MSH3-Deficiency Initiates EMAST without Oncogenic Transformation of Human Colon Epithelial Cells

<div><h3>Background/Aim</h3><p>Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency.</p> <h3>Methods</h3><p>HCT116 and HCT116+chr3 (both MSH3-deficient) and primary human colon epithelial cells (HCEC, MSH3-wildtype) were stably transfected with an EGFP-based reporter plasmid for the detection of frameshift mutations within an [AAAG]17 repeat. MSH3 was silenced by shRNA and changes in protein expression were analyzed by shotgun proteomics. Colony forming assay was used to determine oncogenic transformation and double strand breaks (DSBs) were assessed by Comet assay.</p> <h3>Results</h3><p>Despite differential MLH1 expression, both HCT116 and HCT116+chr3 cells displayed comparable high mutation rates (about 4×10⁻⁴) at [AAAG]17 repeats. Silencing of MSH3 in HCECs leads to a remarkable increased frameshift mutations in [AAAG]17 repeats whereas [CA]13 repeats were less affected. Upon MSH3-silencing, significant changes in the expression of 202 proteins were detected. Pathway analysis revealed overexpression of proteins involved in double strand break repair (MRE11 and RAD50), apoptosis, L1 recycling, and repression of proteins involved in metabolism, tRNA aminoacylation, and gene expression. MSH3-silencing did not induce oncogenic transformation and DSBs increased 2-fold.</p> <h3>Conclusions</h3><p>MSH3-deficiency in human colon epithelial cells results in EMAST, formation of DSBs and significant changes of the proteome but lacks oncogenic transformation. Thus, MSH3-deficiency alone is unlikely to drive human colon carcinogenesis.</p> </div

MSH3-silencing leads to increased double strand breaks in HCECs.

<p>MSH3 was silenced in HCECs and DSBs were analyzed by SCGE. For each experimental point, three cultures were processed. From each culture three slides were prepared and from each slide 50 cells were analyzed for comet formation. Data represent mean ± SD from one experiment. Stars indicate statistical significance (P<0.05).</p

Characterization of frame-shift reporter-plasmid and -cell lines.

<p>(A) Sequence analysis of pIREShyg2-EGFP-[AAAG]17 plasmid. Genomic DNA was isolated, the EGFP region containing the [AAAG]17-repeat was amplified by PCR and sequenced. (B) Verification of plasmid insertion number by Southern blot analysis. 20µg of total DNA was digested with BamHI, EcoRV, or both (B/E), resolved on a 0.8% agarose gel, and transferred onto a nylon membrane. Complementary EGFP-DNA was labeled with [P-32]-dCTP, hybridized, and the blots were analyzed by autoradiography. The 811bp fragment (harboring the coding region for EGFP with the [AAAG]17 microsatellite] is generated by restriction with BamHI (position 957) and EcoRV (position 1768). (C) Flow cytometric analysis of unsorted HCT116-[AAAG]17 and HCT116+chr3-[AAAG]17 cell clones showing similar accumulation of EGFP-positive (mutated) cells and fluorescence intensities (the geometric mean of the FL1 intensity was expressed as mean +SD for the M1 and the M2 fractions).</p

Mutation spectrum within tetranucleotide repeats.

<p>−2, −1, 1. Change in the number of [AAAG]-units (e.g. −2 =  loss of 8 bp or [AAAG]2).</p><p>−1, 0: Wildtype or frameshift mutant cells without expected EGFP fluorescence.</p><p>−2, 1: In-frame mutant cells with expected EGFP fluorescence.</p><p>−1/−2, 0/1: Heteroduplex mutant cells with expected partial (dim) EGFP fluorescence.</p>*<p>including one mutant with a 2 bp (AA) deletion.</p

Interaction partners of MSH3.

<p>MSH3-Interaction partners were analyzed by string-db-org (<a href="http://string-db.org" target="_blank">http://string-db.org</a>) set to a medium confidence of 0.400 using the active prediction mMethods; Neighborhood, Gene Fusion, Co-occurrence, Co-expression, Experiments, Databases, and Textmining (see legend).</p

Mutation rates within tetra- and dinucleotide repeats.

<p>Data are mean ± SEM.</p><p>Mutation rates are expressed as mutations per microsatellite per generation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050541#pone.0050541-Gasche1" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050541#pone.0050541-Natarajan1" target="_blank">[73]</a>.</p><p>ML. maximum likelihood method.</p><p>MM. method of the mean.</p>*<p>Between mutation rates (MM) of MMR-deficient (HCT116) and MMR-corrected (HCT116+chr3).</p>**<p>Mutation rates may be underrated as clones are a mix of [AAAG]17 and 1-unit deletion mutants (as illustrated in the M0 fraction in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050541#pone-0050541-t002" target="_blank">Table 2</a>).</p><p>n.s. not significant.</p

MMR-protein expression and MSH3-silencing in colon epithelial cells.

<p>(A) Western blot analysis of HCT116, HCT116+chr3 and HCEC-1CT cells. (B) Silencing of MMR-protein MSH3 by gene-specific shRNA in HCEC-1CT-[AAAG]17 cells resulted in 70% repression of MSH3. MSH6 was not affected.</p