Background: Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable CRCs where hypermethylation of the DNA mismatch repair (MMR) gene hMLH1's promoter is causal, the precise cause of EMAST is not clearly defined but points towards hMSH3 deficiency.
Aim: To examine if hMSH3 deficiency causes EMAST, and to explore mechanisms for its deficiency.
Methods: We measured -4 bp framshifts at D8S321 and D20S82 loci within EGFP-containing constructs to determine EMAST formation in MMR-proficient, hMLH1⁻/⁻, hMSH6⁻/⁻, and hMSH3⁻/⁻ CRC cells. We observed the subcellular location of hMSH3 with oxidative stress.
Results: D8S321 mutations occurred 31-and 40-fold higher and D20S82 mutations occurred 82-and 49-fold higher in hMLH1⁻/⁻ and hMSH3⁻/⁻ cells, respectively, than in hMSH6⁻/⁻ or MMR-proficient cells. hMSH3 knockdown in MMR-proficient cells caused higher D8S321 mutation rates (18.14 and 11.14×10⁻⁴ mutations/cell/generation in two independent clones) than scrambled controls (0 and 0.26×10⁻⁴ mutations/cell/generation; p<0.01). DNA sequencing confirmed the expected frameshift mutations with evidence for ongoing mutations of the constructs. Because EMAST-positive tumors are associated with inflammation, we subjected MMR-proficient cells to oxidative stress via H₂O₂ to examine its effect on hMSH3. A reversible nuclear-to-cytosol shift of hMSH3 was observed upon H₂O₂ treatment.
Conclusion: EMAST is dependent upon the MMR background, with hMSH3⁻/⁻ more prone to frameshift mutations than hMSH6⁻/⁻, opposite to frameshift mutations observed for mononucleotide repeats. hMSH3⁻/⁻ mimics complete MMR failure (hMLH1⁻/⁻) in inducing EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, hMSH3-deficiency appears to be the event that commences EMAST. Oxidative stress, which causes a shift of hMSH3's subcellular location, may contribute to an hMSH3 loss-of-function phenotype by sequestering it to the cytosol.