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Molecular Biology and Genetics |
Departments of Pathology [N. S., N. F., A. M., H. M., R. H. H., M. G.], Oncology [C. J. Y., R. H. H., M. G.], Surgery [C. J. Y., J. L. C.], and Medicine [M. G.], The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205-2196
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We used a high-throughput microarray approach to identify genes silenced by DNA methylation in pancreatic cancer and to establish a panel of epigenetic markers for the early detection of this deadly disease. A similar strategy using cDNA microarray has been described recently and used to successfully identify a group of genes that were aberrantly methylated in virtually all colorectal cancers (9) .
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment with 5Aza-dC3
and/or TSA.
Four pancreatic cancer cell lines (AsPC1, Hs766T, MiaPaCa2, and Panc1) were treated with 5Aza-dC (Sigma, St. Louis, MO) and TSA (Sigma), either alone or in combination. Cells were exposed continuously to 5Aza-dC (1 µM) for 4 days or to TSA (1 µM) for 24 h. Because we observed previously (10)
that treatment of these cell lines with 5Aza-dC (1 µM) for 4 days results in marked induction of several genes silenced by aberrant methylation without evidence for cell death, we used this 4 day time point for our analysis. Mock-treated cells were cultured with the equivalent volume of PBS alone. For combined treatment, these cells were cultured in the presence of 5Aza-dC (1 µM) for 3 days and then treated for another 24 h with TSA (0.5 µM).
Oligonucleotide Array Hybridization.
Total RNA was isolated from cultured cells using TRIZOL reagent (Invitrogen, Carlsbad, CA) and purified using RNeasy Mini Kit (Qiagen, Valencia, CA). First- and second-stranded cDNA was synthesized from 10 µg of total RNA using T7-(dT)24 primer (Genset Corp., South La Jolla, CA) and SuperScript Choice system (Invitrogen). Labeled cRNA was synthesized from the purified cDNA by in vitro transcription reaction using the BioArray HighYield RNA Transcript Labeling Kit (Enzo Diagnostics, Inc., Farmingdale, NY) at 37°C for 6 h. The cRNA was fragmented at 94°C for 35 min in a fragmentation buffer [40 mM Tris-acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate]. The fragmented cRNA was then hybridized to the Human Genome U133A chips (Affymetrix, Santa Clara, CA) with 18,462 unique gene/EST transcripts at 45°C for 16 h. The washing and staining procedure was performed in the Affymetrix Fluidics Station according to the manufacturer’s instructions. The probes were then scanned using a laser scanner, and signal intensity for each transcript (background-subtracted and adjusted for noise) was calculated using Microarray Suite Software 5.0 (Affymetrix).
RT-PCR.
Four µg of total RNA were reverse transcribed using Superscript II (Invitrogen). PCR reaction was performed under the following conditions: (a) 95°C for 5 min; (b) 30–35 cycles of 95°C for 20 s, 60°C for 20 s, and 72°C for 20 s; and (c) a final extension of 4 min at 72°C. Primer sequences were 5'-CTCTGTTTAGCACTGATAATG-3' (forward) and 5'-TTTATTAGACTTGAGCTGATTC-3' (reverse) for CDH3, 5'-CATCGAGCTGCTCATCAAC-3' (forward) and 5'-CTGCTCTTGTCCAAGGATC-3' (reverse) for NPTX2, 5'-CTGGCCCGAGATGCTTAAG-3' (forward) and 5'-TATTTTCATCCTCAGTGCAAAC-3' (reverse) for SARP2, 5'-CTTCATGAAGCAGACCATTG-3' (forward) and 5'-ATCATGGGCTGCCTGTATG-3' (reverse) for UCHL1, and 5'-CGGGAGATCAAGCAGAATG-3' (forward) and 5'-AACGGCCTCGTTGTACTTG-3' (reverse) for WNT7A. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also amplified to ensure the cDNA integrity.
MSP.
