Sargis, Timothy

Abstract Purpose: Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel–Lindau tumor suppressor, resulting in activation of HIF-1α and HIF-2α. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2α, and a tumor suppressor role for HIF-1α. 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-1α was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2α and HAF were expressed primarily in tumor cells. TAM-associated HIF-1α was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1α was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2α were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1α in M2-polarized CD163-expressing TAMs. Conclusions: These findings highlight a potential role of TAM HIF-1α in ccRCC progression and support the reevaluation of HIF-1α as a therapeutic target and marker of disease progression.

The hypoxia‐inducible factor (HIF), HIF‐1, is a central regulator of the response to low oxygen or inflammatory stress and plays an essential role in survival and function of immune cells. However, the mechanisms regulating nonhypoxic induction of HIF‐1 remain unclear. Here, we assess the impact of germline heterozygosity of a novel, oxygen‐independent ubiquitin ligase for HIF‐1α: hypoxia‐associated factor (HAF; encoded by SART1). SART1−/− mice were embryonic lethal, whereas male SART1+/− mice spontaneously recapitulated key features of nonalcoholic steatohepatitis (NASH)‐driven hepatocellular carcinoma (HCC), including steatosis, fibrosis, and inflammatory cytokine production. Male, but not female, SART1+/− mice showed significant up‐regulation of HIF‐1α in circulating and liver‐infiltrating immune cells, but not in hepatocytes, before development of malignancy. Additionally, Kupffer cells derived from male, but not female, SART1+/− mice produced increased levels of the HIF‐1‐dependent chemokine, regulated on activation, normal T‐cell expressed and secreted (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: This work establishes a new tumor‐suppressor role for HAF in immune cell function by preventing inappropriate HIF‐1 activation in male mice and identifies RANTES as a novel therapeutic target for NASH and NASH‐driven HCC. (Hepatology 2016;63:1576‐1591)