query: TNFAIP3 is a tumor suppressor in glioblastoma.

positive: Targeting A20 Decreases Glioma Stem Cell Survival and Tumor Growth Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioblastoma stem cells (GSCs). GSCs are regulated by molecular pathways distinct from the bulk tumor that may be useful therapeutic targets. We determined that A20 (TNFAIP3), a regulator of cell survival and the NF-kappaB pathway, is overexpressed in GSCs relative to non-stem glioblastoma cells at both the mRNA and protein levels. To determine the functional significance of A20 in GSCs, we targeted A20 expression with lentiviral-mediated delivery of short hairpin RNA (shRNA). Inhibiting A20 expression decreased GSC growth and survival through mechanisms associated with decreased cell-cycle progression and decreased phosphorylation of p65/RelA. Elevated levels of A20 in GSCs contributed to apoptotic resistance: GSCs were less susceptible to TNFalpha-induced cell death than matched non-stem glioma cells, but A20 knockdown sensitized GSCs to TNFalpha-mediated apoptosis. The decreased survival of GSCs upon A20 knockdown contributed to the reduced ability of these cells to self-renew in primary and secondary neurosphere formation assays. The tumorigenic potential of GSCs was decreased with A20 targeting, resulting in increased survival of mice bearing human glioma xenografts. In silico analysis of a glioma patient genomic database indicates that A20 overexpression and amplification is inversely correlated with survival. Together these data indicate that A20 contributes to glioma maintenance through effects on the glioma stem cell subpopulation. Although inactivating mutations in A20 in lymphoma suggest A20 can act as a tumor suppressor, similar point mutations have not been identified through glioma genomic sequencing: in fact, our data suggest A20 may function as a tumor enhancer in glioma through promotion of GSC survival. A20 anticancer therapies should therefore be viewed with caution as effects will likely differ depending on the tumor type.

negative: Development of a novel mouse glioma model using lentiviral vectors We report the development of a new method to induce glioblastoma multiforme in adult immunocompetent mice by injecting Cre-loxP–controlled lentiviral vectors expressing oncogenes. Cell type- or region-specific expression of activated forms of the oncoproteins Harvey-Ras and AKT in fewer than 60 glial fibrillary acidic protein–positive cells in the hippocampus, subventricular zone or cortex of mice heterozygous for the gene encoding the tumor suppressor Tp53 were tested. Mice developed glioblastoma multiforme when transduced either in the subventricular zone or the hippocampus. However, tumors were rarely detected when the mice were transduced in the cortex. Transplantation of brain tumor cells into naive recipient mouse brain resulted in the formation of glioblastoma multiforme–like tumors, which contained CD133+ cells, formed tumorspheres and could differentiate into neurons and astrocytes. We suggest that the use of Cre-loxP–controlled lentiviral vectors is a novel way to generate a mouse glioblastoma multiforme model in a region- and cell type-specific manner in adult mice.

negative: Dynamic epigenetic regulation of glioblastoma tumorigenicity through LSD1 modulation of MYC expression. The available evidence suggests that the lethality of glioblastoma is driven by small subpopulations of cells that self-renew and exhibit tumorigenicity. It remains unclear whether tumorigenicity exists as a static property of a few cells or as a dynamically acquired property. We used tumor-sphere and xenograft formation as assays for tumorigenicity and examined subclones isolated from established and primary glioblastoma lines. Our results indicate that glioblastoma tumorigenicity is largely deterministic, yet the property can be acquired spontaneously at low frequencies. Further, these dynamic transitions are governed by epigenetic reprogramming through the lysine-specific demethylase 1 (LSD1). LSD depletion increases trimethylation of histone 3 lysine 4 at the avian myelocytomatosis viral oncogene homolog (MYC) locus, which elevates MYC expression. MYC, in turn, regulates oligodendrocyte lineage transcription factor 2 (OLIG2), SRY (sex determining region Y)-box 2 (SOX2), and POU class 3 homeobox 2 (POU3F2), a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Our model suggests epigenetic regulation of key transcription factors governs transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development.

negative: Epidermal growth factor receptor expression identifies functionally and molecularly distinct tumor-initiating cells in human glioblastoma multiforme and is required for gliomagenesis. Epidermal growth factor receptor (EGFR) is a known diagnostic and, although controversial, prognostic marker of human glioblastoma multiforme (GBM). However, its functional role and biological significance in GBM remain elusive. Here, we show that multiple GBM cell subpopulations could be purified from the specimens of patients with GBM and from cancer stem cell (CSC) lines based on the expression of EGFR and of other putative CSC markers. All these subpopulations are molecularly and functionally distinct, are tumorigenic, and need to express EGFR to promote experimental tumorigenesis. Among them, EGFR-expressing tumor-initiating cells (TIC) display the most malignant functional and molecular phenotype. Accordingly, modulation of EGFR expression by gain-of-function and loss-of-function strategies in GBM CSC lines enhances and reduces their tumorigenic ability, respectively, suggesting that EGFR plays a fundamental role in gliomagenesis. These findings open up the possibility of new therapeutically relevant scenarios, as the presence of functionally heterogeneous EGFR(pos) and EGFR(neg) TIC subpopulations within the same tumor might affect clinical response to treatment.

negative: Targeting metabolic remodeling in glioblastoma multiforme. A key aberrant biological difference between tumor cells and normal differentiated cells is altered metabolism, whereby cancer cells acquire a number of stable genetic and epigenetic alterations to retain proliferation, survive under unfavorable microenvironments and invade into surrounding tissues. A classic biochemical adaptation is the metabolic shift to aerobic glycolysis rather than mitochondrial oxidative phosphorylation, regardless of oxygen availability, a phenomenon termed the "Warburg Effect". Aerobic glycolysis, characterized by high glucose uptake, low oxygen consumption and elevated production of lactate, is associated with a survival advantage as well as the generation of substrates such as fatty acids, amino acids and nucleotides necessary in rapidly proliferating cells. This review discusses the role of key metabolic enzymes and their association with aerobic glycolysis in Glioblastoma Multiforme (GBM), an aggressive, highly glycolytic and deadly brain tumor. Targeting key metabolic enzymes involved in modulating the "Warburg Effect" may provide a novel therapeutic approach either singularly or in combination with existing therapies in GBMs.

negative: Ca2+-permeable AMPA receptors regulate growth of human glioblastoma via Akt activation. Evidence has been accumulated that glioblastoma cells release and exploit glutamate for proliferation and migration by autocrine or paracrine loops through Ca2+-permeable AMPA-type glutamate receptors. Here, we show that Ca2+ signaling mediated by AMPA receptor regulates the growth and motility of glioblastoma cells via activation of Akt. Ca2+ supplied through Ca2+-permeable AMPA receptor phosphorylated Akt at Ser-473, thereby facilitating proliferation and mobility. A dominant-negative form of Akt inhibited cell proliferation and migration accelerated by overexpression of Ca2+-permeable AMPA receptor. In contrast, introduction of a constitutively active form of Akt rescued tumor cells from apoptosis induced by the conversion of Ca2+-permeable AMPA receptor to Ca2+-impermeable receptors by the delivery of GluR2 cDNA. Therefore, Akt functions as downstream effectors for Ca2+-signaling mediated by AMPA receptor in glioblastoma cells. The activation of the glutamate-AMPA receptor-Akt pathway may contribute to the high degree of anaplasia and invasive growth of human glioblastoma. This novel pathway might give an alternative therapeutic target.

negative: Experimental approaches for the treatment of malignant gliomas. Malignant gliomas, which include glioblastomas and anaplastic astrocytomas, are the most common primary tumors of the brain. Over the past 30 years, the standard treatment for these tumors has evolved to include maximal safe surgical resection, radiation therapy and temozolomide chemotherapy. While the median survival of patients with glioblastomas has improved from 6 months to 14.6 months, these tumors continue to be lethal for the vast majority of patients. There has, however, been recent substantial progress in our mechanistic understanding of tumor development and growth. The translation of these genetic, epigenetic and biochemical findings into therapies that have been tested in clinical trials is the subject of this review.

negative: MEG3 imprinted gene contribution in tumorigenesis. Maternally expressed gene 3 (MEG3) is a maternally expressed imprinted gene representing a large noncoding RNA in which microRNAs (miRNAs) and small nucleolar RNAs are also hosted. It is capable of interacting with cyclic AMP, p53, murine double minute 2 (MDM2) and growth differentiation factor 15 (GDF15) playing a role in cell proliferation control. MEG3 expression is under epigenetic control, and aberrant CpG methylation has been observed in several types of cancer. Moreover, gene copy number loss has been reported as additional mechanism associated with tumorigenesis. MEG3 deletion seems to upregulate the paternally expressed genes and on the other hand downregulate the expression of downstream maternally expressed genes and tumor suppressor miRNAs, although there are conflicting data on the topic. MEG3 could represent a tumor suppressor gene located in chromosome 14q32 and its association with tumorigenesis is growing every day.

