Comprehensive DNA Methylation Analysis of Benign and Malignant Adrenocortical Tumors
Annabelle L. Fonseca, 1,2 Johan Kugelberg,1,2,3 Lee F. Starker, 1,2 Ute Scholl,4 Murim Choi,4 Per Hellman,5 Göran Åkerström,5 Gunnar Westin,5 Richard P. Lifton,4 Peyman Björklund,1,2,5 and Tobias Carling
“Department of Surgery, Yale University School of Medicine, New Haven, CT
2 Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, New Haven, CT
3Faculty of Health Sciences, Linköping University, Linköping, Sweden
4 Department of Genetics, Yale University School of Medicine, New Haven, CT
5 Department of Surgical Sciences, Uppsala University, Uppsala, Sweden
6Cancer Genetics and Genomics Program, Yale Cancer Center, Yale University School of Medicine, New Haven, CT
The molecular pathogenesis of benign and malignant adrenocortical tumors (ACT) is incompletely clarified. The role of DNA methylation in adrenocortical tumorigenesis has not been analyzed in an unbiased, systematic fashion. Using the Infin- ium HumanMethylation27 BeadChip, the DNA methylation levels of 27,578 CpG sites were investigated in bisulfite-modi- fied DNA from 6 normal adrenocortical tissue samples, 27 adrenocortical adenomas (ACA), and 15 adrenocortical carcinomas (ACC). Genes involved in cell cycle regulation, apoptosis, and transcriptional regulation of known or putative importance in the development of adrenal tumors showed significant and frequent hypermethylation. Such genes included CDKN2A, GATA4, BCL2, DLEC1, HDAC10, PYCARD, and SCGB3A1/HIN1. Comparing benign versus malignant ACT, a total of 212 CpG islands were identified as significantly hypermethylated in ACC. Gene expression studies of selected hypermethy- lated genes (CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, SCGB3A1/HIN1) in 6 normal and 16 neoplastic adrenocortical tis- sues (10 ACA and 6 ACC), displayed reduced gene expression in benign and malignant ACT versus normal adrenocortical tissue. Treatment with 5-aza-2’-deoxycytidine of adrenocortical cancer H-295R cells increased expression of the hyperme- thylated genes CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, and SCGB3A1/HIN1. In conclusion, the current study represents the first unbiased, quantitative, genome-wide study of adrenocortical tumor DNA methylation. Genes with altered DNA methylation patterns were identified of putative importance to benign and malignant adrenocortical tumor devel- opment. @ 2012 Wiley Periodicals, Inc.
INTRODUCTION
Adrenocortical tumors (ACT) are common. Most of these tumors are benign non-functioning adrenocortical adenomas (ACA), and are being increasingly found incidentally through diagnostic imaging for a non-adrenal-related reason, hence the term “adrenal incidentaloma” (Wandoloski et al., 2009). Adrenocortical carcinomas (ACC), on the other hand, are rare malignancies, with an estimated prevalence of 4-12 per million adults (Bertherat and Bertagna, 2009). Functioning ACAs (overproducing either aldosterone or corti- sol) may cause significant morbidity, and ACC is associated with poor prognosis and has an overall 5-year survival of 37-38% (Icard et al., 2001; Abiven et al., 2006). In addition, the histopatho- logical distinction between ACA and ACC may be difficult in the absence of widely invasive or metastatic disease (Weiss, 1984; Aubert et al., 2002; DeLellis, 2004; Pohlink et al., 2004; Bili- moria et al., 2008; de Reyniès et al., 2009). A bet- ter method of distinguishing benign from malignant ACTs is therefore needed and atten-
tion has turned toward a search for molecular markers (Soon et al., 2008).
The molecular pathogenesis of both benign and malignant ACT is incompletely clarified. We recently demonstrated that somatic KCNJ5 muta- tions occur frequently in aldosterone producing ACA (Choi et al., 2011), but has not been identi- fied in other ACTs. Identification of molecular markers of adrenocortical carcinoma has been the focus of much recent research. Some suggested markers of malignant adrenocortical tumors include insulin-like growth factor 2 (IGF2) over
Additional Supporting Information may be found in the online version of this article.
Supported by: NIH (Yale Clinical and Translational Science Award UL1 RR024139).
