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

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 phenol/chloroform extraction (22). separate human 15 k oligoarrays (MWG Biotech, Inc., High Point, NC), 5Aza-2dC and TSA treatment. 5Aza-dC (Sigma, St. Louis, MO) was according to the manufacturer’s recommendation for 18 hours at 42jC. The dissolved in PBS as a 5 mmol/L stock solution and stored in aliquots at microarrays were processed and spotted in the DNA Microarray Facility at À80jC. TSA (Wako, Richmond, VA) was dissolved in absolute ethanol as a FCCC. The Gene list, Gene ID, and Template files can be viewed at http:// 330 Amol/L stock solution and stored at À20jC. The four renal cell lines research.fccc.edu/facilities/microarray. In addition, we did microarray were split to low density and exposed to 5Aza-dC at a final concentration of hybridization with the opposite labels (dye flip) from each cell line. The 5 Amol/L, again 24 hours after the beginning of treatment and, if necessary, hybridized slides were scanned using a GMS 428 Scanner (Affymetrix, Santa further treatment until the cells had undergone at least two doublings. The Clara, CA) to generate high-resolution images for both Cy3 and Cy5 cells were also treated with TSA at a final concentration of 500 nmol/L channels. Image analysis was done using the ImaGene software (Bio- during 24 hours before harvest for RNA extraction. Untreated (mock) cells Discovery, Inc., El Segundo, CA). were cultured over an identical period of time with an equivalent volume of Analysis of expression up-regulation after demethylating treatment. PBS and, for the final 24 hours, with an equivalent volume of ethanol. The genes corresponding to each oligonucleotide spotted on the array Oligonucleotide array hybridization. Total RNA was isolated from were identified using an optimized segmentation algorithm. Spots of poor drug-treated and untreated cultured cells using TRIZOL reagent quality and spots with signal levels indistinguishable from the background

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Table 1. Up-regulated genes identified by intuitive selection (Cont’d)

Function Chromosome location Expression CpG island ALUs Methylation

Apoptosis 11q22.1 Yes Yes No U Antiapoptosis 17q.25.3 Yes Yes Yes Unknown 10q24.32 Yes Yes Yes Inflammatory response 2q36.3 No data Apoptosis 8p21.1 Yes No Extracellular matrix structural constituen 17q21.33 Yes Yes No M Transcription factor 6p2l.32 No data Regulation of cell growth 6q23.2 Yes Yes No U Apoptosis 7p15.3 Yes Yes No U DNA repair 1p31.2 Yes Yes No U Growth factor activity 19p13.11 Yes Yes No M Apoptosis 6p21.33 Yes Yes Yes Insulin-like growth factor binding 16p13.3 No exp Regulation of cell growth 7p13 Yes Yes No M Regulation of cell growth, apoptosis 7p14-p12 Yes Yes No M Inflammatory response 4q13.3 No exp Tumor suppressor, apoptosis 2q35 No exp Apoptosis 14q13 Yes Yes No U DNA modification 14q11.2 Yes Yes No U Signal transduction 10q.22.2 Yes Yes No M Acute phase response 11p15.1 No exp Cell cycle, transcription factor Xq26.2 No data Blood coagulation 7q21.3 No exp Protein signaling pathway 20q11.23 Yes Yes No U Neurogenesis Xp22.31 No data

