Hypoxia-Induced SUMOylation of E3 Ligase HAF Determines Specific Activation of HIF2 in Clear-Cell Renal Cell Carcinoma

Koh, Mei Yee; Nguyen, Vuvi; Lemos, Robert; Darnay, Bryant G.; Kiriakova, Galina; Abdelmelek, Mena; Ho, Thai H.; Karam, Jose A.; Monzon, Federico A.; Jonasch, Eric; Powis, Garth

doi:10.1158/0008-5472.can-13-2190

Cited by 42 publications

(43 citation statements)

References 51 publications

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“…Stable cell lines overexpressing HAF and HAF DM (HAF K94R K141R) were generated by retroviral infection using pMX-IRES-GFP (20,24). 786-0 cells containing homozygous loss of HIF-2a (EPAS1): 786-0 EPAS1 À/À #4, EPAS1 À/À #11, and control (Nic Con) clones were generated using plasmids targeting different regions within exon 2 (sc400647-NIC, sc400647-NIC-2, or control double nickase plasmid sc-437281, respectively, from Santa Cruz Biotechnology).…”

Section: Plasmid Construction and Transfectionsmentioning

confidence: 99%

“…Cells were lysed in cold cell lysis buffer with protease and phosphatase inhibitors, and Western blots were performed as described previously (20). Primary antibodies against phosphorylated or total proteins (EGFR, AKT, VEGFR, MEK, ERK, HIF-1a, HIF-2a) were from Cell Signaling Technology.…”

Section: Western Blots and Immunoprecipitationsmentioning

confidence: 99%

“…ccRCC tumor microarray (TMA) preparation and TCGA data analysis TMAs were prepared from patients with metastatic ccRCC enrolled in phase II trials at the University of Texas MD Anderson Cancer Center (Houston, TX) and were treated with sorafenib or sorafenib plus IFN (25). Three cores were obtained per patient and stained using HAF mAb (20). Images were scanned using an Aperio AT2 slide scanner and HAF nuclear staining was quantitated using Aperio Image Analysis Software (Leica Biosystems Inc).…”

Section: Active Ras Pull-down Assaysmentioning

confidence: 99%

“…pVHL loss-of-function mutations are a frequent initiating event in clear cell renal cell carcinoma (ccRCC), which drives constitutive HIF activation-associated ccRCC progression (18). In contrast to pVHL, which does not distinguish between HIF-1a or HIF-2a isoforms, the hypoxia-associated factor (HAF,also known as SART1) mediates the oxygen-independent degradation of HIF-1a, while promoting HIF-2a transactivation, thus mediating HIFa isoform-specific regulation (19)(20)(21).…”

Section: Introductionmentioning

confidence: 99%

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Hypoxia-Associated Factor (HAF) Mediates Neurofibromin Ubiquitination and Degradation Leading to Ras–ERK Pathway Activation in Hypoxia

Green

Sargis

Reichert

et al. 2019

Molecular Cancer Research

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Low oxygen or hypoxia is a feature of all solid tumors and has been associated with aggressive disease. Here, we describe a novel mechanism for the hypoxia-dependent degradation of the Ras-GTPase-activating protein neurofibromin, by hypoxia-associated factor (HAF). We have previously characterized HAF as an oxygen-independent ubiquitin ligase for HIF-1a.Here, we show that HAF promotes neurofibromin ubiquitination and degradation independently of oxygen and pVHL, resulting in Ras-ERK pathway activation. Hypoxia enhanced HAF:neurofibromin binding independently of HAF-SUMOylation, whereas HAF knockdown increased neurofibromin levels primarily in hypoxia, supporting the role of HAF as a hypoxia-specific neurofibromin regulator. HAF overexpression increased p-ERK levels and promoted resistance of clear cell kidney cancer (ccRCC) cells to sorafenib and sunitinib in both normoxia and hypoxia. However, a greater-fold increase in sorafenib/sunitinib resistance was observed during hypox-ia, particularly in pVHL-deficient cells. Intriguingly, HAFmediated resistance was HIF-2a-dependent in normoxia, but HIF-2a-independent in hypoxia indicating two potential mechanisms of HAF-mediated resistance: a HIF-2a-dependent pathway dominant in normoxia, and the direct activation of the Ras-ERK pathway through neurofibromin degradation dominant in hypoxia. Patients with ccRCC with high HAF transcript or protein levels showed significantly decreased overall survival compared with those with low HAF. Thus, we establish a novel, nonmutational pathway of neurofibromin inactivation through hypoxia-induced HAF-mediated degradation, leading to Ras-ERK activation and poor prognosis in ccRCC.Implications: We describe a novel mechanism of neurofibromin degradation induced by hypoxia that leads to activation of the prooncogenic Ras-ERK pathway and resistance to therapy.