*Correspondence to: Tobias Carling, Department of Surgery, Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, 333 Cedar Street, TMP202, PO 208062, New Haven, CT 06520, USA. E-mail: tobias.carling@yale.edu
Received 12 September 2011; Accepted 10 May 2012 DOI 10.1002/gcc.21978
Published online in Wiley Online Library (wileyonlinelibrary.com).
expression, loss of heterozygosity (LOH) at chro- mosome region 11q13, allelic losses at TP53 locus (17p13), and MKI67 and CCNE over expression (Kjellman et al., 1999; Libè et al., 2007; Soon et al., 2008; de Reyniès et al., 2009). The Wnt/B- catenin pathway has also been implicated in the pathogenesis of adrenocortical tumors. Mutations of the ß-catenin gene (CTNNB1) are common in preferentially non-functioning ACAs and in ACC (Gaujoux et al., 2008; Bonnet et al., 2011). Immu- nohistological studies suggest that ß-catenin delocalization is observed in both benign and ma- lignant adrenocortical tumors, suggesting an abnormal activation of the Wnt/ß-catenin signal- ing pathway (Tissier et al., 2005). Loss of hetero- zygosity within chromosome region 9p21 associated with lack of CDKN2A expression has been shown to be associated with adrenocortical carcinomas (Pilon et al., 1999). The CDKN2A locus codes for two different proteins: p16INK4a and p14ARF. p16INK4a interacts with CDK4 and induces G1 cell cycle arrest by inhibiting Rb phosphorylation, while p14ARF stabilizes TP53 by inhibiting MDM2 and inducing cell cycle arrest. These two proteins thus regulate both the p53 pathway and the retinoblastoma (RB) pathway, two critical cell cycle regulatory pathways. Activa- tion of both p16INK4a and p14ARF thus induces inhibition of cell proliferation, and their inactiva- tion has been reported in numerous carcinomas (Caldas et al., 1994; Bartsch et al., 1995; Liu et al., 1995; Florl et al., 2000; Nosho et al., 2008; Abou-Zeid et al., 2011; Alves et al., 2011; Cul’- bová et al., 2011; Demokan et al., 2011; Starker et al., 2011; Zang et al., 2011). The recent use of large scale analysis of gene expression has led to a large amount of progress in both the under- standing of tumorigenesis and tumor classifica- tion, and has been used to much advantage in adrenocortical tumors (Giordano et al., 2003; de Fraipont et al., 2005). It has recently been dem- onstrated that a unique transcriptome profile is associated with adrenocortical tumors, including over expression of IFG2 (Giordano et al., 2003). CTNNB1 and TP53 alterations have also been found to be associated with poor outcome in ACC (Ragazzon et al., 2010).
There is also growing evidence to suggest that epigenetic abnormalities including DNA methyla- tion and histone modification play a key role, in conjunction with genetic modifications, to cause altered patterns of gene expression, resulting in tumorigenesis. DNA hypermethylation of the promoter cytosine in cytosine phosphate guanine
islands (CpG islands) causing downstream gene silencing has been shown to be intimately involved in tumorigenesis (Das and Singal, 2004; Jones and Baylin, 2007; Wright and Gilbertson, 2010). The role of DNA hypermethylation in ad- renocortical tumorigenesis has been evaluated. Altered DNA methylation of the H19 promoter has been shown to be involved in the abnormal expression of both H19 and IGF2 genes in adre- nocortical carcinomas (Gao et al., 2002). Promoter methylation of TP53, however, has been demon- strated not to be a significant event in the devel- opment of adrenocortical carcinomas (Sidhu et al., 2005).
However, DNA methylation studies thus far have focused on individual genes. The current study represents the first comprehensive genome- wide analysis of DNA methylation in benign and malignant adrenocortical tumors.
MATERIAL AND METHODS
Subjects and Tissues
Normal adrenal tissue, ACAs and ACCs that had been surgically resected in the clinical rou- tine were obtained. Tissues were snap-frozen in liquid nitrogen and stored in -80℃ until further processing. Normal adrenocortical tissue was obtained from patients without hormone overpro- duction undergoing adrenalectomy. ACAs were either non-functional or causing hyperaldosteronism or hypercortisolism. All cases of ACC displayed unequivocal histopathological characteristics of malignancy. All tumors were carefully evaluated and dissected by an experienced endocrine pathologist prior to use in the study. Informed consent and approval by institutional review boards at participating institutions was obtained.