No data No data No data

were excluded. The image data were extracted and used for data analysis. Bisulfite modification of DNA. Genomic DNA (1 Ag) from untreated Data were analyzed using the GeneSight software (BioDiscovery), which cell line cultures and from normal kidney tissue and primary tumors was includes background subtraction, data normalization (Lowess tranforma- denatured by NaOH (0.2 mol/L) for 10 minutes at 37jC and then modified tion), calculation of ratios, and statistical analysis of replicate spots and by hydroquinone and sodium bisulfite treatment at 50jC for 17 hours under slides. a mineral oil layer. Modified DNA was purified using the Wizard DNA Selection of genes for validation. We selected a subset of genes for Clean-Up system (Promega, Madison, WI). Modification was completed by validation by the following criteria. We chose genes that showed at least NaOH (0.3 mol/L) treatment for 5 minutes at room temperature followed by 3-fold up-regulation in at least three of the four cell lines. We excluded precipitation with glycogen, 10mol/L ammonium acetate, and ethanol those genes with no evidence of expression in normal renal tissue precipitation. Bisulfite modification of DNA results in the conversion of according to the Cancer Genome Anatomy Project (CGAP) Serial Analysis unmethylated cytosines to uracil, whereas methylated cytosines are Gene Expression (SAGE) database (http//cgap.nci.nih.gov). We then resistant to modification and remain as cytosine. analyzed the promoter region using the GeneCard web site (http:// Bisulfite sequencing of gene promoter CpG islands. DNA fragments bioinfo.weizmann.ac.il/cards/index.html) to obtain the genomic and cDNA of 258 to 461 bp in size containing the promoter CpG island were PCR sequences from the up-regulated genes. We searched for the CpG island amplified from bisulfite-modified cell line DNAs and normal tissue DNAs most proximal to the transcription start site using the CpG island revealing for each gene analyzed. PCR products were run in a 1.5% agarose gel; the program on WebGene Home Page web site (http://www.itba.mi.cnr.it/ gel slice was purified by Qiaquick (Qiagen); and direct sequencing was webgene/). Criteria for a CpG island was based on Takai and Jones: GC z done on all genes. In addition, some gene products were cloned into a 55%; Obs/Exp z 65, and length >200 bp, at http://cpgislands.usc.edu TOPO vector (Invitrogen), and at least 10colonies from each gene were reported to exclude most Alu-repetitive elements (23). We used the analyzed. Sequencing was done on an ABI 3100A capillary genetic RepeatMasker Web Server (ftp.genome.washington.edu/cgi-bin/Repeat- analyzer, and data were analyzed by Sequencer Version 4.2.2 software. Masker) to examine whether the promoter CpG island contained repetitive Primer sequences used for PCR amplification and sequencing are available elements. upon request. Reverse transcription-PCR. The cDNA template used for reverse Methylation-specific PCR. Bisulfite-modified primary renal tumor transcription-PCR (RT-PCR) was aliquoted from the same cDNA used for DNAs were PCR amplified with primers specific for methylated versus hybridization to the microarray. For each gene examined, primers for the unmethylated DNA for the IGFBP1, IGFBP3, COL1AI, and RASSF1A genes. housekeeping gene GAPDH were included in the RT-PCR reaction mix as The methylation-specific PCR (MSP) primer sequences for IGFBP1, a control for successful amplification. Forward and reverse primers were IGFBP3, and COL1AI are available upon request, and for RASSF1A, were chosen from different exons to avoid amplification of any contaminating as previously described (21). PCR amplification of tumor DNA was done genomic DNA. Primer sequences are available upon request. for 31 to 35 cycles at 95jC denaturing, 58jCto66jC annealing, and 72jC www.aacrjournals.org 5023 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 extension with a final extension step of 5 minutes. In each set of DNAs to determine if each gene was known to be expressed in normal modified and PCR amplified, a cell line with known hypermethylation renal cells and excluded 12 genes with no evidence of expression or from the bisulfite sequencing data was used as a positive control, and no data available. We then analyzed the promoter region of each normal renal tissue DNA as a negative control was included. After PCR, gene for the presence of a CpG island with the characteristics samples were run on a 6% nondenaturing acrylamide gel with appropriate described by Takai and Jones (23) and found 14 of the 15 genes had size markers, and the presence or absence of a PCR product was analyzed. such a CpG island (Table 1) within 500 bp either side of the transcription start site. The CpG islands of three genes contained repetitive elements and were excluded from the initial validation. Results and Discussion