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Promoter Methylation Status of Ras-Association Domain Family Members in Pheochromocytoma

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Promoter Methylation Status of Ras-Association Domain Family Members in Pheochromocytoma ORIGINAL RESEARCH ARTICLE published: 19 February 2015 doi: 10.3389/fendo.2015.00021 Promoter methylation status of Ras-association domain family members in pheochromocytoma Antje M. Richter*,Tobias Zimmermann,Tanja Haag, Sara K. Walesch and Reinhard H. Dammann Institute for Genetics, University of Giessen, Giessen, Germany Edited by: Pheochromocytomas (PCCs) are rare neuroendocrine tumors that arise from the medulla Fabio Coppedè, University of Pisa, of the adrenal gland or the sympathetic ganglia and are characterized by the secretion of Italy catecholamines. In 30–40% of patients, PCCs are genetically determined by susceptibility Reviewed by: Daniela Pasquali, Seconda Università genes as various as RET, VHL, and NF1. We have analyzed the Ras-association domain degli Studi Napoli, Italy family members (RASSFs) in PCCs regarding their inactivating promoter hypermethylation Francesca Peruzzi, LSU Health status. Previously, we reported a promoter methylation in PCC for the first family mem- Sciences Center, USA ber RASSF1A. Promoter hypermethylation of CpG islands leads to the silencing of the *Correspondence: according transcript and is a common mechanism for inactivation of tumor suppressors. In Antje M. Richter, Heinrich-Buff Ring 58, Giessen, Hessen, Germany this study, we observed inactivating DNA modifications for the RASSF members RASSF2, e-mail: [email protected] RASSF5A, RASSF9, and RASSF10, but not for the members RASSF3, RASSF4, RASSF5C, giessen.de RASSF6, RASSF7,and RASSF8.The degree of promoter methylation was 19% for RASSF2, 67% for RASSF5A, 18% for RASSF9, and 74% for RASSF10. Interestingly, the degree of hypermethylation for RASSF10 in hereditary PCCs was 89 vs. 60% in sporadic PCCs. A similar but less dramatic effect was observed in RASSF5A and RASSF9. Including all RASSF members, we found that of 25 PCCs, 92% show promoter methylation in at least in one RASSF member. In 75% of the hereditary PCC samples, we found two or more methy- lated RASSF promoters, whereas in sporadic PCCs only 46% were observed. In summary, we could show that in PCC several RASSF members are strongly hypermethylated in their promoter regions and methylation of more than one RASSF member occurs in the majority of PCCs. This adds the inactivation of genes of the RASSF tumor suppressor family to the already known deregulated genes of PCC. Keywords: pheochromocytoma, tumor suppressor, DNA methylation, epigenetics, RASSF INTRODUCTION neoplasia (8). Our focus lies on the tumor suppressors of the Pheochromocytomas (PCCs) are rare catecholamine-secreting Ras-association domain family (RASSF) containing 10 members tumors derived from chromaffin cells and 10% of PCCs are malig- (RASSF1A to RASSF10), which are frequently inactivated via the nant. Tumors are malign when the capsule is invaded or metastases methylation of their CpG island promoter (9). The RASSFs were occur. The hormone imbalance affects the cardiovascular system first described in 2000, by the characterization of the first family leading to hypertension and other metabolic processes. PCC treat- member RASSF1A (10). The family members are characterized ment involves treatment of hypertension and surgical removal of by the presence of the Ras-association domain (C-terminus or the tumor. (1, 2) The occurrence peaks between the ages of 20– N-terminus) and additional protein–protein interaction domains 40 and up to 30–40% occur due to germline mutations of 11 [SARAH (11) or coiled-coil domains]. Their functions range from known susceptibility genes like HIF2A (hypoxia-inducible fac- microtubule network interaction, RAS interaction, Hippo path- tor 2a) or SDH (succinate dehydrogenase) (3). The syndromes way involvement, cell cycle regulation to apoptosis induction (9, familial multiple endocrine neoplasia (MEN) type 2A/2B, von 12). Interestingly, mice studies exist for several RASSFs and sug- Hippel-Lindau disease, and neurofibromatosis are also associ- gest an involvement in tissue or organ size control (13–17). Due ated with PCC development (4). Regarding RASSF1A, which to our earlier studies of RASSF1A inactivation in PCC (7) and is frequently inactivated in different tumor entities (5, 6), we the known contribution of tumor suppressor inactivation to can- have reported its inactivation in