Affiliation of authors: N. Sato, N. Maehara (Department of Pathology), G. H. Su (Departments of Pathology and Oncology), M. Goggins (Departments of Pathology, Oncology, and Medicine), The Johns Hopkins Medical Institutions, Baltimore, MD.
Correspondence to: Michael Goggins, M.D., Departments of Pathology, Oncology, and Medicine, The Johns Hopkins Medical Institutions, 632 Ross Bldg., 720 Rutland Ave., Baltimore, MD 21205 (e-mail: mgoggins{at}jhmi.edu).
ABSTRACT
To investigate whether DNA methylation and the invasive phenotype of pancreatic adenocarcinoma are associated, we studied the role of methylation in the transcriptional regulation of several matrix metalloproteinases (MMPs) and the effect of 5-aza-2'-deoxycytidine (5Aza-dC), an inhibitor of DNA methylation, on the invasive behavior of pancreatic cancer cells. Using the Boyden chamber in vitro invasion assay, we found a statistically significant increase in invasive potential in four of five pancreatic cancer cell lines after treatment with 5Aza-dC. This enhanced invasiveness was associated with the induction of mRNAs for one or more MMPs critical for tumor invasion, including MMP-1, -2, -3, -7, -9, and -14. Addition of an MMP inhibitor (GM6001, GM1489, doxycycline, or tissue inhibitor of metalloproteinase 2) blocked the 5Aza-dC-induced increase in the number of invading cells. As shown by a methylation-specific polymerase chain reaction, 5' CpG sites in MMP-2, -7, and -9 genes were partially or completely methylated in cell lines that expressed little or no corresponding mRNAs. Thus, DNA methylation influences the expression of MMP genes, and use of methylation inhibitors may stimulate the invasion of pancreatic cancer by reactivating invasion-promoting genes.
Accumulating evidence highlights the important role of DNA methylation in the regulation of various genes that determine the biologic behavior of cancer cells (4). In addition to the role of aberrant hypermethylation in the transcriptional silencing of tumor suppressor genes in pancreatic and other cancers (5,6), we have also found that several genes that are overexpressed in pancreatic cancer, including the metastasis-related gene S100A4, display hypomethylation of 5' CpG dinucleotides [(7) and Sato N, Fukushima N, Maitra A, Matsubayashi H, Yeo CJ, Cameron JL, Hruban RH, Goggins M: unpublished results]. Several MMP genes are overexpressed in pancreatic cancer (810), and we suspected that aberrant DNA hypomethylation could be involved in their overexpression. We therefore investigated the role of methylation in the transcriptional regulation of several MMPs and the effect of 5-aza-2'-deoxycytidine (5Aza-dC), an inhibitor of DNA methylation, on the invasive behavior of pancreatic cancer cells.
Using methylation-specific polymerase chain reaction (PCR) and reverse transcriptionPCR (RTPCR), we first determined the methylation status of 5' CpG sites in MMP-2, -7, and -9 genes and its relationship to mRNA expression of these genes in five pancreatic cancer cell lines. We have previously observed that most aberrant methylation events observed in pancreatic cancer cell lines are also evident in the primary pancreatic cancers from which they were derived (11). Methylation-specific PCR analysis of the MMP-2 gene revealed partial or complete methylation in all five cell lines that was associated with little or no MMP-2 mRNA expression in all lines except for Panc1 (Fig. 1, A and D). In three cell lines (AsPC1, BxPC3, and Panc1) that expressed MMP-7, the 5' CpGs of MMP-7 were largely or completely unmethylated, whereas in two cell lines (Hs766T and Mia PaCa2) that did not express MMP-7, the 5' CpG sites of MMP-7 were almost completely methylated (Fig. 1, B and D
). Similarly, the 5' CpG sites of MMP-9 were completely or predominantly methylated in all cell lines, and these cell lines showed little or no basal expression of MMP-9 mRNA (Fig. 1, C and D
). Importantly, 5Aza-dC treatment of Mia PaCa2 cells, which were completely methylated at MMP-2, -7, and -9, led to partial demethylation at these loci, accompanied by increased expression of MMP-2, -7, and -9 mRNAs (Fig. 1, D
). Thus, DNA methylation may be involved in the regulation of MMP gene expression in pancreatic cancer cells.