We, therefore, selected the remaining 12 genes for validation Determination of the optimal 5Aza-dC dose array-based (Table 1). reactivation of expression. To determine the optimal 5Aza-dC Validation analysis of reactivated genes. We did RT-PCR for dosage for robust detection of transcriptional reactivation on the the 12 genes on untreated and treated cell line RNA to microarray without excessive toxicity to the treated cells, we independently confirm the up-regulation of expression observed examined the reexpression of five tumor suppressor genes by array analysis (Fig. 1; Supplementary Fig. S1). The RT-PCR INK4a (p16 , MLH1, MGMT, RARb, and Timp-3) well characterized results were tightly concordant with the microarray analysis for as hypermethylated with associated silencing in the SW48 all 12 genes examined. For example, COL1A1 and IGFBP3 up- colorectal tumor cell line (8, 12, 24–26), which has a similar cell regulation after treatment is confirmed by RT-PCR in Fig. 1, where doubling time to the RCC lines, at different doses of 5Aza-dC. a strong signal from the four cell lines is seen for COL1A1 and, in RNA was extracted from SW48 cell cultures after treatment with the HRC59 and ACHN cell lines for IGFBP3, after treatment 1, 5, or 10 Amol/L 5Aza-dC and TSA (500 nmol/L); RT-PCR was compared with the signal obtained from the untreated cell lines. done; and the product was labeled and hybridized to the IGFBP3 also showed strong up-regulation in the cell lines 786-0and expression array. Analysis of up-regulation of the five TSG HRC51, although these two cell lines had weak basal expression of showed that treatment with 5 Amol/L 5Aza-dC for at least two the gene before the treatment. This basal expression may represent cell doubling times resulted in reexpression of the five TSG with unmethylated, or less densely methylated, subclones in the cell minimal toxicity as assessed by comparison of cell morphology lines (15, 28). and cell death between control and treated cells. We combined We next analyzed the methylation status of the promoter CpG the 5Aza-dC treatment with TSA because there is good evidence island proximal to the transcription start site for the 12 genes in that the processes of methylation and deacetylation interact to the four RCC cell lines and normal renal epithelial cells by bisulfite silence transcription (14, 27), although it is believed that TSA has sequencing (Fig. 2). Five genes were densely methylated in the less synergistic effect at the relatively higher 5 Amol/L 5Aza-dC dose we used, than when combined with lower doses of 5Aza-dC. The optimal synergistic concentration will also vary with different cell lines depending upon uptake and the epigenomic background. Furthermore, different genes in the same cell line, or the same gene locus in different cell lines, may be more, or less, strongly epigenetically silenced. The use of TSA alone may result in gene reactivation (16) but was not a focus of our present study. Another obvious issue of an epigenetic reactivation and expression array approach is that sensitivity of signal might vary depending upon different baseline expression levels of genes and different types of array. Interestingly, MLH1 showed least up- regulation of the five TSGs examined in SW48, and a similar observation was noted for MLH1 reactivation in the RKO colorectal tumor cell line (16). We then analyzed up-regulation of the 14,802 gene microarray in four RCC lines treated with 5 Amol/L 5Aza-dC and 500 nmol/L TSA. We analyzed two ATCC renal tumor cell lines (786-0and ACHN); in addition, because all ATCC renal tumor lines were derived from clinically advanced tumors, a further two cell lines were established from localized primary renal tumors. After treatment, we found 170(786-0),112 (HRC51), 178 (HRC59), and 111 (ACHN) genes up-regulated at least 3-fold in the RCC cell lines compared with the untreated cells (Supplementary Table S1). To verify the specificity of our screen for genes hypermethylated in renal cancer, we applied selection criteria to the list of up-regulated genes. We first identified 27 genes that were up-regulated in at least three of the four RCC lines on the basis that such genes might be expected to Figure 1. RT-PCR validation of expression up-regulation after drug treatment. be frequently methylated in primary renal cancer. In addition, we Reverse transcribed and PCR amplified untreated (U) and treated (T) DNA examined one gene up-regulated after drug treatment in both clear from the four RCC cell lines analyzed for expression of six genes and the GAPDH control (left). The stronger signal in the majority of the treated samples cell lines but neither of the papillary lines to investigate cell type– confirms the up-regulation of the genes, indicated by the array results, after specific methylation. We next examined the CGAP SAGE database demethylation treatment of the cell lines.

