Discovery of Epigenetically Silenced Genes by Methylated DNA Immunoprecipitation in Colon Cancer Cells

Priority Report

Filipe V. Jacinto, Esteban Ballestar, Santiago Ropero, and Manel Esteller

Cancer Epigenetics Laboratory, Spanish National Cancer Centre (CNIO), Madrid, Spain

Abstract at the insulin-like growth factor II locus, and abrogation of the CpG island promoter hypermethylation of tumor suppressor methylation-mediated silencing of the tumor suppressor genes p16INK4a TIMP-3 genes is a common hallmark of human cancer, and new large- and (5). scale epigenomic technologies might be useful in our attempts Among others, the candidate gene and genomic and pharma- cologic approaches have all been used in the search for new genes to define the complete DNA hypermethylome of tumor cells. that undergo methylation-associated inactivation in cancer cells Here, we report a functional search for hypermethylated CpG (1–3), but the DNMT genetic avenue has not yet been fully explored. islands using the colorectal cancer cell line HCT-116, in which We wondered about the extent of CpG island hypomethylation two major DNA methyltransferases, DNMT1and DNMT3b, events in the DKO cells and whether these cells could be used to have been genetically disrupted (DKO cells). Using methylated find new genes with hypermethylation-associated inactivation in DNA immunoprecipitation methodology in conjunction with human cancer. In our preliminary genomic screenings, we promoter microarray analyses, we found that DKO cells observed CpG island hypomethylation events in putative tumor experience a significant loss of hypermethylated CpG islands. suppressor genes (7). The recent introduction of methylated DNA Further characterization of these candidate sequences shows immunoprecipitation (MeDIP) technology, combined with com- CpG island promoter hypermethylation and silencing of genes prehensive gene promoter arrays (8–10), prompted us to reinves- with potentially important roles in tumorigenesis, such as tigate the DKO cells using this new epigenomic tool. Our results the Ras guanine nucleotide-releasing factor (RASGRF2), the show that cancer cells lacking DNMT1 and DNMT3b undergo apoptosis-associated basic helix-loop transcription factor significant CpG island hypomethylation events that identify new (BHLHB9), and the homeobox gene (HOXD1). Hypermethyla- putative tumor suppressor genes undergoing methylation- tion of these genes occurs in premalignant lesions and associated silencing in human cancer. These data contribute to a accumulates during tumorigenesis. Thus, our results show more complete map of the DNA hypermethylome of malignant the usefulness of DNMT genetic disruption strategies com- cells and provide new hypermethylated markers for putative bined with methylated DNA immunoprecipitation in searching translational use in patients with colorectal cancer. for unknown hypermethylated candidate genes in human cancer that might aid our understanding of the biology of the disease and be of potential translational use. [Cancer Res Materials and Methods 2007;67(24):11481–6] Human cancer cell lines and primary tumor samples. HCT-116 colon cancer cells and double DNMT1À/ÀDNMT3bÀ/À (DKO) cells were grown Introduction as previously described (5). HCT-116 cells were treated with 5-aza-2- deoxycytidine (1 Amol/L) for 72 h. HCT116 and DKO cells were a generous Inactivation of tumor suppressor genes in human cancer occurs gift from Dr. Bert Vogelstein (Johns Hopkins Kimmel Comprehensive very often by hypermethylation of the CpG islands located in Cancer Center, Baltimore, MD). All the other human colon cancer cell lines the promoter regions of these genes (1–3). Global cytosine methyla- (n = 7) were obtained from the American Type Culture Collection. Tissue tion patterns in mammals seem to be established by a complex samples of primary colorectal tumors (n = 72), adenomas (n = 34), and interplay of at least three independently encoded DNA methyl- normal colon (n = 10) were all obtained at the time of the clinically transferases (DNMT): DNMT1, DNMT3a, and DNMT3b (1–3). indicated procedures. Homologous recombination has been used in the colorectal cancer MeDIP and microarray hybridization. The MeDIP assay was developed as previously described (8). Four micrograms of genomic DNA cell line HCT-116 to disrupt DNMT1 or/and DNMT3b (4, 5). Single extracted from HCT116 and DKO nuclei were sonicated to produce random DNMT knockouts had minor changes in DNA methylation (4, 5) fragments ranging in size from 300 to 600 bp. We denatured the DNA for that, in part, might be associated with the presence of recently 10 min at 95jC and immunoprecipitated it overnight at 4jC with 10 ALof identified alternative transcripts arising from the DNMT1 gene (6). monoclonal antibody against 5-methylcytidine (1 mg/mL; Eurogentec). However, most important, the HCT-116 double-knockout cells for Real-time PCR on MeDIP samples was carried out using a 7900HT Fast DNMT1 and DNMT3b (DKO cells; ref. 5) showed a minimal DNA Real-Time PCR system (Applied Biosystems). Immunoprecipitated methyl- methyltransferase activity, a 95% reduction in 5-methylcytosine ated DNA was labeled with Cy5 fluorophere and the input genomic DNA content, demethylation of repeated sequences, loss of imprinting was labeled with Cy3 fluorophere. Labeled DNA from the enriched and the input pools were combined (1–2 Ag) and hybridized to the Human Proximal Promoter Array 44K (Agilent Technologies), which contained 60-mer oligonucleotide probes covering the region from À1 to +0.3 kb relative to Note: Supplementary data for this article are available at Cancer Research Online the transcript start sites for 17,917 annotated human genes. Arrays were (http://cancerres.aacrjournals.org/). then washed and scanned with an Agilent DNA microarray scanner. Data Requests for reprints: Manel Esteller, Cancer Epigenetics Laboratory, Spanish normalization and analysis was developed as described in the Supplemen- National Cancer Centre (CNIO), Melchor Fernandez Almagro 3, 28029 Madrid, Spain. tary Methods. To obtain more information about the biological features of Phone: 34-91-2246949; Fax: 34-91-2246923; E-mail: [email protected]. I2007American Association for Cancer Research. the hypermethylated candidate genes and to check the biological coherence doi:10.1158/0008-5472.CAN-07-2687 of the results obtained, we used the FatiGO program (11). www.aacrjournals.org 11481 Cancer Res 2007; 67: (24). December 15, 2007

