Purpose: The goal of this study was to comprehensively identify CpG island methylation alterations between pancreatic cancers and normal pancreata and their associated gene expression alterations.
Experimental Design: We employed methylated CpG island amplification followed by CpG island microarray, a method previously validated for its accuracy and reproducibility, to analyze the methylation profile of 27,800 CpG islands covering 21 MB of the human genome in nine pairs of pancreatic cancer versus normal pancreatic epithelial tissues and in three matched pairs of pancreatic cancer versus lymphoid tissues from the same individual.
Results: This analysis identified 1,658 known loci that were commonly differentially methylated in pancreatic cancer compared with normal pancreas. By integrating the pancreatic DNA methylation status with the gene expression profiles of the same samples before and after treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine, and the histone deacetylase inhibitor, trichostatin A, we identified dozens of aberrantly methylated and differentially expressed genes in pancreatic cancers including a more comprehensive list of hypermethylated and silenced genes that have not been previously described as targets for aberrant methylation in cancer.
Conclusion: We expected that the identification of aberrantly hypermethylated and silenced genes will have diagnostic, prognostic, and therapeutic applications. Clin Cancer Res; 17(13); 4341–54. ©2011 AACR.
Pancreatic cancer is the 4th leading cause of cancer death in the United States and is characterized by advanced disease at the time of diagnosis and resistance to most therapeutic treatments. Improving our understanding of the molecular mechanisms that contribute to the development of pancreatic cancer at the epigenetic and transcriptional level is crucial for the early diagnosis and therefore the treatment of this deadly disease. In this study, we used cancer cell lines derived from pancreatic ductal adenocarcinoma and compared their DNA methylation profile with pancreatic normal tissue as a reference. We found numerous target genes that are either hypermethylated and silenced or hypomethylated and overexpressed in pancreatic cancer versus normal pancreas and that could be used as building blocks for the development of new diagnosis and prognosis biomarkers, as well as therapeutic tools for pancreatic cancer.
Pancreatic cancer is the 4th most common cause of cancer death in the United States and has the lowest survival rate for any solid cancer. This particularly poor outcome is due in large part to the late presentation of the disease in most patients. Identifying those at risk of developing pancreatic cancer (1, 2) and developing better diagnostic markers of pancreatic neoplasia (3, 4) could improve the early diagnosis of pancreatic cancer and its precursors (5) and allow more patients to undergo curative surgical resection. Previous studies have shown that aberrant gene hyper- and hypomethylation contributes to pancreatic cancer development and progression (6–11). Furthermore, aberrant methylation increases during neoplastic development among the precursor lesions known as PanINs and intraductal papillary mucinous neoplasms (IPMN; refs. 11, 12). For example, aberrantly hypermethylated genes have been identified in pancreatic cancer by comparing gene expression profiles of pancreatic cancer cells before and after DNA methylation inhibitor treatment (8) and by using promoter (13) and SNP arrays (13, 14). The detection of aberrantly methylated loci relative to normal tissues could improve the diagnosis of pancreatic cancer (3) and may also identify key regulatory genes and pathways that merit therapeutic targeting (15).
We evaluated the accuracy and reproducibility of the methylated CpG island amplification coupled with a genome-wide promoter microarray platform (MCAM) in a pilot study by using the pancreatic cancer cell lines Panc-1 and MiaPaca2 (13, 16). In this study, we used MCAM to more comprehensively define the differential methylated genes between pancreatic cancer cells and normal pancreatic tissues. We then compared the methylation profile of our candidate genes with their global gene expression profile in pancreatic cancer cell lines and normal pancreatic epithelial ductal samples, including global gene expression profiles of cell lines before and after treatment with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-Aza-dC) and the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). Our aim was to identify genes commonly differentially methylated in pancreatic cancer and to identify the subset of aberrantly methylated genes with aberrant gene expression that represent candidate genes undergoing functional disruption in pancreatic cancer.
Materials and Methods
Cell lines and tissue samples
Pancreatic adenocarcinoma cell lines AsPC1, Capan2, MiaPaca2, BxPC3, Capan1, CFPAC1, HS766, Panc1, and Su8686 were cultured under recommended conditions. A32-1, A38-5, Panc215, Panc2.5, Panc2.8, Panc3.014, A2-1, A6L, Panc198, Panc486, and Panc8.13 pancreatic ductal adenocarcinoma cell lines were described previously (17). Immortalized human pancreatic ductal epithelial (HPDE) cells derived from normal human pancreatic ductal epithelium were generously provided by Dr. Ming-Sound Tsao (University of Toronto, Canada). Stored frozen tissues (−80°C) of normal lymphoid tissues (lymphocytes or spleen) were obtained from 3 of the patients from whom we had developed a pancreatic cancer cell line (A32-1, A38-5, and Panc215).