Methylation status of the 5' CpG island of each gene was determined by MSP as described previously (11)
. DNA samples were treated with sodium bisulfite (Sigma) for 16 h at 50°C. After purification with the Wizard DNA clean-up system (Promega, Madison, WI), 1 µl of bisulfite-treated DNA was amplified using primers specific for either methylated or unmethylated DNA. Primers were designed to detect the sequence differences between methylated and unmethylated DNA as a result of bisulfite modification, and each primer pair contained at least four CpG sites to provide optimal specificity. Primer sequences for 16 genes analyzed in this study are available at our website.4
PCR conditions were as follows: (a) 95°C for 5 min; (b) 40 cycles of 95°C for 20 s, 60°C-62°C for 20 s, and 72°C for 30 s; and (c) a final extension of 4 min at 72°C. Five µl of each PCR product were loaded onto 3% agarose gels and visualized by ethidium bromide staining.
Data Analysis and Statistical Analysis.
Fold change analysis of signal intensities obtained from oligonucleotide microarrays between the two treatment groups was performed using Data Mining Tool software (Affymetrix). The frequency of aberrant methylation in pancreatic juice samples between patients with pancreatic cancer and those with other pancreatic diseases was compared using Fisher’s exact probability test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.5
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(19)
, and TIMP-3 (3
, 20)
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Identification of Genes Induced by TSA in Pancreatic Cancer Cell Lines.
We next analyzed the global changes in gene expression profiles induced by the histone deacetylase inhibitor TSA in four pancreatic cancer cell lines. Treatment with TSA resulted in a marked (>5-fold) induction of 424 transcripts (2.3% of the 18,462 transcripts analyzed) in AsPC1, 349 transcripts (1.9%) in Hs766T, 207 transcripts (1.1%) in MiaPaCa2, and 459 transcripts (2.5%) in Panc1. Overall, 1,196 transcripts (6.5%) including 965 genes and 231 ESTs were induced (>5-fold) by TSA in one or more of four pancreatic cancer cell lines. These include a large panel of novel targets for silencing by histone deacetylation including several known tumor suppressor genes or cell cycle-regulatory genes (ING1, p57KIP2, CHES1, CHFR, GADD45B, and others).6
Many of the genes induced by TSA treatment were also induced by 5Aza-dC treatment alone, suggesting a role for both DNA methylation and histone deacetylation in the transcriptional regulation of these genes. Interestingly, treatment of Hs766T with TSA but not with 5Aza-dC resulted in a marked increase in expression of many cancer testis antigens (e.g., G antigens), whereas these genes were inducible by treatment with 5Aza-dC but not with TSA in the other three cell lines.
Identification of Genes Induced by Treatment of Pancreatic Cancer Cell Lines with Both Agents.
We also determined the gene expression profiles in four pancreatic cancer cell lines after a combined treatment with 5Aza-dC and TSA. Treatment with both agents resulted in a marked (>5-fold) induction of 422 (2.3%) of the 18,462 transcripts in AsPC1, 304 transcripts (1.6%) in Hs766T, 243 transcripts (1.3%) in MiaPaCa2, and 196 transcripts (1.1%) in Panc1, and in total, 857 transcripts (4.6%) corresponding to 707 genes and 150 ESTs were induced (>5-fold) in at least one of the four pancreatic cancer cell lines. The 707 genes induced by a combined treatment include several genes known to be aberrantly methylated in cancers (e.g., p16 and MLH1; see the table online),7
supporting a previous notion that some of the genes with densely methylated CpG islands are reexpressed by a combined treatment with 5Aza-dC and TSA. Although treatment of all of the four pancreatic cancer cell lines with 5Aza-dC alone or TSA alone did not result in apparent changes in their phenotypes during the treatment period, combined treatment of certain pancreatic cancer cell lines with 5Aza-dC and TSA induced cell death in a small fraction of cells (data not shown).
Expression and Methylation Analysis of Selected Genes in Pancreatic Cancer Cell Lines.
