Epigenetic silencing can mimic genetic mutation by abolishing expression of a gene. We hypothesized that an epimutation could occur in any gene as a germline event that predisposes to disease and looked for examples in tumor suppressor genes in individuals with cancer. Here we report two individuals with soma-wide, allele-specific and mosaic hypermethylation of the DNA mismatch repair gene MLH1. Both individuals lack evidence of genetic mutation in any mismatch repair gene but have had multiple primary tumors that show mismatch repair deficiency, and both meet clinical criteria for hereditary nonpolyposis colorectal cancer. The epimutation was also present in spermatozoa of one of the individuals, indicating a germline defect and the potential for transmission to offspring. Germline epimutation provides a mechanism for phenocopying of genetic disease. The mosaicism and nonmendelian inheritance that are characteristic of epigenetic states could produce patterns of disease risk that resemble those of polygenic or complex traits.

The utility of immunohistochemical detection of DNA mismatch repair (MMR) protein in screening colorectal tumors for hereditary nonpolyposis colorectal cancer (HNPCC) syndrome has been the focus of much intensive research over the last 10 years. Particular attention has been given to the relative usefulness of immunohistochemistry (IHC) versus testing of tumor microsatellite instability (MSI). Earlier work that focused on mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2) has created a false impression that IHC has a lower sensitivity than MSI testing in predicting germline mutation. More recent studies that included postmeiotic segregation increased 2 (PMS2) and MSH6, on the other hand, have demonstrated an IHC predictive value that is virtually equivalent to that of MSI testing. Such added value of PMS2 and MSH6 can be explained by the biological and biochemical properties of the MMR proteins. On the premise that IHC with PMS2 and MSH6 is as sensitive as MSI testing, given that IHC is easily available and generally inexpensive and, importantly, identifies the affected gene, it is reasonable to regard IHC as a more optimal first-line screening tool than MSI testing for identifying HNPCC. MSI testing can provide a fallback position in equivocal situations, while remaining an important research tool. However, for IHC to be used as a first-line screening test requires that both pathologists and clinicians be aware that IHC results may be construed as "genetic information," and that appropriate procedures should be established to ensure patient understanding and consent.

OBJECTIVE

Hereditary nonpolyposis colorectal carcinoma (HNPCC) is caused by a mutated mismatch repair (MMR) gene. The aim of our study was to determine the cumulative risk of developing cancer in a large series of MSH6 mutation carriers.

METHODS

Mutation analysis was performed in 20 families with a germline mutation in MSH6. We compared the cancer risks between MSH6 and MLH1/MSH2 mutation carriers. Microsatellite instability (MSI) analysis and immunohistochemistry (IHC) were performed in the available tumors.

RESULTS

A total of 146 MSH6 mutation carriers were identified. In these carriers, the cumulative risk for colorectal carcinoma was 69% for men, 30% for women, and 71% for endometrial carcinoma at 70 years of age. The risk for all HNPCC-related tumors was significantly lower in MSH6 than in MLH1 or MSH2 mutation carriers (P = 0.002). In female MSH6 mutation carriers, the risk for colorectal cancer was significantly lower (P = 0.0049) and the risk for endometrial cancer significantly higher (P = 0.02) than in MLH1 and MSH2 mutation carriers. In male carriers, the risk for colorectal cancer was lower in MSH6 mutation carriers, but the difference was not significant (P = 0.0854). MSI analysis in colorectal tumors had a sensitivity of 86% in predicting a MMR defect. IHC in all tumors had a sensitivity of 90% in predicting a mutation in MSH6.

CONCLUSIONS

We recommend starting colonoscopic surveillance in female MSH6 mutation carriers from age 30 years. Prophylactic hysterectomy might be considered in carriers older than 50 years. MSI and IHC analysis are sensitive tools to identify families eligible for MSH6 mutation analysis.

BACKGROUND

The identification of mutations in germ-line DNA mismatch-repair genes at the time of diagnosis of colorectal cancer is important in the management of the disease.