negative: Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma. PURPOSE Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

negative: IL-17 promotes tumor development through the induction of tumor promoting microenvironments at tumor sites and myeloid-derived suppressor cells. The role of immune responses in tumor development is a central issue for tumor biology and immunology. IL-17 is an important cytokine for inflammatory and autoimmune diseases. Although IL-17-producing cells are detected in cancer patients and tumor-bearing mice, the role of IL-17 in tumor development is controversial, and mechanisms remain to be fully elucidated. In the current study, we found that the development of tumors was inhibited in IL-17R-deficient mice. A defect in IFN-gammaR increased tumor growth, whereas tumor growth was inhibited in mice that were deficient in both IL-17R and IFN-gammaR compared with wild-type animals. Further experiments showed that neutralization of IL-17 by Abs inhibited tumor growth in wild-type mice, whereas systemic administration of IL-17 promoted tumor growth. The IL-17R deficiency increased CD8 T cell infiltration, whereas it reduced the infiltration of myeloid-derived suppressor cells (MDSCs) in tumors. In contrast, administration of IL-17 inhibited CD8 T cell infiltration and increased MDSCs in tumors. Further analysis indicated that IL-17 was required for the development and tumor-promoting activity of MDSCs in tumor-bearing mice. These data demonstrate that IL-17-mediated responses promote tumor development through the induction of tumor-promoting microenvironments at tumor sites. IL-17-mediated regulation of MDSCs is a primary mechanism for its tumor-promoting effects. The study provides novel insights into the role of IL-17 in tumor development and has major implications for targeting IL-17 in treatment of tumors.

negative: Chromosome 7 gain and DNA hypermethylation at the HOXA10 locus are associated with expression of a stem cell related HOX-signature in glioblastoma BACKGROUND HOX genes are a family of developmental genes that are expressed neither in the developing forebrain nor in the normal brain. Aberrant expression of a HOX-gene dominated stem-cell signature in glioblastoma has been linked with increased resistance to chemo-radiotherapy and sustained proliferation of glioma initiating cells. Here we describe the epigenetic and genetic alterations and their interactions associated with the expression of this signature in glioblastoma. RESULTS We observe prominent hypermethylation of the HOXA locus 7p15.2 in glioblastoma in contrast to non-tumoral brain. Hypermethylation is associated with a gain of chromosome 7, a hallmark of glioblastoma, and may compensate for tumor-driven enhanced gene dosage as a rescue mechanism by preventing undue gene expression. We identify the CpG island of the HOXA10 alternative promoter that appears to escape hypermethylation in the HOX-high glioblastoma. An additive effect of gene copy gain at 7p15.2 and DNA methylation at key regulatory CpGs in HOXA10 is significantly associated with HOX-signature expression. Additionally, we show concordance between methylation status and presence of active or inactive chromatin marks in glioblastoma-derived spheres that are HOX-high or HOX-low, respectively. CONCLUSIONS Based on these findings, we propose co-evolution and interaction between gene copy gain, associated with a gain of chromosome 7, and additional epigenetic alterations as key mechanisms triggering a coordinated, but inappropriate, HOX transcriptional program in glioblastoma.

negative: Metabolic reprogramming of stromal fibroblasts through p62-mTORC1 signaling promotes inflammation and tumorigenesis. The tumor microenvironment plays a critical role in cancer progression, but the precise mechanisms by which stromal cells influence the epithelium are poorly understood. Here we show that p62 levels were reduced in the stroma of several tumors and that its loss in the tumor microenvironment or stromal fibroblasts resulted in increased tumorigenesis of epithelial prostate cancer cells. The mechanism involves the regulation of cellular redox through an mTORC1/c-Myc pathway of stromal glucose and amino acid metabolism, resulting in increased stromal IL-6 production, which is required for tumor promotion in the epithelial compartment. Thus, p62 is an anti-inflammatory tumor suppressor that acts through the modulation of metabolism in the tumor stroma.

negative: An Extensive MicroRNA-Mediated Network of RNA-RNA Interactions Regulates Established Oncogenic Pathways in Glioblastoma By analyzing gene expression data in glioblastoma in combination with matched microRNA profiles, we have uncovered a posttranscriptional regulation layer of surprising magnitude, comprising more than 248,000 microRNA (miR)-mediated interactions. These include ∼7,000 genes whose transcripts act as miR "sponges" and 148 genes that act through alternative, nonsponge interactions. Biochemical analyses in cell lines confirmed that this network regulates established drivers of tumor initiation and subtype implementation, including PTEN, PDGFRA, RB1, VEGFA, STAT3, and RUNX1, suggesting that these interactions mediate crosstalk between canonical oncogenic pathways. siRNA silencing of 13 miR-mediated PTEN regulators, whose locus deletions are predictive of PTEN expression variability, was sufficient to downregulate PTEN in a 3'UTR-dependent manner and to increase tumor cell growth rates. Thus, miR-mediated interactions provide a mechanistic, experimentally validated rationale for the loss of PTEN expression in a large number of glioma samples with an intact PTEN locus.

negative: Molecular characterization of long-term survivors of glioblastoma using genome- and transcriptome-wide profiling. The prognosis of glioblastoma, the most malignant type of glioma, is still poor, with only a minority of patients showing long-term survival of more than three years after diagnosis. To elucidate the molecular aberrations in glioblastomas of long-term survivors, we performed genome- and/or transcriptome-wide molecular profiling of glioblastoma samples from 94 patients, including 28 long-term survivors with >36 months overall survival (OS), 20 short-term survivors with <12 months OS and 46 patients with intermediate OS. Integrative bioinformatic analyses were used to characterize molecular aberrations in the distinct survival groups considering established molecular markers such as isocitrate dehydrogenase 1 or 2 (IDH1/2) mutations, and O(6) -methylguanine DNA methyltransferase (MGMT) promoter methylation. Patients with long-term survival were younger and more often had IDH1/2-mutant and MGMT-methylated tumors. Gene expression profiling revealed over-representation of a distinct (proneural-like) expression signature in long-term survivors that was linked to IDH1/2 mutation. However, IDH1/2-wildtype glioblastomas from long-term survivors did not show distinct gene expression profiles and included proneural, classical and mesenchymal glioblastoma subtypes. Genomic imbalances also differed between IDH1/2-mutant and IDH1/2-wildtype tumors, but not between survival groups of IDH1/2-wildtype patients. Thus, our data support an important role for MGMT promoter methylation and IDH1/2 mutation in glioblastoma long-term survival and corroborate the association of IDH1/2 mutation with distinct genomic and transcriptional profiles. Importantly, however, IDH1/2-wildtype glioblastomas in our cohort of long-term survivors lacked distinctive DNA copy number changes and gene expression signatures, indicating that other factors might have been responsible for long survival in this particular subgroup of patients.

negative: Integrated molecular genetic profiling of pediatric high-grade gliomas reveals key differences with the adult disease. PURPOSE To define copy number alterations and gene expression signatures underlying pediatric high-grade glioma (HGG). PATIENTS AND METHODS We conducted a high-resolution analysis of genomic imbalances in 78 de novo pediatric HGGs, including seven diffuse intrinsic pontine gliomas, and 10 HGGs arising in children who received cranial irradiation for a previous cancer using single nucleotide polymorphism microarray analysis. Gene expression was analyzed with gene expression microarrays for 53 tumors. Results were compared with publicly available data from adult tumors. RESULTS Significant differences in copy number alterations distinguish childhood and adult glioblastoma. PDGFRA was the predominant target of focal amplification in childhood HGG, including diffuse intrinsic pontine gliomas, and gene expression analyses supported an important role for deregulated PDGFRalpha signaling in pediatric HGG. No IDH1 hotspot mutations were found in pediatric tumors, highlighting molecular differences with adult secondary glioblastoma. Pediatric and adult glioblastomas were clearly distinguished by frequent gain of chromosome 1q (30% v 9%, respectively) and lower frequency of chromosome 7 gain (13% v 74%, respectively) and 10q loss (35% v 80%, respectively). PDGFRA amplification and 1q gain occurred at significantly higher frequency in irradiation-induced tumors, suggesting that these are initiating events in childhood gliomagenesis. A subset of pediatric HGGs showed minimal copy number changes. CONCLUSION Integrated molecular profiling showed substantial differences in the molecular features underlying pediatric and adult HGG, indicating that findings in adult tumors cannot be simply extrapolated to younger patients. PDGFRalpha may be a useful target for pediatric HGG, including diffuse pontine gliomas.

negative: The Somatic Genomic Landscape of Glioblastoma We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.

negative: Prostaglandin E2 promotes intestinal tumor growth via DNA methylation Although aberrant DNA methylation is considered to be one of the key ways by which tumor-suppressor and DNA-repair genes are silenced during tumor initiation and progression, the mechanisms underlying DNA methylation alterations in cancer remain unclear. Here we show that prostaglandin E(2) (PGE(2)) silences certain tumor-suppressor and DNA-repair genes through DNA methylation to promote tumor growth. These findings uncover a previously unrecognized role for PGE(2) in the promotion of tumor progression.

