DNA mismatch repair (MMR) function is critical for correcting errors coincident with polymerase-driven DNA replication, and its proteins are frequent targets for inactivation (germline or somatic), generating a hypermutable tumor that drives cancer progression. The biomarker for defective DNA MMR is microsatellite instability-high (MSI-H), observed in ~15% of colorectal cancers, and defined by mono- and dinucleotide microsatellite frameshift mutations. MSI-H is highly correlated with loss of MMR protein expression, is commonly diploid, is often located in the right side of the colon, prognosticates good patient outcome, and predicts poor efficacy with 5-fluorouracil treatment. Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is another form of MSI at tetranucleotide repeats that has been observed in multiple cancers, but its etiology and clinical relevance to patient care has only been recently illuminated. Specifically, EMAST is an acquired somatic defect observed in up to 60% of colorectal cancers and caused by unique dysfunction of the DNA MMR protein MSH3 (and its DNA MMR complex MutSβ, a heterodimer of MSH2-MSH3), and in particular a loss-of-function phenotype due to a reversible shift from its normal nuclear location into the cytosol in response to oxidative stress and the pro-inflammatory cytokine interleukin-6. Tumor hypoxia may also be a contributor. Patients with EMAST colorectal cancers show diminished prognosis compared to patients without the presence of EMAST in their cancer. In addition to defective DNA MMR recognized by tetranucleotide (and di- and tri-nucleotide) frameshifts, loss of MSH3 also contributes to homologous recombination-mediated repair of DNA double stranded breaks, indicating the MSH3 dysfunction is a complex defect for cancer cells that generates not only EMAST but also may contribute to chromosomal instability and aneuploidy. Areas for future investigation for this most common DNA MMR defect among colorectal cancers include relationships between EMAST and chemotherapy response, patient outcome with aneuploid changes in colorectal cancers, target gene mutation analysis, and mechanisms related to inflammation-induced compartmentalization and inactivation for MSH3.
Background & Aims Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is the most common DNA mismatch repair (MMR) defect in colorectal cancers, observed in ~60% of specimens. This acquired genotype correlates with metastasis and poor outcome of patients, and is associated with intra-epithelial inflammation and heterogenous nuclear levels of the MMR protein hMSH3. Inflammation and accompanying oxidative stress can cause hMSH3 to change its intracellular location, but little is known about the source of oxidative stress in cancer cells. We investigated whether cytokines mediate this process. Methods We analyzed levels of interleukin 6 (IL6) and its receptor (IL6R) in human colon and lung cancer cell lines by flow cytometry and ELISA; proteins were localized by immunofluorescence and immunoblot analyses. IL6 signaling was blocked with antibodies against IL6, soluble sgp130Fc fragments, and the STAT3 inhibitor NSC74859; a constitutively active form of STAT3 was expressed in colon and lung cancer cell lines to replicate IL6R signaling. EMAST was detected by DNA fragment analysis. Immunohistochemistry was used to examine levels of IL6 in 20 colorectal tumor and adjacent non-tumor tissues. Results Incubation of colon and lung cancer cell lines with IL6, but not other cytokines, caused hMSH3, but no other MMR proteins, to move from the nucleus to the cytosol after generation of oxidative stress; inhibition of IL6 signaling prevented this shift. Expression of constitutively active STAT3 also caused hMSH3 to translocate from the nucleus to the cytoplasm in cancer cell lines. Incubation of cells with IL6 led to tetranucleotide frameshifts, the signature for EMAST. EMAST-positive colorectal tumors had significantly higher levels of IL6 that EMAST-negative tumors. Conclusions IL6 signaling disrupts the nuclear localization of hMSH3 and DNA repair, leading to EMAST in cancer cell lines. Inflammatory cytokines might therefore promote genetic alterations in human cancer cells.
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.
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