Epigenetic Alterations in Sporadic and Familial Cancers

Public PhD defense on 20th of November 2015, at 12 noon.

Auditorium XIV, University main building, Fabianinkatu 33, Helsinki

Author: M.Sc. Emmi Joensuu

Department of Medical and Clinical Genetics, University of Helsinki

Opponent: Prof. Anne Kallioniemi, MD, PhD

Department of Biomedical Sciences, University of Tampere

Welcome!

Prof. Anne Kallioniemi, Prof. Päivi Peltomäki and M.Sc Emmi Joensuu. Kuvaaja: Annette Gylling

Prof. Anne Kallioniemi, Prof. Päivi Peltomäki and M.Sc Emmi Joensuu. Photograph: Annette Gylling

EPIGENETIC CHARACTERISTICS OF LYNCH SYNDROME-ASSOCIATED AND SPORADIC TUMORIGENESIS

Public PhD defence on 25th of September 2015 at 12 p.m

Folkhälsan, Auritorium Arean, Topeliuksenkatu 20, Helsinki

Author: M.Sc. Johanna Lotsari

Department of Medical and Clinical Genetics, Faculty of Medicine, University of Helsinki

Opponent: Prof. Markus Mäkinen, Department of Pathology, University of Oulu

Welcome!

M.Sc. Johanna Lotsari-Salomaa, Prof. Päivi Peltomäki and Prof. Markus Mäkinen

M.Sc. Johanna Lotsari-Salomaa, Prof. Päivi Peltomäki and Prof. Markus Mäkinen

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

Visit of Vice-President of the European Comission

Kristalina Georgieva, Vice-President of the European Comission visited the laboratory of Päivi Peltomäki at the Department of Medical and Clinical Genetics in February. Prof. Päivi Peltomäki together with Prof. Minna Nyström presented the projects funded by the European Research Council (ERC) during years 2009-2014. After the meeting Kristalina Georgieva took time to tour around the laboratory!

Read more about the visit at the European Comission website.

©European Comission

©European Comission Audiovisual Services

The Annual National HNPCC meeting at Himos

The Annual Meeting of the Finnish collaborative HNPCC reseach groups will take place 6.-7.3.2015 at Himos Centre in Jämsä.

Prof. Pävi Peltomäki together with Ph.D Taina Nieminen and M.Sc Anni Niskakoski will be presenting the on-going projects and highlights from the Peltomäki lab last year. Anni will present her current project related to the epigenetic and genetic changes in non-malignant ovarian and endometrial tissues followed by Taina’s talk about the on-going projects aiming to discover novel predisposing genes for mismatch repair proficient HNPCC.

See you there!

himos

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.