Playing Tag with HIF: The VHL Story

Inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL is the cause of inherited VHL disease and is associated with sporadic kidney cancer. pVHL is found in a multiprotein complex with elongins B/C, Cul2, and Rbx1 forming an E3 ubiquitin ligase complex called VEC. This modular enzyme targets the α subunits of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction. Consequently, tumour cells lacking functional pVHL overproduce the products of HIF-target genes such as vascular endothelial growth factor (VEGF), which promotes angiogenesis. This likely accounts for the hypervascular nature of VHL-associated neoplasms. Although pVHL has been linked to the cell-cycle, differentiation, and the regulation of extracellular matrix assembly, microenvironment pH, and tissue invasiveness, this review will focus on the recent insights into the molecular mechanisms governing the E3 ubiquitin ligase function of VEC

Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis

Ras is phosphorylated on a conserved tyrosine at position 32 within the switch I region via Src kinase. This phosphorylation inhibits the binding of effector Raf while promoting the engagement of GTPase-activating protein (GAP) and GTP hydrolysis. Here we identify SHP2 as the ubiquitously expressed tyrosine phosphatase that preferentially binds to and dephosphorylates Ras to increase its association with Raf and activate downstream proliferative Ras/ERK/MAPK signalling. In comparison to normal astrocytes, SHP2 activity is elevated in astrocytes isolated from glioblastoma multiforme (GBM)-prone H-Ras(12V) knock-in mice as well as in glioma cell lines and patient-derived GBM specimens exhibiting hyperactive Ras. Pharmacologic inhibition of SHP2 activity attenuates cell proliferation, soft-agar colony formation and orthotopic GBM growth in NOD/SCID mice and decelerates the progression of low-grade astrocytoma to GBM in a spontaneous transgenic glioma mouse model. These results identify SHP2 as a direct activator of Ras and a potential therapeutic target for cancers driven by a previously 'undruggable' oncogenic or hyperactive Ras

Somatic Pairing of Chromosome 19 in Renal Oncocytoma Is Associated with Deregulated ELGN2-Mediated Oxygen-Sensing Response

Chromosomal abnormalities, such as structural and numerical abnormalities, are a common occurrence in cancer. The close association of homologous chromosomes during interphase, a phenomenon termed somatic chromosome pairing, has been observed in cancerous cells, but the functional consequences of somatic pairing have not been established. Gene expression profiling studies revealed that somatic pairing of chromosome 19 is a recurrent chromosomal abnormality in renal oncocytoma, a neoplasia of the adult kidney. Somatic pairing was associated with significant disruption of gene expression within the paired regions and resulted in the deregulation of the prolyl-hydroxylase ELGN2, a key protein that regulates the oxygen-dependent degradation of hypoxia-inducible factor (HIF). Overexpression of ELGN2 in renal oncocytoma increased ubiquitin-mediated destruction of HIF and concomitantly suppressed the expression of several HIF-target genes, including the pro-death BNIP3L gene. The transcriptional changes that are associated with somatic pairing of chromosome 19 mimic the transcriptional changes that occur following DNA amplification. Therefore, in addition to numerical and structural chromosomal abnormalities, alterations in chromosomal spatial dynamics should be considered as genomic events that are associated with tumorigenesis. The identification of EGLN2 as a significantly deregulated gene that maps within the paired chromosome region directly implicates defects in the oxygen-sensing network to the biology of renal oncocytoma

An integrative genomics approach identifies Hypoxia Inducible Factor-1 (HIF-1)-target genes that form the core response to hypoxia

The transcription factor Hypoxia-inducible factor 1 (HIF-1) plays a central role in the transcriptional response to oxygen flux. To gain insight into the molecular pathways regulated by HIF-1, it is essential to identify the downstream-target genes. We report here a strategy to identify HIF-1-target genes based on an integrative genomic approach combining computational strategies and experimental validation. To identify HIF-1-target genes microarrays data sets were used to rank genes based on their differential response to hypoxia. The proximal promoters of these genes were then analyzed for the presence of conserved HIF-1-binding sites. Genes were scored and ranked based on their response to hypoxia and their HIF-binding site score. Using this strategy we recovered 41% of the previously confirmed HIF-1-target genes that responded to hypoxia in the microarrays and provide a catalogue of predicted HIF-1 targets. We present experimental validation for ANKRD37 as a novel HIF-1-target gene. Together these analyses demonstrate the potential to recover novel HIF-1-target genes and the discovery of mammalian-regulatory elements operative in the context of microarray data sets

LOSS OF JAK2 REGULATION VIA VHL-SOCS1 E3 UBIQUITIN HETEROCOMPLEX UNDERLIES CHUVASH POLYCYTHEMIA

Chuvash polycythemia (CP) is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the von Hippel-Lindau (VHL) gene whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark features of CP such as hypersensitivity to erythropoietin are unclear. Here, we show that VHL directly binds suppressor of cytokine signalling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated (p)JAK2 for ubiquitin-mediated destruction. In contrast, CP-associated VHL mutants have altered affinity for SOCS1 and fail to engage and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reverses the disease phenotype in vhlR200W/R200W knock-in mice, a model that faithfully recapitulates human CP. These results reveal VHL as a SOCS1-cooperative negative regulator of JAK2 and provide compelling biochemical and preclinical evidence for JAK2- targeted therapy in CP patients

