Notch Regulation in Breast Cancer

Breast cancer affects thousands of Canadian women every year, and ranks as the second most common cause of cancer death. Our group has previously shown that patients whose breast cancers abnormally express genes of the Notch family have poor survival rates. In addition, abnormalities in Notch are most common in the aggressive triple negative (TN) subtype of breast cancer for which there is currently no effective treatment. In order to understand what drives Notch activation in TN breast cancer cells, a novel Notch reporter system was developed and optimized for high throughput screens (HTS). Using the Notch reporter system, a chemical kinase inhibitor screen identified the MAPK-ERK signaling pathway as a predominant regulator of Notch ligand (JAG1) expression and Notch activation. Consistent with this, a kinome siRNA screen identified the pseudokinase Tribbles homolog 3 (TRB3), a known regulator of MAPK-ERK, among the most significant hits. We demonstrated that TRB3 is a master regulator of Notch through the MAPK-ERK and TGFβ pathways. Supporting our findings, in vitro and in vivo studies showed that TRB3 inhibition decreases breast cancer cell proliferation in a JAG1-dependant fashion and reduces the growth of MDA MB231 TN breast cancer xenografts. To understand how TRB3 is regulated, a proteomic screen of TRB3-associated cellular complexes was performed and identified the deubiquitinase USP9x as a novel interacting protein for TRB3. We showed that TRB3 serves as a scaffold for USP9x that regulates JAG1 function through two mechanisms: (1) through TRB3 deubiquitination and stabilization and (2) through deubiquitination and activation of Mind Bomb 1 (MIB1), an E3 ligase required for JAG1 activity. We also demonstrated that USP9x is required for TRB3 and JAG1 up-regulation in response to cellular stress. Taken together, the work presented in this thesis, contributes to a better understanding of Notch regulation in breast cancer and identifies novel pathways that may be therapeutically targeted in TN disease.Ph.D