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Section: Plasmid Construction and Transfectionsmentioning

confidence: 99%

Section: Western Blots and Immunoprecipitationsmentioning

confidence: 99%

Section: Active Ras Pull-down Assaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Hypoxia-Associated Factor (HAF) Mediates Neurofibromin Ubiquitination and Degradation Leading to Ras–ERK Pathway Activation in Hypoxia

Green

Sargis

Reichert

et al. 2019

Molecular Cancer Research

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“…Upstream stimulating factor-2 (USF-2) acts as a coactivator specifically for HIF-2α-dependent genes and is dispensable for the transcriptional induction of HIF-1α target genes (46). More recent work has identified that HIF-2α binding to SUMOlyated hypoxia-associated factor (HAF) increases its transcriptional activity, whereas HAF induces HIF-1α degradation, independently of SUMOlyation (47). In addition to coactivators, yin yang-1 (YY-1), a specific corepressor, directly binds and inhibits the transcriptional activity of HIF-2α (48) (Table 1).…”

Section: Oxygen-sensitive Transcription Factorsmentioning

confidence: 99%

Role of Intestinal HIF-2α in Health and Disease

Ramakrishnan¹,

Shah

2016

Annu. Rev. Physiol.

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The intestine is supported by a complex vascular system that undergoes dynamic and transient daily shifts in blood perfusion, depending on the metabolic state. Moreover, the intestinal villi have a steep oxygen gradient from the hypoxic epithelium adjacent to the anoxic lumen to the relative higher tissue oxygenation at the base of villi. Due to the daily changes in tissue oxygen levels in the intestine, the hypoxic transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α are essential in maintaining intestinal homeostasis. HIF-2α is essential in maintaining proper micronutrient balance, the inflammatory response, and the regenerative and proliferative capacity of the intestine following an acute injury. However, chronic activation of HIF-2α leads to enhanced proinflammatory response, intestinal injury, and colorectal cancer. In this review, we detail the major mechanisms by which HIF-2α contributes to health and disease of the intestine and the therapeutic implications of targeting HIF-2α in intestinal diseases.

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Methuosis Inducer SGI‐1027 Cooperates with Everolimus to Promote Apoptosis and Pyroptosis by Triggering Lysosomal Membrane Permeability in Renal Cancer

Luo,

Guan,

Deng

et al. 2024

Advanced Science

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The mTOR inhibitor everolimus has been approved as a sequential or second‐line therapy for renal cell carcinoma (RCC). However, the development of drug resistance limits its clinical applications. This study aims to address the challenge of everolimus resistance and provide new insights into the treatment of advanced RCC. Here, the cytotoxicity of the DNA methyltransferase 1 (DNMT1) inhibitor SGI‐1027 in inducing cell vacuolation and methuosis is discovered and demonstrated for the first time. Additionally, SGI‐1027 exerts synergistic effects with everolimus, as their combination suppresses the growth, migration, and invasion of renal cancer cells. Mechanistically, apoptosis and GSDME‐dependent pyroptosis triggered by lysosomal membrane permeability (LMP) are observed. The upregulation of GSDME expression and increased lysosomal activity in renal cancer cells provide a therapeutic window for the combination of these two drugs to treat renal cancer. The combination treatment exhibits effective anti‐tumor activity and is well tolerated in a subcutaneous tumor model. Overall, this study validates and reveals the specific cytotoxicity property of SGI‐1027 and its potent synergistic effect with everolimus, offering new insights into advanced RCC therapy and everolimus‐resistance overcoming.

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Hypoxia-Induced SUMOylation of E3 Ligase HAF Determines Specific Activation of HIF2 in Clear-Cell Renal Cell Carcinoma

Cited by 42 publications

References 51 publications

Hypoxia-Associated Factor (HAF) Mediates Neurofibromin Ubiquitination and Degradation Leading to Ras–ERK Pathway Activation in Hypoxia

Hypoxia-Associated Factor (HAF) Mediates Neurofibromin Ubiquitination and Degradation Leading to Ras–ERK Pathway Activation in Hypoxia

Role of Intestinal HIF-2α in Health and Disease

Methuosis Inducer SGI‐1027 Cooperates with Everolimus to Promote Apoptosis and Pyroptosis by Triggering Lysosomal Membrane Permeability in Renal Cancer

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