Genome-wide DNA Methylation Profiling
High molecular weight genomic DNA was iso- lated from normal and neoplastic adrenal tissue using the Qiagen DNeasy Tissue Kit according to manufacturer instructions. For genome-wide DNA methylation profiling, DNA from 48 tissue samples was interrogated (6 normal adrenal cor- tex, 27 ACAs; 9 non-functional, 9 cortisol-produc- ing, 9 aldosterone-producing, and 15 ACCs; 9 non-functional, 6 cortisol-producing). Genomic DNA (500 ng) was bisulfite modified using the Epitect Bisulfite Kit (Qiagen) according to manu- facturer instructions and analyzed using Infinium HumanMethylation27 BeadChip (Illumina, San
Diego, CA). This Infinium HumanMethylation27 BeadChip protocol consists of six steps (whole- genome amplification, fragmentation, hybridiza- tion, washing, counterstaining, and scanning) (Thirlwell et al., 2010), which were carried out at the Yale Center for Genome Analysis at Yale Uni- versity. Details regarding this can be found at the following site: http://www.illumina.com/downloads/ InfMethylation_AppNote.pdf. The Illumina Infin- ium HumanMethylation27 BeadChip platform allows interrogation of 27,578 CpG sites per sam- ple at single-nucleotide resolution. The BeadChip, which can analyze 12 samples per array, targets CpG sites located within the proximal promoter regions of transcription start sites of 14,475 consen- sus coding sequencing (CCDS) in the NCBI Data- base (Bibikova et al., 2006; Killian et al., 2009).
Determination of Gene Expression Analysis Using Quantitative RT-PCR
Total RNA was extracted from adrenocortical tissue (6 normal adrenal cortex, 10 ACAs, and 6 ACCs). cDNA was synthesized using 1 µg of total RNA and iScript cDNA Synthesis Kit (Bio-Rad Laboratories Inc. Hercules, CA, USA). Quantita- tive real-time PCR was performed on StepOne- Plus™M Real-Time PCR systems (Applied Biosystems, Foster City, CA) using assays for CDKN2A (Hs00923894_m1), GATA4(Hs00171403_m1), DLEC1 (Hs00201098_m1), HDAC10(Hs00368899_m1), PYCARD (Hs00203118_m1), and SCGB3A1|HIN1 (Hs00369360_m1) with GAPDH (Hs99999905_m1) used as the housekeeping gene. Results were also verified using three additional housekeeping genes: TBP (Hs00427620_m1), GUSB (Hs00939627_m1), and ACTB (Hs99999903_m1) (All from Applied Bio- systems, Foster City, CA). Each cDNA sample was analyzed in triplicate. Standard curves for each experiment were established by amplifying a puri- fied PCR fragment covering the sites for probes and primers.
Determination of Baseline Methylation Status Using MS-PCR, In Vitro Cell Cultures, and Treatment with 5-Aza-2’-Deoxycytidine
The adrenocortical cancer cell line H295-R was used (ATCC: The Global Bioresource Center). The cells were cultured in accordance with ATCC protocol as described (Bird et al., 1993; Kanczkowski et al., 2009).
To verify the baseline methylation status of selected genes in the H295R cells, these genes
were analyzed using methylation-specific (MS)- PCR. Primers were designed using the Methyl Primer Express software v.1.0 (Applied Biosys- tems). Primer sequences are presented in Sup- porting Information Table 2. Semiquantitative PCR was performed using SYBR-Green PCR Master Mix and results were analyzed using Ste- pOne Software v2.1 (Applied Biosystems, Foster City, CA, USA).
H295-R cells were treated in triplicate with 5 uM 5-aza-2’-deoxycytidine (Sigma-Aldrich) as described (Starker et al., 2011). Cells were har- vested after 24 and 48 h for DNA and RNA extraction.
Statistical Analysis
The BeadChip was scanned on the Illumina iScan and analyzed with the Beadstudio software (Version 3.2; Illumina). The output of the Bead- studio analysis is a beta-value for each CpG site interrogated. This is a continuous value between 0 and 1 where 0 indicates 0% methylation and 1 indicates 100% methylation at a given CpG site. Therefore, this assay provides quantitative meth- ylation measurement at the single CpG site level. The calculation of the beta-value is performed as described (Thirlwell et al., 2010). Wilcoxon rank test, Student’s unpaired t-test, and Fisher exact test were used for statistical evaluation, with P < 0.05 considered to be significant. All results are expressed as mean ± SEM (standard error of the mean).