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Figure 2. Bisulfite sequencing of the gene promoter CpG islands in tumor and normal DNA. A, sequencing of normal tissue (NK) DNA and RCC cell line DNA (right) after bisulfite modification for sixgenes. Unmethylated cytosines (C) are converted to uracil (T). The presence of C preceding a G in the sites indicated by arrows shows that these cytosines were methylated in the RCC cell line DNA, whereas the presence of T instead of C in the same positions in the normal DNA shows that these C were unmethylated in the normal tissue DNA. GDF15 shows methylation of CG dinucleotides in normal cell DNA and more dense CG methylation in the tumor cell DNA. TGM2 was unmethylated in both the normal and tumor cell line DNAs. B, representative bisulfite sequencing of a RCC cell line (right) for sixgenes with unmethylated promoter regions. Unmethylated cytosines (C) are converted to uracil (T). The absence of C preceding a G in the sites indicated by arrows shows that these cytosines were unmethylated in the tumor cell line DNA.

untreated RCC lines (Fig. 2A), indicating potential epigenetic limited to, or more common in, tumor cell lines, we did MSP regulation of these genes. Importantly, only the individual RCC analysis of 32 primary, mainly organ-confined (stage I or II), renal lines where a particular gene was methylated showed up-regulation tumors of the most common histologic cell types (20clear cell, of expression for the same gene after demethylating treatment by 10papillary, and 2 chromophobe). IGFBP1 was methylated in 10 the microarray and RT-PCR assays, whereas the cell lines where a of 32 (31%), IGFBP3 in 12 of 32 (37%), and COL1A1 in 18 of 32 gene was unmethylated did not show up-regulation after (56%) primary RCC (Fig. 3A; Table 2). Because conventional MSP treatment. Promoter hypermethylation of IGFBP1, IGFBP3, and is not quantitative, we cannot be certain the methylation of the COL1A1 was cancer cell specific because methylated CpGs were genes in the primary renal tumors is clonal, as we observed in seen only in the RCC lines and not in the normal cell DNA the RCC cell lines, without further studies. IGFBP3 showed a specimens sequenced (Fig. 2A). similar percentage of methylation between clear cell (7 of 20, Frequency of hypermethylation of IGFBP1, IGFBP3, and 35%) and papillary (4 of 10, 40%) tumors. IGFBP1 was more COL1A1 in primary RCC. To determine if hypermethylation of frequently methylated in clear cell (7 of 20, 35%) than papillary the three genes was frequent in primary renal cancer and not tumors (2 of 10, 20%) as was COL1A1 (13 of 20, 65% clear cell www.aacrjournals.org 5025 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 versus 4 of 10, 40% papillary). Thus, the three genes were carcinoma (34), where IGFBP3 promoter hypermethylation was frequently methylated in early-stage tumors of the most common also described (35). IGFBP3 was also recently identified as histologic subtypes of RCC, implicating these genes in renal hypermethylated in a mouse skin multistage carcinogenesis tumorigenesis and as novel candidate markers for the molecular model (36). Clearly, methylation-based silencing of IGFBP1 and detection and prognosis of kidney cancer. Further impetus has IGFBP3 could provide growth advantages to the neoplastic cell. been provided by studies showing the feasibility of detecting gene Activation of this pathway may be of therapeutic advantage in hypermethylation in urine, a readily accessible bodily fluid for limiting tumor growth. diagnosis and monitoring of renal cancer (21, 29). COL1A1 is the human gene coding for the a1 chain of type I Aberrant hypermethylation of normally unmethylated promoter collagen, the major extracellular matrix component of skin and regions with associated transcriptional silencing in cancer cells is a bone. Changes in the synthesis of type I collagen are associated with characteristic of TSG (5, 6). The three genes we have identified are, normal growth and tissue repair processes. The related genes therefore, candidate TSGs, although further testing is needed. The COL1A2 and COL1A5 have also been reported to be hypermethylated IGFBP1 gene maps to chromosome 7p13; IGFBP3 maps to 7p14; in cancer cells (37, 38). Alterations in extracellular matrix compo- and COL1A1 maps to 17q21. These chromosomal regions have not sition have been implicated in tumor progression and metastasis. been examined for loss of heterozygosity in renal cancer (30, 31). Both the IGFBP and the COL1A gene families seem prone to High-density single nucleotide polymorphism array analysis will hypermethylation, and