Itkonen HM, Kantelinen J, Vaara M, Parkkinen S, Schlott B, Grosse F, Nyström M, Syväoja JE, Pospiech H.

The high fidelity of genome duplication is ensured by cooperation of the proofreading and mismatch repair (MMR) activities of DNA polymerases. Here, we show that human mismatch recognizing proteins MutS homologue 2 (MSH2) and MSH6 co-purify and interact with replicative Pol α, the replicative primase which replicates DNA with poor fidelity. We show that MSH2 associates with known human replication origins with different dynamics than Pol α. Furthermore, we explored the potential functional role of Pol α in the mismatch repair reaction by using an in vitro mismatch repair assay and observed that Pol α promotes mismatch repair. Taken together, we show that human Pol α interacts with MSH2-MSH6 complex and propose that this interaction occurs during the mismatch repair reaction.

FEBS Letters, 2016

Tricarico R, Kasela M, Mareni C, Thompson BA, Drouet A, Staderini L, Gorelli G, Crucianelli F, Ingrosso V, Kantelinen J, Papi L, De Angioletti M, Berardi M, Gaildrat P, Soukarieh O, Turchetti D, Martins A, Spurdle AB, Nyström M, Genuardi; InSiGHT Variant Interpretation Committee.

Pathogenicity assessment of DNA variants in disease genes to explain their clinical consequences is an integral component of diagnostic molecular testing. The International Society for Gastrointestinal Hereditary Tumors (InSiGHT) has developed specific criteria for the interpretation of mismatch repair (MMR) gene variants. Here, we performed a systematic investigation of 24 MLH1 and MSH2 variants. The assessments were done by analyzing population frequency, segregation, tumor molecular characteristics, RNA effects, protein expression levels, and in vitro MMR activity. Classifications were confirmed for 15 variants and changed for three, and for the first time determined for six novel variants. Overall, based on our results, we propose the introduction of some refinements to the InSiGHT classification rules. The proposed changes have the advantage of homogenizing the InSIGHT interpretation criteria with those set out by the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium for the BRCA1/BRCA2 genes. We also observed that the addition of only few clinical data was sufficient to obtain a more stable classification for variants considered as “likely pathogenic” or “likely nonpathogenic.” This shows the importance of obtaining as many as possible points of evidence for variant interpretation, especially from the clinical setting.

Hum Mutat. 2016

The Role of Chromosomal Instability and Epigenetics in Colorectal Cancers Lacking β-Catenin/TCF Regulated Transcription

Abdel-Rahman WM, Lotsari-Salomaa JE, Kaur S, Niskakoski A, Knuutila S, Järvinen H, Mecklin JP and Peltomäki P.

All colorectal cancer cell lines except RKO displayed active β-catenin/TCF regulated transcription. This feature of RKO was noted in familial colon cancers; hence our aim was to dissect its carcinogenic mechanism. MFISH and CGH revealed distinct instability of chromosome structure in RKO. Gene expression microarray of RKO versus 7 colon cancer lines (with active Wnt signaling) and 3 normal specimens revealed 611 differentially expressed genes. The majority of the tested gene loci were susceptible to LOH in primary tumors with various β-catenin localizations as a surrogate marker for β-catenin activation. The immunohistochemistry of selected genes (IFI16, RGS4, MCTP1, DGKI, OBCAM/OPCML, and GLIPR1) confirmed that they were differentially expressed in clinical specimens. Since epigenetic mechanisms can contribute to expression changes, selected target genes were evaluated for promoter methylation in patient specimens from sporadic and hereditary colorectal cancers. CMTM3, DGKI, and OPCML were frequently hypermethylated in both groups, whereas KLK10, EPCAM, and DLC1 displayed subgroup specificity. The overall fraction of hypermethylated genes was higher in tumors with membranous β-catenin. We identified novel genes in colorectal carcinogenesis that might be useful in personalized tumor profiling. Tumors with inactive Wnt signaling are a heterogeneous group displaying interaction of chromosomal instability, Wnt signaling, and epigenetics.

Gastroenterol Res Pract. 2016

Peltomäki P.

