DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

Mismatch repair (MMR) enzymes have been shown to be deficient in prostate cancer (PCa). MMR can influence the regulation of tumor development in various cancers but their role on PCa has not been investigated. The aim of the present study was to determine the functional effects of the mutL-homolog 1 (MLH1) gene on growth of PCa cells. The DU145 cell line has been established as MLH1-deficient and thus, this cell line was utilized to determine effects of MLH1 by gene expression. Lack of MLH1 protein expression was confirmed by Western blotting in DU145 cells whereas levels were high in normal PWR-1E and RWPE-1 prostatic cells. MLH1-expressing stable transfectant DU145 cells were then created to characterize the effects this MMR gene has on various growth properties. Expression of MLH1 resulted in decreased cell proliferation, migration and invasion properties. Lack of cell growth in vivo also indicated a tumor suppressive effect by MLH1. Interestingly, MLH1 caused an increase in apoptosis along with phosphorylated c-Abl, and treatment with MLH1 siRNAs countered this effect. Furthermore, inhibition of c-Abl with STI571 also abrogated the effect on apoptosis caused by MLH1. These results demonstrate MLH1 protects against PCa development by inducing c-Abl-mediated apoptosis.     

Chromate exposure induces DNA hypermethylation of the mismatch repair gene MLH1 in lung cancer

Hexavalent chromium is recognized as a human carcinogen. Our previous studies revealed that lung cancer (LC) in chromate-exposed workers (chromate LC) had molecular features of frequent microsatellite instability (MSI), repression of MLH1 level, and aberrant DNA methylation of several tumor-suppressor genes, including MLH1. In the present study, we quantitatively investigated MLH1-promoter methylation status using bisulfite pyrosequencing of paired tumorous/nontumorous tissues from chromate and nonchromate LCs to determine the effect of chromate exposure on MLH1-promoter methylation. The methylation level of MLH1 promoter was significantly higher in chromate LC tumors (P < .001) than nonchromate LC tumors and, among chromate LC, significantly higher in tumorous tissue than nontumorous tissue (P = .004). Moreover, the methylation level of MLH1 promoter in normal lung tissue tended to be higher in chromate LC than nonchromate LC (P = .062). In addition, LC with reduced levels of MLH1 showed significantly higher methylation levels of MLH1 promoter than LC exhibiting normal MLH1 levels (P = .019). Moreover, immunohistochemical analyses determined that levels of SUV39H1, an H3K9me2-related methyltransferase, were higher in chromate LC than nonchromate LC (P = .076). Furthermore, we evaluated three DNA double-strand break-repair genes (MRE11, RAD50, and DNA-PKcs) as possible targets of MSI by fragment-length polymorphism analysis, revealing the mutation frequency of RAD50 as significantly higher in chromate LC than nonchromate LC (P = .047). These results suggest that chromate exposure might induce MLH1 hypermethylation in LC as a mechanism of chromate-induced carcinogenesis.     

Functional analysis of human MLH1 variants using yeast and in vitro mismatch repair assays

The functional characterization of nonsynonymous single nucleotide polymorphisms in human mismatch repair (MMR) genes has been critical to evaluate their pathogenicity for hereditary nonpolyposis colorectal cancer. We previously established an assay for detecting loss-of-function mutations in the MLH1 gene using a dominant mutator effect of human MLH1 expressed in Saccharomyces cerevisiae. The purpose of this study is to extend the functional analyses of nonsynonymous single nucleotide polymorphisms in the MLH1 gene both in quality and in quantity, and integrate the results to evaluate the variants for pathogenic significance. The 101 MLH1 variants, which covered most of the reported MLH1 nonsynonymous single nucleotide polymorphisms and consisted of one 3-bp deletion, 1 nonsense and 99 missense variants, were examined for the dominant mutator effect by three yeast assays and for the ability of the variant to repair a heteroduplex DNA with mismatch bases by in vitro MMR assay. There was diversity in the dominant mutator effects and the in vitro MMR activities among the variants. The majority of functionally inactive variants were located around the putative ATP-binding pocket of the NH(2)-terminal domain or the whole region of the COOH-terminal domain. Integrated functional evaluations contribute to a better prediction of the cancer risk in individuals or families carrying MLH1 variants and provide insights into the function-structure relationships in MLH1.     