it is interesting that other global epigenetic establish if allelic deletion occurs at these loci. screens have shown reactivation of gene families [e.g., IFN in bladder Role of the reactivated genes in tumorigenesis. With regard (15) and SFRP family members in colorectal cancer (16)]. to the putative role of these genes in cancer, the insulin-like The fourth reactivated gene found to have promoter methylation growth factor binding protein 1 (IGFBP1) and IGFBP3 are major was a growth differentiation factor gene (GDF15) located on forms of the IGFBP family that can inhibit the growth promoting chromosome 19p. GDF15 showed methylation of CpG dinucleo- activity of both IGF-I and IGF-II. IGFBP3 is known to inhibit tides in normal cell DNA but more dense CG methylation in the cell growth by sequestering IGF-I; however, the mechanism by tumor cell DNA and thus merits further study. Hypermethylation of which IGFBP1 exerts its activity is less well understood. Down- GDF15 in normal cells may be age related as described for myoD1 regulation of IGFBP3 expression has been reported in non–small and other cancer genes (39). The fifth gene (PLAU) was densely cell lung cancer (32), prostate cancer (33), and hepatocellular methylated in one of the four RCC lines (HRC51); however, by RT- PCR analysis, PLAU expression did not seem up-regulated after demethylating treatment of this line. The other three lines did show PLAU up-regulation by RT-PCR, but all had unmethylated promoters. The functional significance of PLAU promoter hypermethylation is therefore unclear. The remaining seven up-regulated genes had unmethylated promoters by bisulfite sequencing analysis (Fig. 2) but could have been activated by an upstream regulatory gene or transcription factor that was reactivated by demethylation. In support of this idea, TGM2 has been reported to be regulated by the candidate TSG RASSF1A (40), known to be frequently methylated in RCC (9–11). RT-PCR confirmed clear up-regulation of TGM2 in the four RCC lines after treatment (Fig. 1). We examined RASSF1A by MSP and found it to be methylated in three of the four RCC lines (Fig. 3b). The same three RCC cell lines showed up-regulation of RASSF1A on the expression array after demethylating treatment. We identified an equal number of methylated and unmethylated genes; however, because <1% of genes in a human tumor cell are methylated, our screen preferentially identified methylated genes. It is also likely that the epigenetic reactivation of particular genes leads to a cascade of up-regulation in diverse pathways and networks. Other genes may be up-regulated as a direct response to the stress of 5Aza-dC treatment. Lastly, although in this initial Figure 3. A, MSP of IGFBP1, IGFBP3, and COL1A1 in primary renal tumors. validation, we did not bisulfite sequence promoter CpG islands that The presence of a PCR product in the methylated lane (M) indicates that contained repetitive elements, the three genes excluded from initial tumors KT28, KT31, and KT78 have methylated alleles for IGFBP1; KT49, KT31, VHL p16INK4a and KT53 for IGFBP3; and KT15, KT19, and KT38 for COL1A1. The PCR validation will require further study because the and product in the unmethylated lane (U) from tumor DNA most likely arises from TSGs are known to contain repetitive elements in, or near, the normal cell contamination of the tumor specimen. Because of the different base promoter CpG island but still to have promoter hypermethylation composition of the unmethylated versus methylated primers and template sequence, MSP products are nonquantitative and do not necessarily reflect the of functional significance (7, 41). Refinement of intuitive selection relative levels of unmethylated to methylated alleles in the primary tumor. Tumor criteria will help to better prioritize genes up-regulated after cell lines 786-0 for IGFBP1 and ACHN for IGFBP3 and COL1A1 as positive controls and normal renal cell DNA as a negative control (far right). B, MSP of treatment for validation in future studies. RASSF1A in the four RCC lines. The presence of a PCR product in the Alternative strategies for global profiling of gene methyla- methylated lane (M) indicates that RASSF1A is hypermethylated in RCC lines tion in cancer. Until recently, global strategies to identify 786-0, HRC59, and ACHN. The absence of a product in lane M of HRC51 indicates that RASSF1A is unmethylated in this line. Normal renal cell DNA as methylated genes in cancer tended to use arrayed CpG island a negative control and tumor cell line MDA231 DNA as a positive control. fragments identified through restriction enzyme recognition

