MSH3 mediates sensitization of colorectal cancer cells to cisplatin, oxaliplatin, and a poly(ADP-ribose) polymerase inhibitor

The MSH3 gene is one of the DNA mismatch repair (MMR) genes that has undergone somatic mutation frequently in MMR-deficient cancers. MSH3, together with MSH2, forms the MutSβ heteroduplex, which interacts with interstrand cross-links (ICLs) induced by drugs such as cisplatin and psoralen. However, the precise role of MSH3 in mediating the cytotoxic effects of ICL-inducing agents remains poorly understood. In this study, we first examined the effects of MSH3 deficiency on cytotoxicity caused by cisplatin and oxaliplatin, another ICL-inducing platinum drug. Using isogenic HCT116-derived clones in which MSH3 expression is controlled by shRNA expression in a Tet-off system, we discovered that MSH3 deficiency sensitized cells to both cisplatin and oxaliplatin at clinically relevant doses. Interestingly, siRNA-induced down-regulation of the MLH1 protein did not affect MSH3-dependent toxicity of these drugs, indicating that this process does not require participation of the canonical MMR pathway. Furthermore, MSH3-deficient cells maintained higher levels of phosphorylated histone H2AX and 53BP1 after oxaliplatin treatment in comparison with MSH3-proficient cells, suggesting that MSH3 plays an important role in repairing DNA double strand breaks (DSBs). This role of MSH3 was further supported by our findings that MSH3-deficient cells were sensitive to olaparib, a poly(ADP-ribose) polymerase inhibitor. Moreover, the combination of oxaliplatin and olaparib exhibited a synergistic effect compared with either treatment individually. Collectively, our results provide novel evidence that MSH3 deficiency contributes to the cytotoxicity of platinum drugs through deficient DSB repair. These data lay the foundation for the development of effective prediction and treatments for cancers with MSH3 deficiency.     

Nrf2 overexpression increases risk of high tumor mutation burden in acute myeloid leukemia by inhibiting MSH2

Nuclear factor erythroid 2-related factor 2 (Nrf2, also called NFE2L2) plays an important role in cancer chemoresistance. However, little is known about the role of Nrf2 in tumor mutation burden and the effect of Nrf2 in modulating DNA mismatch repair (MMR) gene in acute myeloid leukemia (AML). Here we show that Nrf2 expression is associated with tumor mutation burden in AML. Patients with Nrf2 overexpression had a higher frequency of gene mutation and drug resistance. Nrf2 overexpression protected the AML cells from apoptosis induced by cytarabine in vitro and increased the risk of drug resistance associated with a gene mutation in vivo. Furthermore, Nrf2 overexpression inhibited MutS Homolog 2 (MSH2) protein expression, which caused DNA MMR deficiency. Mechanistically, the inhibition of MSH2 by Nrf2 was in a ROS-independent manner. Further studies showed that an increased activation of JNK/c-Jun signaling in Nrf2 overexpression cells inhibited the expression of the MSH2 protein. Our findings provide evidence that high Nrf2 expression can induce gene instability-dependent drug resistance in AML. This study demonstrates the reason why the high Nrf2 expression leads to the increase of gene mutation frequency in AML, and provides a new strategy for clinical practice.Subject terms: Cancer genomics, Apoptosis     

The role of the human DNA mismatch repair gene hMSH2 in DNA repair, cell cycle control and apoptosis: implications for pathogenesis, progression and therapy of cancer

The cellular DNA mismatch repair (MMR) pathway, involving the DNA mismatch repair genes MLH1 and MSH2, detects and repairs DNA replication errors. Defects in MSH2 and MLH1 account for most cases of hereditary non-polyposis colorectal cancer as well as for sporadic colorectal tumors. Additionally, increased expression of MSH2 RNA and/or protein has been reported in various malignancies. Loss of DNA MMR in mammalian cells has been linked to resistance to certain DNA damaging agents including clinically important cytotoxic chemotherapeutics. Due to other functions besides its role in DNA repair, that include regulation of cell proliferation and apoptosis, MSH2 has recently been shown to be of importance for pathogenesis and progression of cancer. This review summarizes our present understanding of the function of MSH2 for DNA repair, cell cycle control, and apoptosis and discusses its importance for pathogenesis, progression and therapy of cancer.     

Cloning and expression of the Xenopus and mouse Msh2 DNA mismatch repair genes.