RESULTS
Genome-wide DNA Methylation Profiling of Adrenal Tumors
Using the Illumina Infinium HumanMethyla- tion27 BeadChip, a genome-wide DNA methyla- tion platform that interrogates 27,578 cytosine- guanine (CpG) sites, we analyzed the DNA methylome in normal adrenal cortex tissue (n = 6), ACAs (n = 27), and ACCs (n = 15). Compar- ing normal adrenal cortex and ACCs, genes known to be involved in tumorigenesis such as CDKN2A, GATA4, SCGB3A1, PYCARD, and DLEC1 were highly and significantly hyperme- thylated in ACC. The top 50 hypermethylated genes in adrenocortical carcinomas versus normal adrenal cortex are presented in Table 1. Several genes displayed significant hypermethylation in ACCs versus normal adrenal cortex at multiple CpG sites, including CDKN2A, ZNF154, GATA4,
| Gene symbol | p value | ß-Value difference | Function |
|---|---|---|---|
| CDKN2A* | 0.018 | 0.363 | Tumor suppressor |
| LEP | 0.037 | 0.344 | Cell cycle regulation |
| CYBA | 0.017 | 0.330 | Microbicidal oxidase system |
| ZNF154* | 0.016 | 0.316 | Transcriptional regulator |
| PAX3 | 0.034 | 0.310 | Transcription factor |
| GATA4* | 0.022 | 0.304 | Transcription regulator |
| ABHD9 | 0.021 | 0.297 | Tumor suppressor gene |
| ALX4* | 0.028 | 0.294 | Homeobox gene |
| DOCK2 | 0.023 | 0.289 | Transcriptional activator |
| C1QTNF5 | 0.026 | 0.282 | Cell adhesion |
| LAMA5 | 0.015 | 0.279 | Basement membrane protein |
| MEGF11 | 0.014 | 0.279 | Cell membrane protein |
| FERD3L | 0.038 | 0.273 | Transcriptional inhibitor |
| OTOP3 | 0.014 | 0.269 | Basement membrane protein |
| FARP1 | 0.017 | 0.265 | Transcriptional regulator |
| ISYNA1 | 0.019 | 0.261 | Cell signaling molecule |
| MPP2 | 0.024 | 0.260 | Cell cycle regulation |
| MAP4K1 | 0.035 | 0.258 | Hematopoietic cell growth regulator |
| SPAG6 | 0.018 | 0.256 | Cell structural integrity |
| PYCARD* | 0.016 | 0.254 | Tumor suppressor |
| PCDHGC4 | 0.023 | 0.251 | Cell membrane protein |
| NEUROG1 | 0.012 | 0.247 | Cell differentiation |
| FLJ20245 | 0.040 | 0.244 | Basement membrane protein |
| DLX5 | 0.041 | 0.244 | Homeobox gene |
| DLEC1* | 0.023 | 0.243 | Tumor suppressor |
| CD9 | 0.029 | 0.243 | Cell adhesion |
| LAD1 | 0.031 | 0.242 | Basement membrane protein |
| C20orf100 | 0.022 | 0.240 | Transcriptional activator |
| MGC22793 | 0.024 | 0.239 | Hypothetical protein |
| ENTPD3 | 0.025 | 0.237 | Basement membrane protein |
| PRRT1 | 0.028 | 0.237 | Transmembrane protein |
| DCHS2 | 0.020 | 0.236 | Cell adhesion |
| ARHGEF1 | 0.018 | 0.234 | GTPase regulator |
| MPP2 | 0.028 | 0.231 | Cell cycle regulation |
| HDAC10 | 0.016 | 0.229 | Chromatin modulator |
| CNN1 | 0.039 | 0.228 | Muscle contraction modulation |
| TCF15 | 0.027 | 0.228 | Transcription regulator |
| NEFH | 0.024 | 0.227 | Neuronal intracellular transport |
| ATP8A2 | 0.039 | 0.227 | Cellular membrane protein |
| ST6GALNAC2 | 0.025 | 0.227 | Cell adhesion |
| HIST IH4] | 0.016 | 0.226 | Transcription regulation |
| TUBB6 | 0.017 | 0.226 | Microtubule assembly |
| BCL2 | 0.031 | 0.226 | Apoptosis |
| BLVRA | 0.053 | 0.225 | Porphyrin metabolism |
| FLJ32569 | 0.028 | 0.225 | Metal ion binding activity |
| ADRA2B | 0.026 | 0.225 | Cell membrane protein |
| AFAR3 | 0.047 | 0.224 | Transcription factor |
| NFATC2 | 0.023 | 0.223 | Transcription factor |
| AMT | 0.026 | 0.222 | Mitochondrial enzyme |
| CHAD | 0.025 | 0.221 | Cell adhesion |
*indicates genes where multiple CpG islands were differentially hypermethylated. Genes in bold were analyzed in Cohort 2 of adrenocortical tumors. B-Value difference is the difference between the raw B-values of adrenocortical carcinoma and normal adrenal cortex.