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Table 2. Clinicopathologic data of primary RCC

Age (y)/sexCell type Size of tumor (cm) Grade Stage IGFBPI IGFBP3 COL1A1

1 43/M Clear Cell 3 1 I U U M 2 57/F Clear Cell 3 2 III U M M 3 59/M Clear Cell 3.5 3 I U U M 4 78/M Clear Cell 2.7 4 I U U U 5 69/M Clear Cell 3 3 I M U M 6 52/M Clear Cell 4 4 I M U U 7 70/M Clear Cell 4.5 2 I M U M 8 78/M Clear Cell 5.5 2-3 IV U U M 9 59/M Clear Cell 4.5 4 I M M M 1059/M Clear Cell 9.5 3 III M M U 11 61/F Clear Cell 6.5 2 I U U M 12 61/M Clear Cell 15 1 II U M U 13 72/F Clear Cell 4 2 I U U U 14 62/M Clear Cell 6 1-2 I U U M 15 57/M Clear Cell 5.5 2 I M M M 16 66/M Clear Cell 5.5 2 I U M M 17 60/F Clear Cell 3.5 2 I U M M 18 62/M Clear Cell 3.5 1-2 I M U M 19 59/M Clear Cell 4 3-4 I U U U 2066/M Clear Cell 2 1 I U U U 21 30/F Papillary 4 3 I U U M 22 34/M Papillary 7.5 2 II U U U 23 39/F Papillary 8.5 4 III M U M 24 69/M Papillary 3 3 III M M U 25 74/M Papillary 12.5 2 II U U U 26 64/F Papillary 4.5 2 I U M U 27 57/M Papillary 5 3 I U U U 28 67/F Papillary 1.2 1 I U M M 29 73/M Papillary 3 1 I U U M 306S/F Papillary 4 1 I U M U 31 66/M Chromophobe 3.5 1 I U M M 32 65/F Chromophobe 2 1 I M U U

NOTE: Grade is based on the American Joint Committee on Cancer and stage is based on the American Joint Committee on Cancer stage grouping. Abbreviations: M, male; F, female; U, unmethylated gene; M, methylated gene.

sequences (42, 43). One issue with such an approach is that many normal renal cells but hypermethylated in cancer, PLAU to be CpG islands are located outside promoter regions, and methyla- methylated but with a weak correlation to expression status, and tion of such islands does not have a functional effect upon TGM2 to be unmethylated but known to be regulated by transcription (16). A microarray-based screen of genes reexpressed another gene methylated in renal cancer cells. Thus, importantly, after demethylation treatment can address this issue by using at least 6 of 12 genes could be shown to be under epigenetic reactivation of transcription, rather than the presence of a CpG control in support of the ability of our screen to preferentially island, as the identifying determinant. There have now been identify new hypermethylated genes in renal cancer. The three several other global epigenetic studies highlighting the advantages novel hypermethylated genes are candidate tumor suppressor and issues of this approach (15–20). The SPINT2 gene was recently genes and are frequently methylated in primary renal tumors. identified as methylated in 40% of papillary and 30% of clear cell Their potential use in current molecular diagnostic or prognostic renal tumors after demethylation treatment of RCC lines (44). strategies can now be examined (21, 29). As a gene methylation These epigenetic reactivation profiles may in part also be array signature is further developed for renal cancer, we will likely type dependent and sensitive to different 5Aza-dC doses and gain important insights into the biology and progression of this treatment times. One disadvantage of a drug-based reactivation disease. approach is that unlike CpG island array screening (42), use is essentially restricted to cell cultures and is not amenable to examination of primary tumors. Acknowledgments We validated 12 genes up-regulated after demethylating drug Received 9/19/2005; revised 3/14/2006; accepted 3/21/2006. treatment and found IGFBP1, IGFBP3, and COL1A1 to have Grant support: NIH Early Detection Research Network grant 1 U01 CA111242-01. The costs of publication of this article were defrayed in part by the payment of page cancer cell specific hypermethylation, which correlated with charges. This article must therefore be hereby marked advertisement in accordance epigenetic reactivation of expression: GDF15 to be methylated in with 18 U.S.C. Section 1734 solely to indicate this fact. www.aacrjournals.org 5027 Cancer Res 2006; 66: (10). May 15, 2006

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