Olga Roche

VHL Promotes E2 Box-Dependent E-Cadherin Transcription by HIF-Mediated Regulation of SIP1 and Snail

ABSTRACT The product of the von Hippel-Lindau gene (VHL) acts as the substrate-recognition component of an E3 ubiquitin ligase complex that ubiquitylates the catalytic α subunit of hypoxia-inducible factor (HIF) for oxygen-dependent destruction. Although emerging evidence supports the notion that deregulated accumulation of HIF upon the loss of VHL is crucial for the development of clear-cell renal cell carcinoma (CC-RCC), the molecular events downstream of HIF governing renal oncogenesis remain unclear. Here, we show that the expression of a homophilic adhesion molecule, E-cadherin, a major constituent of epithelial cell junctions whose loss is associated with the progression of epithelial cancers, is significantly down-regulated in primary CC-RCC and CC-RCC cell lines devoid of VHL. Reintroduction of wild-type VHL in CC-RCC (VHL−/−) cells markedly reduced the expression of E2 box-dependent E-cadherin-specific transcriptional repressors Snail and SIP1 and concomitantly restored E-cadherin expression. RNA interference-mediated knockdown of HIFα in CC-RCC (VHL−/−) cells likewise increased E-cadherin expression, while functional hypoxia or expression of VHL mutants incapable of promoting HIFα degradation attenuated E-cadherin expression, correlating with the disengagement of RNA polymerase II from the endogenous E-cadherin promoter/gene. These findings reveal a critical HIF-dependent molecular pathway connecting VHL, an established “gatekeeper” of the renal epithelium, with a major epithelial tumor suppressor, E-cadherin.

Regulation of endocytosis via the oxygen-sensing pathway

Tumor hypoxia is associated with disease progression, resistance to conventional cancer therapies and poor prognosis. Hypoxia, by largely unknown mechanisms, leads to deregulated accumulation of and signaling via receptor tyrosine kinases (RTKs) that are critical for driving oncogenesis. Here, we show that hypoxia or loss of von Hippel–Lindau protein—the principal negative regulator of hypoxia-inducible factor (HIF)—prolongs the activation of epidermal growth factor receptor that is attributable to lengthened receptor half-life and retention in the endocytic pathway. The deceleration in endocytosis is due to the attenuation of Rab5-mediated early endosome fusion via HIF-dependent downregulation of a critical Rab5 effector, rabaptin-5, at the level of transcription. Primary kidney and breast tumors with strong hypoxic signatures show significantly lower expression of rabaptin-5 RNA and protein. These findings reveal a general role of the oxygen-sensing pathway in endocytosis and support a model in which tumor hypoxia or oncogenic activation of HIF prolongs RTK-mediated signaling by delaying endocytosis-mediated deactivation of receptors.

Hypoxia promotes ligand-independent EGF receptor signaling via hypoxia-inducible factor–mediated upregulation of caveolin-1

Caveolin-1 (CAV1) is an essential structural constituent of caveolae, specialized lipid raft microdomains on the cell membrane involved in endocytosis and signal transduction, which are inexplicably deregulated and are associated with aggressiveness in numerous cancers. Here we identify CAV1 as a direct transcriptional target of oxygen-labile hypoxia-inducible factor 1 and 2 that accentuates the formation of caveolae, leading to increased dimerization of EGF receptor within the confined surface area of caveolae and its subsequent phosphorylation in the absence of ligand. Hypoxia-inducible factor–dependent up-regulation of CAV1 enhanced the oncogenic potential of tumor cells by increasing the cell proliferative, migratory, and invasive capacities. These results support a concept in which a crisis in oxygen availability or a tumor exhibiting hypoxic signature triggers caveolae formation that bypasses the requirement for ligand engagement to initiate receptor activation and the critical downstream adaptive signaling during a period when ligands required to activate these receptors are limited or are not yet available.