negative: Deleted in liver cancer-1 (DLC-1): a tumor suppressor not just for liver. Deleted in liver cancer 1 (DLC-1), as its name implied, was originally isolated as a potential tumor suppressor gene often deleted in hepatocellular carcinoma. Further studies have indicated that down-expression of DLC-1 either by genomic deletion or DNA methylation is associated with a variety of cancer types including lung, breast, prostate, kidney, colon, uterus, ovary, and stomach. Re-expression of DLC-1 in cancer cells regulates the structure of actin cytoskeleton and focal adhesions and significantly inhibits cell growth, supporting its role as a tumor suppressor. This tumor suppressive function relies on DLC-1's RhoGTPase activating protein (RhoGAP) activity and steroidogenic acute regulatory (StAR)-related lipid transfer (START) domain, as well as its focal adhesion localization, which is recruited by the Src Homology 2 (SH2) domains of tensins in a phosphotyrosine-independent fashion. Therefore, the expression and subcellular localization of DLC-1 could be a useful molecular marker for cancer prognosis, whereas DLC-1 and its downstream signaling molecules might be therapeutic targets for the treatment of cancer.

negative: Inhibition of LSD1 sensitizes glioblastoma cells to histone deacetylase inhibitors. Glioblastoma multiforme (GBM) is a particularly aggressive brain tumor and remains a clinically devastating disease. Despite innovative therapies for the treatment of GBM, there has been no significant increase in patient survival over the past decade. Enzymes that control epigenetic alterations are of considerable interest as targets for cancer therapy because of their critical roles in cellular processes that lead to oncogenesis. Several inhibitors of histone deacetylases (HDACs) have been developed and tested in GBM with moderate success. We found that treatment of GBM cells with HDAC inhibitors caused the accumulation of histone methylation, a modification removed by the lysine specific demethylase 1 (LSD1). This led us to examine the effects of simultaneously inhibiting HDACs and LSD1 as a potential combination therapy. We evaluated induction of apoptosis in GBM cell lines after combined inhibition of LSD1 and HDACs. LSD1 was inhibited by targeted short hairpin RNA or pharmacological means and inhibition of HDACs was achieved by treatment with either vorinostat or PCI-24781. Caspase-dependent apoptosis was significantly increased (>2-fold) in LSD1-knockdown GBM cells treated with HDAC inhibitors. Moreover, pharmacologically inhibiting LSD1 with the monoamine oxidase inhibitor tranylcypromine, in combination with HDAC inhibitors, led to synergistic apoptotic cell death in GBM cells; this did not occur in normal human astrocytes. Taken together, these results indicate that LSD1 and HDACs cooperate to regulate key pathways of cell death in GBM cell lines but not in normal counterparts, and they validate the combined use of LSD1 and HDAC inhibitors as a therapeutic approach for GBM.

negative: Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. The Cancer Genome Atlas Network recently cataloged recurrent genomic abnormalities in glioblastoma multiforme (GBM). We describe a robust gene expression-based molecular classification of GBM into Proneural, Neural, Classical, and Mesenchymal subtypes and integrate multidimensional genomic data to establish patterns of somatic mutations and DNA copy number. Aberrations and gene expression of EGFR, NF1, and PDGFRA/IDH1 each define the Classical, Mesenchymal, and Proneural subtypes, respectively. Gene signatures of normal brain cell types show a strong relationship between subtypes and different neural lineages. Additionally, response to aggressive therapy differs by subtype, with the greatest benefit in the Classical subtype and no benefit in the Proneural subtype. We provide a framework that unifies transcriptomic and genomic dimensions for GBM molecular stratification with important implications for future studies.

negative: Phosphoinositide 3-kinase signalling pathways in tumor progression, invasion and angiogenesis. AIMS AND BACKGROUND The PI3 kinase signalling pathway is now accepted as being at least as important as the ras-MAP kinase pathway in cell survival and proliferation, and hence its potential role in cancer is of great interest. The purpose of this review is briefly to examine evidence for an involvement of PI3K in human cancers, discuss the mechanisms by which its activation promotes tumor progression, and consider its utility as a novel target for anticancer therapy. METHODS AND STUDY DESIGN A Medline review of recent literature concerning the role of PI3 kinase in tumor progression--mechanisms of action and clinical implications. RESULTS Evidence is presented that misregulation of the PI3 kinase pathway is a feature of many common cancers, either by loss of the suppressor protein PTEN, or by constitutive activation of PI3 kinase isoforms or downstream elements such as AKT and mTOR. This activation potentiates not only cell survival and proliferation, but also cytoskeletal deformability and motility; key elements in tumor invasion. In addition the PI3K pathway is implicated in many aspects of angiogenesis, including upregulation of angiogenic cytokines due to tumor hypoxia or oncogene activation and endothelial cell responses to them. These cytokines signal though receptors such as VEGF-R, FGF-R and Tie-2 and potentiate processes essential for neoangiogenesis including cell proliferation, migration, differentiation into tubules and "invasion" of these capillary sprouts into extracellular matrix (ECM). CONCLUSIONS A more complete understanding of the role of the PI3 kinase pathway in cancer will lead the way to the development of more potent and selective inhibitors which should be a useful adjunct to conventional therapies, potentially interfering with tumor progression at several pivotal points; in particular cell survival, invasion and angiogenesis.

negative: Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. BACKGROUND Glioblastoma, the most common primary brain tumor in adults, is usually rapidly fatal. The current standard of care for newly diagnosed glioblastoma is surgical resection to the extent feasible, followed by adjuvant radiotherapy. In this trial we compared radiotherapy alone with radiotherapy plus temozolomide, given concomitantly with and after radiotherapy, in terms of efficacy and safety. METHODS Patients with newly diagnosed, histologically confirmed glioblastoma were randomly assigned to receive radiotherapy alone (fractionated focal irradiation in daily fractions of 2 Gy given 5 days per week for 6 weeks, for a total of 60 Gy) or radiotherapy plus continuous daily temozolomide (75 mg per square meter of body-surface area per day, 7 days per week from the first to the last day of radiotherapy), followed by six cycles of adjuvant temozolomide (150 to 200 mg per square meter for 5 days during each 28-day cycle). The primary end point was overall survival. RESULTS A total of 573 patients from 85 centers underwent randomization. The median age was 56 years, and 84 percent of patients had undergone debulking surgery. At a median follow-up of 28 months, the median survival was 14.6 months with radiotherapy plus temozolomide and 12.1 months with radiotherapy alone. The unadjusted hazard ratio for death in the radiotherapy-plus-temozolomide group was 0.63 (95 percent confidence interval, 0.52 to 0.75; P<0.001 by the log-rank test). The two-year survival rate was 26.5 percent with radiotherapy plus temozolomide and 10.4 percent with radiotherapy alone. Concomitant treatment with radiotherapy plus temozolomide resulted in grade 3 or 4 hematologic toxic effects in 7 percent of patients. CONCLUSIONS The addition of temozolomide to radiotherapy for newly diagnosed glioblastoma resulted in a clinically meaningful and statistically significant survival benefit with minimal additional toxicity.

negative: A Sleeping Beauty mutagenesis screen reveals a tumor suppressor role for Ncoa2/Src-2 in liver cancer. The Sleeping Beauty (SB) transposon mutagenesis system is a powerful tool that facilitates the discovery of mutations that accelerate tumorigenesis. In this study, we sought to identify mutations that cooperate with MYC, one of the most commonly dysregulated genes in human malignancy. We performed a forward genetic screen with a mouse model of MYC-induced liver cancer using SB-mediated mutagenesis. We sequenced insertions in 63 liver tumor nodules and identified at least 16 genes/loci that contribute to accelerated tumor development. RNAi-mediated knockdown in a liver progenitor cell line further validate three of these genes, Ncoa2/Src-2, Zfx, and Dtnb, as tumor suppressors in liver cancer. Moreover, deletion of Ncoa2/Src-2 in mice predisposes to diethylnitrosamine-induced liver tumorigenesis. These findings reveal genes and pathways that functionally restrain MYC-mediated liver tumorigenesis and therefore may provide targets for cancer therapy.

negative: Expression profile of genes modulated by Aloe emodin in human U87 glioblastoma cells. Glioblastoma, the most aggressive and malignant form of glioma, appears to be resistant to various chemotherapeutic agents. Hence, approaches have been intensively investigated to targeti specific molecular pathways involved in glioblastoma development and progression. Aloe emodin is believed to modulate the expression of several genes in cancer cells. We aimed to understand the molecular mechanisms underlying the therapeutic effect of Aloe emodin on gene expression profiles in the human U87 glioblastoma cell line utilizing microarray technology. The gene expression analysis revealed that a total of 8,226 gene alterations out of 28,869 genes were detected after treatment with 58.6 μg/ml for 24 hours. Out of this total, 34 genes demonstrated statistically significant change (p<0.05) ranging from 1.07 to 1.87 fold. The results revealed that 22 genes were up-regulated and 12 genes were down-regulated in response to Aloe emodin treatment. These genes were then grouped into several clusters based on their biological functions, revealing induction of expression of genes involved in apoptosis (programmed cell death) and tissue remodelling in U87 cells (p<0.01). Several genes with significant changes of the expression level e.g. SHARPIN, BCAP31, FIS1, RAC1 and TGM2 from the apoptotic cluster were confirmed by quantitative real-time PCR (qRT-PCR). These results could serve as guidance for further studies in order to discover molecular targets for the cancer therapy based on Aloe emodin treatment.