DNA methylation analysis of candidate genes. The CpG Island representation of all the common hallmark pathways of cancer Searcher Program (12) was used to determine which genes had a CpG island cells (14), such as DNA repair, and cell death, although ¶ in their 5 -ends. DNA methylation status was established by PCR analysis of transcriptional regulators were clearly overrepresented (Fig. 1D). bisulfite-modified genomic DNA. Using two procedures, this induces the This latter observation is of particular interest because many chemical conversion of unmethylated, but not methylated, cytosine to hypermethylated promoter CpG islands in cancer cells correspond uracil. First, methylation status was analyzed by bisulfite genomic sequencing of both strands of the corresponding CpG islands. The second to genes with a critical role in the regulation of transcription, such procedure used methylation-specific PCR involving primers specific to as SFRPs-1, GATAs, HIC-1, DKK-1, and TWIST2 (1–3, 15). either the methylated or modified unmethylated DNA (Supplementary To gain further knowledge of the different DNA methylation Table S1). patterns of the genes enriched after MeDIP in HCT-116 and DKO Semiquantitative reverse transcription PCR expression analysis. cells, we randomly selected nine of these gene-associated CpG We reverse-transcribed total RNA (2 Ag) treated with DNAse I (Ambion) islands for further characterization by bisulfite genomic sequenc- using oligo(dT)12 to 18 primer with Superscript II reverse transcriptase ing of multiple clones. The genes selected were the Ras protein– (Life Technologies). We used 100 ng of cDNA for PCR amplification, and we specific guanine nucleotide-releasing factor 2 (RASGRF2), the amplified all of the genes with multiple cycle numbers (20–35 cycles) to sodium channel non–voltage-gated 1h (SCNN1B), the homeobox determine the appropriate conditions for obtaining semiquantitative D1 (HOXD1), the Polo-like kinase 2 (PLK2), the basic helix-loop- differences in their expression levels. Reverse transcription PCR primers BHLB9 were designed between different exons to avoid any amplification of DNA helix domain containing class B9 ( ), ubiquitin-conjugating UBE2V2 ELAC2 (Supplementary Table S1). enzyme E2 variant 2 ( ), the ElaC homologue 2 ( ), the BH3-interacting domain death agonist (BID), and the PH domain PLEKHE1 Results and Discussion and leucine-rich repeat protein phosphatase 1 ( ). We observed that 78% (seven of nine) of these CpG islands were densely The MeDIP microarray profile of wild-type HCT-116 colon methylated in HCT-116 cells and fully unmethylated in DKO cells cancer cells and DNA methyltransferase-deficient cells (DKO). (Figs. 1C and 2A). The genes were RASGRF2, SCNN1B, HOXD1, MeDIP has been used in conjunction with genomic microarrays in PLK2, BHLB9, and UBE2V2. In the three remaining genes, ELAC2, transformed and normal cells to outline the DNA methylation BID, and PLEKHE1, the 5¶-associated CpG islands were unmethy- differences associated with tumorigenesis in several recent, lated in both HCT-116 and DKO cells (Supplementary Fig. S1). We promising studies (8–10). We have applied the MeDIP approach wanted to focus on cancer-specific DNA hypermethylation, and so to a 44K human proximal promoter array to evaluate the CpG we analyzed the DNA methylation status of the six CpG islands hypomethylation changes in the DNMT1/DNMT3b double knock- found to be hypermethylated in HCT-116 cells by bisulfite genomic out HCT-116 cells (DKO) in relation to the wild-type HCT-116 to sequencing in a collection of normal colon tissues (n = 10). We reveal newly hypermethylated genes in colorectal tumors. The observed that 67% (four of six) of these CpG islands were always A methodology is summarized in Fig. 1 . The entire set of raw and unmethylated in the normal tissues, and thus, their methylation preprocessed microarray data is deposited in the National Center was cancer-specific: this was the case for RASGRF2, SCNN1B, 1 for Biotechnology Information Gene Expression Omnibus (acces- HOXD1, and PLK2 (Fig. 2A and B). 