Understanding oncoprotein networks in cancer cells using knock - in and knock-out AP - MS

摘要

We have focused on biological significant Wnt signaling pathways in this study on selecting important oncogen e/oncoprotein targets. Adenomatous polyposis coli (APC) is a key component of Wnt pathways that have been found mutated in about 80% of colorectal cancers. Knock- in cell lines APC were constructed as previously described [2]. Beta - catenin (β - catenin) is ano ther Wnt signaling oncoprotein encoded by CTNNB1 gene, which has one allele being mutated in colorectal cancer cells HCT116. Knock -out cells of either wild type or mutant allele of CTNNB1 have been generated, resulting in either β- catenin mutant -only or wild type - only HCT116 cells. We then confirmed that the APC endogenous proteins were expressed at comparable levels to the triple FLAG tagged APC proteins. We have also confirmed that wild type or mutant β - catenin have been expressed at comparative level in colorectal cancer HCT116 cells. Protein complexes were then recovered using affinity purifications using either anti- FLAG or anti- β- catenin antibodies from the total protein extracts or subcellular fractionated protein pools from the cells, which in turn increased the specificity of identifying these bait proteins and their interactors by LC/MS - MS. Novel APC interactors such as POLR2E, RQCD1, KPNB1, TPM1, and RAN have been identified. We also identified FLII and ERBB2IP as novel APC interactors by means of AP- MS in the subcellular fractions, indicating the sensitivity of AP- MS can be further increased by subcellular fractionation. In addition, we are able to map mutant oncoprotein β - catenin network comparing mutant β - catenin protein interactome to wild type β- catenin interactome in colorectal cancer cells HCT116. Our results show that combining gene knock- in and knock-out strategies to AP- MS platform allows in- depth analyses of oncoprotein networks in cancer cells, which will unravel molecular mechanism of certain diseases to protein - protein interaction level and facilitate their therapies in the long term.