To identify novel targets for aberrant methylation in pancreatic cancer, we further studied 16 candidate genes that have been reported to be cancer associated or considered functionally important from the list of 475 genes identified as markedly (>5-fold) up-regulated by 5Aza-dC treatment in one or more of pancreatic cancer cell lines but not in the nonneoplastic HPDE cells. These included cadherin 3 (CDH3), reprimo, claudin 5 (CLDN5), death receptor 3 (DR3), forkhead box E1 (FOXE1), leucine zipper down-regulated in cancer 1 (LDOC1), LIM homeobox protein 1 (LHX1), neurofilament heavy polypeptide (NEFH), neuronal pentraxin II (NPTX2), p53-induced protein (PIG11), secreted apoptosis-related protein 2 (SARP2), suppression of tumorigenicity 14 (ST14), the SWI/SNF-related gene (SMARCA1), tight junction protein 2 (TJP2), ubiquitin carboxyl-terminal esterase L1 (UCHL1), and WNT7A. Literature search using PubMed revealed that 14 of the 16 genes have not been implicated for aberrant methylation in any tumor type, whereas SARP2 (also termed SFRP1) and TJP2 (also termed ZO-2) have been recently reported to be frequently methylated in colorectal and pancreatic cancers, respectively (9
, 21)
. All of the 16 genes were identified as having CpG-rich sequences fulfilling the criteria of CpG island [GC content > 50%, CpG:GpC ratio > 0.6, and minimum length (200 bp)] in their 5' regions (22)
.
We first performed RT-PCR on 5 (CDH3, NPTX2, SARP2, UCHL1, and WNT7A) of these 16 genes in two pancreatic cancer cell lines (AsPC1 and MiaPaCa2) to compare the results with the corresponding microarray data and found concordant results (Fig. 1A)
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. The most frequently methylated was UCHL1 (methylated in 100%), followed by CLDN5 (95%), NPTX2 (95%), SARP2 (91%), reprimo (91%), LHX1 (77%), FOXE1 (64%), TJP2 (59%), WNT7A (59%), CDH3 (18%), and ST14 (9%). The number of aberrantly methylated genes varied among individual cell lines, and the average number of aberrantly methylated loci was 7.6 (range, 4–11) per cell line (Fig. 2)
. There was no significant correlation between the number of methylated loci and the genetic profile of cell lines with regard to the presence or absence of mutations in the K-ras, p53, and/or SMAD4 genes.
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. We found that the most common pattern was weak or moderate induction after 5Aza-dC treatment and strong (synergistic) induction after combined treatment (e.g., NPTX2 and UCHL1 in MiaPaCa2). However, we also found that some of these genes were also induced by TSA treatment alone (e.g., UCHL1 in Hs766T and Panc1). Furthermore, in some cases, reexpression of genes completely methylated in an individual cell line was not observed after 5Aza-dC or combined treatment (e.g., reprimo in MiaPaCa2 and SARP2 in Hs766T). One possible explanation is that primers used in this study may not cover the critical region for transcriptional regulation. It is also possible that other mechanisms besides DNA methylation and histone deacetylation could be involved in the silencing of these genes.
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Methylation Analysis of Selected Genes in a Larger Panel of Primary Pancreatic Carcinomas and in Pancreatic Juice Samples.
To test the diagnostic potential of genes we identified as methylated in pancreatic cancer, we selected three genes (NPTX2, SARP2, and CLDN5) that were frequently (>90%) methylated in pancreatic cancer and not methylated in any of the normal ductal epithelia studied. To confirm the high prevalence of aberrant methylation at these loci, we further analyzed the methylation status of these genes in an expanded series of 43 surgically resected, primary pancreatic cancers. Aberrant methylation of NPTX2, SARP2, and CLDN5 was detected in 42 (98%), 41 (95%), and 35 (81%) of these 43 primary pancreatic cancers (Fig. 4)
, and hypermethylation of at least one of these loci was found in 100% of the primary tumors tested.
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. We detected aberrant methylation of NPTX2 in 67%, of SARP2 in 46%, and of CLDN5 in 42% of the 24 juice samples from patients with pancreatic cancer (Fig. 4)
. Overall, 75% (18 of 24) of juice samples from pancreatic cancer patients exhibited aberrant methylation of at least one gene, whereas none of the 13 juice samples from patients with benign pancreatic diseases harbored hypermethylation of any of these genes (P < 0.0001). Importantly, aberrantly methylated DNA was detected in the five juice samples from patients with small pancreatic cancers (tumor < 2 cm in diameter).