negative: Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53 The inefficient clearance of dying cells can lead to abnormal immune responses, such as unresolved inflammation and autoimmune conditions. We show that tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, Death Domain1α (DD1α), which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α appears to function as an engulfment ligand or receptor that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages, unlike other typical scavenger receptors that recognize phosphatidylserine on the surface of dead cells. DD1α-deficient mice showed in vivo defects in clearing dying cells, which led to multiple organ damage indicative of immune dysfunction. p53-induced expression of DD1α thus prevents persistence of cell corpses and ensures efficient generation of precise immune responses.

negative: Different response of human glioma tumor-initiating cells to epidermal growth factor receptor kinase inhibitors. Because a subpopulation of cancer stem cells (tumor-initiating cells, TICs) is believed to be responsible for the development, progression, and recurrence of many tumors, we evaluated the in vitro sensitivity of human glioma TICs to epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) and possible molecular determinants for their effects. Cells isolated from seven glioblastomas (GBM 1-7) and grown using neural stem cell permissive conditions were characterized for in vivo tumorigenicity, expression of tumor stem cell markers (CD133, nestin), and multilineage differentiation properties, confirming that these cultures are enriched in TICs. TIC cultures were challenged with increasing concentrations of erlotinib and gefitinib, and their survival was evaluated after 1-4 days. In most cases, a time- and concentration-dependent cell death was observed, although GBM 2 was completely insensitive to both drugs, and GBM 7 was responsive only to the highest concentrations tested. Using a radioligand binding assay, we show that all GBM TICs express EGFR. Erlotinib and gefitinib inhibited EGFR and ERK1/2 phosphorylation/activation in all GBMs, irrespective of the antiproliferative response observed. However, under basal conditions GBM 2 showed a high Akt phosphorylation that was completely insensitive to both drugs, whereas GBM 7 was completely insensitive to gefitinib, and Akt inactivation occurred only for the highest erlotinib concentration tested, showing a precise relationship with the antiproliferative effects of the drug. Interestingly, in GBM 2, phosphatase and tensin homolog expression was significantly down-regulated, possibly accounting for the insensitivity to the drugs. In conclusion, glioma TICs are responsive to anti-EGFR drugs, but phosphatase and tensin homolog expression and Akt inhibition seem to be necessary for such effect.

negative: Reverse engineering of TLX oncogenic transcriptional networks identifies RUNX1 as tumor suppressor in T-ALL The TLX1 and TLX3 transcription factor oncogenes have a key role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). Here we used reverse engineering of global transcriptional networks to decipher the oncogenic regulatory circuit controlled by TLX1 and TLX3. This systems biology analysis defined T cell leukemia homeobox 1 (TLX1) and TLX3 as master regulators of an oncogenic transcriptional circuit governing T-ALL. Notably, a network structure analysis of this hierarchical network identified RUNX1 as a key mediator of the T-ALL induced by TLX1 and TLX3 and predicted a tumor-suppressor role for RUNX1 in T cell transformation. Consistent with these results, we identified recurrent somatic loss-of-function mutations in RUNX1 in human T-ALL. Overall, these results place TLX1 and TLX3 at the top of an oncogenic transcriptional network controlling leukemia development, show the power of network analyses to identify key elements in the regulatory circuits governing human cancer and identify RUNX1 as a tumor-suppressor gene in T-ALL.

negative: Epigenetic therapy activates type I interferon signaling in murine ovarian cancer to reduce immunosuppression and tumor burden. Ovarian cancer is the most lethal of all gynecological cancers, and there is an urgent unmet need to develop new therapies. Epithelial ovarian cancer (EOC) is characterized by an immune suppressive microenvironment, and response of ovarian cancers to immune therapies has thus far been disappointing. We now find, in a mouse model of EOC, that clinically relevant doses of DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi, respectively) reduce the immune suppressive microenvironment through type I IFN signaling and improve response to immune checkpoint therapy. These data indicate that the type I IFN response is required for effective in vivo antitumorigenic actions of the DNMTi 5-azacytidine (AZA). Through type I IFN signaling, AZA increases the numbers of CD45+ immune cells and the percentage of active CD8+ T and natural killer (NK) cells in the tumor microenvironment, while reducing tumor burden and extending survival. AZA also increases viral defense gene expression in both tumor and immune cells, and reduces the percentage of macrophages and myeloid-derived suppressor cells in the tumor microenvironment. The addition of an HDACi to AZA enhances the modulation of the immune microenvironment, specifically increasing T and NK cell activation and reducing macrophages over AZA treatment alone, while further increasing the survival of the mice. Finally, a triple combination of DNMTi/HDACi plus the immune checkpoint inhibitor α-PD-1 provides the best antitumor effect and longest overall survival, and may be an attractive candidate for future clinical trials in ovarian cancer.

negative: Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival Neutrophil apoptosis is a highly regulated process essential for inflammation resolution, the molecular mechanisms of which are only partially elucidated. In this study, we describe a survival pathway controlled by proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repairing of proliferating cells. We show that mature neutrophils, despite their inability to proliferate, express high levels of PCNA exclusively in their cytosol and constitutively associated with procaspases, presumably to prevent their activation. Notably, cytosolic PCNA abundance decreased during apoptosis, and increased during in vitro and in vivo exposure to the survival factor granulocyte colony-stimulating factor (G-CSF). Peptides derived from the cyclin-dependent kinase inhibitor p21, which compete with procaspases to bind PCNA, triggered neutrophil apoptosis thus demonstrating that specific modification of PCNA protein interactions affects neutrophil survival. Furthermore, PCNA overexpression rendered neutrophil-differentiated PLB985 myeloid cells significantly more resistant to TNF-related apoptosis-inducing ligand- or gliotoxin-induced apoptosis. Conversely, a decrease in PCNA expression after PCNA small interfering RNA transfection sensitized these cells to apoptosis. Finally, a mutation in the PCNA interdomain-connecting loop, the binding site for many partners, significantly decreased the PCNA-mediated antiapoptotic effect. These results identify PCNA as a regulator of neutrophil lifespan, thereby highlighting a novel target to potentially modulate pathological inflammation.

negative: IL-33, a recently identified interleukin-1 gene family member, is expressed in human adipocytes. Inflammation occurs in adipose tissue in obesity. We have examined whether IL-33, a recently identified IL-1 gene family member, and its associated receptors are expressed in human adipocytes. IL-33, IL-1RL1 and IL-1RAP gene expression was observed in human visceral white fat, in preadipocytes and in adipocytes (SGBS cells). Treatment with TNFalpha for 24h induced a 6-fold increase in IL-33 mRNA level in preadipocytes and adipocytes. Time-course studies with adipocytes showed that the increase in IL-33 mRNA with TNFalpha was maximal (>55-fold) at 12h. This response was markedly different to IL-1beta (peak mRNA increase at 2h; 5.4-fold) and 1L-18 (peak mRNA increase at 6h; >1500-fold). Exposure of adipocytes to hypoxia (1% O(2), 24h) did not alter IL-33 mRNA level; in preadipocytes, however, there was a 3-fold increase. Human adipocytes and preadipocytes express IL-33, but the various IL-1 family members exhibit major differences in responsiveness to TNFalpha.

negative: GSK-3-mediated phosphorylation enhances Maf-transforming activity. The Maf oncoproteins are b-Zip transcription factors of the AP-1 superfamily. They are involved in developmental, metabolic, and tumorigenic processes. Maf proteins are overexpressed in about 50% of human multiple myelomas. Here, we show that Maf-transforming activity is controlled by GSK-3-dependent phosphorylation and that phosphorylation by GSK-3 can increase the oncogenic activity of a protein. Using microarray analysis, we identify a gene-expression subprogram regulated by GSK-3-mediated Maf phosphorylation involved in extracellular matrix remodeling and relevant to cancer progression. We also demonstrate that GSK-3 triggers MafA sequential phosphorylation on residues S61, T57, T53, and S49, inducing its ubiquitination and degradation. Paradoxically, this phosphorylation increases MafA-transcriptional activity through the recruitment of the coactivator P/CAF. We further demonstrate that P/CAF protects MafA from ubiquitination and degradation, suggesting that, upon the release of the coactivator complex, MafA becomes polyubiquitinated and degraded to allow the response to terminate.

negative: NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations. Phosphatidylinositol-3-kinase (PI3K) pathway deregulation is a common event in human cancer, either through inactivation of the tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 or activating mutations of p110-alpha. These hotspot mutations result in oncogenic activity of the enzyme and contribute to therapeutic resistance to the anti-HER2 antibody trastuzumab. The PI3K pathway is, therefore, an attractive target for cancer therapy. We have studied NVP-BEZ235, a dual inhibitor of the PI3K and the downstream mammalian target of rapamycin (mTOR). NVP-BEZ235 inhibited the activation of the downstream effectors Akt, S6 ribosomal protein, and 4EBP1 in breast cancer cells. The antiproliferative activity of NVP-BEZ235 was superior to the allosteric selective mTOR complex inhibitor everolimus in a panel of 21 cancer cell lines of different origin and mutation status. The described Akt activation due to mTOR inhibition was prevented by higher doses of NVP-BEZ235. NVP-BEZ235 reversed the hyperactivation of the PI3K/mTOR pathway caused by the oncogenic mutations of p110-alpha, E545K, and H1047R, and inhibited the proliferation of HER2-amplified BT474 cells exogenously expressing these mutations that render them resistant to trastuzumab. In trastuzumab-resistant BT474 H1047R breast cancer xenografts, NVP-BEZ235 inhibited PI3K signaling and had potent antitumor activity. In treated animals, there was complete inhibition of PI3K signaling in the skin at pharmacologically active doses, suggesting that skin may serve as surrogate tissue for pharmacodynamic studies. In summary, NVP-BEZ235 inhibits the PI3K/mTOR axis and results in antiproliferative and antitumoral activity in cancer cells with both wild-type and mutated p110-alpha.

