Contribution of Epigenetic Silencing of Tumor Necrosis Factor–Related Apoptosis Inducing Ligand Receptor 1 (DR4) to TRAIL Resistance and Ovarian Cancer
Total Page：16
File Type：pdf, Size：1020Kb
Contribution of Epigenetic Silencing of Tumor Necrosis Factor–Related Apoptosis Inducing Ligand Receptor 1 (DR4) to TRAIL Resistance and Ovarian Cancer Peter Horak,1 Dietmar Pils,1 Griet Haller,1 Ingrid Pribill,1 Max Roessler,1 Sandra Tomek,1 Reinhard Horvat,3 Robert Zeillinger,2 Christoph Zielinski,1 and Michael Krainer1 1Clinical Division of Oncology, Department of Medicine I; 2Department of Gynecology and Obstetrics; and 3Department of Clinical Pathology, University Hospital Vienna, Vienna, Austria Abstract Introduction Dysregulation of apoptosis may support tumorigenesis Defects in programmed cell death (apoptosis) mechanisms by allowing cells to live beyond their normally intended play an important role in tumor pathogenesis by enabling life span. The various receptors for tumor necrosis neoplastic cells to escape from cell autonomous or immuno- factor–related apoptosis-inducing ligand (TRAIL) are logic growth control mechanisms (1). Players in the various located on chromosome 8p21.2, a region frequently apoptotic pathways are well-known oncogenes but also deleted in ovarian cancer. Lack of expression of TRAIL intriguing candidates for tumor suppressor genes. A consider- receptor 1 (death receptor 4, DR4) correlates with able amount of effort has been put into the mutational analysis resistance to TRAIL-induced apoptosis in ovarian of receptors for apoptosis-inducing proteins, including those for cancer cells. Reconstitution of DR4 in the tumor necrosis factor (TNF)–related apotosis inducing ligand TRAIL-resistant A2780 ovarian cancer cell line was (TRAIL), but definite mutations are rare. The functional investigated with the demethylating agent TRAIL receptors death receptors 4 (DR4, TRAIL-R1) and 5 5-aza-2V-deoxycytidine and transient gene transfer. (DR5, TRAIL-R2/TRICK2/KILLER), and decoy receptors 1 Regulation of other genes in the TRAIL pathway by (DcR1, TRAIL-R3/TRID)and2(DcR2, TRAIL-R4/ 5-aza-2V-deoxycytidine was assessed in DNA GeneChip TRUNDD) with thus far little known physiologic function experiments. Primary ovarian cancers were analyzed by are all located on chromosome 8p21.2 according to the methylation-specific PCR and immunohistochemical University of California Santa Cruz Human Genome Project analysis of a tissue microarray. Regulation of DR4 Working Draft June 2002 (http://genome.cse.ucsc.edu). This expression by demethylation or transient transfection is finding suggests evolutionary multiplication of one gene in this of functional relevance for TRAIL resistance in an region and subsequent gain or loss of function as mediator or ovarian cancer cell line. Hypermethylation of the DR4 inhibitor of apoptosis, respectively. Besides other common promoter could be found in 10 of 36 (27.7%) DNAs areas of genetic variation in ovarian cancer (reviewed in ref. 2), isolated from ovarian cancer tissue. In an independent 8p21.2 is a region frequently deleted in this malignancy (3, 4). set of 68 ovarian cancer cases, a complete loss or More recently, it was observed that ovarian cancers have a down-regulation of DR4 protein expression was higher TRAIL expression than normal ovarian epithelial observed 10.3% and 8.8% patients, respectively. samples and that high expression of TRAIL is associated with A significant (P = 0.019) majority of these patients was prolonged survival (5), additionally implicating a potential role below 50 years of age. Our findings show a functional of the TRAIL system in the pathogenesis of ovarian cancer. relevance of the level of DR4 expression in ovarian However, definite mutations in TRAIL receptors in ovarian cancer and suggest a substantial contribution of DR4 cancer could not be detected (6). Therefore, we investigated a hypermethylation and consequent loss of DR4 number of ovarian cancer cell lines for expression of players in expression to ovarian cancer pathogenesis, particularly the TRAIL pathway and could show that the lack of expression inpremenopausalpatients. (MolCancerRes2005; of DR4 correlated with resistance to TRAIL, whereas the 3(6):335–43) expression of other receptors did not (7). This phenomenon has also been described by other groups in different tumor entities as part of their efforts to characterize TRAIL as a potential Received 8/1/04; revised 2/28/05; accepted 4/1/05. therapeutic agent (8, 9). We wanted to further explore our Grant support: Fonds zur Fo¨rderung der wissenschaftlichen Forschung grant 13473. observation for its true physiologic relevance on a more The costs of publication of this article were defrayed in part by the payment of mechanistic level, and test whether the lack of expression of page charges. This article must therefore be hereby marked advertisement in DR4 in ovarian cancer cell lines is a genetic or epigenetic accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Michael Krainer, Clinical Division of Oncology, phenomenon, because a variety of tumor suppressor genes have Department of Medicine I, University Hospital, Wa¨hringer Gu¨rtel 18-20, been shown to be silenced in human cancers (10). Epigenetic A-1090 Vienna, Austria. Phone: 43-1-40400-4449; Fax: 43-1-40400-4451. E-mail: [email protected] silencing of TRAIL receptors is common in some cancer Copyright D 2005 American Association for Cancer Research. entities (11, 12), although hypermethylation of DR4 and DR5 Mol Cancer Res 2005;3(6). June 2005 335 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. 