Purpose: Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel-Lindau tumor suppressor, resulting in activation of HIF-1a and HIF-2a. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2a, and a tumor suppressor role for HIF-1a. However, few studies have comprehensively examined the clinical relevance of this paradigm. Furthermore, the hypoxia-associated factor (HAF), which regulates the HIFs, has not been comprehensively evaluated in ccRCC.Experimental Design: To assess the involvement of HAF/HIFs in ccRCC, we analyzed their relationship to tumor grade/stage/ outcome using tissue from 380 patients, and validated these associations using tissue from 72 additional patients and a further 57 patients treated with antiangiogenic therapy for associations with response. Further characterization was performed using single-cell mRNA sequencing (scRNA-seq), RNA-in situ hybridization (RNA-ISH), and IHC.Results: HIF-1a was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2a and HAF were expressed primarily in tumor cells. TAM-associated HIF-1a was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1a was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2a were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1a in M2-polarized CD163-expressing TAMs.Conclusions: These findings highlight a potential role of TAM HIF-1a in ccRCC progression and support the reevaluation of HIF-1a as a therapeutic target and marker of disease progression.

The hypoxia inducible factor, HIF-1 is a central regulator of the response to low oxygen or inflammatory stress and plays an essential role in the survival and function of immune cells. However, the mechanisms regulating non-hypoxic induction of HIF-1 remain unclear. Here, we assess the impact of germline heterozygosity of a novel, oxygen independent ubiquitin ligase for HIF-1α; the hypoxia associated factor, HAF (encoded by SART1). SART1−/− mice were embryonic lethal, whereas male SART1+/− mice spontaneously recapitulated key features of non-alcoholic steatohepatitis (NASH) driven HCC including steatosis, fibrosis and inflammatory cytokine production. Male but not female SART1+/− mice showed significant upregulation of HIF-1α in circulating and liver-infiltrating immune cells but not in hepatocytes prior to the development of malignancy. Additionally, Kupffer cells derived from male but not female SART1+/− mice produced increased levels of the HIF-1 dependent chemokine, RANTES, compared to wild-type. This was associated with increased liver-neutrophilic infiltration whereas infiltration of lymphocytes and macrophages were not significantly different. Neutralization of circulating RANTES decreased liver neutrophilic infiltration, and attenuated HCC tumor initiation/growth in SART1+/− mice. Conclusion: We establish a new tumor suppressor role for HAF in immune cell function by preventing inappropriate HIF-1 activation in male mice, and identify RANTES as a novel therapeutic target for NASH and NASH-driven HCC.

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.