the tumor entity of PCC (7). cer development (8), we were interested in the possibility that Tumor suppressors act antiproliferatively and are known to be inactivation of more than one RASSF member occurs in PCC. negatively regulated by their CpG island promoter hypermethy- Their inactivation could contribute to tumor formation of PCC lation, leading to the inactivation of the according transcript in and we therefore used the combined bisulfite restriction analysis (COBRA) methylation analysis to study the promoter methyla- tion status of the RASSF members RASSF2, RASSF3, RASSF4, Abbreviations: COBRA, combined bisulfite restriction analysis; PCC, pheochro- RASSF5A, RASSF5C, RASSF6, RASSF7, RASSF8, RASSF9, and mocytoma; RASSF, Ras-association domain family; TSG, tumor suppressor gene. RASSF10. www.frontiersin.org February 2015 | Volume 6 | Article 21 | 1 Richter et al. RASSF methylation in pheochromocytoma EXPERIMENTAL was purified using MSB Spin PCRapace (STRATEC Molecular, CpG ISLAND PREDICTION, PCR PRODUCT SIZE, AND DIGESTION Berlin, Germany), eluted in 50 ml H2O and followed by 10 min PRODUCTS incubation with 5 ml of 3 M NaOH at 37°C. DNA was then pre- Promoter regions of RASSF2, RASSF3, RASSF4, RASSF5A, cipitated with 100% ethanol and 7.5 M ammonium acetate and RASSF5C, RASSF6, RASSF7, RASSF8, RASSF9, and RASSF10 were resolved in 1× TE buffer. Two hundred nanograms were subse- analyzed for their CpG islands by CpG plot http://www.ebi.ac.uk/ quently used for 25 ml PCR reaction with COBRA primers. The Tools/seqstats/emboss_cpgplot/ and ucsc genome browser https: PCR product was either mock digested or digested with 0.5 ml //genome.ucsc.edu/. Except for RASSF6 and RASSF9, all members of TaqI (Fermentas GmbH, St.Leon-Rot, Germany) 1 h at 65°C show prominent CpG islands. Primers for COBRA methylation and resolved on 2% TBE gel together with mock control (18, analysis were designed to frame at least one restriction site for 19). Digestion of the PCR product indicates an underlying CpG TaqI enzyme on bisulfite treated and therefore fully converted methylation at the analyzed restriction site. We used an in vitro DNA (unmethylated cytosine to uracil). The resulting PCR prod- methylated (ivm) DNA as a positive control for COBRA. For ucts lengths, digestion products, and PCR conditions are listed in in vitro methylation of genomic DNA, we used M.SssI methylase Table S1 in Supplementary Material. (NEB, Frankfurt, Germany) (20). Human fibroblasts (HF) were used as a normal unmethylated control cell line. PHEOCHROMOCYTOMA AND CONTROLS RESULTS Primary tissues were previously published (7). All patients signed informed consent at initial clinical investigation. The study was In the current study, we have analyzed a total of 25 PCC approved by a local ethic committee (Martin-Luther University, patient samples of which 13 were sporadic and 12 hereditary Halle, Germany). (Table 1). We have studied the promoter methylation status of RASSF2, RASSF3, RASSF4, RASSF5A, RASSF5C, RASSF6, METHYLATION ANALYSIS BY COBRA RASSF7, RASSF8, RASSF9, and RASSF10. DNA promoter methy- DNA was isolated by phenol-chloroform extraction. Two micro- lation is an indicator of epigenetic inactivation of tumor suppres- m grams of genomic DNA from PCC was bisulfite treated (12 l of sor genes (TSGs) (8). The CpG island sizes are 1.1 kb for RASSF2, m 0.1 M hydroquinone, 208 l of 1.9 M sodium metabisulfite, and 1.1 kb for RASSF3, 0.5 kb for RASSF4, 1.2 kb for RASSF5A, 0.5 kb pH 5.5 with NaOH) and incubated over night at 50°C. Then DNA for RASSF5C, 0.2 kb for RASSF6, 1 kb for RASSF7, 1.2 kb for Table 1 | RASSF promoter methylation in sporadic (SP) and hereditary (MP) pheochromocytoma. Nr Age Sex Status R1A R2 R3 R4 R5A R5C R6 R7 R8 R9 R10 1 SP17 49 f b 1 n.a. 0 0 0 0 0 n.a. n.a. n.a. n.a. 2 SP2 36 f b 1 0 0 0 1 0 0 0 0 0 n.a. 3 SP1 72 m b 1 1 0 0 1 0 0 0 0 0 1 4 SP25 54 m m 1 1 0 0 1 0 0 0 0 0 0 5 SP4 49 m b 1 n.a. n.a. n.a. n.a. 0 n.a. 0 0 0 n.a. 6 SP11 52 f m 0 0 0 0 0 0 0 0 0 0 0 7 SP6 34 m m 0 0 0 0 1 0 0 0 0 0 0 8 SP13 44 m b 0 n.a. n.a. n.a. 1 0 n.a. 0 0 1 1 9 SP15 20 f b 0 0 n.a. n.a. 1 0 n.a. 0 0 1 0 10 SP5 42 f b 0 0 0 0 0 0 0 0 0 0 1 11 SP22 44 m b 0 0 0 0 0 0 0 0 0 0 1 12 SP26 64 f b 0 n.a. 0 0 n.a. n.a. 0 0 0 0 1 13 SP14 75 f b 0 0 0 0 1 0 0 0 0 0 1 14 MP19 38 f b 1 1 n.a. 0 0 0 0 0 0 1 1 15 MP24 31 m b 1 0 n.a. n.a. 1 0 n.a. 0 0 1 1 16 MP18 22 f b 1 n.a. 0 0 0 0 0 0 0 0 1 17 MP8 45 m b 1 n.a. 0 0 1 0 0 0 0 0 1 18 MP23 31 f b 1 0 0 0 0 0 0 0 0 0 n.a. 19 MP21 29 f b 1 0 0 0 1 0 0 0 0 0 n.a.
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