negative: Comparison of survival between cerebellar and supratentorial glioblastoma patients: surveillance, epidemiology, and end results (SEER) analysis. BACKGROUND Cerebellar glioblastoma multiforme (cGBM) is rare, and although there is a general belief that these tumors have a worse prognosis than supratentorial GBM (sGBM), few studies have been published to support this belief. OBJECTIVE To investigate the effect of cerebellar location on survival through a case-control design comparing overall survival time of cGBM and sGBM patients. METHODS The Surveillance, Epidemiology, and End Results (SEER) registry was used to identify 132 patients with cGBM (1973-2008). Each cGBM patient was matched with an sGBM patient from among 20,848 sGBM patients on the basis of age, extent of resection, decade of diagnosis, and radiation therapy using propensity score matching. RESULTS Within the cGBM, 37% were older than 65 years of age, 62% were men, and 87% were white. Most patients underwent surgery and radiation (74%), whereas only 26% underwent surgical resection only. The median survival time for the cGBM and sGBM matched cohort was 8 months; however, the survival distributions differed (log-rank P = .04). Survival time for cGBM vs sGBM at 2 years was 21.5% vs 8.0%, and 12.7% vs 5.3% at 3 years. Multivariate analysis of survival among cGBM patients showed that younger age (P < .0001) and having radiation therapy (P < .0001) were significantly associated with reduced hazard of mortality. Among all patients, multivariate analysis showed that tumor location (P = .03), age (P < .0001), tumor size (P = .009), radiation (P < .0001), and resection (P < .0001) were associated with survival time in the unmatched cohort. CONCLUSION Median survival time for cGBM and sGBM patients was 8 months, but cGBM patients had a survival time advantage as the study progressed. These findings suggest that cGBM patients should be treated as aggressively as sGBM patients with surgical resection and radiation therapy.


query: Proteins synthesized at the growth cone are ubiquitinated at a higher rate than proteins from the cell body.

positive: Coupled local translation and degradation regulate growth cone collapse Local translation mediates axonal responses to Semaphorin3A (Sema3A) and other guidance cues. However, only a subset of the axonal proteome is locally synthesized, whereas most proteins are trafficked from the soma. The reason why only specific proteins are locally synthesized is unknown. Here we show that local protein synthesis and degradation are linked events in growth cones. We find that growth cones exhibit high levels of ubiquitination and that local signalling pathways trigger the ubiquitination and degradation of RhoA, a mediator of Sema3A-induced growth cone collapse. Inhibition of RhoA degradation is sufficient to remove the protein-synthesis requirement for Sema3A-induced growth cone collapse. In addition to RhoA, we find that locally translated proteins are the main targets of the ubiquitin-proteasome system in growth cones. Thus, local protein degradation is a major feature of growth cones and creates a requirement for local translation to replenish proteins needed to maintain growth cone responses.

negative: Ubiquitinylation of the cytosolic domain of a type I membrane protein is not required to initiate its dislocation from the endoplasmic reticulum. Human cytomegalovirus US2 and US11 target newly synthesized class I major histocompatibility complex (MHC) heavy chains for rapid degradation by the proteasome through a process termed dislocation. The presence of US2 induces the formation of class I MHC heavy chain conjugates of increased molecular weight that are recognized by a conformation-specific monoclonal antibody, W6/32, suggesting that these class I MHC molecules retain their proper tertiary structure. These conjugates are properly folded glycosylated heavy chains modified by attachment of an estimated one, two, and three ubiquitin molecules. The folded ubiquitinated class I MHC heavy chains are not observed in control cells or in cells transfected with US11, suggesting that US2 targets class I MHC heavy chains for dislocation in a manner distinct from that used by US11. This is further supported by the fact that US2 and US11 show different requirements in terms of the conformation of the heavy chain molecule. Although ubiquitin conjugation may occur on the cytosolic tail of the class I MHC molecule, replacement of lysines in the cytosolic tail of heavy chains with arginine does not prevent their degradation by US2. In an in vitro system that recapitulates US2-mediated dislocation, heavy chains that lack these lysines still occur in an ubiquitin-modified form, but in the soluble (cytoplasmic) fraction. Such ubiquitin conjugation can only occur on the class I MHC lumenal domain and is likely to take place once class I MHC heavy chains have been discharged from the endoplasmic reticulum. We conclude that ubiquitinylation of class I MHC heavy chain is not required during the initial step of the US2-mediated dislocation reaction.

negative: Thioredoxin mediates oxidation-dependent phosphorylation of CRMP2 and growth cone collapse. Semaphorin3A (Sema3A) is a repulsive guidance molecule for axons, which acts by inducing growth cone collapse through phosphorylation of CRMP2 (collapsin response mediator protein 2). Here, we show a role for CRMP2 oxidation and thioredoxin (TRX) in the regulation of CRMP2 phosphorylation and growth cone collapse. Sema3A stimulation generated hydrogen peroxide (H2O2) through MICAL (molecule interacting with CasL) and oxidized CRMP2, enabling it to form a disulfide-linked homodimer through cysteine-504. Oxidized CRMP2 then formed a transient disulfide-linked complex with TRX, which stimulated CRMP2 phosphorylation by glycogen synthase kinase-3, leading to growth cone collapse. We also reconstituted oxidation-dependent phosphorylation of CRMP2 in vitro, using a limited set of purified proteins. Our results not only clarify the importance of H2O2 and CRMP2 oxidation in Sema3A-induced growth cone collapse but also indicate an unappreciated role for TRX in linking CRMP2 oxidation to phosphorylation.

negative: The Ulp1 SUMO isopeptidase distinct domains required for viability, nuclear envelope localization, and substrate specificity Protein modification by the ubiquitin-like SUMO protein contributes to many cellular regulatory mechanisms. In Saccharomyces cerevisiae, both sumoylating and desumoylating activities are essential for viability. Of its two known desumoylating enzymes, Ubl-specific protease (Ulp)1 and Ulp2/Smt4, Ulp1 is specifically required for cell cycle progression. A ∼200-residue segment, the Ulp domain (UD), is conserved among Ulps and includes a core cysteine protease domain that is even more widespread. Here we demonstrate that the Ulp1 UD by itself can support wild-type growth rates and in vitro can cleave SUMO from substrates. However, in cells expressing only the UD of Ulp1, many SUMO conjugates accumulate to high levels, indicating that the nonessential Ulp1 NH2-terminal domain is important for activity against a substantial fraction of sumoylated targets. The NH2-terminal domain also includes sequences necessary and sufficient to concentrate Ulp1 at nuclear envelope sites. Remarkably, NH2-terminally deleted Ulp1 variants are able, unlike full-length Ulp1, to suppress defects of cells lacking the divergent Ulp2 isopeptidase. Thus, the NH2-terminal regulatory domain of Ulp1 restricts Ulp1 activity toward certain sumoylated proteins while enabling the cleavage of others. These data define key functional elements of Ulp1 and strongly suggest that subcellular localization is a physiologically significant constraint on SUMO isopeptidase specificity.

negative: Vps22/EAP30 in ESCRT-II mediates endosomal sorting of growth factor and chemokine receptors destined for lysosomal degradation. The ubiquitin-binding protein Hrs and endosomal sorting complex required for transport (ESCRT)-I and ESCRT-III are involved in sorting endocytosed and ubiquitinated receptors to lysosomes for degradation and efficient termination of signaling. In this study, we have investigated the role of the ESCRT-II subunit Vps22/EAP30 in degradative protein sorting of ubiquitinated receptors. Vps22 transiently expressed in HeLa cells was detected in endosomes containing endocytosed epidermal growth factor receptors (EGFRs) as well as Hrs and ESCRT-I and ESCRT-III. Depletion of Vps22 by small interfering RNA, which was accompanied by decreased levels of other ESCRT-II subunits, greatly reduced degradation of EGFR and its ligand EGF as well as the chemokine receptor CXCR4. EGFR accumulated on the limiting membranes of early endosomes and aberrantly small multivesicular bodies in Vps22-depleted cells. Phosphorylation and nuclear translocation of extracellular-signal-regulated kinase1/2 downstream of the EGF-activated receptor were sustained by depletion of Hrs or the ESCRT-I subunit Tsg101. In contrast, this was not the case when Vps22 was depleted. These results indicate an important role for Vps22 in ligand-induced EGFR and CXCR4 turnover and suggest that termination of EGF signaling occurs prior to ESCRT-II engagement.

