Beltrami et al. Clinical Epigenetics (2017) 9:45 DOI 10.1186/s13148-017-0346-2 RESEARCH Open Access Integrated data analysis reveals potential drivers and pathways disrupted by DNA methylation in papillary thyroid carcinomas Caroline Moraes Beltrami1†, Mariana Bisarro dos Reis1,2†, Mateus Camargo Barros-Filho1, Fabio Albuquerque Marchi1, Hellen Kuasne1,2, Clóvis Antônio Lopes Pinto3, Srikant Ambatipudi4, Zdenko Herceg4, Luiz Paulo Kowalski1,5 and Silvia Regina Rogatto2,6* Abstract Background: Papillary thyroid carcinoma (PTC) is a common endocrine neoplasm with a recent increase in incidence in many countries. Although PTC has been explored by gene expression and DNA methylation studies, the regulatory mechanisms of the methylation on the gene expression was poorly clarified. In this study, DNA methylation profile (Illumina HumanMethylation 450K) of 41 PTC paired with non-neoplastic adjacent tissues (NT) was carried out to identify and contribute to the elucidation of the role of novel genic and intergenic regions beyond those described in the promoter and CpG islands (CGI). An integrative and cross-validation analysis were performed aiming to identify molecular drivers and pathways that are PTC-related. Results: The comparisons between PTC and NT revealed 4995 methylated probes (88% hypomethylated in PTC) and 1446 differentially expressed transcripts cross-validated by the The Cancer Genome Atlas data. The majority of these probes was found in non-promoters regions, distant from CGI and enriched by enhancers. The integrative analysis between gene expression and DNA methylation revealed 185 and 38 genes (mainly in the promoter and body regions, respectively) with negative and positive correlation, respectively. Genes showing negative correlation underlined FGF and retinoic acid signaling as critical canonical pathways disrupted by DNA methylation in PTC. BRAF mutation was detected in 68% (28 of 41) of the tumors, which presented a higher level of demethylation (95% hypomethylated probes) compared with BRAF wild-type tumors. A similar integrative analysis uncovered 40 of 254 differentially expressed genes, which are potentially regulated by DNA methylation in BRAFV600E-positive tumors. The methylation and expression pattern of six selected genes (ERBB3, FGF1, FGFR2, GABRB2, HMGA2, and RDH5) were confirmed as altered by pyrosequencing and RT-qPCR. Conclusions: DNA methylation loss in non-promoter, poor CGI and enhancer-enriched regions was a significant event in PTC, especially in tumors harboring BRAFV600E. In addition to the promoter region, gene body and 3’UTR methylation have also the potential to influence the gene expression levels (both, repressing and inducing). The integrative analysis revealed genes potentially regulated by DNA methylation pointing out potential drivers and biomarkers related to PTC development. Keywords: Papillary thyroid cancer, DNA methylation, Integrative analysis, FGF signaling pathway, Retinoic acid pathway, BRAFV600E mutation * Correspondence: [email protected]; [email protected] †Equal contributors 2Department of Urology, Faculty of Medicine, UNESP, Sao Paulo State University, Botucatu, São Paulo, Brazil 6Department of Clinical Genetics, Vejle Hospital and Institute of Regional Health Research, University of Southern Denmark, Kabbeltoft 25, Vejle 7100, Denmark Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Beltrami et al. Clinical Epigenetics (2017) 9:45 Page 2 of 11 Background retrospectively. Forty one papillary thyroid carcinomas of Thyroid cancer is the most common tumor of the head patients treated with total thyroidectomy followed by and neck region, with the highest incidence among the radioiodine therapy and matched non-neoplastic adjacent endocrine neoplasias [1]. Papillary thyroid cancer (PTC) is tissues (NT) samples were included in this study. Cases the histological subtype with higher incidence (80% of with incomplete clinical data in medical records or diag- cases) worldwide [2]. nosed with previous or synchronic malignancies were The thyroid carcinogenesis involves a constitutive excluded (except basal skin cell carcinoma). Clinical and activation of two major pathways associated to tyrosine- histopathological data are summarized in Table 1. kinase, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) [3]. The activation Nucleic acids extraction and analysis of somatic of these pathways occurs mainly due to point mutations in