. Its a huge headache to install all the games.
May 3, 2011
awsome game 5/5this game is one need if you have a steam account.if you dont have this game you dont deserve to have a steam account.amazing game and a must have.cheers
roshan sam
Jun 16, 2012
Sweet dealOkay, many people are cribbing about the Steam issues. I do agree with them and i too was a bit anxious about the installation. However, it turned out pretty fine and it didnt install the whole thing from the net. Either the Valve people have updated Steam to install from disc by default or i was plain lucky. :P I'll tell you what i did.
Rohit Basu
Sep 23, 2011
BEST BUNDLE BEST SERVICEAt this price this bundle is simply a steal, 2 DVD`s and once activated in Steam u can use your account to play all the games and download them again as well in case u loose the data,


text:
Separate names with a comma.
What is the fastest dns # for ipad 4 on Verizon fios
Y can't I use time capsule to save movies, music or anything else
I like racing,fighting and action games
I have changed to iPhone 4 and just got ipad 4 woooooohhoooooo lovin life now
I am not complaning just new to the ipad world this is a great tablet have always been a droid so I will never go back ipad forever. Woooohooooo
That sure doesnt seem like a lot
How much ram does ipad4 have


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Lithuanian Bank to Decide on Snoras Restructuring on Nov. 24
AB Bankas Snoras's state-apointed administrator will release its final report on the alternatives for the bank's restructuring on Nov. 23, the central bank said.
The Lietuvos Bankas, based in the capital, Vilnius, will give its resolution on the Snoras's future operations on Nov. 24., it said after a board meeting late yesterday.
The government took over Snoras on Nov. 16 after the central bank discovered that about 300 million euros ($405 million) of assets may be missing and the lender was at risk of insolvency. Some securities reported as assets by the bank don't exist, the regulator said. The country's Prosecutor General opened an investigation into possible fraud and embezzlement.
Lawmakers approved legislation on Nov. 17 allowing the government to split Snoras into two banks, with good and bad assets. The bad bank is planned to file for protection from creditors, while the government will seek an investor for the good bank with healthy assets and insured deposits "as soon as possible," the Finance Ministry said on Nov. 18.
No public money will be required to save Snoras because the bank has sufficient assets to cover insured deposits, the ministry said. Uninsured liabilities such as bonds will receive "a substantial haircut," it added. Insured liabilities account for 1.4 billion euros, compared with a total of 2.36 billion euros in assets at the end of September, it said.


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Algeria EgyptLibyaSudan
AfghanistanChinaCentral AsiaIndia-PakistanIndonesiaKoreaMyanmarNepalPhilippinesMicronesiaSri LankaThailand
Potential HotspotsTerrorismTop Ten Conflicts
The Kashmir elections have been completed. Foreign observers agreed the elections were free and fair. Rebel violence attempting to stop the elections left at least 830 dead. The party that had dominated Kashmir politics for half a century was voted out and replaced by a coalition that is keen on peace talks with separatists and rebels.
Elections in Pakistan, to elect a parliament that will be mainly for advising and criticizing self-appointed dictator Musharraf. Islamic fundamentalist parties increased their strength, but the traditional major parties still dominate. There were 7.054 candidates running for 342 National Assembly seats. There are 72 political parties.
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Bombay Brasserie
Houston, TX 77005
- Mon-Fri 11am-2:30pm, Sat-Sun 11am-3pm, Mon-Thu, Sun 5:30pm-10pm, Fri-Sat 5:30pm-10:30pm
- $$
- Catering, Delivery, Patio/Sidewalk Dining, Private Party, Takeout
- Brunch, Dinner, Lunch
- Free Parking
- Reservations Accepted, Reservations Accepted, Reservations Recommended
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Cooking & Recipes
Fava beans - known as broad beans in the UK and much of the rest of the world - are among the oldest of all cultivated crops. Along with chickpeas, lentils and peas, they are thought to have entered the eastern Mediterranean diet in about 6,000 BC, not long after the invention of agriculture. Originating in North Africa, fava beans provided sustenance to the pyramid builders in ancient Egypt; and, until the Spanish brought the "common bean," Phaseolus vulgaris back from the New World in the 15th century, favas were the only beans in the European diet.
Large, pale-green beans, and they have a meaty, starchy texture and a bold, earthy taste. They're versatile too:
- In Egypt and most Arab countries, favas are cooked, then puréed or mashed and served with olive oil, parsley, lemon juice, garlic, onions and cumin in a breakfast dish called ful medames.
- In many Latin American countries such as Peru, Colombia and Mexico, they are often either fried or dried, then salted to create a crunchy street-food snack.
- In the Sichuan province of China, favas are combined with soybeans and chili peppers in a fermented bean paste called doubanjiang.
When in season, fava beans can also be eaten while young and tender, either raw or lightly blanched, shelled or unshelled, and sometimes even in the pod! Fresh fava beans are often enjoyed in Greece, stewed with artichokes (note that the Greek word fáva actually refers to yellow split peas, which are also popular there, and not to fava/broad beans).
Fava beans are high in protein and fiber. They are also especially rich in vitamin A, vitamin C, potassium and iron. Favas even contain L-dopa, a precursor to dopamine, a neurotransmitter in the brain responsible for regulating mood and libido. However, please be aware that the ingestion of fava beans by some individuals with the hereditary glucose-6-phosphate dehydrogenase (G6PD) deficiency can cause a disorder known as favism, which can result in hemolytic anemia and kidney failure.
Cooking time: 60-120 minutes
Liquid per cup of legume: 3 cups
How to cook fava beans: Soak overnight. Drain water. If your fava beans were not already shelled, you should be able to slip the outer skins off after soaking by squeezing the beans between your fingers. Once favas are shelled, fill pot with fresh, cold water for cooking. Place on stove and bring to a boil in a pot with a lid. Once boiling, reduce to a simmer, tilting the lids slightly to allow steam to escape, and leave to cook for one to two hours, until tender or desired consistency.
Related Topics
Cooking With Legumes
Pinto Beans
Anasazi Beans
Black Beans
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