negative: Evolution and function of ubiquitin-like protein-conjugation systems Ubiquitin functions by covalently modifying other proteins. In the past few years, a surprising number of other proteins have been identified that, despite often being only slightly similar to ubiquitin, can also be attached to proteins. Newly discovered parallels between the activation of ubiquitin and the biosynthesis of certain enzyme cofactors now hint at the possible evolutionary origins of the ubiquitin system.

negative: Maternal Protein Restriction Affects Postnatal Growth and the Expression of Key Proteins Involved in Lifespan Regulation in Mice We previously reported that maternal protein restriction in rodents influenced the rate of growth in early life and ultimately affected longevity. Low birth weight caused by maternal protein restriction followed by catch-up growth (recuperated animals) was associated with shortened lifespan whereas protein restriction and slow growth during lactation (postnatal low protein: PLP animals) increased lifespan. We aim to explore the mechanistic basis by which these differences arise. Here we investigated effects of maternal diet on organ growth, metabolic parameters and the expression of insulin/IGF1 signalling proteins and Sirt1 in muscle of male mice at weaning. PLP mice which experienced protein restriction during lactation had lower fasting glucose (P = 0.038) and insulin levels (P = 0.046) suggesting improved insulin sensitivity. PLP mice had higher relative weights (adjusted by body weight) of brain (P = 0.0002) and thymus (P = 0.031) compared to controls suggesting that enhanced functional capacity of these two tissues is beneficial to longevity. They also had increased expression of insulin receptor substrate 1 (P = 0.021) and protein kinase C zeta (P = 0.046). Recuperated animals expressed decreased levels of many insulin signalling proteins including PI3 kinase subunits p85alpha (P = 0.018), p110beta (P = 0.048) and protein kinase C zeta (P = 0.006) which may predispose these animals to insulin resistance. Sirt1 protein expression was reduced in recuperated offspring. These observations suggest that maternal protein restriction can affect major metabolic pathways implicated in regulation of lifespan at a young age which may explain the impact of maternal diet on longevity.

negative: Function and regulation of SUMO proteases Covalent attachment of small ubiquitin-like modifier (SUMO) to proteins is highly dynamic, and both SUMO–protein conjugation and cleavage can be regulated. Protein desumoylation is carried out by SUMO proteases, which control cellular mechanisms ranging from transcription and cell division to ribosome biogenesis. Recent advances include the discovery of two novel classes of SUMO proteases, insights regarding SUMO protease specificity, and revelations of previously unappreciated SUMO protease functions in several key cellular pathways. These developments, together with new connections between SUMO proteases and the recently discovered SUMO-targeted ubiquitin ligases (STUbLs), make this an exciting period to study these enzymes.

negative: Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization The p53 tumour suppressor is a short-lived protein that is maintained at low levels in normal cells by Mdm2-mediated ubiquitination and subsequent proteolysis. Stabilization of p53 is crucial for its tumour suppressor function. However, the precise mechanism by which ubiquitinated p53 levels are regulated in vivo is not completely understood. By mass spectrometry of affinity-purified p53-associated factors, we have identified herpesvirus-associated ubiquitin-specific protease (HAUSP) as a novel p53-interacting protein. HAUSP strongly stabilizes p53 even in the presence of excess Mdm2, and also induces p53-dependent cell growth repression and apoptosis. Significantly, HAUSP has an intrinsic enzymatic activity that specifically deubiquitinates p53 both in vitro and in vivo. In contrast, expression of a catalytically inactive point mutant of HAUSP in cells increases the levels of p53 ubiquitination and destabilizes p53. These findings reveal an important mechanism by which p53 can be stabilized by direct deubiquitination and also imply that HAUSP might function as a tumour suppressor in vivo through the stabilization of p53.

negative: The role of protein clearance mechanisms in organismal ageing and age-related diseases. The ability to maintain a functional proteome, or proteostasis, declines during the ageing process. Damaged and misfolded proteins accumulate with age, impairing cell function and tissue homeostasis. The accumulation of damaged proteins contributes to multiple age-related diseases such as Alzheimer's, Parkinson's or Huntington's disease. Damaged proteins are degraded by the ubiquitin-proteasome system or through autophagy-lysosome, key components of the proteostasis network. Modulation of either proteasome activity or autophagic-lysosomal potential extends lifespan and protects organisms from symptoms associated with proteostasis disorders, suggesting that protein clearance mechanisms are directly linked to ageing and age-associated diseases.

negative: FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation FBW7 (F-box and WD repeat domain-containing 7) is the substrate recognition component of an evolutionary conserved SCF (complex of SKP1, CUL1 and F-box protein)-type ubiquitin ligase. SCFFBW7 degrades several proto-oncogenes that function in cellular growth and division pathways, including MYC, cyclin E, Notch and JUN. FBW7 is also a tumour suppressor, the regulatory network of which is perturbed in many human malignancies. Numerous cancer-associated mutations in FBW7 and its substrates have been identified, and loss of FBW7 function causes chromosomal instability and tumorigenesis. This Review focuses on structural and functional aspects of FBW7 and its role in the development of cancer.

negative: Attachment of the ubiquitin-related protein Urm1p to the antioxidant protein Ahp1p. Urm1p is a ubiquitin-related protein that serves as a posttranslational modification of other proteins. Urm1p conjugation has been implicated in the budding process and in nutrient sensing. Here, we have identified the first in vivo target for the urmylation pathway as the antioxidant protein Ahp1p. The attachment of Urm1p to Ahp1p requires the E1 for the urmylation pathway, Uba4p. Loss of the urmylation pathway components results in sensitivity to a thiol-specific oxidant, as does loss of Ahp1p, implying that urmylation has a role in an oxidative-stress response. Moreover, treatment of cells with thiol-specific oxidants affects the abundance of Ahp1p-Urm1p conjugates. These results suggest that the conjugation of Urm1p to Ahp1p could regulate the function of Ahp1p in antioxidant stress response in Saccharomyces cerevisiae.

negative: The anaphase promoting complex/cyclosome: a machine designed to destroy The anaphase promoting complex/cyclosome (APC/C) is a ubiquitin ligase that has essential functions in and outside the eukaryotic cell cycle. It is the most complex molecular machine that is known to catalyse ubiquitylation reactions, and it contains more than a dozen subunits that assemble into a large 1.5-MDa complex. Recent discoveries have revealed an unexpected multitude of mechanisms that control APC/C activity, and have provided a first insight into how this unusual ubiquitin ligase recognizes its substrates.

negative: A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth Cell growth, an increase in mass and size, is a highly regulated cellular event. The Akt/mTOR (mammalian target of rapamycin) signalling pathway has a central role in the control of protein synthesis and thus the growth of cells, tissues and organisms. A striking example of a physiological context requiring rapid cell growth is tissue repair in response to injury. Here we show that keratin 17, an intermediate filament protein rapidly induced in wounded stratified epithelia, regulates cell growth through binding to the adaptor protein 14-3-3σ. Mouse skin keratinocytes lacking keratin 17 (ref. 4) show depressed protein translation and are of smaller size, correlating with decreased Akt/mTOR signalling activity. Other signalling kinases have normal activity, pointing to the specificity of this defect. Two amino acid residues located in the amino-terminal head domain of keratin 17 are required for the serum-dependent relocalization of 14-3-3σ from the nucleus to the cytoplasm, and for the concomitant stimulation of mTOR activity and cell growth. These findings reveal a new and unexpected role for the intermediate filament cytoskeleton in influencing cell growth and size by regulating protein synthesis.

negative: Ubiquitin-dependent intramembrane rhomboid protease promotes ERAD of membrane proteins. The ER-associated degradation (ERAD) pathway serves as an important cellular safeguard by directing incorrectly folded and unassembled proteins from the ER to the proteasome. Still, however, little is known about the components mediating ERAD of membrane proteins. Here we show that the evolutionary conserved rhomboid family protein RHBDL4 is a ubiquitin-dependent ER-resident intramembrane protease that is upregulated upon ER stress. RHBDL4 cleaves single-spanning and polytopic membrane proteins with unstable transmembrane helices, leading to their degradation by the canonical ERAD machinery. RHBDL4 specifically binds the AAA+-ATPase p97, suggesting that proteolytic processing and dislocation into the cytosol are functionally linked. The phylogenetic relationship between rhomboids and the ERAD factor derlin suggests that substrates for intramembrane proteolysis and protein dislocation are recruited by a shared mechanism.

negative: Serum amyloid A: An acute-phase protein involved in tumour pathogenesis The synthesis of acute-phase protein serum amyloid A (SAA) is largely regulated by inflammation- associated cytokines and a high concentration of circulating SAA may represent an ideal marker for acute and chronic inflammatory diseases. However, SAA is also synthesized in extrahepatic tissues, e.g. human carcinoma metastases and cancer cell lines. An increasing body of in vitro data supports the concept of involvement of SAA in carcinogenesis and neoplastic diseases. Accumulating evidence suggests that SAA might be included in a group of biomarkers to detect a pattern of physiological events that reflect the growth of malignancy and host response. This review is meant to provide a broad overview of the many ways that SAA could contribute to tumour development, and accelerate tumour progression and metastasis, and to gain a better understanding of this acute-phase reactant as a possible link between chronic inflammation and neoplasia.