The frozen primary pancreatic cancer tissues, normal pancreatic, and spleen tissues were obtained from patients the time of their pancreatic resection at Johns Hopkins Hospital. Normal pancreatic duct epithelial cells were isolated by using laser capture microdissection from the resected pancreata of 3 patients (mean age, 64 years; range, 59–72 years) who underwent pancreatic resection for benign conditions. Additional normal pancreatic tissues were obtained from patients with pancreatic adenocarcinomas or neuroendocrine neoplasms. DNA was isolated from xenografts of primary pancreatic cancers as previously described (8, 9). Specimens were collected and analyzed with the approval of the Johns Hopkins Committee for Clinical Investigation.
Methylated CpG island amplification procedure and Agilent CpG island microarrays
Methylated CpG island amplification (MCA) was done as described previously (13). The Human CpG Island ChIP-on-Chip Microarray 244K chip interrogates 27,800 CpG islands covering 21 MB with an average of 8 probes per island. Array hybridization was done by the Johns Hopkins SKCCC Microarray Core Facility. Briefly, 2 μg of MCA amplicon were labeled with either Cy3-dUTP or Cy5-dUTP (Perkin Elmer) by using the BioPrime DNA Labeling System (Invitrogen). These dye-labeled amplicons were then mixed and cohybridized to 244K CpG island microarrays, washed, dried, and scanned by using the Agilent G2505B scanner.
Bisulfite treatment and DNA amplification were done as described previously (13). Primer sequences are available in Supplementary Table S5.
Affymetrix exon arrays
Cells were treated with 1 μmol/L of 5-Aza-dC (Sigma) for 4 days and 1 μmol/L of the HDAC inhibitor TSA (Sigma) either alone or in combination for 24 hours as previously described (8, 9). Total RNA from cell lines was extracted by using mirVana miRNA Isolation Kit (Ambion) per manufacturer's protocol before DNAase treatment (Ambion).
The Affymetrix Exon Array ST1.0 (Affymetrix) was used to analyze gene expression profiles in untreated and 5-Aza-dC or TSA-treated cell lines as previously described (18, 19).
For MCAM, data were extracted with Agilent Feature Extraction 9.1 software. Methylation-specific sites were called with Agilent Genomic Workbench Standard Edition 5.0.14 software for methylation analysis, which calculates the normalized log2-signal ratios (NLR) and combined Z scores for each probe. For Affymetrix Exon Array ST1.0, statistical analysis of gene expression array data was completed with Partek Genomic Suite 6.4 software. Raw Affymetrix intensity measurements of all probe sets were background corrected and normalized by the Robust Multichip Average method. Sample relationships were examined by using principal component analysis to reveal any technical effects that would encumber the subsequent analysis. Gene expression intensities were summarized by the 1-step Tukey's biweight method. Two-way ANOVA analysis was done to identify significant expression changes between pancreatic cancer and noncancer samples and between 5-Aza-dC and/or TSA-treated and untreated cells. Gene network and pathway analysis were done by using Ingenuity Pathways Analysis (Ingenuity systems; http://www.ingenuity.com) software. We were in compliance with the Minimum Information about a Microarray Experiment guidelines and have submitted our microarray data set to the Gene Expression Omnibus of National Center for Biotechnology Information. They are accessible through GEO Series accession number GSE21163 and GSE29723.
The expression of ZBTB16 protein was examined by immunohistochemical labeling of tissue microarrays with anti-ZBTB16 (Invitrogen 34-3,700; 1:100) polyclonal antibody, counterstained with hematoxylin. Immunohistochemical labeling was scored based on intensity; 0 (negative), 1 (weak), or 2 (strong), and on the percentage of positive epithelial cells as 0 (<5%), 1 (6–25%), 2 (26–50%), 3 (51–75%), or 4 (>76%), respectively. A histoscore was generated as the product of intensity multiplied by area and mean histoscores of pancreatic ductal adenocarcinoma and normal ductal epithelial cells were compared by using Paired Student's t test.
