USP9X integrates TGF-ß and hypoxia signalings to promote ovarian cancer chemoresistance via HIF-2a-maintained stemness

Widespread intraperitoneal metastases and chemoresistance render ovarian cancer the leading cause of gynecological malignancy–related deaths, wherein TGF-ß signaling plays the pivotal role by promoting cancer stem cells (CSCs) activity. The activation mechanism and key protumorigeneic events downstream of TGF-ß signaling remain incompletely understood. Here, we identify hypoxic tumor microenvironment as an initiator of TGF-ß signaling to promote HIF-2a positive CSC-mediated chemoresistance in high-grade serous ovarian cancer (HGSOC). Mechanistically, deubiquitinase USP9X, as a TGF-ß downstream effector, stabilizes HIF-2? in a hydroxylation- and ubiquitylation-dependent manner, thus promoting stemness reprogramming. Hypoxia and TGF-ß signals converge on USP9X-HIF-2? axis via multi-level regulations, which in turn facilitates Smad/HIF responses. Clinically, USP9X expression correlates with TGF-ß signatures, CSCs characteristics, EMT behaviors, and chemotherapy responsiveness, along with HIF-2?. Antagonizing USP9X efficiently represses tumor formation, metastasis, CSCs occurrence, while increasing chemosensitivity in orthotopic tumors, patient derived xenograft (PDX), organoid, and chemoresistant cell models, in part via restricting TGF-ß and hypoxia activities. This study deciphers the critical role of hypoxic niche in stimulating TGF-ß signaling, and a downstream USP9X-HIF-2? proteostatic regulatory axis in priming the HGSOC stemness, thereby provides novel targeting venues to counteract TGF-ß signaling in CSCs and meliorate clinical chemoresistance. Overall design: mRNA profiles of mouse ovarian cancer cell line ID8 Control and USP9X knocked-down were generated by deep sequencing, in duplicate, using Illumina novaseq6000.