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DISCUSSION |
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Recently, Suzuki et al. (9) have described a cDNA microarray-based approach to screen for genes epigenetically silenced in colorectal cancer. They studied gene expression profiles in a colorectal cancer cell line (RKO) treated with 5Aza-dC and/or TSA and successfully identified a number of genes harboring CpG island hypermethylation in colorectal cancer cell lines and in primary tumors; however, some of these genes were also methylated in normal colonic tissues (9) . Although slight methylation was occasionally noted in only a small number of samples from normal pancreatic ductal epithelia, most of the genes we identified as aberrantly methylated in pancreatic cancer were completely unmethylated in a panel of normal pancreatic ductal epithelia.
An advantage of using high-throughput oligonucleotide microarray data from multiple cell lines is the ability to identify a substantial number of candidate genes targeted for aberrant methylation in human cancers. Such data also enabled us to provide a conservative estimate of the number of genes directly affected by aberrant methylation in pancreatic cancers. In our analysis, we found that treatment of pancreatic cancer cell lines with 5Aza-dC induced an average of 
200 transcripts (range, 116–251 transcripts) per cell line. We selected 16 genes with CpG islands from the list of genes induced by 5Aza-dC in pancreatic cancer cell lines but not in nonneoplastic HPDE cells, and we confirmed that 70% (11 of 16) of these genes were aberrantly methylated in pancreatic cancer. Therefore, an average of 140 genes (70% of 200 genes) may be aberrantly methylated in a pancreatic cancer cell line, of which 60 would be expected to be CpG islands [one previous study has estimated that 60% of genes induced by 5Aza-dC do not have CpG islands within their 5' regions (13)
]. We consider 60 aberrantly methylated CpG islands in a pancreatic cancer to be a minimum estimate for several reasons: our analysis did not include a large fraction of ESTs on the Affymetrix U133B chip; expression of many genes that harbor aberrantly methylated CpG islands may be unaffected by 5Aza-dC treatment (25)
; and because we used a stringent 5-fold induction of expression as a cutoff for identifying genes induced by 5Aza-dC. Previously, Costello et al. (25)
studied a panel of cancers using RLGS and estimated that 600 CpG islands were aberrantly methylated in a given cancer. Their estimate is higher than ours for a number of reasons. RLGS also identifies methylated CpG islands that are unrelated to genes (22% of CpG islands in their study). Treatment with 5Aza-dC induced the expression of only one-third of the CpG islands they identified as hypermethylated in tumors. In addition, RLGS may also identify methylated CpG islands in tumors when corresponding normal tissue has a low level methylation. This study and our results highlight the fact that in human cancers, a substantial number of genes are silenced by aberrant methylation. Similarly, the large number of genes induced by TSA is consistent with previous reports that have found between 2% and 10% of genes are induced in cancer cells by TSA treatment (26)
.
We observed variability in the gene expression response of individual cell lines to 5Aza-dC and to TSA. Some cell lines harboring methylation of CpG island at a specific locus had induction of gene expression after 5Aza-dC treatment, whereas others did not. The same observation was true for TSA treatment. Surprisingly, none of the genes analyzed in this study showed induction (>5-fold) after 5Aza-dC treatment in all of the four pancreatic cancer cell lines, even when a gene was methylated in each of these cell lines. This may partly reflect our use of a >5-fold cutoff as an indicator of a significant induction of expression. It is also likely that differences in CpG island methylation density and different levels of transcriptional cofactors between different cell lines contribute to differences in gene expression responses to 5Aza-dC and to TSA. Because a panel of genes induced by 5Aza-dC treatment in even one of the four pancreatic cancer cell lines tested usually led us to identify aberrant CpG methylation of these genes in other pancreatic cancer cell lines, we thought it would be more helpful to provide a list of all genes induced 5-fold or greater by 5Aza-dC treatment.5
A number of genes without 5' CpG islands were identified that were up-regulated after 5Aza-dC treatment. These findings imply that even genes with poor CpG promoters can be regulated by DNA methylation. In keeping with this notion, we and others have observed that relatively CpG-poor genes such as 14-3-3 are aberrantly methylated in cancer (19)
. We also identified genes induced by 5Aza-dC that are known to be overexpressed in pancreatic and other cancers (for example, kallikrein 10). Interestingly, kallikrein 10 has previously been shown to be methylated in certain cancers (27)
. This observation suggests that alterations in methylation patterns may be responsible for the overexpression, as well as the underexpression, of many affected genes in cancer.