ALX4, PYCARD, and DLEC1. Comparing benign versus malignant ACT, a total of 212 CpG islands were identified as significantly hypermethylated in ACC (P < 0.05). The top 50 hypermethylated genes in ACCs versus ACAs are presented in Table 2. The top 50 hypermethylated genes in
ACAs versus normal adrenal cortex are presented in Supporting Information Table 1. Multiple CpG sites were hypermethylated in the ALX4, CDKN2A, ZNF154, DLX5, MPP2, CNTN2, BCOR, HCG9, PYCARD, and DLEC1 genes in ACC ver- sus ACA. Comparing non-functional and
DNA METHYLATION IN ADRENOCORTICAL TUMORS
| Gene symbol | p value | ß-Value difference | Function |
|---|---|---|---|
| ALX4* | 0.014 | 0.395 | Homeobox gene |
| CDKN2A* | 0.031 | 0.368 | Tumor suppressor |
| CYBA | 0.020 | 0.365 | Microbicidal oxidase system |
| ABHD9 | 0.022 | 0.341 | Tumor suppressor |
| MEGF11 | 0.030 | 0.340 | Cell membrane protein |
| AMT | 0.022 | 0.332 | Mitochondrial enzyme |
| ISYNA1 | 0.021 | 0.327 | Cell signaling molecule |
| SPAG6 | 0.028 | 0.318 | Cell structural integrity |
| TMEM106A | 0.023 | 0.317 | Transmembrane protein |
| C19orf35 | 0.023 | 0.316 | Hypothetical protein |
| ZNF154* | 0.018 | 0.316 | Zinc finger protein |
| ADRA2B | 0.014 | 0.313 | Cell membrane protein |
| LAMA5 | 0.035 | 0.311 | Basement membrane protein |
| PDE9A | 0.014 | 0.308 | Cell signal transduction |
| PAX3 | 0.020 | 0.304 | Transcription factor |
| PGLYRP1 | 0.019 | 0.303 | Transcription factor |
| HSU79303 | 0.025 | 0.303 | Promotes degradation of p53 |
| CSPG5 | 0.035 | 0.301 | Growth factor in neurogenesis |
| WNT10A | 0.016 | 0.298 | Proto-oncogene |
| LEP | 0.016 | 0.296 | Cell signaling pathway |
| TMEM130 | 0.022 | 0.293 | Transcription factor |
| TUBB6 | 0.012 | 0.287 | Scaffold for cell shape |
| BAPX1 | 0.037 | 0.286 | Homeobox gene |
| DLX5* | 0.022 | 0.283 | Homeobox gene |
| CHAD | 0.021 | 0.282 | Cell adhesion |
| NEUROG1 | 0.029 | 0.280 | Cell differentiation |
| DNASE1L2 | 0.018 | 0.274 | Deoxyribonuclease |
| CD9 | 0.022 | 0.273 | Cell adhesion |
| MPP2* | 0.023 | 0.272 | Cell cycle regulation |
| AHNAK | 0.025 | 0.270 | Neuronal cell differentiation |
| FARP1 | 0.025 | 0.269 | Transcriptional regulator |
| FEV | 0.020 | 0.268 | Transcriptional regulator |
| HES6 | 0.018 | 0.266 | Transcriptional repressor |
| KIAA 1446 | 0.027 | 0.265 | Cell membrane protein |
| CGREF1 | 0.016 | 0.265 | Cell growth regulator |
| RAB34 | 0.033 | 0.264 | Proto-oncogene |
| LAD1 | 0.027 | 0.261 | Basement membrane protein |
| OTOP3 | 0.023 | 0.260 | Basement membrane protein |
| CNTN2 ** | 0.037 | 0.260 | Cell adhesion |
| OLFM2 | 0.025 | 0.259 | Nervous system development |
| DOCK2 | 0.016 | 0.258 | Lymphocyte chemotaxis |
| BCOR* | 0.017 | 0.257 | Apoptosis |
| LRRC8C | 0.031 | 0.257 | Endoplasmic reticulum membrane protein |
| ENTPD2 | 0.043 | 0.257 | Membrane protein |
| ST6GALNAC2 | 0.025 | 0.254 | Cell adhesion |
| SH3TC2 | 0.026 | 0.254 | Adaptor/docking molecule |
| HCG9* | 0.041 | 0.253 | Noncoding gene |
| ATP8A2 | 0.023 | 0.253 | ATPase activity, phospholipid transport |
| TGIF2 | 0.033 | 0.252 | Homeobox gene |
| GATA4* | 0.027 | 0.246 | Transcription regulator |
| PYCARD* | 0.023 | 0.237 | Tumor suppressor |
| HDAC10 | 0.019 | 0.233 | Chromatin modulator |
| DLEC1* | 0.034 | 0.229 | Tumor suppressor |
| SCGB3A1/HIN1 | 0.031 | 0.229 | Tumor suppressor |
*indicates genes where multiple CpG islands were differentially hypermethylated. Genes in bold were analyzed in Cohort 2 of adrenocortical tumors. B-Value difference is the difference between the raw B-values of adrenocortical carcinoma and adrenocortical.