Understanding the Warburg effect and the prognostic value of stromal caveolin-1 as a marker of a lethal tumor microenvironment

Cancer cells show a broad spectrum of bioenergetic states, with some cells using aerobic glycolysis while others rely on oxidative phosphorylation as their main source of energy. In addition, there is mounting evidence that metabolic coupling occurs in aggressive tumors, between epithelial cancer cells and the stromal compartment, and between well-oxygenated and hypoxic compartments. We recently showed that oxidative stress in the tumor stroma, due to aerobic glycolysis and mitochondrial dysfunction, is important for cancer cell mutagenesis and tumor progression. More specifically , increased autophagy/mitophagy in the tumor stroma drives a form of parasitic epithelial-stromal metabolic coupling. These findings explain why it is effective to treat tumors with either inducers or inhibitors of autophagy, as both would disrupt this energetic coupling. We also discuss evidence that glutamine addiction in cancer cells produces ammonia via oxidative mitochondrial metabolism. Ammonia production in cancer cells, in turn, could then help maintain autophagy in the tumor stromal compartment. In this vicious cycle, the initial glutamine provided to cancer cells would be produced by autophagy in the tumor stroma. Thus, we believe that parasitic epithelial-stromal metabolic coupling has important implications for cancer diagnosis and therapy, for example, in designing novel metabolic imaging techniques and establishing new targeted therapies. In direct support of this notion, we identified a loss of stromal caveolin-1 as a marker of oxidative stress, hypoxia, and autophagy in the tumor microenvironment, explaining its powerful predictive value. Loss of stromal caveolin-1 in breast cancers is associated with early tumor recurrence, metastasis, and drug resistance, leading to poor clinical outcome

Posttranscriptional regulation of ICAM-1 gene expression

Cell-cell adhesion is critical for the generation of effective immune responses and is dependent upon the expression of a variety of cell surface receptors. Intercellular adhesion molecule-1 (ICAM- 1; CD54) is an inducible cell surface glycoprotein expressed at a low level on a wide range of cell types. Although its expression is dramatically increased at sites of inflammation, providing important means of regulating cell-cell interactions and thereby inflammatory responses, the intracellular regulatory elements and signaling pathways underlying the inducible expression of ICAM- 1 by proinflammatory cytokines were previously largely unknown. In this thesis, a novel posttranscriptional regulation of ICAM-1 gene expression by two proinflammatory mediators, interferon-γ (IFN-γ) and phorbol myristate acetate (PMA), and the possible role of serine/threonine (ser/thr) phosphorylation pathway in cycloheximide induced ICAM-1 message stabilization were investigated. The results show that (1) constitutively expressed ICAM-1 mRNA has a short half-life; (2) IFN-γ and PMA induce the accumulation of ICAM-1 message, at least in part, by stabilizing the mRNA; (3) the WN-γ responsive element(s) is located within the protein coding region encoding the cytoplasmic domain; (4) the PMA-responsive elements lie within the 3’ untranslated region (UTR) and may even be the AUUUA multimers; (5) cycloheximide, a potent eukaryotic protein synthesis inhibitor that superinduces the expression of many genes by preventing the degradation of otherwise labile mRNAs, also induces the level of ICAM- 1 mRNA by message stabilization; (6) the stabilizing effect of cycloheximide does not depend on the 3’UTR containing the AUUUA sequences; (7) the stabilization of ICAM-1 mRNA by cycloheximide is independent of its translational inhibition; and (8) the ser/thr phosphorylation pathway seems to play a crucial role in the cycloheximide-induced stabilization of ICAM- 1 message. These results demonstrate the existence of distinct destabilizing elements throughout the ICAM- 1 message that are responsive to the actions of various proinflammatory cytokines, and underscore the importance of posttranscriptional regulation of ICAM-1 expression during an inflammatory response.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Ubiquitin Pathway in VHL Cancer Syndrome

The physiologic response to changes in cellular oxygen tension is ultimately governed by a heterodimeric transcription factor called hypoxia-inducible factor (HIF), which, in adaptation to compromised oxygen availability, transactivates a myriad of genes, including those responsible for de novo vascularization, production of oxygen-carrying red blood cells, and anaerobic metabolism. Accumulation of HIF is observed in most types of solid tumors and is frequently associated with poor prognosis and disease progression, underscoring the importance and relevance of HIF in cancer. The protein stability and, thereby, the activity of HIF are principally regulated by the von Hippel-Lindau (VHL) tumor-suppressor-containing E3 ubiquitin ligase complex (ECV) that targets the catalytic subunit HIFα for oxygen-dependent ubiquitin-mediated destruction. Individuals who inherit germline VHL mutation develop VHL disease, which is characterized by the development of hypervascular tumors in multiple yet specific organs. This review will examine recent progress in our understanding of the molecular mechanisms governing the function of ECV and the significance of consequential regulation of HIF in oncogenesis