Results and Discussion
Identification of loci differentially methylated between pancreatic cancer and normal pancreatic tissues
We used Agilent 244K CpG island microarrays to conduct 9 MCAM experiments comparing pancreatic cancer cell lines with normal pancreas. The pancreatic cancer cell lines were compared with pooled laser-captured microdissected normal pancreatic ductal tissues (A32-1, A38-5, Panc215, Panc2.5, Panc2.8, and Panc3.014), to normal pancreatic tissue (MiaPaca2 and Capan2), or to the nonneoplastic pancreatic ductal line HPDE (AsPC1).
To identify differentially methylated probes, we used the previously validated cutoff (13) of ∼4-fold (NLR ≥ 2 or ≤−2) differential methylation between pancreatic cancers and normal pancreata (Fig. 1). This cutoff was established by using the same MCAM strategy comparing the pancreatic cancer cell line Panc-1 to the epithelial line, HPDE, and validating the methylation status of 31 genes by using bisulfite sequencing and methylation-specific PCR (MSP). By this criterion, there were 41,606 probes, representing 10,012 loci with differential methylation in at least one of the 9 MCAM experiments, a considerably more extensive list than we identified in our pilot MCA experiment by using the 44K array (13). To identify from this list genes commonly methylated in pancreatic cancers, we then calculated the mean of differential methylation ratios of the 9 cancer/normal pairs and the combined Z scores for each of these 41,606 probes and filtered out probes for which the mean of the 9 NLRs was −2 or more but 2 or less (note: Z scores reflects how far a probe log-ratio value is in relation to the Gaussian distributions of probes with similar melting temperatures on the Agilent array). This criterion allowed us to identify 4,672 probes for which the mean combined Z score was either −4.67 or less or 5.1 or more. To refine our gene list, we then filtered out outlier probes for which the mean combined Z scores was −5 or more but 5 or less which left 4,662 probes, corresponding to 1,658 known loci (Supplementary Table S1). Thus, the gene lists identified by using the mean NLR of −2 or more but 2 or less and the mean combined Z scores was −5 or more but 5 or less were almost identical. Among these 1,658 loci, 1,226 were hypermethylated (1,206 genes, Supplementary Table S2) and 423 were hypomethylated (379 genes, Supplementary Table S3) in the pancreatic cancer cell lines compared with normal pancreatic tissue.
We found that 60% of hypermethylated probes were located inside known gene bodies, whereas 31.3% were located in their promoters, and 8.42% were located downstream of known genes (Fig. 2B, left). These proportions were very similar among the hypermethylated genes whose expression was downregulated (Fig. 2B, right) and to the overall distribution of probes on the Agilent chip (Fig. 2A). We also found a very similar distribution of hypomethylated probes (Fig. 2C, left). However, when we included only hypomethylated probes that were associated with upregulated genes, we found that 48.5% of probes were located in the promoter region (Fig. 2C, right). This analysis is consistent with the observation that hypermethylation within the gene body and the promoter can result in downregulation, whereas gene upregulation is associated promoter hypomethylation but not gene body hypomethylation.
Because cancer-associated CpG island hypermethylation tends to extensively involve the affected CpG island, we used MSP to confirm differential CpG island methylation identified by MCAM. Specifically, to validate our MCAM results, we used MSP to examine the methylation status of 24 aberrantly hypermethylated genes in 69 pancreatic cancers [43 xenografts of primary pancreatic cancers, 15 primary pancreatic cancer tissues, and 11 cell lines (i.e., BxPC3, Capan1, CFPAC1, HS766, Panc1, Su8686, A2-1, A6L, Panc198, Panc486, and Panc8.13)] and 16 normal pancreatic tissue. We confirmed the differential methylation of 21 of the 24 genes tested (Supplementary Table S4), showing the robustness of our MCAM strategy.
Several genes were methylated in more than 70% of pancreatic cancers and 10% or less of normal pancreata by MSP including BMI1, ID4, CACNA1H, IRF5, and SMOC2.