Interactions of the DNA mismatch repair proteins MLH1 and MSH2 with c-MYC and MAX

MSH2 and MLH1 have a central role in correcting mismatches in DNA occurring during DNA replication and have been implicated in the engagement of apoptosis induced by a number of cytotoxic anticancer agents. The function of MLH1 is not clearly defined, although it is required for mismatch repair (MMR) and engagement of apoptosis after certain types of DNA damage. In order to identify other partners of MLH1 that may be involved in signalling MMR or apoptosis, we used human MLH1 in yeast two-hybrid screens of normal human breast and ovarian cDNA libraries. As well as known partners of MLH1 such as PMS1, MLH3 and MBD4, we identified the carboxy terminus of the human c-MYC proto-oncogene as an interacting sequence. We demonstrate, both in vitro by yeast two-hybrid and GST-fusion pull-down experiments, as well as in vivo by coimmunoprecipitation from human tumour cell extracts, that MLH1 interacts with the c-MYC protein. We further demonstrate that the heterodimeric partner of c-MYC, MAX, interacts with a different MMR protein, MSH2, both in vitro and in vivo. Using an inducible c-MYC-ER fusion gene, we show that elevated c-MYC expression leads to an increased HGPRT mutation rate of Rat1 cells and an increase in the number of frameshift mutants at the HGPRT locus. The effect on HGPRT mutation rate is small (2-3-fold), but is consistent with deregulated c-MYC expression partially inhibiting MMR activity.     

MLH1 promoter hypermethylation predicts poorer prognosis in mismatch repair deficiency endometrial carcinomas

ObjectiveThe antitumor effects of anti-PD-1 antibody against mismatch repair deficiency (MMR-D)-associated cancers have been reported. MMR-D is found in approximately 20%–30% of endometrial carcinomas (ECs) and frequently occurs due to MLH1 promoter hypermethylation (MLH1-PHM). ECs with MLH1-PHM are classified according to the molecular screening of Lynch syndrome (LS), but few detailed reports are available. The purpose of this study was to clarify the clinical features of EC with MLH1-PHM.MethodsImmunohistochemistry of MMR proteins (MLH1, MSH2, MSH6, and PMS2) was performed on specimens from 527 ECs treated at our university hospital from 2003 to 2018. MLH1 methylation analysis was added to cases with MLH1/PMS2 loss. ECs were classified as follows: cases that retained MMR proteins as “MMR-proficient;” cases with MLH1/PMS2 loss and MLH1-PHM as “met-EC;” and cases with other MMR protein loss and MLH1/PMS2 loss without MLH1-PHM as “suspected-LS.” The clinical features, including long-term prognosis, of each group, were analyzed.ResultsAccordingly, 419 (79.5%), 65 (12.3%), and 43 (8.2%) cases were categorized as “MMR-proficient,” “suspected-LS,” and “met-EC,” respectively. Significantly, “met-EC” had a lower proportion of grade 1 tumors (37.5%) and a higher proportion of stage III/IV tumors (37.2%) than the other groups. The overall and progression-free survival of “met-EC” were significantly worse than those of “suspected-LS” in all cases.ConclusionIn ECs with MMR-D, “met-ECs” were a subgroup with a poorer prognosis than “suspected-LS.” “Met-ECs” would be the main target for anti-PD-1 antibody treatment, and its clinical susceptibility should be verified individually.     

