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Integrative Epigenomic and Genomic Analysis of Malignant Pheochromocytoma

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Integrative Epigenomic and Genomic Analysis of Malignant Pheochromocytoma EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 42, No. 7, 484-502, July 2010 Integrative epigenomic and genomic analysis of malignant pheochromocytoma Johanna Sandgren1,2* Robin Andersson3*, pression examination in a malignant pheochromocy- Alvaro Rada-Iglesias3, Stefan Enroth3, toma sample. The integrated analysis of the tumor ex- Goran̈ Akerstro̊ m̈ 1, Jan P. Dumanski2, pression levels, in relation to normal adrenal medulla, Jan Komorowski3,4, Gunnar Westin1 and indicated that either histone modifications or chromo- somal alterations, or both, have great impact on the ex- Claes Wadelius2,5 pression of a substantial fraction of the genes in the in- vestigated sample. Candidate tumor suppressor 1Department of Surgical Sciences genes identified with decreased expression, a Uppsala University, Uppsala University Hospital H3K27me3 mark and/or in regions of deletion were for SE-75185 Uppsala, Sweden 2 instance TGIF1, DSC3, TNFRSF10B, RASSF2, HOXA9, Department of Genetics and Pathology Rudbeck Laboratory, Uppsala University PTPRE and CDH11. More genes were found with in- SE-75185 Uppsala, Sweden creased expression, a H3K4me3 mark, and/or in re- 3The Linnaeus Centre for Bioinformatics gions of gain. Potential oncogenes detected among Uppsala University those were GNAS, INSM1, DOK5, ETV1, RET, NTRK1, SE-751 24 Uppsala, Sweden IGF2, and the H3K27 trimethylase gene EZH2. Our ap- 4Interdisciplinary Centre for Mathematical and proach to associate histone methylations and DNA Computational Modelling copy number changes to gene expression revealed ap- Warsaw University parent impact on global gene transcription, and en- PL-02-106 Warszawa, Poland abled the identification of candidate tumor genes for 5Corresponding author: Tel, 46-18-471-40-76; further exploration. Fax, 46-18-471-48-08; E-mail, [email protected] *These authors contributed equally to this work. Keywords: histone code; DNA copy number chang- DOI 10.3858/emm.2010.42.7.050 es; gene expression; oncogenes; pheochromocyto- ma; tumor suppressor genes Accepted 3 June 2010 Available Online 8 June 2010 Introduction Abbreviations: MEN, multiple endocrine neoplasia; PGL, paragan- glioma syndromes; VHL, von Hippel Lindau disease Pheochromocytoma is a rare catecholamine-pro- ducing tumor that arises from chromaffin cells of the adrenal medulla or at extra-adrenal location Abstract (paraganglioma). The tumors mostly occur spora- dically although the hereditary syndromes multiple Epigenomic and genomic changes affect gene ex- endocrine neoplasia (MEN) 2A and 2B, von Hippel pression and contribute to tumor development. The Lindau disease (VHL), neurofibromatosis type 1 histone modifications trimethylated histone H3 lysine (NF1) and paraganglioma syndromes (PGL) 1, 2 4 (H3K4me3) and lysine 27 (H3K27me3) are epigenetic and 3 are all associated with the development of regulators associated to active and silenced genes, re- pheochromocytoma/paraganglioma. Mutations in spectively and alterations of these modifications have the proto-oncogene RET or the tumor suppressor genes VHL, NF1, SDHD, SDHC, and SDHB predi- been observed in cancer. Furthermore, genomic aber- sposes to tumor development in these disorders rations such as DNA copy number changes are com- (Xu et al., 1992; Latif et al., 1993; Mulligan et al., mon events in tumors. Pheochromocytoma is a rare en- 1993; Baysal et al., 2000; Niemann and Muller docrine tumor of the adrenal gland that mostly occurs 2000; Astuti et al., 2001a, 2001b). Approximately sporadic with unknown epigenetic/genetic cause. The 10-25% of apparently sporadic tumors harbor majority of cases are benign. Here we aimed to combine mutation in one of these predisposing genes the genome-wide profiling of H3K4me3 and (Neumann et al., 2002; Bryant et al., 2003). H3K27me3, obtained by the ChIP-chip methodology, However, the genetic/epigenetic cause(s) of the and DNA copy number data with global gene ex- majority of apparently sporadic tumors is still Combinatorial analysis in malignant pheochromocytoma 485 unknown. Although most pheochromocytomas are Mikkelsen et al., 2007; Pan et al., 2007; Zhao et al., benign, the prevalence of malignancy in sporadic 2007; Araki et al., 2009; Ke et al., 2009; Wei et al., adrenal pheochromocytomas is up to 10 % (Bravo 2009). However, global profiling of H3K27me3 and and Tagle 2003; Elder et al., 2005). The presence H3K4me3 and their suggested effects on gene of metastases or invasive behaviour is the only expression has not been extensively studied in criterium to determine malignancy. The diagnostic tumor tissue. H3K27me3-mediated tumor suppre- distinction between benign and malignant disease ssor gene silencing as well