Aberrantly methylated and differentially expressed genes
We have previously examined gene expression in a set of 6 pancreatic cancer cell lines (A32-1, A38-5, Panc215, Panc2.5, Panc2.8, and Panc3.014) and the pancreatic nonneoplastic line, HPDE, before and after epigenetic treatment (5-Aza-dC, TSA, and the combination) by using Affymetrix ST1.0 Exon Arrays (18). Here, we merged this analysis with the results of our MCAM experiments and compared the baseline and posttreatment gene expression profiles of these 6 pancreatic cancer cell lines focusing on genes shown to be hypermethylated or hypomethylated in our MCAM experiments. We used the 2-way ANOVA analysis to identify expression changes between cancer cells and the nonneoplastic line HPDE. We defined genes as differentially expressed based on a fold-change criteria of ±2-fold. Among the hypermethylated genes, 96 genes were underexpressed in pancreatic cancer cells compared with HPDE cells and the expression of 675 were induced by 5-Aza-dC and/or TSA treatment either alone or in combination (P value <0.05; Supplementary Table S2). 77 genes were both underexpressed and responsive to epigenetic treatment (Table 1). If we employed a more stringent cutoff for underexpressed genes (>3-fold reduction in expression), 37 genes were both underexpressed and responsive to epigenetic treatment (Table 1, bold genes). Among the hypomethylated genes, 24 genes were overexpressed in the pancreatic cancer cell lines compared with nonneoplastic pancreatic duct HPDE cells (fold-change ≥2; Table 2) and 243 genes were significantly induced by epigenetic treatment but not necessarily differentially expressed (P < 0.05 with a fold-change >2; Supplementary Table S3).
Even by a modest criteria of ±2-fold for differential expression, only a minority of genes displayed methylation-associated alterations in expression compared with those that underwent aberrant methylation (Tables 1 and 2). We previously made this same observation in an MCAM analysis of the Panc-1 versus HPDE cell lines (13). However, when we next examined the relationship between global methylation and gene expression by comparing mean expression levels in the 6 pancreatic cancer cell lines, we found that hypermethylated genes (mean of NLR >1) were significantly less expressed in the pancreatic cancer cells than nonhypermethylated genes (mean of NLR <1, Fig. 3A and B). And conversely, we found that the expression of hypomethylated genes (mean of NLR <−1) was significantly higher than nonhypomethylated genes (mean of NLR >−1, Fig. 3A and B). These results support the general role of hypermethylation as a mechanism of aberrant silencing in cancer and reinforcing the association between localized promoter hypomethylation and gene overexpression.
In addition, we checked the expression profile of our newly identified hypermethylated and hypomethylated genes previously analyzed by the serial analysis of gene expression (SAGE) method (17). The advantage of this SAGE analysis is that it quantified 1 to 2 million SAGE tags in each of the 24 pancreatic cancer samples 2 laser capture microdissected normal pancreatic duct samples and the nonneoplastic pancreatic duct cell line, HPDE. We defined silenced genes, neoexpressed genes, underexpressed, and overexpressed genes. Silenced genes were defined as genes for which the number of tags was simultaneously less than 1 in more than 50% of the 24 pancreatic cancer samples and more than 10 in both normal samples and were added to Table 1. Underexpressed genes were those with a 2-fold decrease in the number of tags in pancreatic cancer compared with either one of the 2 normal samples (the percentage of pancreatic cancers with underexpression is noted in the SAGE columns of Supplementary Table S2). Neoexpressed genes were defined as genes for which the number of tags was simultaneously more than 10 in more than 50% of the 24 pancreatic cancer samples and less than 1 in both normal samples and were added to Table 2. Overexpressed genes were defined as those with a 2-fold increase in the number of tags in pancreatic cancer compared with either one of the 2 normal samples (the percentage of pancreatic cancer samples with overexpression is noted in Supplementary Table S3).
Genes known to be hypermethylated in pancreatic cancer
Among the list of 1,206 candidate hypermethylated genes, were virtually all of the genes we previously identified by using MCAM on the Agilent 44K microarray and validated by bisulfite sequencing and MSP in Panc-1 cancer cells, that is, BAI1, KCNV1, EYA4, BNC1, HOXA5, PAX7, SOX14, TLX3, NRXN1, CNTNAP2, PKP1, ACTA1, MDFI, EVC2, LIN28, NRN1, PENK, FAM84A, and ZNF415 (13).
We also confirmed the differential methylation profiles of genes whose epigenetic silencing was revealed by our group and others. These genes include NPTX2, CLDN5, LHX1, WNT7A, FOXE1, PAX6, BNIP3, GADD45B (8), HIC1, HS3ST2, TWIST1 (14), IRF7, CCNA1, ALPP, CEBPA (20), CACNA1G (21), CCND2 (22), and TFPI-2 (23).
Our list included the tumor suppressor genes STK11 and WT1, the transcription factor RUNX3, the transcriptional repressor CBFA2T3, the secreted glycoprotein and chemorepulsive factor SLIT2 and the aryl-hydrocarbon receptor repressor, AHRR.