METHODS

Without preselection and regardless of family history, we recruited 870 patients under the age of 55 years soon after they received a diagnosis of colorectal cancer. We studied these patients for germ-line mutations in the DNA mismatch-repair genes MLH1, MSH2, and MSH6 and developed a two-stage model by multivariate logistic regression for the prediction of the presence of mutations in these genes. Stage 1 of the model incorporated only clinical variables; stage 2 comprised analysis of the tumor by immunohistochemical staining and tests for microsatellite instability. The model was validated in an independent population of patients. We analyzed 2938 patient-years of follow-up to determine whether genotype influenced survival.

RESULTS

There were 38 mutations among the 870 participants (4 percent): 15 mutations in MLH1, 16 in MSH2, and 7 in MSH6. Carrier frequencies in men (6 percent) and women (3 percent) differed significantly (P<0.04). The addition of immunohistochemical analysis in stage 2 of the model had a sensitivity of 62 percent and a positive predictive value of 80 percent. There were 35 mutations in the validation series of 155 patients (23 percent): 19 mutations in MLH1, 13 in MSH2, and 3 in MSH6. The performance of the model was robust among a wide range of cutoff probabilities and was superior to that of the Bethesda and Amsterdam criteria for hereditary nonpolyposis colorectal cancer. Survival among carriers was not significantly different from that among noncarriers.

CONCLUSIONS

We devised and validated a method of identifying patients with colorectal cancer who are carriers of mutations in DNA repair genes. Survival was similar among carriers and noncarriers.

Initial results by Le and colleagues, which were published in the June 25, 2015 issue of the New England Journal of Medicine, report significant responses of cancers with microsatellite instability (MSI) to anti-PD-1 inhibitors in patients who failed conventional therapy. This finding fits into a broader body of research associating somatic hypermutation and neoepitope formation with response to immunotherapy, with the added benefit of relying on a simple, widely used diagnostic test. This review surveys the pathogenesis and prognostic value of MSI, diagnostic guidelines for detecting it, and the frequency of MSI across tumors, with the goal of providing a reference for its use as a biomarker for PD-1 blockade. MSI usually arises from either germline mutations in components of the mismatch repair (MMR) machinery (MSH2, MSH6, MLH1, PMS2) in patients with Lynch syndrome or somatic hypermethylation of the MLH1 promoter. The result is a cancer with a 10- to 100-fold increase in mutations, associated in the colon with poor differentiation, an intense lymphocytic infiltrate, and a superior prognosis. Diagnostic approaches have evolved since the early 1990s, from relying exclusively on clinical criteria to incorporating pathologic features, PCR-based MSI testing, and immunohistochemistry for loss of MMR component expression. Tumor types can be grouped into categories based on the frequency of MSI, from colorectal (20%) and endometrial (22%-33%) to cervical (8%) and esophageal (7%) to skin and breast cancers (0%-2%). If initial results are validated, MSI testing could have an expanded role as a tool in the armamentarium of precision medicine.

Hereditary non-polyposis colon cancer (HNPCC), the most common form of hereditary colon cancer, is a syndrome of deficient DNA mismatch repair (MMR). Five, possibly six, human MMR genes have been identified that, when mutated in the germline, cause susceptibility to this syndrome. To date, more than 300 different predisposing mutations are known, mainly affecting the MMR genes MLH1 ( approximately 50%), MSH2 ( approximately 40%) and MSH6 ( approximately 10%). Genetically predisposed individuals carry a defective copy of an MMR gene in every cell. Somatic inactivation of the remaining wild-type copy in a target tissue, typically colon, gives rise to a profound repair defect, progressive accumulation of mutations and cancer. Instability at short tandem repeat sequences, microsatellites, is a typical manifestation of MMR deficiency and apart from HNPCC tumors, occurs in approximately 15% of sporadic colon and other tumors. The majority of the latter cases are attributable to one particular MMR gene, MLH1, and unlike HNPCC, an epigenetic rather than a genetic mechanism plays an important role in the inactivation of this gene. The present review provides an update of the genetics of HNPCC and more generally, of cancer development driven by deficient MMR. Recent discoveries suggest that apart from post-replication repair, MMR proteins have several other functions that are highly relevant to carcinogenesis. Knowledge of the complex interplay between the MMR system and other cellular pathways allows us to better understand the phenotypic manifestations of HNPCC and other cancers with deficient MMR.