functional ACTs, no significant differences in the DNA methylation profile was detected. When examining the known function of annotated
genes showing significantly altered DNA methyl- ation levels, genes involved in regulation of apo- ptosis, transcriptional, and cell cycle control
showed significant and frequent hypermethyl- ation. These genes were also significantly hyper- methylated in ACCs versus normal adrenal tissue. In addition, the methylation mapping patterns of the CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, and SCGB3A1|HIN1genes were ana- lyzed. The DNA methylation ratio (relative DNA methylation; ß value) comparing normal adreno- cortical tissue versus adrenocortical carcinoma (ACC) and adenoma (ACA), respectively) of indi- vidual CpG sites in the CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, and SCGB3A1|HIN1 genes are presented in Supporting Information Table 3.
Hypermethylated Genes Show Reduced mRNA Expression in Benign and Malignant Adrenocortical Tumors
Six genes were selected for further study based on data obtained from the genome-wide DNA methylation profiling of adrenocortical tumors. These genes were selected based on their known or putative role in the pathogenesis of other malignancies as well as known protein function. We studied the genes CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, and SCGB3A1|HIN1 to deter- mine whether the hypermethylation was related to gene expression in ACT. Total RNA was extracted from normal adrenal cortex tissue (n = 6), ACAs (n = 10), and ACCs (n = 6) and gene expression was analyzed using quantitative RT- PCR. Gene expression of CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, and SCGB3A1|HIN1 was significantly reduced in both ACAs and ACCs compared to normal adrenal tissue (Fig. 1). In addition, gene expression was also significantly reduced in ACC versus ACA (P < 0.05).
Treatment of H295R Adrenocortical Cancer Cells with 5-Aza-2’-Deoxycytidine Restores Expression of Hypermethylated Genes
To investigate whether transcriptional repres- sion of these genes was due to reversible epige- netic alterations, the adrenocortical cancer H295R cell line was analyzed. To determine the methyl- ation status of the selected genes CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, and SCGB3A1|HIN1 in the H295R cells, MS-PCR was performed as detailed in the methods sec- tion. All four genes were found to be significantly methylated (Methylation % > 99%) by previously
validated formula (Cottrell et al., 2007) as pre- sented in Supporting Information Figure 1.
Cells were then treated with 5-aza-2’-deoxycy- tidine, a demethylating agent, and cells were har- vested at 24 and 48 h after treatment. Total RNA was prepared and gene expression of CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, and SCGB3A1|HIN1 genes was examined using quan- titative RT-PCR. Treatment with 5-aza-2’-deoxy- cytidine clearly increased expression of the hypermethylated genes in Figure 2.
DISCUSSION
The influence of epigenetic events on tumori- genesis has been well illustrated in the develop- ment of a variety of solid tumors including breast and colon cancer, leukemia, and other hemato- logic malignancies (Esteller et al., 2001; Catteau and Morris, 2002; Leone et al., 2002). Of all epi- genetic modifications, DNA hypermethylation which involves a covalent chemical modification resulting in the addition of a methyl group to the carbon 5 position of the cytosine ring in the pro- moter region of CpG islands (short regions of DNA approximately 0.5-5kb long that are unme- thylated and rich in CG contents) has been the most extensively studied. This results in the repression of transcription of the promoter regions of tumor suppressor genes, leading to gene silenc- ing (Jones and Laird, 1999). This has been pro- posed as one of the two hits in Knudson’s two hits hypothesis for oncogenic transformation (Jones and Laird, 1999).