mutations BRAF and RAS and chromosomal rearrangements in RET Genomic DNA extraction was performed according to [3]. MAPK signaling pathway activated by genetic alter- conventional protocol using enzymatic degradation with ations is frequently involved in cell proliferation, growth, proteinase K followed by purification with organic solvents and survival [4]. (phenol/chloroform). RNA was isolated as previously Approximately 60% of PTC cases are characterized by reported [26]. T1799A BRAF transversion nucleotide change (over 95% Somatic point mutations of BRAF (codon 599), KRAS of the mutations), resulting in V600E mutant protein (codon 12/13), HRAS (codon 61), and NRAS (codon 61) with constitutive activation of BRAF kinase [5–7]. BRAF were evaluated by pyrosequencing using a Pyromark Q96 mutation has been associated with unfavorable prognosis ID system (Qiagen, Valencia, CA, USA). RET rearrange- including large primary tumors, lymph node and vascu- ments (RET/PTC1 and RET/PTC3) were detected by lar invasion, advanced stage, extrathyroidal extension, RT-qPCR on a 7500 Real Time PCR System (Applied distant metastases, and recurrence [8, 9]. However, there Biosystems, Foster, CA, USA) (detailed in Additional file 1). are no consensus in literature, since many studies have not found this association [10–12]. The methylation pattern of several genes has been DNA methylation and gene expression profiling assessed in PTC, and most of them plays a role in thyroid Five hundred nanograms of DNA (Qubit® dsDNA BR gland function (TSHR) [13] and iodine metabolism (NIS Assay no Qubit® 2.0 Fluorometer (Life Technologies, and SLC26A4) [14] or acts as a tumor suppressor gene Carlsbad, CA, USA) were bisulfite-modified using (RASSF1A, TIMP3,andRARβ2) [15, 16]. In addition, an EZ-DNA Methylation-Gold Kit (Zymo Research, Irvine, association between BRAFV600E mutation and aberrant CA, USA) according to the manufacturer’s recommen- DNA methylation profile has been reported in thyroid dations. Converted DNA was used for the genome-wide cancer [17–21]. methylation assays (Infinium Human Methylation450 Recently, large-scale approaches were used to investigate BeadChip array-Illumina, San Diego, CA, USA). Arrays the methylation profile of thyroid cancer [17–23]. These were scanned by HiScan system (Illumina), and methylation technologies allow the assessment of not only CpG islands data were analyzed as β values. Genome-wide DNA methy- (CGI) and promoter regions, as previously reported, but lation data processing was done as reported previously [27] unveiled novel regions involved in neoplastic process, (detailed in Additional file 1). Limma package [28] was used such as CGI shores/shelf, non-CGI promoter and to identify significant probes adopting adjusted (Bonferroni) enhancers [24, 25]. However, the methylation as a regula- p value <0.05 and mean delta β value (Δβ)of0.15asa tory mechanism of gene expression is poorly explored in threshold for differential DNA methylation. A hypergeo- PTC, even in the multiplatform robust study performed metric test (p < 0.05) was performed from phyper function by The Cancer Genome Atlas (TCGA) [21]. of STATS package in R language to compare differentially To our knowledge, the current study is the first to assess methylated probes in relation to genomic regions (Illumina a substantial matched-sample subset with methylation 450K array annotation). and expression data addressing the available data from Gene expression data were obtained from our previously TCGA study to cross-validate the results. The genes reported study (GEO accession number GSE50901) [26]. signature potentially regulated by methylation inferred the The unsupervised hierarchical clustering analysis was role of this epigenetic event in PTC development. performed using the most variable probes (interquartile range >0.2 to methylation and >1.0 to gene expression). Methods Euclidean distance with average linkage method was used Sample population in all clustering analysis by BRB array tool software Snap frozen PTC samples stored at tissue biobank of the (https://brb.nci.nih.gov/BRB-ArrayTools/download.html). A.C. Camargo Cancer Center, SP, Brazil, were obtained Student t test was assessed to verify the association Beltrami et al. Clinical Epigenetics (2017) 9:45 Page 3 of 11 Table 1 Clinicopathological features of 41 patients diagnosed Table 1 Clinicopathological features of 41 patients diagnosed with papillary