Abstract
The MSH3 gene is one of the DNA mismatch repair (MMR) genes that has undergone somatic mutation frequently in MMR-deficient cancers. MSH3, together with MSH2, forms the MutSβ heteroduplex, which interacts with interstrand cross-links (ICLs) induced by drugs such as cisplatin and psoralen. However, the precise role of MSH3 in mediating the cytotoxic effects of ICL-inducing agents remains poorly understood. In this study, we first examined the effects of MSH3 deficiency on cytotoxicity caused by cisplatin and oxaliplatin, another ICL-inducing platinum drug. Using isogenic HCT116-derived clones in which MSH3 expression is controlled by shRNA expression in a Tet-off system, we discovered that MSH3 deficiency sensitized cells to both cisplatin and oxaliplatin at clinically relevant doses. Interestingly, siRNA-induced down-regulation of the MLH1 protein did not affect MSH3-dependent toxicity of these drugs, indicating that this process does not require participation of the canonical MMR pathway. Furthermore, MSH3-deficient cells maintained higher levels of phosphorylated histone H2AX and 53BP1 after oxaliplatin treatment in comparison with MSH3-proficient cells, suggesting that MSH3 plays an important role in repairing DNA double strand breaks (DSBs). This role of MSH3 was further supported by our findings that MSH3-deficient cells were sensitive to olaparib, a poly(ADP-ribose) polymerase inhibitor. Moreover, the combination of oxaliplatin and olaparib exhibited a synergistic effect compared with either treatment individually. Collectively, our results provide novel evidence that MSH3 deficiency contributes to the cytotoxicity of platinum drugs through deficient DSB repair. These data lay the foundation for the development of effective prediction and treatments for cancers with MSH3 deficiency.
Highlights
The DNA mismatch repair (MMR)6 system, composed of several proteins such as MLH1, MSH2, MSH6, MSH3, and PMS2, eliminates replication errors and maintains genomic stability
MSH3 Expression Is Controlled by Doxycycline in MSH3-deficient Clones—We first determined whether MSH3 expression in G1, G2, and G5 cell clones of HCT116 colorectal cancer (CRC) cells is controlled by doxycycline
We confirmed sustained levels of 53BP1 in MSH3-deficient G5 cells after oxaliplatin treatment (Fig. 5, C and D). These results suggest that the higher sensitivity of MSH3-deficient cells to oxaliplatin may in part be due to reduced DNA double strand break (DSB) repair efficiency, rather than a quantitative difference in the burden of DNA damage induced by treatment
Summary
The DNA mismatch repair (MMR) system, composed of several proteins such as MLH1, MSH2, MSH6, MSH3, and PMS2, eliminates replication errors and maintains genomic stability. The MutL complex, mainly MutL␣, an MLH1/ PMS2 heterodimer, forms a ternary complex with a MutS heterodimer that binds to DNA mismatches during replication and leads to recruitment of other proteins to complete the process of DNA MMR. MutS recognizes interstrand cross-links (ICLs) generated by DNA cross-linkers such as psoralen and cisplatin. The level of homologous recombination (HR) that repairs ICLs is dependent on MutS but not on MutS␣ or MLH1. These results suggest that MutS may cooperate with the nucleotide excision repair, HR and Fanconi anemia proteins for repairing psoralen-induced ICLs [15]. The detailed molecular mechanisms involved in repair and the cytotoxic effects of oxaliplatin-induced adducts, especially ICLs, have not been explored extensively
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