The clinical classification of hereditary sequence variants identified in disease-related genes directly affects clinical management of patients and their relatives. The International Society for Gastrointestinal Hereditary Tumours (InSiGHT) undertook a collaborative effort to develop, test and apply a standardized classification scheme to constitutional variants in the Lynch syndrome-associated genes MLH1, MSH2, MSH6 and PMS2. Unpublished data submission was encouraged to assist in variant classification and was recognized through microattribution. The scheme was refined by multidisciplinary expert committee review of the clinical and functional data available for variants, applied to 2,360 sequence alterations, and disseminated online. Assessment using validated criteria altered classifications for 66% of 12,006 database entries. Clinical recommendations based on transparent evaluation are now possible for 1,370 variants that were not obviously protein truncating from nomenclature. This large-scale endeavor will facilitate the consistent management of families suspected to have Lynch syndrome and demonstrates the value of multidisciplinary collaboration in the curation and classification of variants in public locus-specific databases.

OBJECTIVE

Multiple-gene sequencing is entering practice, but its clinical value is unknown. We evaluated the performance of a customized germline-DNA sequencing panel for cancer-risk assessment in a representative clinical sample.

METHODS

Patients referred for clinical BRCA1/2 testing from 2002 to 2012 were invited to donate a research blood sample. Samples were frozen at -80° C, and DNA was extracted from them after 1 to 10 years. The entire coding region, exon-intron boundaries, and all known pathogenic variants in other regions were sequenced for 42 genes that had cancer risk associations. Potentially actionable results were disclosed to participants.

RESULTS

In total, 198 women participated in the study: 174 had breast cancer and 57 carried germline BRCA1/2 mutations. BRCA1/2 analysis was fully concordant with prior testing. Sixteen pathogenic variants were identified in ATM, BLM, CDH1, CDKN2A, MUTYH, MLH1, NBN, PRSS1, and SLX4 among 141 women without BRCA1/2 mutations. Fourteen participants carried 15 pathogenic variants, warranting a possible change in care; they were invited for targeted screening recommendations, enabling early detection and removal of a tubular adenoma by colonoscopy. Participants carried an average of 2.1 variants of uncertain significance among 42 genes.

CONCLUSIONS

Among women testing negative for BRCA1/2 mutations, multiple-gene sequencing identified 16 potentially pathogenic mutations in other genes (11.4%; 95% CI, 7.0% to 17.7%), of which 15 (10.6%; 95% CI, 6.5% to 16.9%) prompted consideration of a change in care, enabling early detection of a precancerous colon polyp. Additional studies are required to quantify the penetrance of identified mutations and determine clinical utility. However, these results suggest that multiple-gene sequencing may benefit appropriately selected patients.

BACKGROUND

Alterations in DNA methylation in cancer include global hypomethylation and gene-specific hypermethylation. It is not clear whether these two epigenetic errors are mechanistically linked or occur independently. This study was performed to determine the relationship between DNA hypomethylation, hypermethylation and microsatellite instability in cancer.

