A common early mutation in colorectal cancer is the deregulation of the Wnt/ -catenin growth factor signaling pathway. This pathway controls the activity of cellular growth control genes through stabilization or degradation of the protein -catenin, a dual transcription factor and adhesion protein. In particular, 80% of colorectal cancers deregulate Wnt signaling by truncating the scaffolding protein Adenomatous Polyposis Coli (APC) in a region called the Mutational Cluster Region (MCR). Despite over 20 years of study, there remains a critical gap in understanding how this truncation disrupts the regulated proteolytic destruction of -catenin. Understanding why this truncation leads to cessation of -catenin proteolysis could enable new treatments for colorectal cancers. My overall objective for this project is to obtain training in biochemical and biophysical techniques to understand specifically why APC truncation leads to interruption of -catenin ubiquitination and proteolytic destruction in Wnt signaling. My central hypothesis is that the Catenin Inhibitory Domain (CID) within the MCR region of APC interacts with different ubiquitination proteins throughout the cell cycle, and that this interaction is mediated by phosphorylation events within the CID.
I aim to identify the ubiquitin transfer mechanism controlling -catenin proteolysis in the cytoplasm and nucleus during the cell cycle and Wnt signaling following the hypothesis that the DDB1/Cul4 complex acts in parallel with the SKP1/Cul1 complex to destroy -catenin during homeostasis and Wnt deactivation. I also plan to identify how phosphorylation of the CID region in APC affects -catenin proteolysis. I hypothesize the APC phosphorylation site T1438 is required for aE-catenin binding and enhances -catenin proteolysis by scaffolding the aE-catenin/-catenin heterodimer to position -catenin for recognition by the ubiquitin ligase -TrCP. Fulfillment of this project will provide me with extensive training in biophysical techniques. Furthermore, they will elucidate two specific molecular interactions between APC and -catenin proteolysis machinery. These data will provide insight into why colorectal cancers usually truncate APC in the same region, and may provide new targets for cancer treatments and diagnostics. The expected outcomes are the identification and validation of new ubiquitin proteins that interact with -catenin proteolysis. Additionally, this project will define a new role for phosphorylation n the MCR region and relate it to -catenin proteolysis.
Colorectal cancer is one of the leading causes of cancer deaths in the US today, accounting for over 50,000 citizens per year. Yet, the molecular mechanism of one of the most common causes of this disease, the mutation of the protein APC, remains poorly understood. In this proposal, I intend to explore, at a molecular level, how this mutation causes cells to grow uncontrollably. Understanding this process will be important for developing new diagnostic markers and new treatments of colorectal cancer.