as H3K4me3 and may however be difficult in the absence of metas- H3K27me3 enrichment associated to differential tases. Novel molecular markers with prognostic gene expression have been observed in prostate relevance are needed. cancer cell lines (Kondo et al., 2008; Ke et al., Loss of heterozygocity and metaphase/array-com- 2009). The importance of this modification in tumor parative genomic hybridization (CGH) analyses of biology is further supported by reports that have both sporadic and familial pheochromocytomas demonstrated pre-marking by H3K27me3 of de have shown frequent losses on particularly chro- novo- DNA methylated genes in a colon cancer cell mosome arms 1p, 3p/q, 11p/q, 17p and 22q and line (Schlesinger et al., 2007) and frequent obser- gains on chromosome arms 1q, 12q, 17q, 19p/q and vations of polycomb protein EZH2 overexpression in 20q (Bender et al., 2000; August et al., 2004; several cancers (Simon and Lange 2008; Ke et al., Cascon et al., 2005; Aarts et al., 2006; Jarbo et al., 2009). The latter finding may be associated to the 2006; van Nederveen et al., 2009; Sandgren et al., polycomb repression signature of metastatic prostate 2010). DNA copy number changes in tumors may cells that was related to cancer outcome (Yu et al., reflect an important cause of altered gene expre- 2007). EZH2 is part of the polycomb repressive ssion and by locating these changes one may complex 2 that specifically trimethylates H3K27 on identify individual genes that have implications in target genes (Kirmizis et al., 2004). Furthermore, tumor development and progression. Global gene overexpression of the H3K4-specific methyltrans- expression profiling of pheochromocytomas has ferase SMYD3 has been observed in colorectal, mainly focused on, and revealed significant diffe- hepatic and prostate cancer cells (Hamamoto et al., rences between familial and subsets of sporadic 2004; Ke et al., 2009), indicative of H3K4 forms (Eisenhofer et al., 2004; Dahia et al., 2005) as hypermethylation at promoters of oncogenes. A role well as between malignant and benign cases for H3K4me3 in tumorigenesis was also suggested (Brouwers et al., 2006; Thouennon et al., 2007). by the finding that knockdown of SMYD3 by RNA Epigenetic changes are important in cancer interference inhibited cervical carcinoma cell growth biology since they may affect gene transcription. and invasion in vitro (Wang et al., 2008a). Epigenetic profiling of pheochromocytoma has been Here we have applied chromatin immunoprecipi- restricted so far to investigations of promoter tation coupled to oligonucleotide arrays with 35 bp CpG-methylation of selected genes (Geli et al., resolution (ChIP-chip), to genome-wide profile 2007, 2008; Margetts et al., 2008). Thus, it is of H3K4me3 and H3K27me3 occupancy in a malig- considerable interest to characterize genome-wide nant pheochromocytoma. Gene expression profiling the epigenetic mechanisms involved in benign and in relation to normal adrenal medulla was also malignant pheochromocytoma development. performed as well as array-CGH analysis to detect Posttranslational modifications of histones are tumor-associated DNA copy number changes. additional major epigenetic transcriptional control Comprehensive genome wide array-CGH results for mechanisms that establish domains of active and a large set of pheochromocytomas including the inactive chromatin. These modifications include present sample were recently described (Sandgren methylation of lysine residues that can serve as et al., 2010). Epigenetic profiling in tumor tissues is either repressive or active marks. Specifically, trime- preferred to tumor cell lines (Meissner et al., 2008) thylation on lysine 27 (H3K27me3) and lysine 4 and so far global H3K4me3/H3K27me3 profiling has (H3K4me3) on histone H3 are associated with silent been done only in a limited number of cases using and active gene expression, respectively (Kirmizis fresh or frozen tissues (Yu et al., 2007; Acevedo et et al., 2004; Bernstein et al., 2005; Barski et al., al., 2008; Rada-Iglesias et al., 2009; Zhang et al., 2007; Mikkelsen et al., 2007; Pan et al., 2007). 2009). This study provides an integrated and Several recent studies have reported global distri- detailed view of the genomic and epigenomic status bution patterns of these histone modifications in of a malignant pheochromocytoma, and investigates various cell types and their association to gene the impact of copy number changes and histone expression, mostly in embryonic stem (ES) cells of marks on gene expression in the search for human and mouse origin, but also in human and candidate tumor genes. mouse T cells and prostate cells (Barski et al., 2007; 486 Exp. Mol. Med. Vol. 42(7), 484-502, 2010 Figure 1. Distribution of histone modifications relative to genes. (A) The number (and fractions of total) of histone modification regions located
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