RESULTS

We examined 61 cancer cell lines and 60 colorectal carcinomas and their adjacent tissues using LINE-1 bisulfite-PCR as a surrogate for global demethylation. Colorectal carcinomas with sporadic microsatellite instability (MSI), most of which are due to a CpG island methylation phenotype (CIMP) and associated MLH1 promoter methylation, showed in average no difference in LINE-1 methylation between normal adjacent and cancer tissues. Interestingly, some tumor samples in this group showed increase in LINE-1 methylation. In contrast, MSI-showed a significant decrease in LINE-1 methylation between normal adjacent and cancer tissues (P<0.001). Microarray analysis of repetitive element methylation confirmed this observation and showed a high degree of variability in hypomethylation between samples. Additionally, unsupervised hierarchical clustering identified a group of highly hypomethylated tumors, composed mostly of tumors without microsatellite instability. We extended LINE-1 analysis to cancer cell lines from different tissues and found that 50/61 were hypomethylated compared to peripheral blood lymphocytes and normal colon mucosa. Interestingly, these cancer cell lines also exhibited a large variation in demethylation, which was tissue-specific and thus unlikely to be resultant from a stochastic process.

CONCLUSIONS

Global hypomethylation is partially reversed in cancers with microsatellite instability and also shows high variability in cancer, which may reflect alternative progression pathways in cancer.

The Saccharomyces cerevisiae genome encodes four MutL homologs. Of these, MLH1 and PMS1 are known to act in the MSH2-dependent pathway that repairs DNA mismatches. We have investigated the role of MLH3 in mismatch repair. Mutations in MLH3 increased the rate of reversion of the hom3-10 allele by increasing the rate of deletion of a single T in a run of 7 Ts. Combination of mutations in MLH3 and MSH6 caused a synergistic increase in the hom3-10 reversion rate, whereas the hom3-10 reversion rate in an mlh3 msh3 double mutant was the same as in the respective single mutants. Similar results were observed when the accumulation of mutations at frameshift hot spots in the LYS2 gene was analyzed, although mutation of MLH3 did not cause the same extent of affect at every LYS2 frameshift hot spot. MLH3 interacted with MLH1 in a two-hybrid system. These data are consistent with the idea that a proportion of the repair of specific insertion/deletion mispairs by the MSH3-dependent mismatch repair pathway uses a heterodimeric MLH1-MLH3 complex in place of the MLH1-PMS1 complex.

Inactivation of mismatch repair (MMR) is the cause of the common cancer predisposition disorder Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer (HNPCC), as well as 10-40% of sporadic colorectal, endometrial, ovarian, gastric, and urothelial cancers. Elevated mutation rates (mutator phenotype), including simple repeat instability [microsatellite instability (MSI)] are a signature of MMR defects. MicroRNAs (miRs) have been implicated in the control of critical cellular pathways involved in development and cancer. Here we show that overexpression of miR-155 significantly down-regulates the core MMR proteins, hMSH2, hMSH6, and hMLH1, inducing a mutator phenotype and MSI. An inverse correlation between the expression of miR-155 and the expression of MLH1 or MSH2 proteins was found in human colorectal cancer. Finally, a number of MSI tumors with unknown cause of MMR inactivation displayed miR-155 overexpression. These data provide support for miR-155 modulation of MMR as a mechanism of cancer pathogenesis.

Persons who have hypermethylation of one allele of MLH1 in somatic cells throughout the body (a germ-line epimutation) have a predisposition for the development of cancer in a pattern typical of hereditary nonpolyposis colorectal cancer. By studying the families of two such persons, we found evidence that the epimutation was transmitted from a mother to her son but was erased in his spermatozoa. The affected maternal allele was inherited by three other siblings from these two families, but in those offspring the allele had reverted to the normal active state. These findings demonstrate a novel pattern of inheritance of cancer susceptibility and are consistent with transgenerational epigenetic inheritance.

BACKGROUND

Identifying families at high risk for the Lynch syndrome (ie, hereditary nonpolyposis colorectal cancer) is critical for both genetic counseling and cancer prevention. Current clinical guidelines are effective but limited by applicability and cost.

OBJECTIVE

To develop and validate a genetic counseling and risk prediction tool that estimates the probability of carrying a deleterious mutation in mismatch repair genes MLH1, MSH2, or MSH6 and the probability of developing colorectal or endometrial cancer.

