Cellular Stress Induces TRB3/USP9x-dependent Notch Activation in Cancer; Therapeutic Implications

Compared to other subtypes of breast cancer (BC), triple-negative breast cancer (TNBC) has a poor prognosis and limited therapeutic options. Activation of the Notch developmental signaling pathway is a defining feature of TNBC and is an independent predictor of outcome in this malignancy. For this reason, anti-Notch agents have been explored as potential anti-cancer therapeutics. Although Notch inhibitors have demonstrated efficacy in pre-clinical studies of breast cancer, owing to the ubiquity of Notch in normal tissues, dose-limiting toxicities in humans have limited their clinical usefulness. Thus, there is a need to devise novel approaches to block Notch signaling specifically in cancer cells. We have shown that through its control of the MAPK and TGF signaling pathways, the pseudokinase Tribbles homolog 3 (TRB3), regulates the expression of the Notch ligand, JAG1. As a stress-sensor, TRB3 is markedly upregulated during hypoxia, nutrient deprivation and ER stress (ERS), prominent features of TNBC associated with tumor progression and relapse. Through its scaffolding properties, TRB3 promotes enzyme-substrate interactions, signaling pathways, and transcriptional outputs. To further understand how TRB3 is regulated in TNBC, a proteomic screen was conducted which identified the deubiquitinase USP9x as a binding partner of TRB3. USP9x activates the Notch pathway in two ways: (1) as a deubiquitinase it protects stress-generated TRB3 from ubiquitin-mediated proteasomal degradation, resulting in JAG1 up-regulation in cancer cells and (2) USP9x regulates cancer-intrinsic Notch activation by stabilizing Mind Bomb 1 (MIB1), an E3 ligase required for JAG1 ubiquitination and stabilization. Thus, in stressed TNBC cells USP9x promotes JAG1-mediated Notch activation via both the transcriptional and post-transcriptional mechanisms. Next, we used murine models of TNBC to explore TRB3/USP9x as potential therapeutic avenues to block Notch-dependent TNBC progression. While knock-out of TRB3 showed modest anti-tumor efficacy, USP9x knock-out significantly reduced Notch activation and tumor growth. This was accompanied by a switch of the tumor immune microenvironment from a “cold” immunosuppressive to a “hot” immunoreactive, tumoricidal phenotype. Consistent with these findings, pharmaceutical inhibition of USP9x occurs without appreciable toxicity in mouse models of TNBC. This work identifies a novel therapeutic approach to block cancer-intrinsic Notch activation without affecting Notch-dependent tissue homeostasis.Ph.D