Note that our selection criterion was designed to identify genes that are commonly differentially methylated in pancreatic cancer. Thus, several important genes that are infrequently methylated in pancreatic cancer did not meet our commonly methylated filter including p16/CDKN2A, E-cadherin/CDH1, and hMLH1 because these genes are uncommon targets in pancreatic cancer (24, 25). Among our 9 pancreatic cancer/normal pancreas methylomes examined, 3 had 1 hypermethylated probe in the CDKN2A promoter. Other genes hypermethylated in pancreatic cancer (RELN; 7; MMP1 and MMP14; 26) did not meet our filter for commonly methylated genes, although several MCAM probes covering these genes were significant for hypermethylation. Finally, some genes hypermethylated in pancreatic cancer were not identified because their promoters were not interrogated by the MCAM method (e.g., SPARC, MMP3, MMP2, MMP9, and MMP7).
Genes involved in stem cell pluripotency are hypermethylated in pancreatic cancer
We found DNA hypermethylation of genes involved in stem cell pluripotency, such as the intestinal stem cell marker and chromatin structure regulator BMI1, the genes encoding bone morphogenetic proteins BMP3, BMP6, the transcription factors FOXD3, CDX2, UTF1, and T, as well as NR5A1, NR5A2, NR2F1, NTRK1, NTRK2, NTRK3, NODAL, SALL4, and SPHK1 genes. In addition, hypermethylation of FOXA1/A2, BMP7, IGF2BP1, HOXB1, and NR2F2 was associated with gene relative underexpression in pancreatic cancer by SAGE analysis (Supplementary Table S2; ref. 17).
The WNT signaling pathway is a target of hypermethylation in pancreatic cancer
Interestingly, numerous actors and mediators of the WNT signaling pathway were found to be hypermethylated in the majority of our pancreatic cancer samples and account for an enrichment ratio of 0.167 in our ingenuity pathway analysis. As expected, we found hypermethylation involving genes previously shown to be epigenetically silenced both in pancreatic and other types of cancer (14, 27) and encoding either the WNT ligands WNT5A, WNT7A, and WNT9A, or the cell-surface receptor FZD9, as well as the cytoplasmic transducer APC2, the nuclear factors SOX1, SOX7, SOX14, and SOX17, and the pathway inhibitors FRZB, SFRP1, SFRP2, KREMEN2, NKD2, and WIF1. Finally, we also found hypermethylation of the tumor suppressor candidate HIC1, which encodes a transcriptional repressor responsible for abnormal survival circuits through nuclear release of WNT signaling pathway transcription factors (28).
We also found DNA hypermethylation surrounding genes of the WNT pathway and overexpressed in different types of cancer, either in the promoter region (WNT3 and SOX3), within the gene body (FZD1, FZD2, FZD7, SOX15, and SOX18), or both (FZD10, SOX11, and MDFI).
Several hypermethylated WNT pathway genes showed differential gene expression in multiple pancreatic cancer cell lines versus HPDE either by Affymetrix Exon Array (WNT5A, FZD2, SFRP1, and SOX15) or by SAGE analysis (WNT10A, SOX17, SOX18, NKD2, WNT18, FZD1, and FZD7; Supplementary Table S2; refs. 17, 18).
Eleven other WNT pathway genes showed no differential expression by Affymetrix Microarray or SAGE analysis (Supplementary Table S2; refs. 17, 18), although 5-Aza-dC treatment increased expression of 7 of these 11 genes (FZD9, APC2, SOX1, SOX3, SOX11, FRZB, and KREMEN2; Supplementary Table S2), suggesting that DNA hypermethylation influences their expression.
Among the WNT Pathway hypermethylated gene members WNT5A, SFRP1, and SOX15 are of particular interest because they had evidence of epigenetic silencing (loss of expression in pancreatic cancers relative to normal pancreas and induced expression with 5-Aza-dC treatment; Supplementary Fig. S1). SFRP1 is epigenetically inactivated in colorectal and other cancers (29) and suspected to facilitate tumorigenesis by abrogating WNT signaling.
WNT5A is a member of the nontransforming WNT protein family frequently inactivated by tumor-specific methylation in colorectal cancer (30), although opposing roles of WNT5A in cancer have been described (31). For instance, one study found WNT5A overexpression in pancreatic cancers (32) whereas our gene expression profiling found underexpression of WNT5A compared with HPDE cells (2.9-fold by Affymetrix Exon Array) and compared with normal pancreatic ductal epithelium by SAGE. Another identified hypermethylated and downregulated gene implicated in pancreatic differentiation was SOX15 (33). To our knowledge, hypermethylation and silencing of SOX15 have not been previously reported in pancreatic cancer.