METHODS

External validation of the MMRpro model was conducted on 279 individuals from 226 clinic-based families in the United States, Canada, and Australia (referred between 1993-2005) by comparing model predictions with results of highly sensitive germline mutation detection techniques. MMRpro models the autosomal dominant inheritance of mismatch repair mutations, with parameters based on meta-analyses of the penetrance and prevalence of mutations and of the predictive values of tumor characteristics. The model's prediction is tailored to each individual's detailed family history information on colorectal and endometrial cancer and to tumor characteristics including microsatellite instability.

METHODS

Ability of MMRpro to correctly predict mutation carrier status, as measured by operating characteristics, calibration, and overall accuracy.

RESULTS

In the independent validation, MMRpro provided a concordance index of 0.83 (95% confidence interval, 0.78-0.88) and a ratio of observed to predicted cases of 0.94 (95% confidence interval, 0.84-1.05). This results in higher accuracy than existing alternatives and current clinical guidelines.

CONCLUSIONS

MMRpro is a broadly applicable, accurate prediction model that can contribute to current screening and genetic counseling practices in a high-risk population. It is more sensitive and more specific than existing clinical guidelines for identifying individuals who may benefit from MMR germline testing. It is applicable to individuals for whom tumor samples are not available and to individuals in whom germline testing finds no mutation.

Somatic PIK3CA mutations are often present in colorectal cancer. Mutant PIK3CA activates AKT signaling, which up-regulates fatty acid synthase (FASN). Microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) are important molecular classifiers in colorectal cancer. However, the relationship between PIK3CA mutation, MSI and CIMP remains uncertain. Using Pyrosequencing technology, we detected PIK3CA mutations in 91 (15%) of 590 population-based colorectal cancers. To determine CIMP status, we quantified DNA methylation in eight CIMP-specific promoters [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by real-time polymerase chain reaction (MethyLight). PIK3CA mutation was significantly associated with mucinous tumors [P = .0002; odds ratio (OR) = 2.44], KRAS mutation (P < .0001; OR = 2.68), CIMP-high (P = .03; OR = 2.08), phospho-ribosomal protein S6 expression (P = .002; OR = 2.19), and FASN expression (P = .02; OR = 1.85) and inversely with p53 expression (P = .01; OR = 0.54) and beta-catenin (CTNNB1) alteration (P = .004; OR = 0.43). In addition, PIK3CA G-to-A mutations were associated with MGMT loss (P = .001; OR = 3.24) but not with MGMT promoter methylation. In conclusion, PIK3CA mutation is significantly associated with other key molecular events in colorectal cancer, and MGMT loss likely contributes to the development of PIK3CA G>A mutation. In addition, Pyrosequencing is useful in detecting PIK3CA mutation in archival paraffin tumor tissue. PIK3CA mutational data further emphasize heterogeneity of colorectal cancer at the molecular level.

DNA mismatch repair (MMR) deficiency results in a strong mutator phenotype and high-frequency microsatellite instability (MSI-H), which are the hallmarks of tumors arising within Lynch syndrome. MSI-H is characterized by length alterations within simple repeated sequences, microsatellites. Lynch syndrome is primarily due to germline mutations in one of the DNA MMR genes; mainly hMLH1 or hMSH2 and less frequently hMSH6 and rarely hPMS2. Germline hemiallelic methylation of MLH1, termed epimutation, has been reported to be a new cause of Lynch syndrome. MSI-H is also observed in approximately 15% of colorectal, gastric and endometrial cancers and in lower frequencies in a minority of other tumors, where it is associated with the hypermethylation of the promoter region of hMLH1. MSI-H underlies a distinctive tumorigenic pathway because cancers with MSI-H exhibit many differences in genotype and phenotype relative to cancers without MSI-H, irrespective of their hereditary or sporadic origins. Genetic, epigenetic and transcriptomic differences exist between cancers with and those without the MSI-H. The BRAF V600E mutation is associated with sporadic MSI-H colorectal cancers (CRCs) harboring hMLH1 methylation but not Lynch syndrome-related CRCs. The differences in genotype and phenotype between cancers with and those without MSI-H are likely to be causally linked to their differences in biological and clinical features. Therefore, the diagnosis of MSI-H in cancers is thus considered to be of increasing relevance, because MSI-H is a useful screening marker for identifying patients with Lynch syndrome, a better prognostic factor and could affect the efficacy of chemotherapy. This review addresses recent advances in the field of microsatellite instability research.