One of the novel findings of our present study is that TSA alone could induce the expression of 4 of the 11 genes whose CpG islands were identified as aberrantly methylated in pancreatic cancer. In addition, several genes previously characterized as having methylated CpG islands (such as p57KIP2 and CACNA1G) were also reexpressed after treatment with TSA alone. Previous studies have found that TSA alone is not sufficient to induce the expression of genes with densely methylated CpG islands, although it can facilitate induction of gene expression when combined with 5Aza-dC (9 , 28) . Recently, El-Osta et al. (29) have reported that methyl-CpG-binding protein 2 is involved in methylation-dependent silencing of the MDR1 gene and that treatment with 5-azacytidine but not TSA can release methyl-CpG-binding protein 2 from the heavily methylated promoter, thereby leading to a partial relief of the transcriptional repression. Although the mechanisms underlying the correlation between DNA methylation and histone deacetylation in the control of gene expression are still under investigation, our results provide evidence that treatment with TSA alone can, at least in some cases, relieve the silencing of methylated genes in cancer cells.
Changes in methylation patterns play a crucial role in cancer development and progression (30)
. A number of genes we identified as aberrantly methylated in pancreatic cancer have known important properties involved in cell cycle regulation (reprimo), apoptosis (SARP2), cell adhesion (CDH3), and tight junction barrier (CLDN5 and TJP2). Aberrant methylation and associated silencing of these genes may be functionally important for pancreatic carcinogenesis. For example, reprimo, which displayed frequent hypermethylation in pancreatic cancer, is a downstream mediator of p53-induced G2 cell cycle arrest (31)
. When overexpressed, reprimo induces cell cycle arrest at the G2 phase, suggesting it has tumor suppressor function (31)
. Because functional abrogation of the p53 tumor suppressor gene and its downstream mediators, such as 14-3-3, is central to the development of human cancers (19
, 32)
, it is likely that aberrant methylation of reprimo could lead to defects in cell cycle control and contribute to pancreatic neoplastic progression.
We also show that SARP2 is a frequent target for aberrant methylation in pancreatic cancer. SARP2 is a member of SARP gene families that counteract the Wnt oncogenic signaling pathway, and this gene is considered to be involved in the regulation of apoptosis (33) . Breast cancer cells transfected with SARP2 show an increased sensitivity to different proapoptotic stimuli (33) . Therefore, inactivation of SARP2 by aberrant methylation may provide a growth advantage to cancer cells through increasing the cellular resistance to apoptosis. Interestingly, SARP2 has recently been identified as frequently hypermethylated in colorectal and gastric cancer (9) , thus suggesting general involvement of this gene in tumorigenesis of digestive organs.
Although a growing number of genes have been identified as aberrantly methylated in various cancers, to date few genes have been reported that are aberrantly methylated in a large majority of cancers (6) . Our approach identified five genes (UCHL1, NPTX2, SARP2, CLDN5, and reprimo), each of which was aberrantly methylated in >80% of a panel of pancreatic cancer cell lines. Furthermore, all of the genes we found methylated in pancreatic cancer cell lines were also methylated in primary pancreatic carcinomas. This supports our previous observation that aberrantly methylated genes identified in cancer cell lines are often present in the primary cancers from which they were derived (23) . Genes that are aberrantly methylated at a high frequency in a given cancer are particularly suitable for early cancer detection strategies.
