Mechanisms of Protein Regulation and Chemoresistance in Mismatch Repair Defective Cells

Abstract

Lynch Syndrome is a hereditary cancer syndrome associated with mutations in MSH2, a highly conserved DNA mismatch repair (MMR) gene. Lynch Syndrome individuals have a ~90% chance of getting cancer and these tumors with defective mismatch repair are largely resistant to conventional chemotherapeutics such as cisplatin. Our laboratory employs yeast to characterize clinically-identified MSH2 missense mutations. Previously we have shown that one of the primary causes of defective MMR among these variants is due to the accelerated turnover of variant forms of Msh2 via the ubiquitin-mediated degradation pathway. The ubiquitin ligase San1 targets most of the Msh2 low-level variants for degradation; however, it does not target the low-level variant Msh2R542P. By transforming ubiquitin ligase deletion strains with plasmids encoding MSH2 and msh2-R542P and quantifying Msh2 steady-state levels, we were able to identify Not4 as the ubiquitin ligase responsible for mediating the breakdown of Msh2R542P. Because Msh2 missense variants such as Msh2R542P frequently demonstrate resistance to traditional chemotherapy drugs, we utilized growth curve assays to explore the mechanism of chemoresistance in mismatch repair defective cells. After undergoing preliminary experiments to establish the optimal parameters for cisplatin sensitivity growth curve assays, we explored the possibility of utilizing a yeast two-hybrid system to characterize the ways in which MMR proteins mediate a response to DNA damage. Upon investigating the ability of Msh2 variants to interact with ATR-like (Mec1 in yeast) and BRCA-1 like (Rad9 in yeast) proteins, it appears as if a two-hybrid growth curve assay is a viable means to characterize the molecular mechanism of chemoresistance; however further exploration still needs to be done to define the optimal experimental parameters. Similarly, to develop a more comprehensive understanding of mechanism of resistance to cisplatin, we examined the differing ability of Msh2 missense variants to grow in the presence of cisplatin using growth curve assays. Using an R based program to obtain doubling times from the slopes of the lines of best fit for the exponential growth phase of a given variant upon treatment with a specific cisplatin dosage, we have characterized nine different missense mutations to exhibit wild-type sensitivity, heightened sensitivity or resistance to cisplatin.