A study of the mechanism and method to control the degradation of Mad1 by regulating ubiquitin ligase activity of c-IAP1

Inhibitors of Apoptosis Proteins (IAPs) are known as important regulators of apoptosis. Human X-chromosome-linked IAP (XIAP) directly binds to caspases and inhibits their activities. However, evidence is currently lacking for a role for cellular IAP protein 1 (c-IAP1) in caspase regulation. Up-regulated in many human cancers, c-IAP1 cooperates with c-Myc by an unknown mechanism to promote tumorigenesis in a human live cancer model. Since c-IAP1 is an E3 ubiquitin ligase, we hypothesize that c-IAP1 exerts its oncogenic functions by promoting the ubiquitination and proteasomemediated degradation of certain tumor suppressors.; The Myc/Mad/Max transcription factor family contains important regulators of cell proliferation and apoptosis. Onco-protein Myc forms heterodimers with its partner Max to activate gene transcription and cell proliferation, which can be repressed by its antagonist Mad1 (Max-dimerization protein 1) through competing Max. Mad1 has been reported as a tumor suppressor with reduced expression in breast cancers.; In the chapter 2 of this dissertation, I present the identification of Mad1 as a substrate of c-IAP1-mediated ubiquitination and proteasomal degradation. The expression of c-IAP1 reduces Mad1 protein levels in cells. Knocking down c-IAP1 expression stabilizes Mad1 and increases its protein levels. The ubiquitin ligase activity of c-IAP1 is crucial for its inhibition of Mad1, which in turn cooperate with c-Myc to promote cell proliferation. My study suggests a mechanism for c-IAP1 and Myc cooperation in cells by promoting Mad1 ubiquitination and degradation. My study provides a novel mechanism to inhibit Myc-mediated tumorigenesis by inhibiting E3 ubiquitin ligase activity of c-IAP1.; Dr. Jidong Zhu in our laboratory identified a compound, Degrastatin, which inhibits c-IAP1 auto-ubiquitination. In the chapter 3 of this dissertation, I present my collaborative project on characterization of Degrastatin. Some data from Dr. Zhu will also be presented to his credit. Although further studies are needed to pinpoint its mechanism, Degrastatin inhibits Mad1 ubiquitination by c-IAP1 and stabilizes Mad1 proteins in cells. Treating multiple cell lines with Degrastatin increases endogenous Mad1 and inhibits cell proliferation. Our research provides a proof of principle for inhibiting the ubiquitin ligase activity of c-IAP1 as a novel anti-cancer method.