2 sion number GSE9267) and is also accessible at our ftp repository. For the two remaining genes, the CpG island UBE2V2 was To test the specificity and efficiency of MeDIP, we compared the consistently methylated in all normal colon samples studied (data relative enrichment of known methylated and unmethylated not shown), but the case of the second gene, BHLB9, was genomic sequences using real-time PCR. MeDIP-enriched methyl- particularly interesting due to its chromosomal location on the ated DNA, as exemplified by the cancer-specific promoter hyper- X-chromosome (Xq23), that, in females, is randomly inactivated by methylation of the retinoic acid receptor B2 (RARB2; refs. 1–3) and DNA methylation in one copy. Thus, the normal tissues in which the imprinting control regions of H19 and GPR109A (refs. 8, 13; we found BHLB9 CpG island hypermethylation were all from B Fig. 1 ), in comparison with unmethylated CpG sequences, such as female donors whereas normal tissues from male donors were B the histone H3B promoter (ref. 10; Fig. 1 ). Most importantly, DKO always unmethylated (Supplementary Fig. S2). Most importantly, cells showed markedly depleted MeDIP enrichment in comparison because the HCT-116 cancer cells originated from a male (16), and with HCT-116 cells for the methylated DNA sequences, such as thus had not undergone X-inactivation and its associated BHLB9 B RARB2, H19, and GPR109A (Fig. 1 ). Only 28 promoters of 17,917 CpG island hypermethylation, the presence of BHLB9 hyper- annotated human genes (0.15%) retained MeDIP enrichment in methylation in the HCT-116 malignant cells could be considered a DKO cells in comparison to HCT-116 (Supplementary Table S2). cancer-specific hypermethylation event, similar to those described The false discovery rate had a value of 0.06. for the other four newly identified candidates (RASGRF2, SCNN1B, From the global genomic perspective, we observed abundant HOXD1, and PLK2). DNA demethylation events in DKO cells in comparison with wild- CpG island hypermethylation in the newly identified type HCT-116 cells. Of the 17,917 printed promoters in the array, we candidate genes is associated with transcriptional inactiva- observed significant 5-methylcytosine DNA immunoprecipitation tion. It is critical to establish the effect of the detected 5¶-end CpG C losses in 126 candidate genes in the DKO cells (Fig. 1 ; island hypermethylation events on the expression of the contigu- Supplementary Table S3). Using the CpG Island Searcher Program ous genes. The presence of PLK2 hypermethylation-associated (11), we estimated that 103 (82%) of these candidate genes had a silencing has recently been described (17), and for this reason, we ¶ C CpG island in their 5 -ends (Fig. 1 ). Gene ontology analyses of undertook no further studies. For the four remaining hyper- these 103 candidate hypermethylated genes revealed a broad methylated cancer-specific genes (RASGRF2, SCNN1B, HOXD1, and BHLB9), we addressed the association between DNA methylation and expression analyzed by semiquantitative reverse transcription 1 http://www.ncbi.nlm.nih.gov/geo/ PCR. For all four genes, hypermethylated HCT-116 cells showed 2 http://www.cnio.es/es/index.asp loss of expression of their respective transcripts (Fig. 2C). Most