MutL homolog 3 (Mlh3) is a member of a family of proteins conserved during evolution and having dual roles in DNA mismatch repair and meiosis. The pathway in eukaryotes consists of the DNA-binding components, which are the homologs of the bacterial MutS protein (MSH 2 6), and the MutL homologs, which bind to the MutS homologs and are essential for the repair process. Three of the six homologs of MutS that function in these processes, Msh2, Msh3 and Msh6, are involved in the mismatch repair of mutations, frameshifts and replication errors, and two others, Msh4 and Msh5, have specific roles in meiosis. Of the four MutL homologs, Mlh1, Mlh3, Pms1 and Pms2, three are involved in mismatch repair and at least two, Pms2 and Mlh1, are essential for meiotic progression in both yeast and mice. To assess the role of Mlh3 in mammalian meiosis, we have generated and characterized Mlh3(-/-) mice. Here we show that Mlh3(-/-) mice are viable but sterile. Mlh3 is required for Mlh1 binding to meiotic chromosomes and localizes to meiotic chromosomes from the mid pachynema stage of prophase I. Mlh3(-/-) spermatocytes reach metaphase before succumbing to apoptosis, but oocytes fail to complete meiosis I after fertilization. Our results show that Mlh3 has an essential and distinct role in mammalian meiosis.

Gastric cancers with mismatch repair (MMR) inactivation are characterised by microsatellite instability (MSI). In this study, the transcriptional profile of 38 gastric cancers with and without MSI was analysed. Unsupervised analysis showed that the immune and apoptotic gene networks efficiently discriminated these two cancer types. Hierarchical clustering analysis revealed numerous gene expression changes associated with the MSI phenotype. Amongst these, the p53-responsive genes maspin and 14-3-3 sigma were significantly more expressed in tumours with than without MSI. A tight immunosurveillance coupled with a functional p53 gene response is consistent with the better prognosis of MSI cancers. Frequent silencing of MLH1 and downregulation of MMR target genes, such as MRE11 and MBD4, characterised MSI tumours. The downregulation of SMUG1 was also a typical feature of these tumours. The DNA repair gene expression profile of gastric cancer with MSI is of relevance for therapy response.

MSH5 (MutS homologue 5) is a member of a family of proteins known to be involved in DNA mismatch repair. Germline mutations in MSH2, MLH1 and GTBP (also known as MSH6) cause hereditary non-polyposis colon cancer (HNPCC) or Lynch syndrome. Inactivation of Msh2, Mlh1, Gtmbp (also known as Msh6) or Pms2 in mice leads to hereditary predisposition to intestinal and other cancers. Early studies in yeast revealed a role for some of these proteins, including Msh5, in meiosis. Gene targeting studies in mice confirmed roles for Mlh1 and Pms2 in mammalian meiosis. To assess the role of Msh5 in mammals, we generated and characterized mice with a null mutation in Msh5. Msh5-/- mice are viable but sterile. Meiosis in these mice is affected due to the disruption of chromosome pairing in prophase I. We found that this meiotic failure leads to a diminution in testicular size and a complete loss of ovarian structures. Our results show that normal Msh5 function is essential for meiotic progression and, in females, gonadal maintenance.