Bacterial MutS protein and its yeast and human homologs MSH2 trigger the mismatch repair process by their initial binding to mispaired and unpaired bases in DNA. We describe the cloning and sequencing of genes from Xenopus laevis and Mus musculus encoding the homolog of the Saccharomyces cerevisiae MSH2 (the major DNA mismatch binding protein). Mutations in the human homolog of this gene have recently been implicated in microsatellite instability and DNA mismatch repair deficiency in tumour cells from patients with the most common hereditary predisposition to cancer (Lynch syndrome, or hereditary non-polyposis colorectal cancer, HNPCC), as well as in a significant percentage of sporadic tumours. Expression of the amphibian and murine Msh2 gene in different tissues appears to be ubiquitous. The Xenopus gene is highly expressed in eggs, a model system for the biochemistry of DNA mismatch repair. Expression of the murine gene is low in all tissues examined, and is relatively high in a rapidly dividing cell line. These data are suggestive of a role for MSH2 during DNA replication.     

Restoration of Mismatch Repair Functions in Human Cell Line Nalm-6, Which Has High Efficiency for Gene Targeting

Gene targeting is a powerful approach in reverse genetics. The approach has been hampered in most of human cell lines, however, by the poor targeting efficiency. Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, exhibits exceptionally high gene targeting efficiency and is used in DNA repair and the related research fields. Nonetheless, usage of the cell line is still limited partly because it lacks expression of MSH2, a component of mismatch repair complex, which leads to increased genome instability. Here, we report successful restoration of MSH2 expression in Nalm-6 cells and demonstrate that the recovery does not affect the high targeting efficiency. We recovered the expression by introduction of cDNA sequences corresponding to exons 9 to 16 at downstream of exon 8 of the MSH2 gene. Endogenous exons 9 to 16 were deleted in the cell line. The MSH2 expression substantially reduced spontaneous HPRT mutation frequency. Moreover, gene targeting efficiency in the MSH2-expressing cells was similar to that in the MSH2-lacking cells. In fact, we generated heterozygously REV3L knockout and the catalytically dead mutants in the MSH2-proficient Nalm-6 cells with efficiency of 20–30%. The established cell line, Nalm-6-MSH+, is useful for reverse genetics in human cells.     

Deletions account for 17% of pathogenic germline alterations in MLH1 and MSH2 in hereditary nonpolyposis colorectal cancer (HNPCC) families

Hereditary nonpolyposis colorectal cancer (HNPCC) is due to defects in DNA mismatch repair (MMR) genes MSH2, MLH1, MSH6, and to a lesser extent PMS2. Of 466 suspected HNPCC families, we defined 54 index patients with either tumors of high microsatellite instability (MSI-H) and/or loss of expression for either MLH1, MSH2, and/or MSH6, but without a detectable pathogenic point mutation in these genes. This study cohort was augmented to 64 patients by 10 mutation-negative index patients from Amsterdam families where no tumors were available. Deletion/duplication screening using the multiplex ligation-dependent probe amplification (MLPA) revealed 12 deletions in MSH2 and two deletions in MLH1. These deletions constitute 17% of pathogenic germline alterations but elucidate the susceptibility to HNPCC in only 22% of the mutation-negative study cohort, pointing towards other mutation mechanisms for an inherited inactivation of MLH1 or MSH2. We describe here four novel deletions. One novel and one known type of deletion were found for three and two unrelated families, respectively. MLPA analysis proved a reliable method for the detection of genomic deletions in MLH1 and MSH2; however, sequence variations in the ligation-probe binding site can mimic single exon deletions.     

Hypermutation in Ig V genes from mice deficient in the MLH1 mismatch repair protein

During somatic hypermutation of Ig V genes, mismatched nucleotide substitutions become candidates for removal by the DNA mismatch repair pathway. Previous studies have shown that V genes from mice deficient for the MSH2 and PMS2 mismatch repair proteins have frequencies of mutation that are comparable with those from wild-type (wt) mice; however, the pattern of mutation is altered. Because the absence of MSH2 and PMS2 produced different mutational spectra, we examined the role of another protein involved in mismatch repair, MLH1, on the frequency and pattern of hypermutation. MLH1-deficient mice were immunized with oxazolone Ag, and splenic B cells were analyzed for mutations in their V kappa Ox1 light chain genes. Although the frequency of mutation in MLH1-deficient mice was twofold lower than in wt mice, the pattern of mutation in Mlh1-/- clones was similar to wt clones. These findings suggest that the MLH1 protein has no direct effect on the mutational spectrum.     

The Prevention of Repeat-Associated Deletions in Saccharomyces Cerevisiae by Mismatch Repair Depends on Size and Origin of Deletions

We have investigated the effects of mismatch repair on 1- to 61-bp deletions in the yeast Saccharomyces cerevisiae. The deletions are likely to involve unpaired loop intermediates resulting from DNA polymerase slippage. The mutator effects of mutations in the DNA polymerase δ (POL3) gene and the recombinational repair RAD52 gene were studied in combination with mismatch repair defects. The pol3-t mutation increased up to 1000-fold the rate of extended (7-61 bp) but not of 1-bp deletions. In a rad52 null mutant only the 1-bp deletions were increased (12-fold). The mismatch repair mutations pms1, msh2 and msh3 did not affect 31- and 61-bp deletions in the pol3-t but increased the rates of 7- and 1-bp deletions. We propose that loops less than or equal to seven bases generated during replication are subject to mismatch repair by the PMS1, MSH2, MSH3 system and that it cannot act on loops >=31 bases. In contrast to the pol3-t, the enhancement of 1-bp deletions in a rad52 mutant is not altered by a pms1 mutation. Thus, mismatch repair appears to be specific to errors of DNA synthesis generated during semiconservative replication.     