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Figure 1. Unmasking of epigenetically silenced genes using MeDIP. A, explanatory illustration of the MeDIP approach used. DNA methylation levels are calculated as the average of oligonucleotide ratios between immunoprecipitated 5-methylcytosine (IP a-5mC) versus input. The confidence of binding calls is represented as a P value. In the graph, the probes marked with a red square (set P < 0.001) were considered to be potentially methylated (Bound). B, validation by real-time PCR of the enriched DNAs obtained from the MeDIP assays. Highly methylated promoters from the imprinted genes H19 and GPR109 and the tumor suppressor gene RARh2 (hypermethylated in HCT-116) were selected as positive controls to measure the enrichment levels obtained after MeDIP. The graph shows a specific and efficient enrichment of methylated DNA over an unmethylated promoter (H3b) used as a negative control. C, schematic strategy used to identify cancer-specific promoter hypermethylation in colon cancer cells using MeDIP. D, gene ontology categories of the 126 hypermethylated candidate genes obtained from the MeDIP approach. Ontology terms (Y-axis), percentage of enrichment (X-axis). www.aacrjournals.org 11483 Cancer Res 2007; 67: (24). December 15, 2007

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Cancer Research importantly, restoration of expression was observed upon treat- transfecting the HCT-116 cell line with the RASGRF2 gene or the ment with the demethylating agent 5-aza-2-deoxycytidine and was empty vector (Supplementary Fig. S3). RASGRF2 reexpression also observed in DKO cells (Fig. 2C). revealed tumor suppressor activity whereby there was a reduction Furthermore, to determine whether these genes display putative of 56% in colony formation density with respect to the empty tumor suppressor features, we examined the effect of the ectopic vector (Supplementary Fig. S3). expression of RASGRF2 in the growth of wild-type HCT-116 cells. In The presence of hypermethylation of RASGRF2, SCNN1B, HOXD1, the colony formation assay, we used G418 selection after and BHLB9 was not a unique feature of the HCT-116 colon cancer

