Methyltransferase expression and tumor suppressor gene methylation in sporadic and familial colorectal cancer

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 methylation increases with dysplasia in Lynch syndrome colorectal adenomas and carcinomas

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.

Lynch syndrome (LS) is associated with germline mutations in DNA mismatch repair (MMR) genes. The first “hit” to inactivate one allele of the predisposing MMR gene is present in every cell, contributing to accelerated tumorigenesis. Less information is available of the nature, timing, and order of other molecular “hits” required for tumor development. To this end, MMR protein expression and coordinated promoter methylation were examined in colorectal specimens prospectively collected from LS mutation carriers (n = 55) during colonoscopy surveillance (10/2011-5/2013), supplemented with retrospective specimens.

Loss of MMR protein corresponding to the gene mutated in the germline increased with dysplasia, with frequency of 0 % in normal mucosa, 50-68 % in low-grade dysplasia adenomas, and 100 % in high-grade dysplasia adenomas and carcinomas. Promoter methylation as a putative “second hit” occurred in 1/56 (2 %) of tumors with silenced MMR protein. A general hypermethylation tendency was evaluated by two gene sets, eight CpG island methylator phenotype (CIMP) genes, and seven candidate tumor suppressor genes linked to colorectal carcinoma (CRC). Hypermethylation followed the same trend as MMR protein loss and was present in some low-grade dysplasia adenomas that still expressed MMR protein suggesting the absence of a “second hit.” To assess prospectively collected normal mucosa for carcinogenic “fields,” the specimen donors were stratified according to age at biopsy (50 years or below vs. above 50 years) and further according to the absence vs. presence of a (previous or concurrent) diagnosis of CRC. In mutation carriers over 50 years old, two markers from the candidate gene panel (SFRP1 and SLC5A8) revealed a significantly elevated average degree of methylation in individuals with CRC diagnosis vs. those without.

Our findings emphasize the importance and early appearance of epigenetic alterations in LS-associated tumorigenesis. The results serve early detection and assessment of progression of CRC.

Clinical Epigenetics, 2015

Subgroup-specific miRNA patterns in sporadic and Lynch syndrome-associated colorectal and endometrial carcinoma

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

DNA methylation analysis of sporadic and Lynch-associated ovarian cancers

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.

A Proteomic study on Inherited Cancer Predisposition and Western style diet effects

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.

Using the in vitro MMR assay to recognize reduced MMR gene expression levels

Assessing how reduced expression levels of the mismatch repair genes MLH1, MSH2 and MSH6 affect repair efficiency

Minttu Kansikas, Mariann Kasela, Jukka Kantelinen and Minna Nyström

Lynch syndrome (LS), the most common familial colon cancer, is associated with mismatch repair (MMR) malfunction. As mutation carriers inherit one normal and one defected MMR gene allele, cancer risk can be considered as limited amount of normal MMR gene product. How reductions in different MMR gene expressions affect MMR capability is, however, not known. The in vitro MMR assay is a method for the pathogenicity assessment of MMR gene variants causing functional or expressional defects and thus also suitable to evaluate the effects of reduced expression of normal mRNA. Here, the assay was applied to quantify repair efficiencies of human cells retaining varying expression levels (25%/50%/75%) of the main LS susceptibility genes MLH1,MSH2 or MSH6. Compared to the shRNA knockdown control, already a 50% reduction in mRNA levels could be detected as decreased MMR function although without statistical significance in MLH1. In MSH2 and MLH1, total loss of MMR was achieved with 25% expression, while in MSH6 and MSH2 the repair capability decreased significantly already with 75% expression. Our results provide a preliminary indication of relative expressions required for wild-type function and suggest that the in vitro MMR assay could be used to recognize expression levels indicative of LS.

Human Mutation, 2014.

A Novel Pathogenic 132 kb Deletion in the APC Promoter 1B Recognized as a Rare Cause for Mutation-negative FAP

Promoter-Specific Alterations of APC are a Rare Cause for Mutation-Negative Familial Adenomatous Polyposis

Walter Pavicic, Taina T. Nieminen, Annette Gylling, Juha-Pekka Pursiheimo, Asta Laiho, Attila Gyenesel, Heikku J. Järvinen and Päivi Peltomäki

In familial adenomatous polyposis (FAP), 20% of classical and 70% of attenuated/atypical (AFAP) cases remain mutation-negative after routine testing; yet, allelic expression imbalance may suggest an APC alteration. Our aim was to determine the proportion of families attributable to genetic or epigenetic changes in the APC promoter region. We studied 51 unrelated families/cases (26 with classical FAP and 25 with AFAP) with no point mutations in the exons and exon/intron borders and no rearrangements by multiplex ligation-dependent probe amplification (MLPA, P043-B1). Promoter-specific events of APC were addressed by targeted re-sequencing, MLPA (P043-C1), methylation-specific MLPA, and Sanger sequencing of promoter regions. A novel 132-kb deletion encompassing the APC promoter 1B and upstream sequence occurred in a classical FAP family with allele-specific APC expression. No promoter-specific point mutations or hypermethylation were present in any family. In conclusion, promoter-specific alterations are a rare cause for mutation-negative FAP (1/51, 2%). The frequency and clinical correlations of promoter 1B deletions are poorly defined. This investigation provides frequencies of 1/26 (4%) for classical FAP, 0/25 (0%) for AFAP, and 1/7 (14%) for families with allele-specific expression of APC. Clinically, promoter 1B deletions may associate with classical FAP without extracolonic manifestations.

Genes, Chromosomes and Cancer, 2014.


New publication in Gastroenterology

Germline mutation of RPS20, a ribosomal protein gene, predisposes to hereditary nonpolyposis colorectal carcinoma without DNA mismatch repair deficiency

Taina T. Nieminen, Marie-Françoise O’Donohue, Yunpeng Wu, Hannes Lohi, Stephen W. Scherer, Andrew D. Paterson, Pekka Ellonen, Wael M. Abdel-Rahman, Satu Valo,Jukka-Pekka Mecklin, Heikki J. Järvinen, Pierre-Emmanuel Gleizes, and Päivi Peltomäki

The predisposing genes for FCCX, hereditary nonpolyposis colorectal carcinoma with no mismatch repair defects, are largely unknown. Genetic linkage analysis, exome sequencing, tumor studies, and functional investigations of a four-generation FCCX family resulted in the identification of a truncating germline mutation in RPS20, a novel colon cancer predisposition gene encoding a component (S20) of the small ribosomal subunit. The mutation was associated with a defect in pre-rRNA maturation. Our findings show that a mutant ribosomal protein gene can underlie hereditary predisposition to microsatellite-stable colon cancer. Evaluation of additional FCCX families for mutations in RPS20 and other ribosome-associated genes is warranted.

Gastroenterology, 2014.