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Because 5Aza-dC treatment increased the expression of several MMPs, we hypothesized that this drug could affect the invasiveness of pancreatic cancer cells. We tested this hypothesis by analyzing the in vitro invasiveness of five 5Aza-dC-treated pancreatic cancer cell lines in a modified Boyden chamber assay (12), a model that predicts in vivo invasive behaviors (13). Cell culture inserts equipped with a filter membrane containing 8-µm pores (BD Biosciences, Franklin Lakes, NJ) were coated with 20 µg of Matrigel. Cells were treated with 5Aza-dC as indicated or with solvent (phosphate-buffered saline [PBS]) alone for 48 hours, harvested by trypsinization, and placed into the upper chamber at 2 x 105 cells per cm2. After a 24- or 48-hour incubation, cells attached to the upper side of the filter were mechanically removed, the filters were stained with hematoxylin and eosin, and the number of cells that had invaded through Matrigel and migrated to the undersurface of the membrane was counted in five randomly selected microscopic fields in each sample. Because the numbers of invading cells were distributed normally, we used a Student's t test to compare the data between mock-treated and 5Aza-dC-treated groups, as previously described (14,15). All statistical tests were two-sided, and P values of .05 or less were considered statistically significant. Invasion assays were performed in triplicate for each cell line. Cultures of AsPC1 cells treated with 5Aza-dC at 0.5, 1, or 2 µM had statistically significantly higher numbers of invading cells than cultures of untreated cells (P<.001; Fig. 2, A and B). The maximum increase in invasive potential was observed at 1 µM 5Aza-dC (4.3-fold, 95% confidence interval [CI] = 3.9-fold to 4.9-fold). Exposure of other cell lines to 1 µM 5Aza-dC led to increased invasion as follows: for BxPC3 cells, 2.8-fold (95% CI = 2.5-fold to 3.1-fold); for Hs766T cells, 2.7-fold (95% CI = 2.4-fold to 3.3-fold); and for Mia PaCa2 cells, 1.8-fold (95% CI = 1.6-fold to 1.9-fold) (P<.001, for each cell line; Fig. 2, C
). In Panc1 cells, however, treatment with 1 µM 5Aza-dC resulted in a statistically significant decrease (0.5-fold, 95% CI = 0.4-fold to 0.6-fold) in the number of invading cells (P<.001). We next determined whether the increased invasiveness after drug treatment was maintained for generations in vitro. After a 4-day exposure to 1 µM 5Aza-dC, AsPC1 cells were cultured in drug-free medium, passaged on a weekly basis, and assayed for their invasion capacity. The AsPC1 cells that had been treated with 5Aza-dC at passages 1 (14 days after treatment) and 3 (28 days after treatment) still retained a higher (1.7-fold and 2.2-fold, respectively) invasion capacity than untreated control cells, indicating that the altered invasive behavior after 5Aza-dC treatment is "heritable" after several passages in culture.
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We finally examined the effect of 5Aza-dC on the proliferation of five pancreatic cancer cell lines. Treatment of AsPC1 cells with 0.110 µM 5Aza-dC caused a dose-dependent inhibition of cell proliferation (Fig. 2, E). Similarly, exposure of other cell lines to 1 µM 5Aza-dC for 4 days inhibited cell proliferation by 40%88% (Fig. 2, F
). These results are consistent with a previous report that 5Aza-dC treatment suppresses the growth of human tumor cells (16).
Thus, despite its growth-inhibitory effects, 5Aza-dC treatment accelerated in vitro invasion of certain pancreatic cancer cell lines. A previous report (17) described the acquisition of a metastatic phenotype in murine tumor cells in vivo after 5Aza-dC exposure. Because invasion is a complex process and because many genes are induced by 5Aza-dC in cancer cell lines (18,19), it is likely that the expression of other genes in addition to MMPs contributes to the enhanced invasion by 5Aza-dC treatment in vitro. However, because MMP inhibitors suppressed the 5Aza-dC-induced increased invasiveness, induction of MMP expression may be an important mediator of these effects.
The important role of aberrant methylation in the silencing of tumor-suppressor and growth-regulatory genes during tumorigenesis has led to the hypothesis that this epigenetic alteration may be an attractive therapeutic target in human cancers (20). Several clinical trials have been developed to test the efficacy of DNA methylation inhibitors for the treatment of different types of malignant tumors (2124). The use of methylation inhibitors could, at least in some cases, stimulate the invasive growth and/or metastasis of pancreatic cancer through reactivation of certain invasion-promoting genes silenced by promoter methylation. This potential adverse effect of 5Aza-dC on tumor progression should be carefully evaluated in clinically relevant in vivo models.
NOTES
Supported by Specialized Projects of Oncology Research Excellence (SPORE) in Gastrointestinal Malignancies grant CA62924 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; by the Lustgarten Foundation for Pancreatic Cancer Research; and by the Michael Rolfe Foundation. N. Sato is a research fellow of the Japan Society for Promotion of Science.
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Manuscript received June 24, 2002; revised December 4, 2002; accepted December 11, 2002.
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