Histone Deacetylase 10 Regulates DNA Mismatch Repair and May Involve the Deacetylation of MutS Homolog 2

MutS homolog 2 (MSH2) is an essential DNA mismatch repair (MMR) protein. It interacts with MSH6 or MSH3 to form the MutSα or MutSβ complex, respectively, which recognize base-base mispairs and insertions/deletions and initiate the repair process. Mutation or dysregulation of MSH2 causes genomic instability that can lead to cancer. MSH2 is acetylated at its C terminus, and histone deacetylase (HDAC6) deacetylates MSH2. However, whether other regions of MSH2 can be acetylated and whether other histone deacetylases (HDACs) and histone acetyltransferases (HATs) are involved in MSH2 deacetylation/acetylation is unknown. Here, we report that MSH2 can be acetylated at Lys-73 near the N terminus. Lys-73 is highly conserved across many species. Although several Class I and II HDACs interact with MSH2, HDAC10 is the major enzyme that deacetylates MSH2 at Lys-73. Histone acetyltransferase HBO1 might acetylate this residue. HDAC10 overexpression in HeLa cells stimulates cellular DNA MMR activity, whereas HDAC10 knockdown decreases DNA MMR activity. Thus, our study identifies an HDAC10-mediated regulatory mechanism controlling the DNA mismatch repair function of MSH2.     

DNA repair gene polymorphisms and risk of early onset colorectal cancer in Lynch syndrome

DNA repair plays a pivotal role in maintaining genomic integrity with over 130 genes involved in various repair pathways that include base excision repair, nucleotide excision repair, double strand break repair and DNA mismatch repair. Polymorphisms within genes that are involved in these processes have been widely reported to be associated with cancer susceptibility in an extensive range of malignancies that include colorectal cancer (CRC). Lynch syndrome is caused by inherited germline mutations in DNA mismatch repair genes, predominantly in MLH1 and MSH2, that predispose to a variety of epithelial malignancies, most notably CRC. Despite being a relatively well understood hereditary cancer syndrome there remain several questions in relation to genetic influences on disease expression. Since Lynch syndrome is associated with a breakdown in DNA mismatch repair variation in other DNA repair genes may influence disease expression. In this report we have genotyped 424 Australian and Polish Lynch syndrome participants for eight common DNA repair gene polymorphisms to assess any association with the age of CRC onset. The DNA repair gene SNPs included in the study were: BRCA2 (rs11571653), MSH3 (rs26279), Lig4 (rs1805386), OGG1 (rs1052133), XRCC1 (rs25487), XRCC2 (rs3218536 and rs1799793) and XRCC3 (rs861539). Cox multi-variant regression modelling failed to provide any convincing evidence of an effect in any of the polymorphisms analysed. The data suggest that polymorphisms in DNA repair genes do not contribute to cancer risk in a population of CRC patients who are at increased risk of disease as a result in a deficiency of DNA mismatch repair.     

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) triggers MSH2 and Cdt2 protein-dependent degradation of the cell cycle and mismatch repair (MMR) inhibitor protein p21Waf1/Cip1

p21(Waf1/Cip1) protein levels respond to DNA damage; p21 is induced after ionizing radiation, but degraded after UV. p21 degradation after UV is necessary for optimal DNA repair, presumably because p21 inhibits nucleotide excision repair by blocking proliferating cell nuclear antigen (PCNA). Because p21 also inhibits DNA mismatch repair (MMR), we investigated how p21 levels respond to DNA alkylation by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which triggers the MMR system. We show that MNNG caused rapid degradation of p21, and this involved the ubiquitin ligase Cdt2 and the proteasome. p21 degradation further required MSH2 but not MLH1. p21 mutants that cannot bind PCNA or cannot be ubiquitinated were resistant to MNNG. MNNG induced the formation of PCNA complexes with MSH6 and Cdt2. Finally, when p21 degradation was blocked, MNNG treatment resulted in reduced recruitment of MMR proteins to chromatin. This study describes a novel pathway that removes p21 to allow cells to efficiently activate the MMR system.     