Four main DNA mismatch repair (MMR) genes have been identified, MLH1, MSH2, MSH6, and PMS2, which when mutated cause susceptibility to Lynch syndrome (LS). LS is one of the most prevalent hereditary cancer syndromes in man and accounts for 1-3 % of unselected colorectal carcinomas and some 15 % of those with microsatellite instability and/or absent MMR protein. The International Society for Gastrointestinal Hereditary Tumours (InSiGHT) maintains a database for LS-associated mutations since 1996. The database was recently reorganized to efficiently gather published and unpublished data and to classify the variants according to a five-tiered scheme linked to clinical recommendations. This review provides an update of germline mutations causing susceptibility to LS based on information available in the InSiGHT database and the latest literature. MMR gene mutation profiles, correlations between genotype and phenotype, and possible mechanisms leading to the characteristic spectrum of tumors in LS are discussed in light of the different functions of MMR proteins, many of which directly serve cancer avoidance.

Review in Fam cancer, 2016.

Fig. Distributions of the types of germline variants across each MMR gene (Peltomäki P, Fam Cancer 2016).

Emmi Joensuu, Taina Nieminen, Johanna Lotsari, Walter Pavicic, Wael Abdel-Rahman and Päivi Peltomäki

Molecular mechanisms underlying coordinated hypermethylation of multiple CpG islands in cancer remain unclear and studies of methyltransferase enzymes have arrived at conflicting results. We focused on DNMT1 and DNMT3B, DNA methyltransferases responsible for (de novo) methylation, and EZH2, histone (H3K27) methyltransferase, and examined their roles in tumor suppressor gene (TSG) methylation patterns we have previously established in sporadic and familial cancers. Our investigation comprised 165 tumors, stratified by tissue of origin (117 colorectal and 48 endometrial carcinomas) and sporadic vs. familial disease (57 sporadic vs. 60 familial, mainly Lynch syndrome, colorectal carcinomas). By immunohistochemical evaluation, EZH2 protein expression was associated with a TSG methylator phenotype. DNMT1, DNMT3B, and EZH2 were expressed at significantly higher levels in tumor vs. normal tissues. DNMT1 and EZH2 expression were positively correlated and higher in microsatellite-unstable vs. microsatellite-stable tumors, whether sporadic or hereditary. Ki-67 expression mirrored the same pattern. Promoter methylation of the methyltransferase genes themselves was addressed as a possible cause behind their altered expression. While DNMT1 or EZH2 did not show differential methylation between normal and tumor tissues, DNMT3B analysis corroborated the regulatory role of a distal promoter region. Our study shows that methyltransferase expression in cancer depends on the tissue of origin, microsatellite-instability status, cellular proliferation, and-in the case of DNMT3B-promoter methylation of the respective gene. Translation of methyltransferase expression into DNA methylation appears complex as suggested by the fact that except for EZH2, no clear association between methyltransferase protein expression and TSG methylation was observed.

Genes Chromosomes Cancer, 2015

DNA hypermethylation appears early and shows increased frequency with dysplasia in Lynch syndrome-associated colorectal adenomas and carcinomas

Satu Valo, Sippy Kaur, Ari Ristimäki, Laura Renkonen-Sinisalo, Heikki Järvinen, Jukka-Pekka Mecklin, Minna Nyström & Päivi Peltomäki.

Identification of subgroup-specific miRNA patterns by epigenetic profiling of sporadic and Lynch syndrome-associated colorectal and endometrial carcinoma.

Sippy Kaur*, Johanna Lotsari*, Sam Al-Sohaily, Janindra Warusavitarne, Maija Kohonen-Corish and Päivi Peltomäki

Altered expression of microRNAs (miRNAs) commonly accompanies colorectal (CRC) and endometrial carcinoma (EC) development, but the underlying mechanisms and clinicopathological correlations remain to be clarified. We focused on epigenetic mechanisms and aimed to explore if DNA methylation patterns in tumors depend on DNA mismatch repair (MMR) status, sporadic vs. Lynch-associated disease, and geographic origin (Finland vs. Australia). Treatment of cancer cell lines with demethylating agents revealed 109 significantly upregulated miRNAs. Seven met our stringent criteria for possible methylation-sensitive miRNAs and were used to screen patient specimens (205 CRCs and 36 ECs) by methylation-specific multiplex ligation-dependent probe amplification.