The finding that the WNT signaling pathway is a common target of DNA methylation in colon and other cancers led Jones and Baylin (34) to propose that loss of epigenetic gatekeepers such as WNT signaling pathway inhibitors locks cells into stem-cell like states that foster abnormal clonal expansion. The pancreatic cancer genome project provided evidence for aberrant WNT pathway signaling during pancreatic tumorigenesis as somatic mutations in the pathway were occasionally identified in pancreatic cancers (17). In this study, the enrichment of aberrant methylation of the WNT pathway gene members supports a significant role for alterations of this pathway during pancreatic tumorigenesis.
Hypermethylation of cell adhesion molecules during pancreatic carcinogenesis
Numerous genes involved in cell adhesion and junctions were also hypermethylated in pancreatic cancer samples, including members of the cadherin superfamily CDH2, CDH4, CDH11, PCDH1, PCDH10, PCDH11Y, PCDH17, PCDH8, PCDHB7, PCDHGA6, PCDHGB1, PCDHGB6, PCDHGC4, and RET. Interestingly, the pancreatic cancer genome project identified occasional somatic mutations in cadherins and protocadherins in pancreatic cancers (17). Of these genes, hypermethylation of CDH4 and PCDHGB6 was associated with gene underexpression in pancreatic cancers by Affymetrix Array or SAGE data, respectively (Table 1 and Supplementary Table S2).
Hypermethylation of gene clusters and families as a common feature of human cancer
We found that numerous families of genes were simultaneously hypermethylated in our pancreatic cancer samples compared with normal pancreas (Supplementary Table S2, bold genes). These include families of transcription factors and, in particular, homeobox genes clustered on chromosome 7p15.2 (EVX1, HOXA10, HOXA11, HOXA4, HOXA5, HOXA7, and HOXA9), on 17q21.32 (HOXB1, HOXB3, HOXB5, HOXB6, and HOXB9), on 12q13.13 (HOXC11, HOXC12, HOXC13, HOXC4, and HOXC5), on 2q31.1 (EVX2, HOXD1, HOXD10, HOXD11, HOXD12, HOXD4, HOXD8, and HOXD9; Supplementary Fig. S2). These also include chromobox genes on 17q25.3 (CBX2, CBX4, and CBX8), Iroquois homeobox genes on 16q12.2 (IRX3, IRX5, and IRX6) and on 5p15.33 (IRX1, IRX2, and IRX4), and SIX homeobox genes on 2p21 (SIX2 and SIX3).
Hypermethylation of clusters of homeobox genes has been reported in other types of cancer, such as lung cancer and astrocytomas (35, 36), but not in pancreatic cancers. Hypermethylation of a gene region such as the homeobox genes is most likely related to deregulation of the well-orchestrated epigenetic machinery involved in the spatiotemporal regulation of these developmental genes. This finding supports other evidence that gene neighborhood is required for an efficient coordination of chromatin modifications during carcinogenesis and developmental processes (37, 38). An important contributor to these epigenetic marks is the Polycomb group of proteins, which has been shown to be responsible for the concerted silencing of homeobox gene clusters. From our hypermethylated candidate gene list, we found 288 known targets of Polycomb proteins (Supplementary Table S2), including families of highly homologous genes that do not belong to clusters, such as LIM homeobox, NK2/3/6 homeobox, paired box, POU class homeobox, sex-determining region Y-box, and T-box transcription factors, which confirms the very tight link between specific hypermethylation and Polycomb protein complexes occupancy in cancer. However, we found aberrant hypermethylation of several other gene family members including members of the G-protein–coupled receptor superfamily, adenylate cyclases, and voltage-dependant calcium and potassium channels. Sequence similarities between these family members are likely responsible for their similar cancer epigenetic patterns. For example, SINE and LINE repetitive elements are enriched at sites of aberrant hypermethylation (38). These sequence-based contributions to aberrant methylation indicate that selection driven by gene silencing need is not responsible for causing methylation marks to arise during tumorigenesis. The fact that only a relatively small proportion of hypermethylated genes showed evidence of downregulation or silencing (HOXD9, HOXD8, CBX2, IRX2, IRX4, TBX18, SOX15, SOX17, SOX18, and PAX8; Supplementary Table S2) supports this hypothesis.