缺氧对人小细胞肺癌H446细胞DNA错配修复基因MLH1、MSH2表达的影响及其机制探讨

背景与目的:缺氧对DNA错配修复系统(mismatch repair, MMR)活性的调控是肿瘤细胞遗传不稳定的重要原因,但其机制尚不完全清楚.本研究拟观察缺氧状态下人小细胞肺癌H446细胞DNA错配修复基因MLH1、MSH2的表达变化,初步探讨DNA甲基化在其中的作用.方法:应用RT-PCR、Western blot等方法检测H446细胞在缺氧状态下以及甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-CdR)处理后MLH1、MSH2基因的表达水平,同时,采用甲基化特异性PCR(MSP)方法检测MLH1、MSH2基因启动子CpG岛甲基化状态.结果:缺氧状态下,H446细胞MLH1、MSH2基因在转录和翻译水平均显著性降低.同时,随着缺氧时间延长,MLH1基因启动子逐渐由非甲基化状态、部分甲基化状态转变为完全甲基化状态,而MSH2基因启动子则直接由非甲基化状态转变为完全甲基化状态.甲基转移酶抑制剂5-Aza-CdR可使MLH1、MSH2基因表达水平有所恢复,但去除5-Aza-CdR后其表达再次下调.结论:启动子甲基化可能是缺氧诱导H446细胞显著性下调MLH1、MSH2基因表达的重要机制,甲基转移酶抑制剂5-Aza-CdR可恢复其表达.     

Myc down-regulation sensitizes melanoma cells to radiotherapy by inhibiting MLH1 and MSH2 mismatch repair proteins.

PURPOSE: Melanoma patients have a very poor prognosis with a response rate of <1% due to advanced diagnosis. This type of tumor is particularly resistant to conventional chemotherapy and radiotherapy, and the surgery remains the principal treatment for patients with localized melanoma. For this reason, there is particular interest in the melanoma biological therapy. EXPERIMENTAL DESIGN: Using two p53 mutant melanoma models stably expressing an inducible c-myc antisense RNA, we have investigated whether Myc protein down-regulation could render melanoma cells more susceptible to radiotherapy, reestablishing apoptotic p53-independent pathway. In addition to address the role of p53 in the activation of apoptosis, we studied the effect of Myc down-regulation on radiotherapy sensitivity also in a p53 wild-type melanoma cell line. RESULTS: Myc down-regulation is able per se to induce apoptosis in a fraction of the cell population (approximately 40% at 72 hours) and in combination with gamma radiation efficiently enhances the death process. In fact, approximately 80% of apoptotic cells are evident in Myc down-regulated cells exposed to gamma radiation for 72 hours compared with approximately 13% observed after only gamma radiation treatment. Consistent with the enhanced apoptosis is the inhibition of the MLH1 and MSH2 mismatch repair proteins, which, preventing the correction of ionizing radiation mismatches occurring during DNA replication, renders the cells more prone to radiation-induced apoptosis. CONCLUSIONS: Data herein reported show that Myc down-regulation lowers the apoptotic threshold in melanoma cells by inhibiting MLH1 and MSH2 proteins, thus increasing cell sensitivity to gamma radiation in a p53-independent fashion. Our results indicate the basis for developing new antitumoral therapeutic strategy, improving the management of melanoma patients.     

Endometrial cancer risk is associated with variants of the mismatch repair genes MLH1 and MSH2.