Loss of DNA mismatch repair because of hypermethylation of the hMLH1 gene promoter occurs at a high frequency in a number of human tumors. A role for loss of mismatch repair (MMR) in resistance to a number of clinically important anticancer drugs has been shown. We have investigated whether the demethylating agent 2'-deoxy-5-azacytidine (DAC) can be used in vivo to sensitize MMR-deficient, drug-resistant ovarian (A2780/cp70) and colon (SW48) tumor xenografts that are MLH1 negative because of gene promoter hypermethylation. Treatment of tumor-bearing mice with the demethylating agent DAC at a nontoxic dose induces MLH1 expression. Re-expression of MLH1 is associated with a decrease in hMLH1 gene promoter methylation. DAC treatment alone has no effect on the growth rate of the tumors. However, DAC treatment sensitizes the xenografts to cisplatin, carboplatin, temozolomide, and epirubicin. Sensitization is comparable with that obtained by reintroduction of the hMLH1 gene by chromosome 3 transfer. Consistent with loss of MMR having no effect on sensitivity in vitro to Taxol, DAC treatment has no effect on the Taxol sensitivity of the xenografts. DAC treatment does not sensitize xenografts of HCT116, which lacks MMR because of hMLH1 mutation. Because there is emerging data on the role of loss of MMR in clinical drug resistance, DAC could have a role in increasing the efficacy of chemotherapy for patients whose tumors lack MLH1 expression because of hMLH1 promoter methylation.

MutL homologs are crucial for mismatch repair and genetic stability, but their function is not well understood. Human MutLalpha (MLH1-PMS2 heterodimer) harbors a latent endonuclease that is dependent on the integrity of a PMS2 DQHA(X)2E(X)4E motif (Kadyrov, F. A., Dzantiev, L., Constantin, N., and Modrich, P. (2006) Cell 126, 297-308). This sequence element is conserved in many MutL homologs, including the PMS1 subunit of Saccharomyces cerevisiae MutLalpha, but is absent in MutL proteins from bacteria like Escherichia coli that rely on d(GATC) methylation for strand directionality. We show that yeast MutLalpha is a strand-directed endonuclease that incises DNA in a reaction that depends on a mismatch, yMutSalpha, yRFC, yPCNA, ATP, and a pre-existing strand break, whereas E. coli MutL is not. Amino acid substitution within the PMS1 DQHA(X)2E(X)4E motif abolishes yMutLalpha endonuclease activity in vitro and confers strong genetic instability in vivo, but does not affect yMutLalpha ATPase activity or the ability of the protein to support assembly of the yMutLalpha.yMutSalpha.heteroduplex ternary complex. The loaded form of yPCNA may play an important effector role in directing yMutLalpha incision to the discontinuous strand of a nicked heteroduplex.

Genetic amplification, mutation, and translocation are known to play a causal role in the upregulation of an oncogene in cancer cells. Here, we report an emerging role of microRNA, the epigenetic deregulation of which may also lead to this oncogenic activation. SOX4, an oncogene belonging to the SRY-related high mobility group box family, was found to be overexpressed (P < 0.005) in endometrial tumors (n = 74) compared with uninvolved controls (n = 20). This gene is computationally predicted to be the target of a microRNA, miR-129-2. When compared with the matched endometria, the expression of miR-129-2 was lost in 27 of 31 primary endometrial tumors that also showed a concomitant gain of SOX4 expression (P < 0.001). This inverse relationship is associated with hypermethylation of the miR-129-2 CpG island, which was observed in endometrial cancer cell lines (n = 6) and 68% of 117 endometrioid endometrial tumors analyzed. Reactivation of miR-129-2 in cancer cells by pharmacologic induction of histone acetylation and DNA demethylation resulted in decreased SOX4 expression. In addition, restoration of miR-129-2 by cell transfection led to decreased SOX4 expression and reduced proliferation of cancer cells. Further analysis found a significant correlation of hypermethylated miR-129-2 with microsatellite instability and MLH1 methylation status (P < 0.001) and poor overall survival (P < 0.039) in patients. Therefore, these results imply that the aberrant expression of SOX4 is, in part, caused by epigenetic repression of miR-129-2 in endometrial cancer. Unlike the notion that promoter hypomethylation may upregulate an oncogene, we present a new paradigm in which hypermethylation-mediated silencing of a microRNA derepresses its oncogenic target in cancer cells.