Loss of JAK2 regulation via a heterodimeric VHL-SOCS1 E3 ubiquitin ligase underlies Chuvash polycythemia

Chuvash polycythemia is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the VHL (von Hippel-Lindau) gene, whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark abnormalities of Chuvash polycythemia, such as hypersensitivity to erythropoietin, are unclear. Here we show that VHL directly binds suppressor of cytokine signaling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated JAK2 (pJAK2) for ubiquitin-mediated destruction. In contrast, Chuvash polycythemia–associated VHL mutants have altered affinity for SOCS1 and do not engage with and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reversed the disease phenotype in VhlR200W/R200W knock-in mice, an experimental model that recapitulates human Chuvash polycythemia. These results show that VHL is a SOCS1-cooperative negative regulator of JAK2 and provide biochemical and preclinical support for JAK2-targeted therapy in individuals with Chuvash polycythemia.

Chemotherapy induces NEDP1-mediated destabilization of MDM2.

MDM2 is an E3 ligase that promotes ubiquitin-mediated destruction of p53. Cellular stresses such as DNA damage can lead to p53 activation due in part to MDM2 destabilization. Here, we show that the stability of MDM2 is regulated by an ubiquitin-like NEDD8 pathway and identify NEDP1 as a chemotherapy-induced isopeptidase that deneddylates MDM2, resulting in MDM2 destabilization concomitant with p53 activation. Concordantly, RNAi-mediated knockdown of endogenous NEDP1 blocked diminution of MDM2 levels and increased chemoresistance of tumor cells. These findings unveil the regulation of MDM2 stability through NEDP1 as a common molecular determinant governing chemotherapy-induced p53-dependent cell death.

SATB2 augments ΔNp63α in head and neck squamous cell carcinoma

ΔNp63α is a critical pro‐survival protein overexpressed in 80% of head and neck squamous cell carcinomas (HNSCCs) where it inhibits TAp73β transcription of p53‐family target genes, which is thought to increase HNSCC resistance to chemotherapy‐induced cell death. However, the mechanisms governing ΔNp63α function are largely unknown. In this study, we identify special AT‐rich‐binding protein 2 (SATB2) as a new ΔNp63α‐binding protein that is preferentially expressed in advanced‐stage primary HNSCC and show that SATB2 promotes chemoresistance by enhancing ΔNp63α‐mediated transrepression by augmenting ΔNp63α engagement to p53‐family responsive elements. Furthermore, SATB2 expression positively correlates with HNSCC chemoresistance, and RNA interference‐mediated knockdown of endogenous SATB2 re‐sensitizes HNSCC cells to chemotherapy‐ and γ‐irradiation‐induced apoptosis, irrespective of p53 status. These findings unveil SATB2 as a pivotal modulator of ΔNp63α that governs HNSCC cell survival.

Deregulation of E2-EPF Ubiquitin Carrier Protein in Papillary Renal Cell Carcinoma

Molecular pathways associated with pathogenesis of sporadic papillary renal cell carcinoma (PRCC), the second most common form of kidney cancer, are poorly understood. We analyzed primary tumor specimens from 35 PRCC patients treated by nephrectomy via gene expression analysis and tissue microarrays constructed from an additional 57 paraffin-embedded PRCC samples via immunohistochemistry. Gene products were validated and further studied by Western blot analyses using primary PRCC tumor samples and established renal cell carcinoma cell lines, and potential associations with pathologic variables and survival in 27 patients with follow-up information were determined. We show that the expression of E2-EPF ubiquitin carrier protein, which targets the principal negative regulator of hypoxia-inducible factor (HIF), von Hippel-Lindau protein, for proteasome-dependent degradation, is markedly elevated in the majority of PRCC tumors exhibiting increased HIF1α expression, and is associated with poor prognosis. In addition, we identified multiple hypoxia-responsive elements within the E2-EPF promoter, and for the first time we demonstrated that E2-EPF is a hypoxia-inducible gene directly regulated via HIF1. These findings reveal deregulation of the oxygen-sensing pathway impinging on the positive feedback mechanism of HIF1-mediated regulation of E2-EPF in PRCC.