negative: Breaking the chains: structure and function of the deubiquitinases Ubiquitylation is a reversible protein modification that is implicated in many cellular functions. Recently, much progress has been made in the characterization of a superfamily of isopeptidases that remove ubiquitin: the deubiquitinases (DUBs; also known as deubiquitylating or deubiquitinating enzymes). Far from being uniform in structure and function, these enzymes display a myriad of distinct mechanistic features. The small number (<100) of DUBs might at first suggest a low degree of selectivity; however, DUBs are subject to multiple layers of regulation that modulate both their activity and their specificity. Due to their wide-ranging involvement in key regulatory processes, these enzymes might provide new therapeutic targets.

negative: Three-dimensional structure of the AAH26994.1 protein from Mus musculus, a putative eukaryotic Urm1. We have used NMR spectroscopy to determine the solution structure of protein AAH26994.1 from Mus musculus and propose that it represents the first three-dimensional structure of a ubiquitin-related modifier 1 (Urm1) protein. Amino acid sequence comparisons indicate that AAH26994.1 belongs to the Urm1 family of ubiquitin-like modifier proteins. The best characterized member of this family has been shown to be involved in nutrient sensing, invasive growth, and budding in yeast. Proteins in this family have only a weak sequence similarity to ubiquitin, and the structure of AAH26994.1 showed a much closer resemblance to MoaD subunits of molybdopterin synthases (known structures are of three bacterial MoaD proteins with 14%-26% sequence identity to AAH26994.1). The structures of AAH26994.1 and the MoaD proteins each contain the signature ubiquitin secondary structure fold, but all differ from ubiquitin largely in regions outside of this fold. This structural similarity bolsters the hypothesis that ubiquitin and ubiquitin-related proteins evolved from a protein-based sulfide donor system of the molybdopterin synthase type.

negative: Two-tiered coupling between flowing actin and immobilized N-cadherin/catenin complexes in neuronal growth cones. Neuronal growth cones move forward by dynamically connecting actin-based motility to substrate adhesion, but the mechanisms at the individual molecular level remain unclear. We cultured primary neurons on N-cadherin-coated micropatterned substrates, and imaged adhesion and cytoskeletal proteins at the ventral surface of growth cones using single particle tracking combined to photoactivated localization microscopy (sptPALM). We demonstrate transient interactions in the second time scale between flowing actin filaments and immobilized N-cadherin/catenin complexes, translating into a local reduction of the actin retrograde flow. Normal actin flow on micropatterns was rescued by expression of a dominant negative N-cadherin construct competing for the coupling between actin and endogenous N-cadherin. Fluorescence recovery after photobleaching (FRAP) experiments confirmed the differential kinetics of actin and N-cadherin, and further revealed a 20% actin population confined at N-cadherin micropatterns, contributing to local actin accumulation. Computer simulations with relevant kinetic parameters modeled N-cadherin and actin turnover well, validating this mechanism. Such a combination of short- and long-lived interactions between the motile actin network and spatially restricted adhesive complexes represents a two-tiered clutch mechanism likely to sustain dynamic environment sensing and provide the force necessary for growth cone migration.

negative: GSK-3-mediated phosphorylation enhances Maf-transforming activity. The Maf oncoproteins are b-Zip transcription factors of the AP-1 superfamily. They are involved in developmental, metabolic, and tumorigenic processes. Maf proteins are overexpressed in about 50% of human multiple myelomas. Here, we show that Maf-transforming activity is controlled by GSK-3-dependent phosphorylation and that phosphorylation by GSK-3 can increase the oncogenic activity of a protein. Using microarray analysis, we identify a gene-expression subprogram regulated by GSK-3-mediated Maf phosphorylation involved in extracellular matrix remodeling and relevant to cancer progression. We also demonstrate that GSK-3 triggers MafA sequential phosphorylation on residues S61, T57, T53, and S49, inducing its ubiquitination and degradation. Paradoxically, this phosphorylation increases MafA-transcriptional activity through the recruitment of the coactivator P/CAF. We further demonstrate that P/CAF protects MafA from ubiquitination and degradation, suggesting that, upon the release of the coactivator complex, MafA becomes polyubiquitinated and degraded to allow the response to terminate.

negative: Subversion of Systemic Glucose Metabolism as a Mechanism to Support the Growth of Leukemia Cells. From an organismal perspective, cancer cell populations can be considered analogous to parasites that compete with the host for essential systemic resources such as glucose. Here, we employed leukemia models and human leukemia samples to document a form of adaptive homeostasis, where malignant cells alter systemic physiology through impairment of both host insulin sensitivity and insulin secretion to provide tumors with increased glucose. Mechanistically, tumor cells induce high-level production of IGFBP1 from adipose tissue to mediate insulin sensitivity. Further, leukemia-induced gut dysbiosis, serotonin loss, and incretin inactivation combine to suppress insulin secretion. Importantly, attenuated disease progression and prolonged survival are achieved through disruption of the leukemia-induced adaptive homeostasis. Our studies provide a paradigm for systemic management of leukemic disease.

negative: Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Selective autophagy can be mediated via receptor molecules that link specific cargoes to the autophagosomal membranes decorated by ubiquitin-like microtubule-associated protein light chain 3 (LC3) modifiers. Although several autophagy receptors have been identified, little is known about mechanisms controlling their functions in vivo. In this work, we found that phosphorylation of an autophagy receptor, optineurin, promoted selective autophagy of ubiquitin-coated cytosolic Salmonella enterica. The protein kinase TANK binding kinase 1 (TBK1) phosphorylated optineurin on serine-177, enhancing LC3 binding affinity and autophagic clearance of cytosolic Salmonella. Conversely, ubiquitin- or LC3-binding optineurin mutants and silencing of optineurin or TBK1 impaired Salmonella autophagy, resulting in increased intracellular bacterial proliferation. We propose that phosphorylation of autophagy receptors might be a general mechanism for regulation of cargo-selective autophagy.

negative: Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA. Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Emu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc-overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap-dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (also known as Cdc2l and PITSLRE), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Emu-Myc/+ mice. When accurate translational control is re-established in Emu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level.

negative: Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase. Ubiquitination controls a broad range of cellular functions. The last step of the ubiquitination pathway is regulated by enzyme type 3 (E3) ubiquitin ligases. E3 enzymes are responsible for substrate specificity and catalyze the formation of an isopeptide bond between a lysine residue of the substrate (or the N terminus of the substrate) and ubiquitin. MIR1 and MIR2 are two E3 ubiquitin ligases encoded by Kaposi's sarcoma-associated herpesvirus that mediate the ubiquitination of major histocompatibility complex class I (MHC I) molecules and subsequent internalization. Here, we found that MIR1, but not MIR2, promoted down-regulation of MHC I molecules lacking lysine residues in their intracytoplasmic domain. In the presence of MIR1, these MHC I molecules were ubiquitinated, and their association with ubiquitin was sensitive to beta2-mercaptoethanol, unlike lysine-ubiquitin bonds. This form of ubiquitination required a cysteine residue in the intracytoplasmic tail of MHC I molecules. An MHC I molecule containing a single cysteine residue in an artificial glycine and alanine intracytoplasmic domain was endocytosed and degraded in the presence of MIR1. Thus, ubiquitination can occur on proteins lacking accessible lysines or an accessible N terminus.

negative: Metabolism of glucose, glutamine, long-chain fatty acids and ketone bodies by murine macrophages. Maximum activities of some key enzymes of metabolism were studied in elicited (inflammatory) macrophages of the mouse and lymph-node lymphocytes of the rat. The activity of hexokinase in the macrophage is very high, as high as that in any other major tissue of the body, and higher than that of phosphorylase or 6-phosphofructokinase, suggesting that glucose is a more important fuel than glycogen and that the pentose phosphate pathway is also important in these cells. The latter suggestion is supported by the high activities of both glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. However, the rate of glucose utilization by 'resting' macrophages incubated in vitro is less than the 10% of the activity of 6-phosphofructokinase: this suggests that the rate of glycolysis is increased dramatically during phagocytosis or increased secretory activity. The macrophages possess higher activities of citrate synthase and oxoglutarate dehydrogenase than do lymphocytes, suggesting that the tricarboxylic acid cycle may be important in energy generation in these cells. The activity of 3-oxoacid CoA-transferase is higher in the macrophage, but that of 3-hydroxybutyrate dehydrogenase is very much lower than those in the lymphocytes. The activity of carnitine palmitoyltransferase is higher in macrophages, suggesting that fatty acids as well as acetoacetate could provide acetyl-CoA as substrate for the tricarboxylic acid cycle. No detectable rate of acetoacetate or 3-hydroxybutyrate utilization was observed during incubation of resting macrophages, but that of oleate was 1.0 nmol/h per mg of protein or about 2.2% of the activity of palmitoyltransferase. The activity of glutaminase is about 4-fold higher in macrophages than in lymphocytes, which suggests that the rate of glutamine utilization could be very high. The rate of utilization of glutamine by resting incubated macrophages was similar to that reported for rat lymphocytes, but was considerably lower than the activity of glutaminase.