Figure 2. CpG island DNA methylation and expression analyses of the cancer-specific hypermethylated genes found in the MeDIP approach. A, bisulfite genomic sequencing analyses of SCNN1B, RASGRF2, BHLHB9, and HOXD1 CpG island methylation status in HCT116, DKO and normal colon. CpG dinucleotides (short vertical lines). The transcriptional start site (long black arrow) and the location of bisulfite genomic sequencing PCR primers (white arrows). Ten single clones are represented for each sample. The presence of a methylated (black squares) or unmethylated (white squares) cytosine. The four CpG islands are hypermethylated in HCT-116 cells, but unmethylated in DKO and normal colon. B, illustrative methylation-specific PCR analyses for SCNN1B, RASGRF2, BHLHB9, and HOXD1 gene in human normal colon samples (NC 1–5). The presence of a PCR band indicates methylated (lane M) or unmethylated (lane U) genes. DNA from normal lymphocytes (NL) and in vitro methylated DNA (IVD) were used as positive and negative controls for methylated DNA, respectively. For BHLHB9, only colon samples from male donors were used. The four CpG islands were unmethylated in normal colon. C, expression analyses for SCNN1B, RASGRF2, BHLHB9, and HOXD1 using reverse transcription PCR. Hypermethylated HCT-116 cells show loss of expression of the respective transcripts and restoration of expression is observed upon treatment with the demethylating agent 5-aza-2-deoxycytidine (DAC) and in DKO cells. The water reaction and normal colon are shown as negative and positive controls, respectively.

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Figure 3. CpG island DNA methylation and expression analyses of SCNN1B, RASGRF2, BHLHB9, and HOXD1 in human colon cancer cell lines, primary colorectal tumors, and adenomas. A, methylation-specific PCR analysis of SCNN1B, RASGRF2, BHLHB9, and HOXD1 in human colon cancer cell lines. The presence of a PCR band indicates methylated (lane M) or unmethylated (lane U) genes. Normal colon (NCOLON) and normal lymphocytes (NL) were used as positive controls for unmethylated DNA and in vitro methylated DNA (IVD) was used as a positive control for methylated DNA. B, expression analyses of SCNN1B, RASGRF2, BHLHB9, and HOXD1 by reverse transcription PCR in human colon cancer cell lines. The hypermethylated genes in A are not expressed, whereas unmethylated genes are expressed. Two illustrative normal colon cDNAs (N Colon 1 and N Colon 2) are positive controls. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. C, representative methylation-specific PCR analyses of SCNN1B, RASGRF2, BHLHB9, and HOXD1 (left) in primary colorectal tumors (C 1–5) showing unmethylated and methylated samples. Representative reverse transcription PCR expression analyses of SCNN1B, RASGRF2, BHLHB9, and HOXD1 (right) in the primary colorectal tumors in A. D, representative methylation-specific PCR analyses of SCNN1B, RASGRF2, BHLHB9, and HOXD1 in primary adenomas (AD 1–5) showing unmethylated and methylated samples. cell line; upon analyzing a collection of colorectal cancer cell lines methylation of each gene and loss of expression shown above in (n = 7), we observed the presence of these epigenetic alterations HCT-116 cells, was also found in this panel of cancer cell lines (Fig. 3A; Table 1). The only exception was HOXD1, which was (Fig. 3B). only hypermethylated in HCT-116. Because all cell lines used CpG island hypermethylation profile for the newly identified originated from male colorectal patients, except SW48, the normal candidate genes in human colorectal tumorigenesis. The X-chromosome–related hypermethylation of BHLB9 in females presence of RASGRF2, SCNN1B, HOXD1, and BHLB9 hypermethy- was not an issue. The association between CpG island hyper- lation was not an in vitro cell culture phenomenon, but when a

Table 1. CpG island hypermethylation distribution of the MeDIP-identified candidate genes

Genes Colorectal cancer cell lines Colorectal primary tumors Adenomas Normal colon

SCNN1B 88% (7/8) 20% (11/56) 13% (4/30) 0% (0/10) RASGRF2 38% (3/8) 45% (29/65) 35% (12/34) 0% (0/10) BHLHB9* 42% (3/7) 33% (18/54) 33% (9/27) 0% (0/7) HOXD1 13% (1/8) 4% (2/52) 0% (0/33) 0% (0/10)