336 Horak et al. in ovarian cancer has not been studied to date. Hyper- expression (Fig. 1B). In contrast, DR4 hypermethylation could methylation of the promoters of these genes could be causally not be found in any of the remaining DR4-expressing cell lines. involved in tumorigenesis of ovarian cancer. A reversion of DR5 expression was rather uniformly present throughout the DR4 loss in ovarian cancer by demethylation, together with the cell lines and hypermethylation of the DR5 promoter was not observation that methylation of DR4 can be found in a observed (data not shown). significant number of clinical cases, would provide an important piece of the puzzle in the poorly understood field Ovarian Cancer Cells Are Resistant to TRAIL of ovarian cancer biology. This observations would also guide We tested the nine available cell lines for TRAIL sensitivity. the use of TRAIL as one of the emerging tools for future The cell lines with low DR4 expression levels showed to be apoptosis-targeted cancer therapies. more resistant to the apoptotic effects of TRAIL at concen- trations of 1,000 ng/mL (Fig. 1C). Ovarian cancer cell line Results A2780 was completely resistant to TRAIL-induced apoptosis DR4 and DR5 Expression in Ovarian Cancer Cell Lines and previous experiments prompted us to assume that TRAIL We examined a panel of ovarian cancer cell lines and resistance is caused by lack of DR4 expression (6). Incubation determined the DR4 and DR5 mRNA expression using real- of A2780 cells with 5-aza-2V-deoxycytidine (AzadC) restored time PCR. We found that two cell lines (A2780 and MZ-37) DR4 expression and sensitized A2780 cells to subsequent had no detectable DR4 expression and two other cell lines had treatment with TRAIL (Fig. 2A). In accordance with these two a very low DR4 expression (MZ-4 and MZ-15; Fig. 1A). Then, facts, AzadC induces time-dependent DR4 mRNA reexpression the methylation status was determined and correlated with the in the A2780 cell line, reaching a peak of expression expression pattern. We observed DR4 promoter hypermethy- comparable with the constitutively DR4-expressing, TRAIL- lation in 3 of 4 (75%) cell lines displaying low or missing DR4 sensitive cell lines after 96 hours (Fig. 2B). FIGURE 1. AzadC treatment demethylates hypermethylated CpG islands of the DR4 promoter and induces DR4 reexpression, thus sensitizing A2780 cells to TRAIL-induced apoptosis. A. Apoptosis assay. Combined treatment with AzadC and TRAIL leads to an increase in apoptosis as measured with APO- Direct kit. B. Time-dependent up-regulation of DR4 mRNA by AzadC (relative quantitative reverse transcription-PCR) in the ovarian cancer cell line A2780. C. Apoptosis after TRAIL treatment (100 ng/mL) in A2780 ovarian cancer cells transfected with either truncated or full-length DR4 compared with mock-treated cells. Mol Cancer Res 2005;3(6). June 2005 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Hypermethylation of DR4 in Ovarian Cancer 337 FIGURE 2. A. Real-time DR4 and DR5 expression related to an internal calibrator (set to 1). PCR reactions of the A2780 and MZ-37 cell lines yielded no detectable product after 40 PCR cycles. B. Methylation status of the DR4 promoter in ovarian cancer cell lines. C. TRAIL sensitivity of ovarian cancer cell lines. TRAIL (1,000 ng/mL) – induced apoptosis was assessed as follows: negative (À) for <10% positively stained cells; positive (+) for 10% to 50% apoptosis; and strongly positive (++) for >50% apoptotic cells after 24-hour incubation. Microarray Analysis of AzadC-Treated Cells demethylation by AzaD (Fig. 2C). Interestingly, also, the Regulation of other players in the TRAIL pathway besides overexpression of the truncated variant of the DR4 protein— DR4 can also be observed and may, in addition, be responsible missing the highly conserved death domain but retaining the for the reconstitution of TRAIL sensitivity in the A2780 cell possibility to form heterodimers with other receptors—exhibited line. To investigate these genes, we did a global gene expression some residual proapoptotic effects. analysis comparing mock- and AzadC-treated A2780 cells by standard microarray technology. We focused on the expression DR4 Methylation and Expression in Primary Cancer of genes involved in the known TRAIL pathways and checked Samples all players for their representation on the Affymetrix HG-U133A Tumor DNA of 36 ovarian cancer cases were investigated and HG-U133B GeneChip (Table 1). All genes of interest were and 10 of 36 (27.8%) patients showed methylation of DR4 in well represented except, interestingly, DR4, where only 2 of 11 their tumor (Fig.