We performed a comprehensive genome wide study of the DNA methylome of benign and ma- lignant adrenocortical tumors. We demonstrate that genes such as CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, and SCGB3A1|HIN1 are fre- quently hypermethylated in these tumors. Fur- thermore, hypermethylation of CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, and SCGB3A1|HIN1 was associated with decreased gene expression, which was reversible by treatment of ACC cells with a demethylating agent, in vitro.
This study does have its limitations, we were limited to tissue where fresh frozen tissue was available for the gene expression experiments, and hence we used two separate cohorts of tissue: one for the initial microarray studies, and the sec- ond for the gene expression studies. However, this was also beneficial in that we could confirm that hypermethylation was associated with decreased gene expression in a different set of
CDKN2A (P<0.0001)
GATA4 (P<0.0001)
Relative Gene Expression
1.4
1.6
Relative Gene Expression
1.2
1.4
1
1.2
0.8
1
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
0
Normal
Adenoma
Carcinoma
Normal
Adenoma
Carcinoma
DLEC1 (P=0.001)
HDAC10 (P=0.003)
Relative Gene Epression
1.4
Relative Gene Expression
1.4
1.2
1.2
1
1
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
T
0
Normal
Adenoma
Carcinoma
Normal
Adenoma
Carcinoma
PYCARD (P<0.0001)
SCGB3A1 (P<0.0001)
Relative Gene Expression
1.4
Relative Gene Expression
1.2
1.2
1
1
0.8
0.8
0.6
0.6
0.4
0.4
T
0.2
0.2
0
I
0
Normal
Adenoma
Carcinoma
Normal
Adenoma
Carcinoma
primary tumors, which suggests that these find- ings are indeed universally applicable.
CDKN2A (Cyclin-dependent kinase inhibitor 2A) is a known tumor suppressor gene, located on chromosome region 9p21.3 and plays an impor- tant role in cell cycle regulation. Hypermethyl- ation of this gene has been shown to be associated with the development of hepatocellu- lar, pancreatic, gastric and colorectal carcinoma, transitional cell carcinoma of the bladder, mela-
noma, primary head and neck cancer, ovarian and parathyroid tumors (Caldas et al., 1994; Bartsch et al., 1995; Liu et al., 1995; Florl et al., 2000; Nosho et al., 2008; Abou-Zeid et al., 2011; Alves et al., 2011; Cul’bová et al., 2011; Demokan et al., 2011; Starker et al., 2011; Zang et al., 2011). Inactivation of CDKN2A as well as reduced gene and protein expression has also been shown to be associated with adrenocortical carcinomas (Pilon et al., 1999). GATA4 (GATA binding
CDKN2A (P<0.0001)
GATA4 (P<0.0001)
4
Relative Gene Expression
4.5
3.5
Relative Gene Expression
4
3
3.5
2.5
3
2
2.5
1.5
2
1.5
1
1
0.5
0.5
0
0
0 hours
24 hours
48 hours
0 hours
24 hours
48 hours
DLEC1 (P<0.0001)
HDAC10 (P<0.0001)
3.5
Relative Gene Expression
3.5
3
Relative Geen Expression
3
2.5
2.5
2
2
1.5
1.5
1
1
0.5
0.5
0
0
0 Hours
24 Hours
48 Hours
0 Hours
24 Hours
48 Hours
PYCARD (P=0.016)
SCGB3A1 (P<0.0001)
Relative Gene Expression
3.50
3.5
3.00
Relative Gene Expression
3
2.50
2.5
2.00
2
1.50
1.5
1.00
1
0.50
0.5
0.00
0
0 Hours
24 Hours
48 Hours
0 Hours
24 Hours
48 Hours
protein 4) is a gene located on chromosome region 8p23.1-p22 which encodes a member of the GATA family of zinc-finger transcription fac- tors. Hypermethylation of GATA4 has been shown to be associated with breast, esophageal, gastric, and colorectal carcinoma, non-small cell lung carcinoma and malignant human astrocytoma (Akiyama et al., 2003; Guo et al., 2004; Guo et al., 2006; Hellebrekers et al., 2009; Agnihotri et al., 2011). It has also been shown to be
silenced by other epigenetic modifications such as histone modification in ovarian and breast car- cinoma (Caslini et al., 2006; Cai et al., 2009; Hua et al., 2009). DLEC1 (Deleted in lung and esoph- ageal cancer) is a known tumor suppressor gene, located on chromosome region 3p22-p21.3. It is known to be involved in carcinogenesis of lung, esophagus, colon, liver, and kidney (Kwong et al., 2007; Qiu et al., 2008; Ying et al., 2009), and hy- permethylation of this gene is a marker of breast