Two systems are essential in humans for genome integrity, DNA repair and apoptosis. Cells that are defective in DNA repair tend to accumulate excess DNA damage. Cells defective in apoptosis tend to survive with excess DNA damage and thus allow DNA replication past DNA damages, causing mutations leading to carcinogenesis. It has recently become apparent that key proteins which contribute to cellular survival by acting in DNA repair become executioners in the face of excess DNA damage. Five major DNA repair pathways are homologous recombinational repair (HRR), non-homologous end joining (NHEJ), nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). In each of these DNA repair pathways, key proteins occur with dual functions in DNA damage sensing/repair and apoptosis. Proteins with these dual roles occur in: (1) HRR (BRCA1, ATM, ATR, WRN, BLM, Tip60 and p53); (2) NHEJ (the catalytic subunit of DNA-PK); (3) NER (XPB, XPD, p53 and p33(ING1b)); (4) BER (Ref-1/Ape, poly(ADP-ribose) polymerase-1 (PARP-1) and p53); (5) MMR (MSH2, MSH6, MLH1 and PMS2). For a number of these dual-role proteins, germ line mutations causing them to be defective also predispose individuals to cancer. Such proteins include BRCA1, ATM, WRN, BLM, p53, XPB, XPD, MSH2, MSH6, MLH1 and PMS2.

The yeast genome encodes four proteins (Pms1 and Mlh1-3) homologous to the bacterial mismatch repair component, MutL. Using two hybrid-interaction and coimmunoprecipitation studies, we show that these proteins can form only three types of complexes in vivo. Mlh1 is the common component of all three complexes, interacting with Pms1, Mlh2, and Mlh3, presumptively as heterodimers. The phenotypes of single deletion mutants reveal distinct functions for the three heterodimers during meiosis: in a pms1 mutant, frequent postmeiotic segregation indicates a defect in the correction of heteroduplex DNA, whereas the frequency of crossing-over is normal. Conversely, crossing-over in the mlh3 mutant is reduced to approximately 70% of wild-type levels but correction of heteroduplex is normal. In a mlh2 mutant, crossing-over is normal and postmeiotic segregation is not observed but non-Mendelian segregation is elevated and altered with respect to parity. Finally, to a first approximation, the mlh1 mutant represents the combined single mutant phenotypes. Taken together, these data imply modulation of a basic Mlh1 function via combination with the three other MutL homologs and suggest specifically that Mlh1 combines with Mlh3 to promote meiotic crossing-over.

Preclinical studies have shown that hypomethylating agents reverse platinum resistance in ovarian cancer. In this phase II clinical trial, based upon the results of our phase I dose defining study, we tested the clinical and biologic activity of low-dose decitabine administered before carboplatin in platinum-resistant ovarian cancer patients. Among 17 patients with heavily pretreated and platinum-resistant ovarian cancer, the regimen induced a 35% objective response rate (RR) and progression-free survival (PFS) of 10.2 months, with nine patients (53%) free of progression at 6 months. Global and gene-specific DNA demethylation was achieved in peripheral blood mononuclear cells and tumors. The number of demethylated genes was greater (P < 0.05) in tumor biopsies from patients with PFS more than 6 versus less than 6 months (311 vs. 244 genes). Pathways enriched at baseline in tumors from patients with PFS more than 6 months included cytokine-cytokine receptor interactions, drug transporters, and mitogen-activated protein kinase, toll-like receptor and Jak-STAT signaling pathways, whereas those enriched in demethylated genes after decitabine treatment included pathways involved in cancer, Wnt signaling, and apoptosis (P < 0.01). Demethylation of MLH1, RASSF1A, HOXA10, and HOXA11 in tumors positively correlated with PFS (P < 0.05). Together, the results of this study suggest that low-dose decitabine altered DNA methylation of genes and cancer pathways, restoring sensitivity to carboplatin in patients with heavily pretreated ovarian cancer and resulting in a high RR and prolonged PFS.