Newly identified hypermethylated and underexpressed candidate genes
Of the 81 hypermethylated and underexpressed genes identified by our analysis, 33 have, to our knowledge, not been previously identified as hypermethylated either in pancreatic or other cancers. The differential methylation of several of these genes including ID4, FOXF2, FOXG1, IRF5, SIM1, SMOC2, and ZBTB16 was confirmed by MSP (Supplementary Table S4). Twelve genes showed gene silencing in pancreatic cancer compared with normal pancreatic duct by SAGE (including HOXD8, BNIP3, FOXF2, and ID4), many of which have not been identified as differentially methylated in cancer (Table 1).
Many hypermethylated and underexpressed genes identified have been implicated in tumorigenesis including tumor suppression (RINT1; ref. 39), proliferation (GAS6, CDT1; ref. 40), apoptosis (IRF5; ref. 41; C16ORF5/CDIP; ref. 42), cell adhesion (CLDN10, ICAM4, LAMA1, AMOTL1), or transcriptional regulation (FOXG1, INSM2). Interestingly, although SEMA5A was recently proposed as a marker for aggressive pancreatic cancers, we find most pancreatic cancers underexpressed SEMA5A (43). Other genes of note include the type IV collagen genes COL4A1 and COL4A2 which have not been described as aberrantly methylated in cancer.
Finally, to further evaluate genes identified as both hypermethylated and underexpressed by array analysis, we searched for commercially available antibodies to proteins corresponding to genes on our list (Table 1) and selected an antibody to ZBTB16 for further analysis. The transcription factor ZBTB16 (a.k.a PLZF), has been shown to regulate apoptosis and proliferation, as well as the Hox family of genes during development, and has been proposed as a candidate tumor suppressor gene in acute promyelocytic leukemias. By MSP, we confirmed ZBTB16 promoter methylation in 35.2% of the pancreatic cancer samples tested, whereas none of the DNAs from 16 normal pancreas samples showed methylation. By immunohistochemistry, ZBTB16 protein expression was not detectable in 38 (15.4%) of 247 pancreatic cancers evaluated. Reduced expression relative to normal pancreas was found in the majority of pancreatic cancers (193/211, 94%; mean histoscore 2.7 vs. 6.9, P < 0.0001; Supplementary Fig. S3). Interestingly, aberrant methylation and silencing of ZBTB16 in pancreatic cancers may be functionally significant as it functions to repress miR-221 and miR-222 which is overexpressed in pancreatic cancer cells (44). MiR-221/222 expression is associated with enhanced proliferation and dedifferentiation in melanoma cells (45).
Newly identified hypomethylated and overexpressed candidate genes
Because hypomethylation is associated with gene overexpression in pancreatic and other cancers (9), we also identified candidate hypomethylated genes by using the MCAM assay. Of the 379 unique hypomethylated candidate genes identified, 31 have been described as aberrantly methylated in different cancers (Supplementary Table S3). This list confirmed the aberrant hypomethylation of NDN and SMARCA1 genes, previously described in pancreatic cancer (14). Similarly to hypermethylated genes, some known hypomethylated genes did not meet our criteria for frequent hypomethylation. However, when we examined the NLR for probes covering MUC4 and MUC2, genes previously identified as hypomethylated in pancreatic cancer (46), there was evidence for hypomethylation (NLR ≤−2) in at least one of the 9 differential methylomes of pancreatic cancer/normal pancreas studied. The promoter regions of other hypomethylated genes in pancreatic cancers (S100A4, LCN2, TFF2, MSLN CLDN4, SFN, and PSCA; 9) were not interrogated by our MCAM method.
Interestingly, while approximately half of the hypermethylated candidate genes had multiple probes meeting criteria for hypermethylation, the vast majority (84.7%) of hypomethylated gene candidates harbored only 1 hypomethylated probe (mean of the 9 NLR ≤−2 and mean combined Z scores was ≤−5; Supplementary Table S3).
Our hypomethylated gene list contained chromatin modifiers, including the histone methyltransferases SETD8 (lysine) and PRMT1 (arginine), the histone ubiquitin ligase CTR9, the histone demethylase KDM6A, the chromatin assembly protein HIRIP3, and the histone acetyltransferase EP400. These genes were overexpressed in pancreatic cancer samples compared with HPDE cells and/or normal ductal epithelium by SAGE, and HIRIP3 was found to be neoexpressed in 41% of pancreatic cancer samples (Supplementary Table S3; ref. 17).
