41 Nuclear Ikkß Phosphorylates ATM in Response to Genotoxic Stimuli, Which Helps Cancer Cells to Survive DNA Damage

Background and Aims. Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in up to 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable colorectal cancers, where hypermethylation of the major mismatch repair (MMR) gene hMLH1 drives multiple target gene mutations, the cause of EMAST is unknown but was recently associated with reduced expression of the minor MMR protein hMSH3 in CRCs. We assessed experimentally whether hMSH3 deficiency is a cause of EMAST.Methods. We constructed plasmids containing D8S321 and D20S82 tetranucleotide loci (12 and 16 repeats of AAAG respectively) that are used to define EMAST. Sequences were cloned +1 bp out of frame immediately after the start codon of the EGFP gene. A -4 bp frameshift deletion of one AAAG unit would allow in-frame expression of EGFP. Mutationresistant counterpart plasmids were constructed by changing 2 nucleotide sequences in every 3 units of AAAG, preventing frameshift mutations. First, we created MMR proficient, hMLH1, hMSH6, and hMSH3 stable cell lines carrying the plasmids. Subsequently, we reduced hMSH3 expression via RNAi in MMR proficient cells harboring the EMAST constructs. Non-fluorescent cells were sorted and cultured for flow cytometry analysis. Mutations were examined by DNA-sequencing. Results. Sequencing confirmed frameshift mutations in fluorescent cells containing D8S321 and D20S82. Such mutations included contraction and expansion of microsatellites. D8S321 mutations occurred 31-and 40-fold higher in hMLH1 and hMSH3 cells compared to hMSH6 cells, respectively. D20S82 mutations occurred 82-and 49-fold higher in hMLH1 and hMSH3 cells compared to hMSH6 cells, respectively. When hMSH3 expression levels were reduced in MMR proficient cells upon hMSH3 shRNA transfection, significantly higher mutation rates were detected in hMSH3 knockdown cells for D8S321 (18.14 x 10-4) and D20S82 (11.14 x 10-4) compared to their individual scramble control cells (0 and 0.26 x 10-4 separately). Conclusions. EMAST is dependent upon MMR background, with hMSH3 more prone to frameshift mutations than hMSH6, opposite to frameshift mutation observed at mononucleotide repeats. hMSH3 mimics complete MMR failure (hMLH1) in inducing EMAST. Furthermore, knock down of hMSH3 expression alone was able to elicit EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, loss of hMSH3 function appears to be the cause of EMAST.