Three miRNAs (129-2, 345, and 132) with low methylation levels in normal tissue and frequent hypermethylation in tumors were of particular interest. Hypermethylation of miR-345 and miR-132 associated with MMR deficiency in CRC regardless of geographic origin, and hypermethylation of miR-132 distinguished sporadic MMR-deficient CRC from Lynch-CRC. Finally, hypermethylation of miRNAs stratified 49 endometrial hyperplasias into low-methylator (simple hyperplasia) and high-methylator groups (complex hyperplasia with or without atypia) and suggested that miR-129-2 methylation in particular could serve as a marker of progression in early endometrial tumorigenesis.

Our study identifies miR-345 and miR-132 as novel differentially methylated miRNAs in CRC, thereby facilitating sub-classification of CRC and links miR-129-2 methylation to early endometrial tumorigenesis.

Clinical Epigenetics, 2015

Epigenetic analysis of sporadic and Lynch-associated ovarian cancers reveals histology-specific patterns of DNA methylation

Anni Niskakoski, Sippy Kaur, Synnove Staff, Laura Renkonen-Sinisalo, Heini Lassus, Heikki Järvinen, Jukka-Pekka Mecklin, Ralf Bützow and Päivi Peltomäki

Diagnosis and treatment of epithelial ovarian cancer is challenging due to the poor understanding of the pathogenesis of the disease. Our aim was to investigate epigenetic mechanisms in ovarian tumorigenesis and, especially, whether tumors with different histological subtypes or hereditary background (Lynch syndrome) exhibit differential susceptibility to epigenetic inactivation of growth regulatory genes. Gene candidates for epigenetic regulation were identified from the literature and by expression profiling of ovarian and endometrial cancer cell lines treated with demethylating agents. Thirteen genes were chosen for methylation-specific multiplex ligation-dependent probe amplification assays on 104 (85 sporadic and 19 Lynch syndrome-associated) ovarian carcinomas. Increased methylation (i.e., hypermethylation) of variable degree was characteristic of ovarian carcinomas relative to the corresponding normal tissues, and hypermethylation was consistently more prominent in non-serous than serous tumors for individual genes and gene sets investigated. Lynch syndrome-associated clear cell carcinomas showed the highest frequencies of hypermethylation. Among endometrioid ovarian carcinomas, lower levels of promoter methylation of RSK4, SPARC, and HOXA9 were significantly associated with higher tumor grade; thus, the methylation patterns showed a shift to the direction of high-grade serous tumors. In conclusion, we provide evidence of a frequent epigenetic inactivation of RSK4, SPARC, PROM1, HOXA10, HOXA9, WT1-AS, SFRP2, SFRP5, OPCML, and MIR34B in the development of non-serous ovarian carcinomas of Lynch and sporadic origin, as compared to serous tumors. Our findings shed light on the role of epigenetic mechanisms in ovarian tumorigenesis and identify potential targets for translational applications.

                                                                                                        Epigenetics, 2014.

Inherited cancer predisposition sensitizes colonic mucosa to address Western diet effects and putative cancer-predisposing changes on mouse proteome

Denis Đermadi Bebek, Satu Valo, Marjaana Pussila, Nima Reyhani, Laura Sarantaus, Maciej Lalowski, Marc Baumann, Minna Nyström

Human epidemiological evidence and previous studies on mice have shown that Western style diet (WD) may predispose gut mucosa to colorectal cancer (CRC). The mechanisms, which mediate the effects of diet on tumorigenesis are largely unknown. To address putative cancer predisposing events available for early detection, we quantitatively analyzed the proteome of histologically normal colon of a wild type (Mlh1+/+) and an Mlh1+/- mouse after a long term feeding experiment with WD and AIN-93G control diet. The Mlh1+/- mouse carries susceptibility to colon cancer analogous to a human CRC syndrome (Lynch syndrome). Remarkably, WD seemed to induce expression changes reflecting metabolic disturbances especially in the cancer predisposed colon, while similar changes were not significant in the wild type proteome. Overall, the detected changes constitute a complex interaction network of proteins involved in ATP synthesis coupled proton transport, oxidoreduction coenzyme and nicotinamide nucleotide metabolic processes, important in cell protection against ROS toxicity. Of these proteins, SELENBP1 and LGALS4 are underlined in neoplastic processes, which directly interact with MLH1, suggesting that sensitivity to WD is increased by an Mlh1 mutation. The significance of WD on CRC risk is highlighted by the fact that 5 out of 6 mice with neoplasias were fed with WD.

The Journal of Nutritional Biochemistry, 2014.