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Identification of Novel Target Genes by an Epigenetic Reactivation Screen of Renal Cancer Inmaculada Ibanez De Caceres,1 Essel Dulaimi,1 Amanda M

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Identification of Novel Target Genes by an Epigenetic Reactivation Screen of Renal Cancer Inmaculada Ibanez De Caceres,1 Essel Dulaimi,1 Amanda M Research Article Identification of Novel Target Genes by an Epigenetic Reactivation Screen of Renal Cancer Inmaculada Ibanez de Caceres,1 Essel Dulaimi,1 Amanda M. Hoffman,1 Tahseen Al-Saleem,2 RobertG. Uzzo, 1 and Paul Cairns1,2 Departments of 1Surgical Oncology and 2Pathology, Fox Chase Cancer Center, Philadelphia, Pennsylvania Abstract common mechanism for the silencing of tumor suppressor gene Aberrant promoter hypermethylation is a common mecha- (TSG) in cancer cells (5, 6). Several TSG have been identified as hypermethylated with associated loss of expression in renal cancer nism for inactivation of tumor suppressor genes in cancer INK4a by a candidate approach. The VHL and p16 TSG are inactivated cells. To generate a global profile of genes silenced by hypermethylation in renal cell cancer (RCC), we did an by promoter hypermethylation in up to 20% of clear cell (7) and 10% expression microarray-based analysis of genes reactivated in of all RCC (8), respectively. However, to date, few genes have been found to be frequently hypermethylated in RCC. The RASSF1A the 786-0, ACHN, HRC51, and HRC59 RCC lines after treatment Timp-3 with the demethylating drug 5-aza-2 deoxycytidine and gene is hypermethylated in 27% to 56% (9–11), and the gene histone deacetylation inhibiting drug trichostatin A. Between is hypermethylated in 58% to 78%, of primary RCC (11, 12). By 111 to 170 genes were found to have at least 3-fold up- definition, a candidate gene approach has resulted in the exam- regulation of expression after treatment in each cell line. To ination of a limited number of genes for epigenetic alteration (11). establish the specificity of the screen for identification of Many other tumor suppressor and cancer genes important in renal genes, epigenetically silenced in cancer cells, we validated a tumorigenesis likely remain to be identified. A global approach to the subset of 12 up-regulated genes. Three genes (IGFBP1, IGFBP3, identification of epigenetically silenced genes in renal tumor cells and COL1A1) showed promoter methylation in tumor DNA but could provide methylation signatures for early detection and for were unmethylated in normal cell DNA. One gene (GDF15) was prognostic stratification, identify novel targets for therapy, and lead to further elucidation of the biology of this disease. methylated in normal cells but more densely methylated in tumor cells. One gene (PLAU) showed cancer cell–specific Epigenetic silencing of a gene can be reversed, resulting in methylation that did not correlate well with expression status. reexpression, by drugs, such as 5-aza-2 deoxycytidine (5Aza-dC), The remaining seven genes had unmethylated promoters, which acts by incorporation into the new strand during DNA although at least one of these genes (TGM2) may be regulated replication where it forms a covalent complex with the methyl- by RASSF1A, which was methylated in the RCC lines. Thus, we transferase active sites, depleting methyltransferase activity, which were able to show that up-regulation of at least 6 of the 12 results in generalized demethylation (5). Trichostatin A (TSA) is a genes examined was due to epigenetic reactivation. The histone deacetylase inhibitor agent that can reverse the formation IGFBP1, IGFBP3, and COL1A1 gene promoter regions were of transcriptionally repressive chromatin structure by facilitating an found to be frequently methylated in primary renal cell accumulation of acetylated histones (13). These two drugs have been reported to act in synergy for reactivation of epigenetically tumors, and further study will provide insight into the biology of the disease and facilitate translational studies in renal silenced genes (14). Until recently, global analyses of methylation cancer. (Cancer Res 2006; 66(10): 5021-8) in cancer cells were largely restricted to array or gel-based com- parisons of CpG islands between normal and tumor cell DNA. A microarray-based screen has the advantage of a more global Introduction analysis and, coupled with a reactivation strategy, has the further There will be >25,000 new cases of renal cell cancer (RCC) in 2005, advantage that it should preferentially identify reexpression of and the incidence of this disease has increased by 60% over the last epigenetically silenced genes over methylated CpG islands that do two decades (1). RCCs are heterogeneous in histology, genetics, not influence transcription. The potential of this approach has been and clinical behavior. Clear cell (75-80%), and papillary (10-15%) highlighted in bladder (15), colorectal (16), esophageal (17), and carcinoma are the two most frequent subtypes of RCC (2–4). other cancers (18–20). In the present study, we examined the global Tumorigenesis is a multistep process that results from the reactivation of epigenetically silenced genes in renal cancer by accumulation and interplay of genetic and epigenetic mutations. analysis of a 14,802 gene expression array with RNA from four RCC The epigenetic alteration of aberrant DNA methylation of CpG lines after treatment with 5Aza-dC and TSA. Through validation, we islands in the promoter region of genes is well established as a have shown that the screen preferentially selects for epigenetically silenced genes and identified three genes unmethylated in normal cells but frequently hypermethylated in primary RCC. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Disclosure: P. Cairns is a paid consultant to Oncomethylome Sciences. The terms Materials and Methods of this arrangement are being managed by Fox Chase Cancer Center in accordance with its conflict of interest policies. Cell lines and tissue specimens. The clear cell renal tumor line 786-0 Requests for reprints: Paul Cairns, Fox Chase Cancer Center, 333 Cottman Avenue, and the papillary renal tumor cell line ACHN were obtained from the Philadelphia, PA 19111. Phone: 215-7285635; Fax: 215-7282487; E-mail: Paul.Cairns@ American Type Culture Collection (ATCC, Manassas, VA). The HRC51 cell fccc.edu. I2006 American Association for Cancer Research. line was established from an organ-confined primary clear cell renal tumor doi:10.1158/0008-5472.CAN-05-3365 (T1b, grade 1), and the HRC59 cell line was established from a localized www.aacrjournals.org 5021 Cancer Res 2006; 66: (10). May 15, 2006 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Cancer Research Table 1. Up-regulated genes identified by intuitive selection Gene ID Gene name Gene symbol Function known NM_001165 Baculoviral IAP repeat-containing protein 3 BIRC3 NM_001012270 Hypothetical protein xp_037199 BIRCS NM_024886 Chromosome 10 open reading frame 95 C10orf95 NM_004591 Small inducible cytokine subfamily a CCL2O NM_001831 Clusterin CLU NM_000088 Alpha 1 type I collagen preproprotein COL1A1 AK000928 cAMP responsive element binding protein-like 1 CREBL1 NM_001901 Connective tissue growth factor CTGF NM_018947 Cytochrome c CYCS NM_001924 Growth arrest and DNA damage–inducible, alpha GADD45A NM_004864 Prostate differentiation factor GDF15 AF039067 Anti-death protein IER3 NM_004970 Insulin-like growth factor binding protein, acid labile subunit IGFALS NM_000596 Insulin-like growth factor binding protein 1 IGFBP1 NM_000598 Insulin-like growth factor binding protein 3 IGFBP3 NM_000584 Interleukin 8 IL8 NM_002192 Inhibin beta a subunit precursor INHBA NM_020529 Nuclear factor of kappa light polypeptide gene enhancer in B cell inhibitor, alpha NFKBIA NM_000270 Purine nucleoside phosphorylase NP NM_002658 Plasminogen activator, urokinase PLAU NM_000331 Serum amyloid a1 SAA1 NM_016521 Transcription factor Dp family, member 3 TFDP3 NM_006528 Tissue factor pathway inhibitor 2 TFPI2 NM_004613 Transglutaminase 2 TGM2 NM_013452 Variable charge VCX3A Function unknown AC006070.1.1.161987.6 Ensembl genscan prediction ENSG00000104620 Ensembl prediction BC010467 Unknown NOTE: The initial statistical analysis addressed the question of whether there are genes in all microarrays of the experiment, which are differentially expressed in the treated cells compared with nontreated controls. Twenty-seven genes showed 3-fold up-regulation in at least three of the four cell lines and follow the intuitive selection criteria described. The IGFBP1 gene was selected because of up-regulation in both clear cell lines but neither of the papillary cell lines. Abbreviations: U, unmethylated; M, methylated. papillary renal tumor (T3a, grade 3) at Fox Chase Cancer Center (FCCC). (Invitrogen) and purified with the RNAeasy Mini kit (Qiagen, Valencia, 786-0was grown in RPMI; ACHN was grown in MEM; and HRC51 and 59 CA), combined with DNase treatment. The RNA quality was confirmed by were grown in type IIA (available upon request) medium supplemented with the ratio of 28S and 18S rRNA after agarose gel electrophoresis. Twenty 10% FCS. Primary renal tumors and normal kidney tissue were micro- micrograms of total RNA was reverse transcribed using oligo (dT)24 dissected with the assistance of a pathologist (T. A-S.) as described (21). primer and Superscript II reverse transcriptase (Invitrogen) for 1.5 hours Areas of immune cell infiltration, observed in a minority of tumor at 42jC. specimens, were avoided. DNA was extracted using conventional techniques For each cell line, both treated and untreated cDNA was labeled with Cy3 of digestion with proteinase K (Invitrogen, Carlsbad, CA) followed by or Cy5 (Amersham Biosciences, Piscataway, NJ) and then hybridized to
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