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Genome-Wide Alterations in Gene Methylation by the BRAF V600E Mutation in Papillary Thyroid Cancer Cells

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Genome-Wide Alterations in Gene Methylation by the BRAF V600E Mutation in Papillary Thyroid Cancer Cells Endocrine-Related Cancer (2011) 18 687–697 Genome-wide alterations in gene methylation by the BRAF V600E mutation in papillary thyroid cancer cells Peng Hou, Dingxie Liu and Mingzhao Xing Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology and Metabolism, The Johns Hopkins University School of Medicine, 1830 East Monument Street, Suite 333, Baltimore, Maryland 21287, USA (Correspondence should be addressed to M Xing; Email: [email protected]) Abstract The BRAF V600E mutation plays an important role in the tumorigenesis of papillary thyroid cancer (PTC). To explore an epigenetic mechanism involved in this process, we performed a genome- wide DNA methylation analysis using a methylated CpG island amplification (MCA)/CpG island microarray system to examine gene methylation alterations after shRNA knockdown of BRAF V600E in thyroid cancer cells. Our results revealed numerous methylation targets of BRAF V600E mutation with a large cohort of hyper- or hypo-methylated genes in thyroid cancer cells, which are known to have important metabolic and cellular functions. As hypomethylation of numerous genes by BRAF V600E was particularly a striking finding, we took a further step to examine the selected 59 genes that became hypermethylated in both cell lines upon BRAF V600E knockdown and found them to be mostly correspondingly under-expressed (i.e. they were normally maintained hypomethylated and over-expressed by BRAF V600E in thyroid cancer cells). We confirmed the methylation status of selected genes revealed on MCA/CpG microarray analysis by performing methylation-specific PCR. To provide proof of concept that some of the genes uncovered here may play a direct oncogenic role, we selected six of them to perform shRNA knockdown and examined its effect on cellular functions. Our results demonstrated that the HMGB2 gene played a role in PTC cell proliferation and the FDG1 gene in cell invasion. Thus, this study uncovered a prominent epigenetic mechanism through which BRAF V600E can promote PTC tumorigenesis by altering the methylation and hence the expression of numerous important genes. Endocrine-Related Cancer (2011) 18 687–697 Introduction and oncogenic activation of the BRAF kinase in Papillary thyroid cancer (PTC) is the most common the Ras/Raf/MEK/ERK signaling pathway (MAPK endocrine malignancy, accounting for 80% of all pathway; Davies et al. 2002). Through activating the thyroid cancers (Hundahl et al. 1998, Altekruse et al. MAPK pathway, the BRAF mutation plays a funda- 2010). A strong oncogenic mutation that has been mental role in the tumorigenesis of PTC and promotes characterized in recent years in PTC is the BRAF and predicts its poor clinical outcomes (Xing 2005, mutation, which, by far, is the most common 2007a). The specific molecular mechanisms under- oncogenic genetic event found in this cancer, occurring lying PTC tumorigenesis driven by the BRAF mutation in about 45% of cases on average (Xing 2005). There have not been well understood. This is particularly the are several types of BRAF mutations found in PTC and case in epigenetic aspects. For example, the role of the T1799A transverse point mutation accounts for vast aberrant gene methylation and its extent in this process majority of them (Nikiforov 2008). The T1799A BRAF have not been defined for BRAF mutation in PTC. mutation causes a substitution of valine with glutamic Gene methylation is an epigenetic phenomenon in acid in codon 600 (V600E), resulting in constitutive which a methyl group is covalently added to the fifth Endocrine-Related Cancer (2011) 18 687–697 Downloaded from Bioscientifica.comDOI: 10.1530/ERC-11-0212 at 09/29/2021 10:06:23AM 1351–0088/11/018–687 q 2011 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.orgvia free access P Hou et al.: BRAF mutation and gene methylation in PTC carbon of the cytosine residue in a CpG dinucleotide in to generate lentiviral particles. Cells were then exposed CpG islands, typically located in the promoter area of to lentivirus-containing supernatant for 48 h in the a gene. Promoter methylation often silences a gene and presence of polybrene (8 mg/ml final concentration). aberration in its methylation state can thus seriously After 2 days, transfected cells were serum starved affect its function. As such, hypermethylation can (0.5% FBS) and then harvested for 24 h in RIPA lysis silence tumor suppressor genes and hypomethylation buffer (Santa Cruz Biotechnology, Santa Cruz, CA, can cause over-expression of oncogenes. Conse- USA). Western blotting assays were used to detect quently, aberrant alterations in gene methylation play the protein expression of BRAF, HLX1, KLHL14, a fundamental role in human tumorigenesis (Jones HMGB2, NR4A2, FGD1, and ZBTB10. Cell pools & Baylin 2007, Hsiao et al. 2009, Kondo & Issa with stable transfection were selected using puromycin. 