Women with germ-line mutations in the mismatch repair genes (responsible for hereditary nonpolyposis colorectal cancer) face an increased risk of colonic and endometrial cancer. However, these germ-line mutations are rare and are responsible for fewer than 1% of endometrial cancers. Therefore, we examined whether or not common variants of the hereditary nonpolyposis colorectal cancer-associated genes might also be associated with an increased risk of endometrial cancer. Three single-nucleotide polymorphisms were selected in the MLH1 and MSH2 mismatch repair genes. All the various 672 women with endometrial cancer and 880 controls were genotyped. Each of these three single-nucleotide polymorphisms was associated with an increased risk of endometrial cancer. Carriers of the MLH1 nt-93 A allele were at a 1.5-fold increased risk of developing endometrial cancer compared with controls [95% confidence interval (95% CI), 1.2-2.0; P = 0.001]. The risk was higher for homozygote carriers [odds ratio (OR), 1.9; 95% CI, 1.2-3.2; P = 0.009]. For carriers of the MSH2 rs2303428 C allele, the OR was 1.4 (95% CI, 1.0-1.9; P = 0.05), and for carriers of the MSH2 rs2059520 G allele, the OR was 1.3 (95% CI, 1.0-1.7; P = 0.03). More than 9% of endometrial cancer cases carried a variant allele in both MLH1 and MSH2. For these women, the risk of endometrial cancer was particularly high (OR, 2.1; 95% CI, 1.2-3.6; P = 0.005). For patients younger than 50 years at diagnosis who carried both variants, the risk was even higher (OR, 3.4; 95% CI, 1.7-6.6; P = 0.0005). In summary, two common variant alleles of the MLH1 and MSH2 genes make a substantial contribution to endometrial cancer incidence in Ontario.     

Characterization of MLH1 and MSH2 DNA mismatch repair proteins in cell lines of the NCI anticancer drug screen

Purpose and methods: The lack of a functional DNA mismatch repair (MMR) pathway has been recognized as a common characteristic of several different types of human cancers due to mutation affecting one of the MMR genes or due to promoter methylation gene silencing. These MMR-deficient cancers are frequently resistant to alkylating agent chemotherapy such as DNA-methylating or platinum-containing compounds. To correlate drug resistance with MMR status in a large panel of human tumor cell lines, we evaluated by Western blot the cellular levels of the two MMR proteins most commonly mutated in human cancers, MLH1 and MSH2, in the NCI human tumor cell line panel. This panel consists of 60 cell lines distributed among nine different neoplastic diseases. Results: We found that in most of these cell lines both MLH1 and MSH2 were expressed, although at variable levels. Five cell lines (leukemia CCRF-CEM, colon HCT 116 and KM12 and ovarian cancers SK-OV-3 and IGROV-1) showed complete deficiency in MLH1 protein. MSH2 protein was detected in all 57 cell lines studied. Absence of MLH1 protein was always linked to resistance to the methylating chemotherapeutic agent temozolomide. This resistance was independent of cellular levels of O⁶-alkylguanine DNA alkyltransferase. Based on data available for review in the NCI COMPARE database, cellular levels of MLH1 and MSH2 did not correlate significantly with sensitivity to any standard anticancer drug or with any characterized molecular target already tested against the same panel of cell lines. Conclusion: Based on evaluation of 60 tumor cell lines in the NCI anticancer drug screen, MLH1 deficiency was more common than MSH2 deficiency and was always associated with a high degree of temozolomide resistance. These data will enable correlations with other drug sensitivities and molecular targets in the COMPARE database to evaluate linked processes in tumor drug resistance. Received: 13 December 1999 / Accepted: 27 July 2000     

Characterization of the mismatch repair defect in the human lymphoblastoid MT1 cells

Mutations in mismatch repair (MMR) genes predispose to hereditary nonpolyposis colon cancer. Those leading to truncated proteins bring about a MMR defect, but phenotypes of missense mutations are harder to predict especially if they do not affect conserved residues. Several systems capable of predicting the phenotypes of MMR missense mutations were described. We deployed one of these to study the MMR defect in MT1 cells, which carry mutations in both alleles of the hMSH6 gene. In one, an A-->T transversion brings about an Asp(1213)Val amino acid change in the highly conserved ATP binding site, whereas the other carries a G-->A transition, which brings about a Val(1260)Ile change at a nonconserved site. The hMSH2/hMSH6 (hMutS alpha) heterodimers carrying these mutations were expressed in the baculovirus system and tested in in vitro MMR assays. As anticipated, the Asp(1213)Val mutation inactivated MMR by disabling the variant hMutS alpha from translocating along the DNA. In contrast, the recombinant Val(1260)Ile variant displayed wild-type activity. Interestingly, partial proteolytic analysis showed that this heterodimer was absent from MT1 extracts, although both hMSH6 alleles in MT1 cells could be shown to be transcribed with an efficiency similar to each other and to that seen in control cells. The MMR defect in MT1 cells is thus the compound result of one mutation that inactivates the ATPase function of hMutS alpha and a second mutation that apparently destabilizes the Val(1260)Ile hMSH6 protein in human cells in vivo.     