*For BHLHB9, located in the X-chromosome, only samples from male donors were included.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Cancer Research collection of primary tumor samples from colorectal cancer silencing of other ion channels such as CACNA1G and CALCA1I patients was analyzed, it was also observed (Fig. 3C; Table 1). (1–3, 7) seems to be a common finding in human tumors. These The presence of hypermethylation of any of the described four data, and the newly identified epigenetic silencing of SCNN1B in genes was not associated with any particular clinical stage, age our experiments, are evidence in favor of the proposed role of of the patient, or anatomic location in the colon. Furthermore, sodium, calcium, potassium, and chloride channels in the in 19 colorectal primary tumors in which RNA was available, the regulation of cell proliferation, migration, and invasion (18). Finally, promoter CpG island methylation of RASGRF2, SCNN1B, HOXD1, the identification of CpG island hypermethylation of the BHLHB9 and BHLB9 was associated with the loss of their corresponding gene pinpoints another family of proteins critical to tumorigenesis, transcripts (Fig. 3C). the basic helix-loop (bHLH) factors (19). The bHLH family of tran- To determine whether hypermethylation of the described genes scription factors functions in the coordinated regulation of gene might represent an early lesion in colorectal tumorigenesis, we expression, cell lineage commitment, and cell differentiation in examined the CpG island methylation status of RASGRF2, SCNN1B, most tissues (19). In the case of BHLHB9, a pivotal role in apoptotic HOXD1, and BHLB9 in benign colorectal adenomas, a lesion that is cell death has been proposed (20). Interestingly, BHLHB9 hyper- a precursor to invasive colorectal tumors. We observed a hypermethylation is not an isolated event in its category; other bHLH methylation frequency similar to that of invasive colon carcinomas proteins, such as TWIST2 (15), also undergo methylation-associat- (Fig. 3D; Table 1), which is also present in both small (<15 mm) and ed silencing in human neoplasias. This further underlines the role large adenomas (>15 mm). These findings point to hypermethyla- of this family of transcription factors in cellular transformation. tion of the identified candidate genes as early events in the Thus, overall, we have shown that the use of powerful pathway to full-blown colorectal tumors. epigenomic technologies, such as MeDIP in conjunction with Our results suggest that the genomic disruption of the DNMTs comprehensive promoter microarrays, in cancer cells whose DNMT associated with a MeDIP-promoter microarray approach is a useful genes have been disrupted, could identify new hypermethylated strategy for ‘‘catching’’ new genes undergoing DNA methylation– genes in human colorectal tumorigenesis. These aberrantly associated silencing in human cancer. We have shown that the CpG epigenetically silenced genes are members of the various cellular island hypermethylation of these newly identified genes is not a pathways that contribute to the tumorigenic phenotype and unique feature of HCT-116 cells, but is also common among illustrate the disrupted DNA methylation landscape present in colorectal tumorigenesis, which is also found in other colon cancer cancer cells. cell lines, primary colon tumors, and colon adenomas. The list of epigenetically silenced genes revealed covers most of the disrupted Acknowledgments pathways of cancer cells (14), such as Ras-mediated signal RASGRF2 Received 7/16/2007; revised 10/12/2007; accepted 10/24/2007. transduction and development, exemplified by and Grant support: Health (FIS01-04) and Education and Science (I+D+I MCYT08-03 HOXD1, respectively. These two cases are not isolated epigenetic and Consolider MEC09-05) Departments of the Spanish Government, the European Grants Transfog LSHC-CT-2004-503438 and CANCERDIP (200620), and the Spanish events in their categories, but are added to other Ras-related genes Association Against Cancer. Foundation for Science and Technology of Portugal such as the Ras effectors RASSF1A and NORE1A (1–3, 7), and Fellowship recipient (F.V. Jacinto). homeobox genes such as HOXA9, LMX-1, HOXA5, and DUX-4 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance (1–3, 7). SCNN1B is another interesting case because it codes for with 18 U.S.C. Section 1734 solely to indicate this fact. the h subunit of an epithelial sodium channel and epigenetic We thank Dr. Xose Bustelo for providing us with the RASGRF2 expression vector.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Discovery of Epigenetically Silenced Genes by Methylated DNA Immunoprecipitation in Colon Cancer Cells

Filipe V. Jacinto, Esteban Ballestar, Santiago Ropero, et al.

Cancer Res 2007;67:11481-11486.

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