2010), including thyroid tumorigenesis (Xing 2007b). Given the importance of this epigenetic aberration Western blotting assay in thyroid tumorigenesis, we hypothesize that the Cells were lysed in RIPA buffer using standard protocol. BRAF V600E mutation, through activating the MAPK Protein lysates were separated on 10% SDS–PAGE pathway, may aberrantly affect gene methylation in and transferred onto polyvinylidene fluoride (PVDF) thyroid cancer, as a molecular mechanism in BRAF membranes (Amersham Pharmacia Biotech), followed by mutation-promoted thyroid tumorigenesis. In this incubation with specific primary antibodies. Anti-BRAF study, we performed a genome-wide screening of (sc-166), anti-phospho-ERK (sc-7383), anti-ZBTB10 gene methylation in thyroid cancer cells using a (sc-87402), and anti-actin (sc-1616-R) were purchased methylated CpG island amplification (MCA)/CpG from Santa Cruz (Santa Cruz, CA, USA). Anti-HLX1 island microarray approach (Este´cio et al. 2007) and (AV100839), anti-KLHL14 (AV34677), anti-HMGB2 selectively examined the cellular functions of some (AV31939), anti-NR4A2 (AV38753), and anti-FGD1 genes among which many identified to be altered in (AV31673) were purchased from Sigma. Antigen– the methylation state by BRAF V600E. antibody complexes were visualized using HRP-con- jugated anti-mouse (sc-2005, Santa Cruz) or anti-rabbit Materials and methods (sc-2004, Santa Cruz) IgG antibodies and ECL western blotting analysis system (Amersham Pharmacia). Thyroid cancer cell lines The PTC-derived cell lines BCPAP and OCUT1 were Methylated CpG island amplification from Dr Massimo Santoro (University of Federico II, The MCA assay was performed as described pre- Naples, Italy) and Dr Naoyoshi Onoda (Osaka City viously (Toyota et al. 1999). Briefly, w5 mg of each University Graduate School of Medicine, Osaka, genomic DNA from cells stably transfected with Japan) respectively. We chose these two thyroid BRAF shRNA (termed ‘test’ hereafter) and vector cancer cell lines because both harbor the BRAF (termed ‘control’ hereafter) were digested with 100 V600E mutation. Cells were routinely grown at 37 8C units of methylation-sensitive restriction endonuclease in RPMI 1640 medium containing 10% fetal bovine SmaI (New England Biolabs, Ipswich, MA, USA) for serum (FBS) with standard supplements. Genomic 16 h at 20 8C in a total volume of 100 ml, followed by DNA was isolated by SDS and proteinase K digestion, digestion with 20 units of methylation-insensitive followed by standard phenol–chloroform extraction restriction endonuclease XmaI for 6 h at 37 8C, which and ethanol precipitation. leaves sticky ends (C/CCGGG). DNA fragments were then precipitated with ethanol and their sticky ends Lentivirus-mediated RNA interference ligated with 0.5 nmol of unphosphorylated linkers The lentiviral pSicoR-PGK-puro vectors (Addgene, RXMAK24/K12, as described (Toyota et al. 1999). Inc., Cambridge, MA, USA) encoding seven self- The oligonucleotide sequences were as follows: complementary hairpin RNA sequences were used (RXMAK24) 50-AGC ACT CTC CAG CCT CTC to knock down seven selected genes, including ACC GAC-30 and (RXMAK12) 50-CCG GGT CGG BRAF, HLX1, KLHL14, HMGB2, NR4A2, FGD1, and TGA-30. PCR reactions were performed using the ZBTB10. The sequences are presented in Supplemen- digests as templates and subjected to 20 cycles of tary Table 1, see section on supplementary data given amplification as described previously (Este´cio et al. at the end of this article. Lentiviral vector DNAs and 2007). Ten microliters of PCR product were resolved packaging vectors were transfected into human in 1.5% agarose gel and visualized under u.v. after embryonickidney293cells(ATCC,Manassas,VA,USA) ethidium bromide staining. Successful MCA reactions Downloaded from Bioscientifica.com at 09/29/2021 10:06:23AM via free access 688 www.endocrinology-journals.org Endocrine-Related Cancer (2011) 18 687–697 resulted in amplicon smear ranging from 300 bp to RNA extraction and real-time quantitative 3 kb, with most amplicons at 1 kb. The PCR products RT-PCR analysis were purified and reserved for fluorescence labeling. RNA expression analysis was performed by real-time quantitative PCR using the ABI Prism 7900HT CpG island microarray Sequence Detection System Instrument (Applied Biosystems, Carlsbad, CA, USA). Two micrograms Amino-allyl dUTP (aa-dUTP; Sigma) was incorpor- of total RNA underwent RT using Oligo-dT and ated into 600 ng of ‘test and control’ amplicons using SuperScript II according to the instructions of the the BioPrime DNA labeling system (Life Tech- manufacturer (SuperScript First-Strand Synthesis kit, nologies,
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