Oxygen-independent degradation of HIF-α via bioengineered VHL tumour suppressor complex

Tumour hypoxia promotes the accumulation of the otherwise oxygen‐labile hypoxia‐inducible factor (HIF)‐α subunit whose expression is associated with cancer progression, poor prognosis and resistance to conventional radiation and chemotherapy. The oxygen‐dependent degradation of HIF‐α is carried out by the von Hippel–Lindau (VHL) protein‐containing E3 that directly binds and ubiquitylates HIF‐α for subsequent proteasomal destruction. Thus, the cellular proteins involved in the VHL–HIF pathway have been recognized as attractive molecular targets for cancer therapy. However, the various compounds designed to inhibit HIF‐α or HIF‐downstream targets, although promising, have shown limited success in the clinic. In the present study, we describe the bioengineering of VHL protein that removes the oxygen constraint in the recognition of HIF‐α while preserving its E3 enzymatic activity. Using speckle variance–optical coherence tomography (sv–OCT), we demonstrate the dramatic inhibition of angiogenesis and growth regression of human renal cell carcinoma xenografts upon adenovirus‐mediated delivery of the bioengineered VHL protein in a dorsal skin‐fold window chamber model. These findings introduce the concept and feasibility of ‘bio‐tailored’ enzymes in the treatment of HIF‐overexpressing tumours.

Transcriptional regulation of genes via hypoxia-inducible factor.

Hypoxia-inducible factor (HIF) is the principal transcription factor that regulates adaptive physiologic responses to compromised oxygen tension. von Hippel-Lindau (VHL) tumor-suppressor protein binds and ubiquitylates the catalytic α subunit of HIF in an oxygen-dependent manner for rapid destruction via the 26S proteasome, thereby establishing VHL as a critical negative regulator of HIF. Mutations in VHL cause VHL disease, which is frequently characterized by the overexpression of HIFα and the development of tumors in multiple organ systems, including the central nervous system and the kidney. Here, we describe classical experimental approaches to demonstrate and validate HIF-responsive transcriptional regulation of genes.

Abstract 1973: Caveolin-1 promotes cell proliferation, EGFR activation and tumor growth in kidney cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Tumor hypoxia is associated with disease progression, drug resistance and poor prognosis. It has been shown previously that hypoxia prolongs the activation of epidermal growth factor receptor (EGFR) by delaying clathrin-dependent endocytosis-mediated deactivation of receptors. Recently, caveolin (CAV) mediated signaling and trafficking have been reported to be involved in the oncogenesis of many tumours, including clear cell renal cell carcinoma (CCRCC). However, the role of CAV-1 in the tumor progression is unclear. Here, we show that the loss of von Hippel-Lindau (VHL) protein – the principal negative regulator of hypoxia-inducible factor (HIF) – increases the auto-phosphorylation of epidermal growth factor receptor (EGFR), thus attributing to an enhanced ligand-independent cell signaling pathway, majorly Ras-Raf-MEK-ERK pathway, and leading to cell proliferation. The auto-phosphorylation of EGFR is due to the HIF-dependent overexpression of caveolin-1 (CAV-1) at the level of transcription, which enhances the association between EGFR and CAV-1 and the auto-dimerization of EGFR. Primary CCRCC tumors and numerous hypoxia treated tumor cell lines exhibit significantly higher expression of CAV-1 protein. Using a dorsal skin-fold window chamber on SCID mice, shCAV-1 knockdown 786-O tumor cell line exhibits lower growth than that of shscramble counterpart in xenografts. These findings support a model in which tumour hypoxia or oncogenic activation of HIF prolongs ligand-independent RTK signaling through the over-expression of CAV-1. Given the oncogenic role of CAV-1, it should be considered as a future therapeutic target in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1973. doi:10.1158/1538-7445.AM2011-1973