cancer progression (Park et al., 2011). HDAC10 (Histone Deacetylase 10), a novel Class II His- tone Deacetylase is known to play an important role in transcriptional regulation and cell cycle progression. HDAC10 promoter polymorphism has been shown to be associated with the develop- ment of hepatocellular carcinoma in patients with chronic Hepatitis B virus infection (Park et al., 2007), and epigenetic changes in HDAC10 have been shown to be associated with poor prognosis in patients with non-small cell lung cancer (Osada et al., 2004). PYCARD/TMS1 (PYD and CARD domain containing), located on chromo- some region 16p11.2, is a gene that encodes an adaptor protein that is composed of two protein- protein interaction domains: a N-terminal PYRIN-PAAD-DAPIN domain (PYD) and a C- terminal caspase-recruitment domain (CARD). PYD and CARD domains are members of the 6- helix bundle death-domain fold superfamily, which mediates assembly of complexes that mediate apoptotic and inflammatory signaling pathways (McConnell and Vertino, 2004). It was identified by Conway et al. in 2000 by screening for targets of methylation associated gene silenc- ing (Conway et al., 2000). Methylation mediated silencing of this gene has been described in breast and pancreatic cancer, and reactivation of gene expression in breast cancer cells is thought to contribute to docetaxel sensitivity (Gordian et al., 2009; Ramachandran et al., 2010). It has now been shown to be methylated in a number of other cancers including primary melanoma, ovarian cancer, lung cancer, glioblastoma multi- forme, and prostate cancer (Guan et al., 2003; Virmani et al., 2003; Akahira et al., 2004; Stone et al., 2004; Terasawa et al., 2004; Das et al., 2006; Martinez et al., 2007; Tam et al., 2007). Methylation of this gene has also been proposed as a late stage event in colorectal carcinogenesis (Riojas et al., 2007). SCGB3A1|HIN1 (Secretoglo- bin, Family 3A, Member 1) is located on chro- mosome region 5q35. It is a gene that codes for a growth inhibitory cytokine that plays a role in epithelial cell differentiation. It is known to be frequently silenced by promoter hypermethyl- ation in a multitude of cancers including breast, nasopharyngeal, esophageal, prostate, pancreatic, ovarian, and non-small cell lung cancer, Wilm’s tumor, neuroblastoma, and rhabdomyosarcomas (Krop et al., 2001; Wong et al., 2003; Krop et al., 2004; Marchetti et al., 2004; Shigematsu et al., 2005; Wu et al., 2007; Yang et al., 2007; Guo et al., 2008).
Except for CDKN2A, all the genes we selected for further study have thus been shown to be involved in the pathogenesis of a number of other cancers; however, have not been studied in adrenocortical tumorigenesis. Hypermethylation of these genes was found to correlate with a decrease in gene expression by quantitative RT- PCR. Furthermore treatment of the adrenocorti- cal cancer cell line H-295R cells with a demethy- lating agent restored expression of these genes, suggesting that hypermethylation in adrenocorti- cal tumors is a reversible event. Furthermore, CDKN2A, PYCARD, and DLEC1 hypermethyl- ation may represent markers of endocrine malig- nancy as they are also highly hypermethylated in parathyroid carcinomas versus adenomas (Starker et al., 2011). We failed to identify any overt genes within the WNT/ß-catenin and IGF1R/ AKT pathways displaying frequently altered DNA methylation patterns in ACTs. This does not exclude the importance of these pathways in adrenal tumorigenesis, but rather may reflect alternate ways of inactivation during tumor pro- gression, i.e., a synergy between epigenetic and genetic alterations causing tumorigenesis.
This is the first comprehensive study of DNA methylation in adrenocortical tumors, and pro- vides the framework for understanding the epige- netic mechanisms contributing to the development of these tumors. In addition, the genes identified are potential targets for pharma- cologic therapies of ACC.
ACKNOWLEDGMENTS
The authors thank Sheila Umlauf and Shrikant Mane and the Yale Center for Genome Analysis for assistance in the development of critical methodologies. R.P.L. is an Investigator of the Howard Hughes Medical Institute. T.C. is a Doris Duke-Damon Runyon Clinical Investigator supported in part by the Damon Runyon Cancer Research Foundation and the Doris Duke Chari- table Foundation.
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