The 15-aa Repeat Region of Adenomatous Polyposis Coli: Association with β-catenin and Interplay with Cell Cycle Regulator TopoisomeraseIIα

Abstract

The tumor suppressor Adenomatous polyposis coli (APC) is a large, multi-domain protein with many identified cellular functions. The best characterized role of APC is to scaffold a protein complex that negatively regulates Wnt signaling via β-catenin destruction. This destruction is mediated by β-catenin binding to centrally located 15- and 20-amino acid (aa) repeat regions of APC. Greater than 80% of cancers of the colon and rectum present with an APC mutation. Most carcinomas with mutant APC express a truncated APC protein which retains the ~200-aa long 15-aa repeat region. Understanding why the presence of the 15-aa repeats in truncated APC is retained by cancer cells will help direct future therapeutic intervention strategies for APC mutant tumors. In this work, I show that the 15-aa repeat region of APC is intrinsically disordered. I characterize the binding of the 15-aa repeat region with binding partner β-catenin, the downstream transducer of Wnt signaling. I found that the 15-aa region of APC retains flexibility upon binding β-catenin and that APC does not have a single, observable “highest affinity” binding site for β-catenin. This flexibility has implications for the architecture and assembly of the β-catenin destruction complex. We hypothesize the disorder retained upon association allows β-catenin to be readily captured by APC and then remain accessible to other elements of the destruction complex for subsequent processing. In Chapter 4, I expand upon studies performed previously in our lab, which discovered a novel association between the tumor-suppressor APC and Topoisomerase IIα (TopoIIα). This association has implications in colon cancer progression and initiation. I show that tumors that harbor APC mutation are more resistant to chemotherapeutic compounds targeting topoisomerases than tumors with wild-type APC. I also show that APC does not appear to associate with the β-cat/TCF transcriptional complex, though it does associate with both β-catenin and TopoIIα in the nucleus. I show that TopoIIα contained in the nuclear extract from cells with mutant or no APC has enhanced catalytic activity compared to TopoIIα from cells with wild type APC. This observed effect could explain why cells with altered APC status respond differently to TopoIIα-targeting compounds. Finally, I report generation of a new polyclonal antibody raised in chicken against the central region of APC. This new tool will be valuable for APC researchers worldwide. The work presented here expands our understanding of a critical binding region of APC, and will guide future studies aimed at defining precise molecular mechanisms of Wnt signaling.