Identification of germline MLH1 alterations in familial prostate cancer

Several linkage and loss of heterozygosity (LOH) analyses suggest that the region 3p21-p26, which is a chromosomal location of MLH1, could harbour a susceptibility gene for prostate cancer (PRCA). Furthermore, in a recent candidate single nucleotide polymorphism (SNP) analysis the I219V variation of the MLH1 gene was associated with PRCA. Microsatellite instability (MSI) and germ-line MLH1 mutations were originally demonstrated in hereditary non-polyposis colorectal cancer (HNPCC) but MSI and loss of MLH1 function have also been detected in PRCA. To assess the contribution of MLH1 germline mutations to the development of PRCA in Finland different approaches were used. First, the samples from 11 PRCA-colon cancer patients were screened for MLH1, MSH2 and MSH6 protein expression by immunohistochemistry (IHC). IHC revealed one patient with a putative MLH1 aberration and sequencing of this sample revealed five sequence variants including two missense variants P434L and I219V. Second, the samples from Finnish hereditary prostate cancer (HPC) families were used for the screening of MLH1 mutations which produced twelve MLH1 sequence variants including two missense mutations, I219V, as in the PRCA-colon cancer patient, and V647M. P434L and V647 were both novel, rare variants. Carrier frequencies of the I219V mutation were compared between hereditary prostate cancer (HPC) patients, unselected PRCA cases, patients with benign prostate hyperplasia and controls, but no differences between the sample groups were found. P434L was not present in this study population and V647M was a very rare variant found only in one HPC family. According to the present results, MLH1 does not have a major role in PRCA causation in Finland.     

HNPCC mutations in the human DNA mismatch repair gene hMLH1 influence assembly of hMutLalpha and hMLH1-hEXO1 complexes.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common inherited form of neoplasia caused by germline mutations in DNA mismatch repair (MMR) genes. MMR proteins have been reported to associate with several proteins, including the human exonuclease 1 (hEXO1). We report here novel HNPCC-hMLH1 mutant proteins (T117M, Q426X and 1813insA) in Danish HNPCC patients. We demonstrate that these mutant HNPCC-hMLH1 proteins are unable to form complexes with hEXO1 and hPMS2 in vivo. The results indicate that mutations found in HNPCC gene carriers disrupt hMLH1-hEXO1 complex formation and hMutLalpha heterodimer assembly essential for MMR activity.     

Multivariate analysis of MLH1 c.1664T>C (p.Leu555Pro) mismatch repair gene variant demonstrates its pathogenicity

Genetic testing of an Irish kindred identified an exonic nucleotide substitution c.1664T>C (p.Leu555Pro) in the MLH1 mismatch repair (MMR) gene. This previously unreported variant is classified as a “variant of uncertain significance” (VUS). Immunohistochemical (IHC) analysis and microsatellite instability (MSI) studies, genetic testing, a literature and online MMR mutation database review, in silico phenotype prediction tools, and an in vitro MMR activity assay were used to study the clinical significance of this variant. The MLH1 c.1664T>C (p.Leu555Pro) VUS co-segregated with three cases of classic Lynch syndrome-associated malignancies over two generations, with consistent loss of MLH1 and PMS2 protein expression on IHC, and evidence of the MSI-High mutator phenotype. The leucine at position 555 is well conserved across a number of species, and this novel variant has not been reported as a normal polymorphism in the general population. In silico and in vitro analyses suggest that this variant may have a deleterious effect on the MLH1 protein and abrogate MMR activity. Evidence from clinical, histological, immunohistochemical, and molecular genetic data suggests that MLH1 c.1664T>C (p.Leu555Pro) is likely to be the pathogenic cause of Lynch syndrome in this family.     