negative: Protein restriction during pregnancy affects maternal liver lipid metabolism and fetal brain lipid composition in the rat. Suboptimal developmental environments program offspring to lifelong metabolic problems. The aim of this study was to determine the impact of protein restriction in pregnancy on maternal liver lipid metabolism at 19 days of gestation (dG) and its effect on fetal brain development. Control (C) and restricted (R) mothers were fed with isocaloric diets containing 20 and 10% of casein. At 19 dG, maternal blood and livers and fetal livers and brains were collected. Serum insulin and leptin levels were determinate in mothers. Maternal and fetal liver lipid and fetal brain lipid quantification were performed. Maternal liver and fetal brain fatty acids were quantified by gas chromatography. In mothers, liver desaturase and elongase mRNAs were measured by RT-PCR. Maternal body and liver weights were similar in both groups. However, fat body composition, including liver lipids, was lower in R mothers. A higher fasting insulin at 19 dG in the R group was observed (C = 0.2 +/- 0.04 vs. R = 0.9 +/- 0.16 ng/ml, P < 0.01) and was inversely related to early growth retardation. Serum leptin in R mothers was significantly higher than that observed in C rats (C = 5 +/- 0.1 vs. R = 7 +/- 0.7 ng/ml, P < 0.05). In addition, protein restriction significantly reduced gene expression in maternal liver of desaturases and elongases and the concentration of arachidonic (AA) and docosahexanoic (DHA) acids. In fetus from R mothers, a low body weight (C = 3 +/- 0.3 vs. R = 2 +/- 0.1 g, P < 0.05), as well as liver and brain lipids, including the content of DHA in the brain, was reduced. This study showed that protein restriction during pregnancy may negatively impact normal fetal brain development by changes in maternal lipid metabolism.

negative: Effect of a C/EBP gene replacement on mitochondrial biogenesis in fat cells. CCAAT/enhancer-binding proteins, C/EBPalpha and C/EBPbeta, are required for fat cell differentiation and maturation. Previous studies showed that replacement of C/EBPalpha with C/EBPbeta, generating the beta/beta alleles in the mouse genome, prevents lipid accumulation in white adipose tissue (WAT). In this study, beta/beta mice lived longer and had higher energy expenditure than their control littermates due to increased WAT energy oxidation. The WAT of beta/beta mice was enriched with metabolically active, thermogenic mitochondria known for energy burning. The beta/beta allele exerted its effect through the elevated expression of the G protein alpha stimulatory subunit (Galphas) in WAT. Galphas, when overexpressed in fat-laden 3T3-L1 cells, stimulated mitochondrial biogenesis similar to that seen in the WAT of beta/beta mice, and effectively diminished the stored lipid pool.

negative: Differential regulation of actin microfilaments by human MICAL proteins. The Drosophila melanogaster MICAL protein is essential for the neuronal growth cone machinery that functions through plexin- and semaphorin-mediated axonal signaling. Drosophila MICAL is also involved in regulating myofilament organization and synaptic structures, and serves as an actin disassembly factor downstream of plexin-mediated axonal repulsion. In mammalian cells there are three known isoforms, MICAL1, MICAL2 and MICAL3, as well as the MICAL-like proteins MICAL-L1 and MICAL-L2, but little is known of their function, and information comes almost exclusively from neural cells. In this study we show that in non-neural cells human MICALs are required for normal actin organization, and all three MICALs regulate actin stress fibers. Moreover, we provide evidence that the generation of reactive oxygen species by MICAL proteins is crucial for their actin-regulatory function. However, although MICAL1 is auto-inhibited by its C-terminal coiled-coil region, MICAL2 remains constitutively active and affects stress fibers. These data suggest differential but complementary roles for MICAL1 and MICAL2 in actin microfilament regulation.

negative: Body-mass index and mortality in Korean men and women. BACKGROUND Obesity is associated with diverse health risks, but the role of body weight as a risk factor for death remains controversial. METHODS We examined the association between body weight and the risk of death in a 12-year prospective cohort study of 1,213,829 Koreans between the ages of 30 and 95 years. We examined 82,372 deaths from any cause and 48,731 deaths from specific diseases (including 29,123 from cancer, 16,426 from atherosclerotic cardiovascular disease, and 3362 from respiratory disease) in relation to the body-mass index (BMI) (the weight in kilograms divided by the square of the height in meters). RESULTS In both sexes, the average baseline BMI was 23.2, and the rate of death from any cause had a J-shaped association with the BMI, regardless of cigarette-smoking history. The risk of death from any cause was lowest among patients with a BMI of 23.0 to 24.9. In all groups, the risk of death from respiratory causes was higher among subjects with a lower BMI, and the risk of death from atherosclerotic cardiovascular disease or cancer was higher among subjects with a higher BMI. The relative risk of death associated with BMI declined with increasing age. CONCLUSIONS Underweight, overweight, and obese men and women had higher rates of death than men and women of normal weight. The association of BMI with death varied according to the cause of death and was modified by age, sex, and smoking history.

negative: The Utilization of Extracellular Proteins as Nutrients Is Suppressed by mTORC1 Despite being surrounded by diverse nutrients, mammalian cells preferentially metabolize glucose and free amino acids. Recently, Ras-induced macropinocytosis of extracellular proteins was shown to reduce a transformed cell's dependence on extracellular glutamine. Here, we demonstrate that protein macropinocytosis can also serve as an essential amino acid source. Lysosomal degradation of extracellular proteins can sustain cell survival and induce activation of mTORC1 but fails to elicit significant cell accumulation. Unlike its growth-promoting activity under amino-acid-replete conditions, we discovered that mTORC1 activation suppresses proliferation when cells rely on extracellular proteins as an amino acid source. Inhibiting mTORC1 results in increased catabolism of endocytosed proteins and enhances cell proliferation during nutrient-depleted conditions in vitro and within vascularly compromised tumors in vivo. Thus, by preventing nutritional consumption of extracellular proteins, mTORC1 couples growth to availability of free amino acids. These results may have important implications for the use of mTOR inhibitors as therapeutics.

negative: Metabolism strikes back: metabolic flux regulates cell signaling. Mammalian cells depend on growth factor signaling to take up nutrients; however, coordination of glucose and glutamine uptake has been a mystery. In this issue of Genes & Development, Wellen and colleagues (pp. 2784-2799) show that glucose flux through the hexosamine biosynthesis pathway regulates growth factor receptor glycosylation and enables glutamine consumption. This mechanism ensures that cells do not engage in anabolic metabolism when nutrients are limiting, and highlights how substrate availability for protein modifications can modulate cell signaling.

negative: Oxidative phosphorylation: synthesis of mitochondrially encoded proteins and assembly of individual structural subunits into functional holoenzyme complexes. The bulk of ATP consumed by various cellular processes in higher eukaryotes is normally produced by five multimeric protein complexes (I-V) embedded within the inner mitochondrial membrane, in a process known as oxidative phosphorylation (OXPHOS). Maintenance of energy homeostasis under most physiological conditions is therefore contingent upon the ability of OXPHOS to meet cellular changes in bioenergetic demand, with a chronic failure to do so being a frequent cause of human disease. With the exception of Complex II, the structural subunits of OXPHOS complexes are encoded by both the nuclear and the mitochondrial genomes. The physical separation of the two genomes necessitates that the expression of the 13 mitochondrially encoded polypeptides be co-ordinated with that of relevant nuclear-encoded partners in order to assemble functional holoenzyme complexes. Complex biogenesis is a highly ordered process, and several nuclear-encoded factors that function at distinct stages in the assembly of individual OXPHOS complexes have been identified.

negative: Molecular mechanism of BST2/tetherin downregulation by K5/MIR2 of Kaposi's sarcoma-associated herpesvirus. K3/MIR1 and K5/MIR2 of Kaposi's sarcoma-associated herpesvirus (KSHV) are viral members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family and contribute to viral immune evasion by directing the conjugation of ubiquitin to immunostimulatory transmembrane proteins. In a quantitative proteomic screen for novel host cell proteins downregulated by viral immunomodulators, we previously observed that K5, as well as the human immunodeficiency virus type 1 (HIV-1) immunomodulator VPU, reduced steady-state levels of bone marrow stromal cell antigen 2 (BST2; also called CD317 or tetherin), suggesting that BST2 might be a novel substrate of K5 and VPU. Recent work revealed that in the absence of VPU, HIV-1 virions are tethered to the plasma membrane in BST2-expressing HeLa cells. By targeting BST2, K5 might thus similarly overcome an innate antiviral host defense mechanism. Here we establish that despite its type II transmembrane topology and carboxy-terminal glycosylphosphatidylinositol (GPI) anchor, BST2 represents a bona fide target of K5 that is downregulated during primary infection by and reactivation of KSHV. Upon exit of the protein from the endoplasmic reticulum, lysines in the short amino-terminal domain of BST2 are ubiquitinated by K5, resulting in rapid degradation of BST2. Ubiquitination of BST2 is required for degradation, since BST2 lacking cytosolic lysines was K5 resistant and ubiquitin depletion by proteasome inhibitors restored BST2 surface expression. Thus, BST2 represents the first type II transmembrane protein targeted by K5 and the first example of a protein that is both ubiquitinated and GPI linked. We further demonstrate that KSHV release is decreased in the absence of K5 in a BST2-dependent manner, suggesting that K5 contributes to the evasion of intracellular antiviral defense programs.