Oxidative Stress Induces Nuclear-to-Cytosol Shift of hMSH3, a Potential Mechanism for EMAST in Colorectal Cancer Cells

Abstract

BackgroundElevated 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.AimTo examine if hMSH3 deficiency causes EMAST, and to explore mechanisms for its deficiency.MethodsWe 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.ConclusionEMAST 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.

<i>Fusobacterium nucleatum</i> Infection in Colorectal Cancer: Linking Inflammation, DNA Mismatch Repair and Genetic and Epigenetic Alterations

“…As mentioned above, cumulative evidence supports the idea that the etiology of MSI-L/EMAST formation in cancer genome is the inflammation-induced loss of nuclear MSH3 in dividing cells (10,37,39). Therefore, it is reasonable to speculate that a sub-group of MSI-L/EMAST CRC may be associated with pathogenic infections of microbiota including Fusobacterium .…”

“…The frequent incidence of EMAST in CRCs was confirmed by two other studies (34,35). The fact that EMAST is due to the loss of MSH3 was also proven by other studies using tissue cultured human cells (36,37). Later, Adam et al found patients with bi-allelic inactivation mutations at the MSH3 locus who suffered from colorectal adenoma polyposis syndrome.…”

“…These results suggest that some immunological and inflammatory responses are active in EMAST CRC. Later, Tseng-Rogenski et al demonstrated that in several cancer cell lines (37,39) inflammatory factors including oxidative stress (hydrogen peroxide), interleukin-6 (IL6) and prostaglandin E 2 (PGE 2 ) induce displacement of MSH3 from the nucleus to the cytoplasm, whereas the other MMR proteins do not displace. Repeated treatment of microsatellite stable colon cancer cell lines with IL6 induced EMAST.…”

Differentiating Lynch-Like From Lynch Syndrome

“…While it is important to identify Lynch syndrome from sporadic cases of colorectal cancer as patients with Lynch syndrome have recurring bouts of cancer development, the separation between Lynch-like and Lynch syndrome, to date, is not as clear cut. As shown in the table , there are a number of recognized mechanisms to inactivate DNA MMR function (4-8,10,12-18), several of which do not directly affect the sequence of the DNA MMR genes. These and other mechanisms could be operative in the germline of Lynch-like patients.…”

A Practical Approach to Tumor Heterogeneity in Clinical Research and Diagnostics

“…As recently described, a group of frequent colic tumors with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), usually related to MSH3 gene loss, presents a bad prognosis. EMAST can be associated with any kind of colon cancer, both hypermutated and nonhypermutated, and usually a high metastatic rate [86][87][88].…”