Methylation of the MLH1 gene in hematological malignancies

Hypermethylation of the MLH1 gene has been described in many kinds of human cancers with microsatellite instability (MSI). However, it is not clear whether the same mechanism occurs in hematological malignancies. Genomic DNA was extracted from 31 patients with adult T-cell leukemia/lymphoma (ATL), 9 patients with acute lymphoblastic leukemia (ALL) who had MSI, and 12 leukemia and lymphoma cell lines with MSI. Aberrant methylation of the MLH1 gene was found in 2/31 (6%) ATL patients, and in 1/12 (8%) cell lines with MSI. MLH1 promoter was not methylated in either of the twelve peripheral blood samples from normal individuals or ALL samples. The MLH1 gene was expressed in the normal peripheral blood samples, but not in the MLH1-methylated cell line KCL22. Demethylation with 5-Azacytidine treatment restored MLH1 expression in the KCL22 cell line. Methylation of the MSH2 gene was not found in any of the samples. Our data show that hypermethylation of the MLH1 gene is occasionally involved in the pathogenesis of hematological malignancies, but is not always associated with MSI.     

ＤＮＡ错配修复基因ｈＭＬＨ１与肿瘤及化疗耐药相关性的研究进展

ＤＮＡ是生命活动最重要的遗传物质。ＤＮＡ复制产生的误差（碱基错配）是一种重要的损伤。若ＤＮＡ损伤得不到修复,将会导致基因的突变,其中一部分突变有利于物种的进化,而另一部分将导致细胞恶化和死亡。ＤＮＡ错配修复系统（ｍｉｓｍａｔｃｈｒｅｐａｉｒｓｙｓｔｅｍ,ＭＭＲ）是人体细胞中存在的一种能识别并修复ＤＮＡ碱基错配的安全保障系统。到目前为止,已从人体细胞中共分离克隆到９种ＭＭＲ基因。ｈＭＬＨ１基因是一系列ＤＮＡ错配修复基因中最重要的一种,也是ＭＭＲ系统的重要成分,其甲基化可导致ＭＭＲ缺陷。近年来的研究发现,ｈＭＬＨ１的失活与肿瘤发生发展有关,并可能因此导致肿瘤对某些抗肿瘤药物耐药。     

Cancer risk in mismatch repair gene mutation carriers

Optimally accurate and valid estimates of cancer risk in genetically-defined subgroups requires population-based research. For rare genetic traits, very large studies are needed. Such is the case in hereditary nonpolyposis colorectal cancer syndrome (HNPCC), caused by mutations in mismatch repair (MMR) genes. A potentially more efficient approach is genotyped-proband design (GPD), in which probands are genotyped and the phenotypes of their relatives are investigated. However, to date most information comes from registry-based studies, where ascertainment bias makes interpretation difficult. Development in testing technology will lead to more identified mutation carriers, producing a clinical imperative to estimate risk, despite these problems. We reviewed the available results, and concluded that male mutation carriers have a lifetime colorectal cancer risk of 74% or more; female mutation carriers have a lower risk which is still many times higher than the risk in the general population. Risk is highest between the ages of 40 and 60, but considerable even before age 40. Lifetime endometrial cancer risk is 42% or more; the highest incidence is between age 40 and 60, and diagnosis before the age of 35 is rare. MMR mutation carriers are at elevated risk for ovarian, gastric, urologic tract, small bowel, hepatobiliary tract cancer, and for brain tumors. The risk of these cancer types is much lower than the risk for colorectal and endometrial cancer, but accurate, especially age-related estimates of risk are not available. Prevention strategies depend on estimates of age-specific risk. Clearly, multicenter studies to obtain such estimates are needed.