Several studies have addressed the diagnostic utility of epigenetic markers in detection of cancer. Methylation abnormalities have been detected in blood or sputum of patients with lung cancer, in serum of patients with head and neck cancer, in ductal lavage fluid of patients with breast cancer, and in urine from patients with prostate and bladder cancer (reviewed in Ref. 7 ). In particular, the inclusion of multiple genes in these analyses appears to provide a highly sensitive and specific marker for cancer diagnosis (6 , 34 , 35) . Using three markers, we were able to detect aberrantly methylated DNA in 75% of pancreatic juice samples from patients with pancreatic cancer. Although these results need to be extended in larger clinical studies, the detection of aberrantly methylated genes in pancreatic juice or other secondary fluids may serve as a powerful new tool for pancreatic cancer diagnosis, especially for high-risk individuals such as those with a strong family history of pancreatic cancer (36) .
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FOOTNOTES |
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1 Supported by the SPORE in Gastrointestinal Malignancies (CA62924), the Michael Rolfe Foundation and a gift to support pancreatic cancer research from Susan Gurney. 
2 To whom requests for reprints should be addressed, at Departments of Pathology, Medicine, and Oncology, The Johns Hopkins Medical Institutions, 632 Ross Building, 720 Rutland Avenue, Baltimore, MD. Phone: (410) 955-3511; Fax: (410) 614-0671; E-mail: mgoggins{at}jhmi.edu
3 The abbreviations used are: 5Aza-dC, 5-aza-2'-deoxycytidine; TSA, trichostatin A; RT-PCR, reverse transcription-PCR; MSP, methylation-specific PCR; EST, expressed sequence tag; RLGS, restriction landmark genomic scanning.
4 http://pathology2.jhu.edu/pancreas/primer.pdf.
5 For more information, see http://pathology2.jhu.edu/pancreas/475genes5aza_dc.htm.
6 http://pathology2.jhu.edu/pancreas/TSA.pdf.
7 http://pathology2.jhu.edu/pancreas/combi.pdf.
Received 10/15/02. Accepted 4/28/03.
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 R. A. Winn, L. Marek, S.-Y. Han, K. Rodriguez, N. Rodriguez, M. Hammond, M. Van Scoyk, H. Acosta, J. Mirus, N. Barry, et al. Restoration of Wnt-7a Expression Reverses Non-small Cell Lung Cancer Cellular Transformation through Frizzled-9-mediated Growth Inhibition and Promotion of Cell Differentiation J. Biol. Chem., May 20, 2005; 280(20): 19625 - 19634. [Abstract] [Full Text] [PDF]
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N. Takai, N. Kawamata, C. S. Walsh, S. Gery, J. C. Desmond, S. Whittaker, J. W. Said, L. M. Popoviciu, P. A. Jones, I. Miyakawa, et al. Discovery of Epigenetically Masked Tumor Suppressor Genes in Endometrial Cancer Mol. Cancer Res., May 1, 2005; 3(5): 261 - 269. [Abstract] [Full Text] [PDF]
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H. Matsubayashi, N. Sato, K. Brune, A. L. Blackford, R. H. Hruban, M. Canto, C. J. Yeo, and M. Goggins Age- and Disease-Related Methylation of Multiple Genes in Nonneoplastic Duodenum and in Duodenal Juice Clin. Cancer Res., January 15, 2005; 11(2): 573 - 583. [Abstract] [Full Text] [PDF]
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N. Sato, N. Maehara, and M. Goggins Gene Expression Profiling of Tumor-Stromal Interactions between Pancreatic Cancer Cells and Stromal Fibroblasts Cancer Res., October 1, 2004; 64(19): 6950 - 6956. [Abstract] [Full Text] [PDF]
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Y. Tokumaru, K. Yamashita, M. Osada, S. Nomoto, D.-I. Sun, Y. Xiao, M. O. Hoque, W. H. Westra, J. A. Califano, and D. Sidransky Inverse Correlation between Cyclin A1 Hypermethylation and p53 Mutation in Head and Neck Cancer Identified by Reversal of Epigenetic Silencing Cancer Res., September 1, 2004; 64(17): 5982 - 5987. [Abstract] [Full Text] [PDF]
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J. Okami, D. M. Simeone, and C. D. Logsdon Silencing of the Hypoxia-Inducible Cell Death Protein BNIP3 in Pancreatic Cancer Cancer Res., August 1, 2004; 64(15): 5338 - 5346. [Abstract] [Full Text] [PDF]
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