Three oncogenes, JUNB, MYB, and FOS were hypomethylated in our analysis. Although FOS undergoes hypomethylation during liver tumorigenesis, JUNB and MYB have not been previously reported to be hypomethylated. Interestingly, MYB was found to be neoexpressed in 31% of pancreatic cancer compared with normal samples by SAGE (Supplementary Table S3; ref. 17).
Of the 44 hypomethylated genes identified in our analysis and found to be overexpressed by both ExonArray and SAGE methods, 34 have, to our knowledge, not been described as aberrantly methylated in cancer. Among them, DDHD2, CREB1, and PIM2 have been proposed as oncogenes, whereas ITGB2 (CD18) and MAPRE2 are involved in cell invasion and metastasis. HELLS is a putative stem cell marker, whereas MTRR restores methionine synthase for folate metabolism and polymorphisms have been associated with pancreatic cancer risk.
Differentially methylated loci of pancreatic cancer samples compared with normal lymphoid tissues
To further validate our list of hyper- and hypomethylated candidate markers, we conducted 3 additional MCAM experiments, in which we compared the methylation profiles of pancreatic cancer cell samples with matched normal DNA either from peripheral blood lymphocytes (A32-1 and Panc215) or from normal spleen (A38-5).
We used the cutoffs described above for the NLR and combined Z score to select probes that were hyper- or hypomethylated in pancreatic cancer cells compared with matched lymphocytes or spleen, respectively (Fig. 4).
We found 11,029 and 14,863 differentially methylated probes in Panc215 and A32-1 samples compared with matched lymphocytes DNA of the patient, respectively. We found 13,680 differentially methylated probes in A38-5 cancer cells compared with their matched normal spleen. We then compared these probes with those differentially methylated in Panc215, A32-1, and A38-5 cancer cells versus the same pooled microdissected normal pancreatic ductal tissue. Because some methylation patterns reflect tissue specific differences, we found more differentially methylated probes when comparing pancreatic cancer DNA with matched lymphoid tissue of a patient than to unmatched normal pancreatic duct. We found 4,434 and 6,200 differentially methylated probes both in cancer compared with lymphocytes and in cancer compared with normal pancreatic tissue, for Panc215 and A32-1 samples, respectively. Moreover, we found differentially methylated 2,956 probes in both Panc215 and A32-1 samples, and both in cancer compared with lymphocytes and cancer compared with normal pancreatic tissue, corresponding to 786 known loci (Fig. 4). Among these loci, 669 unique genes were part of our hypermethylated and hypomethylated candidate lists (Supplementary Tables S2 and S3). These data indicate that while more than half of our differentially methylated candidate genes in pancreatic cancers relative to normal pancreata are also differential methylated relative to peripheral blood lymphocytes, many genes that are methylated in pancreatic cancers are normally methylated in other tissues.
We found differentially methylated 6,858 probes that were both in cancer compared with spleen and cancer compared with normal pancreatic tissue (Fig. 4). Among these probes, 1,896 were also differentially methylated in pancreatic cancer cells compared with normal pancreatic tissue and in pancreatic cancer cells compared with lymphocytes. These corresponded to 530 known loci, and 489 genes present in our hypermethylated and hypomethylated gene lists (Supplementary Tables S2 and S3).
Among these 489 genes, 32 genes show DNA hypermethylation associated with downregulation in cancer cells compared with normal cells (Table 1). These latter genes, with a more specific hypermethylation profiles in pancreatic cancer tissues are likely to be better candidates as pancreatic cancer markers. Indeed, several of these genes have already been proposed as candidate biomarkers of prognosis (BNC1; ref. 47; SLIT2; ref. 48), or response to treatment (WNT5A; 49) in other types of cancer.
In summary, we provide a comprehensive list of candidate aberrantly methylated in pancreatic cancers relative to normal pancreatic duct samples, including novel aberrantly methylated and epigenetically silenced genes validated in pancreatic cancer tissue samples, as well as commonly methylated signaling pathways and gene families. We anticipate that our integrated analysis of pancreatic cancer gene methylation and transcription will facilitate the further investigation of pancreatic cancer biomarkers for diagnostic, prognostic, and therapeutic applications.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
This work was supported by the National Cancer Institute grants (CA62924, CA120432, and RC2CA148346) and the Michael Rolfe Foundation.
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 with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
- Received December 24, 2010.
- Revision received April 15, 2011.
- Accepted May 12, 2011.
- ©2011 American Association for Cancer Research.