Characterization of components of the mismatch repair machinery in Trypanosoma brucei

Mismatch repair is one of a number of DNA repair pathways that cells possess to deal with damage to their genome. Mismatch repair is concerned with the recognition and correction of incorrectly paired bases, which can be base-base mismatches or insertions or deletions of a few bases, and appears to have been conserved throughout evolution. Primarily, this is concerned with increasing the fidelity of DNA replication, but also has important roles in the regulation of homologous recombination and the correction of chemical damage. In this study, we describe five genes in the protistan parasite Trypanosoma brucei that are likely to be involved in nuclear mismatch repair. The predicted T. brucei mismatch repair genes are diverged compared with their likely counterparts in the other eukaryotes examined to date. To demonstrate that these do indeed encode a functional nuclear mismatch repair system, we made T. brucei null mutants in two of the genes, MSH2 and MLH1, that are likely to be central to the functioning of the mismatch repair machinery. These mutations resulted in increased rates of sequence variation at a number of microsatellite loci in the parasite genome, and led to increased tolerance to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, both phenotypes consistent with mismatch repair impairment.     

Isolation and Characterization of Point Mutations in Mismatch Repair Genes That Destabilize Microsatellites in Yeast

The stability of simple repetitive DNA sequences (microsatellites) is a sensitive indicator of the ability of a cell to repair DNA mismatches. In a genetic screen for yeast mutants with elevated microsatellite instability, we identified strains containing point mutations in the yeast mismatch repair genes, MSH2, MSH3, MLH1, and PMS1. Some of these mutations conferred phenotypes significantly different from those of null mutations in these genes. One semidominant MSH2 mutation was identified. Finally we showed that strains heterozygous for null mutations of mismatch repair genes in diploid strains in yeast confer subtle defects in the repair of small DNA loops.     

Functional analysis of HNPCC-related missense mutations in MSH2

Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with germline mutations in the human DNA mismatch repair (MMR) genes, most frequently MSH2 and MLH1. The majority of HNPCC mutations cause truncations and thus loss of function of the affected polypeptide. However, a significant proportion of MMR mutations found in HNPCC patients are single amino acid substitutions and the functional consequences of many of these mutations in DNA repair are unclear. We have examined the consequences of seven MSH2 missense mutations found in HNPCC families by testing the MSH2 mutant proteins in functional assays as well as by generating equivalent missense mutations in Escherichia coli MutS and analyzing the phenotypes of these mutants. Here we show that two mutant proteins, MSH2-P622L and MSH2-C697F confer multiple biochemical defects, namely in mismatch binding, in vivo interaction with MSH6 and EXO1, and in nuclear localization in the cell. Mutation G674R, located in the ATP-binding region of MSH2, appears to confer resistance to ATP-dependent mismatch release. Mutations D167H and H639R show reduced mismatch binding. Results of in vivo experiments in E. coli with MutS mutants show that one additional mutant, equivalent of MSH2-A834T that do not show any defects in MSH2 assays, is repair deficient. In conclusion, all mutant proteins (except for MSH2-A305T) have defects; either in mismatch binding, ATP-release, mismatch repair activity, subcellular localization or protein-protein interactions.     

Separation-of-Function Mutations in Saccharomyces cerevisiae MSH2 That Confer Mismatch Repair Defects but Do Not Affect Nonhomologous-Tail Removal during Recombination

Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication. In addition to their role in mismatch repair (MMR), the MSH2 and MSH3 gene products are required to remove 3' nonhomologous DNA tails during genetic recombination. The mismatch repair genes MSH6, MLH1, and PMS1, whose products interact with Msh2p, are not required in this process. We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair. Twenty-four msh2 mutations that conferred a dominant negative phenotype for mismatch repair were isolated. A subset of these mutations mapped to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds. Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the removal of nonhomologous DNA tails during genetic recombination. The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein complexes that are thought to play different roles during DNA repair and genetic recombination.     

Mismatch repair gene mutations in Chinese HNPCC patients

To explore the characteristics of DNA mismatch repair gene mutations in Chinese patients with hereditary non-polyposis colorectal cancer (HNPCC) or Lynch syndrome, the MLH1 and MSH2 genes from probands of 76 HNPCC families were sequenced. By doing so, two frame-shift mutations, three splice-site mutations and fourteen missense mutations (thirteen missense mutations and one nonsense mutation) were identified in the MLH1 gene. In addition, one splice-site mutation and six missense mutations were detected in the MSH2 gene. None of these mutations were detected in 100 matched healthy controls. The remaining mutation-negative cases were subjected to large fragment deletion analysis using multiplex ligation-dependent probe amplification (MLPA). By doing so, five large fragment deletions were detected in the MSH2 gene. No large fragment deletions were detected in the MLH1 gene. We conclude that the MLH1 and MSH2 genes in Chinese HNPCC families exhibit broad mutation spectra.