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Originally published as JCO Early Release 10.1200/JCO.2005.03.4074 on June 26 2006 © 2006 American Society of Clinical Oncology. Overcoming Resistance to Interferon-Induced Apoptosis of Renal Carcinoma and Melanoma Cells by DNA Demethylation
From the Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland; University of Toledo, Toledo, OH; MethylGene Inc, Québec, Canada. Address reprint requests to Ernest C. Borden, MD, The Cleveland Clinic Foundation Taussig Cancer Center, R40, 9500 Euclid Ave, Cleveland, OH 44195; e-mail: bordene{at}ccf.org
Epigenetic editing of gene expression by aberrant methylation of DNA may help tumor cells escape attack from the innate and acquired immune systems. Resistance to antiproliferative effects and apoptosis induction by interferons (IFNs) was postulated to result from silencing of IFN response genes by promoter hypermethylation. Treatment of human ACHN renal cell carcinoma (RCC) and A375 melanoma cells with the DNA demethylating nucleoside analog 5-AZA-2'-deoxycytidine (5-AZA-dC) synergistically augmented antiproliferative effects of IFN- alpha (  ) 2 and IFN-beta (ß). Either 5-AZA-dC or an antisense to DNA methyltransferase 1 (DNMT1) overcame resistance to apoptosis induction by IFNs with up to 85% apoptotic cells resulting from the combinations. No similar potentiation occurred in normal kidney epithelial cells. IFN response genes were augmented more than 10 times in expression by 5-AZA-dC. Demethylation by 5-AZA-dC of the promoter of the prototypic, apoptosis-associated IFN response gene XAF1 was confirmed by methylation-specific polymerase chain reaction. siRNA to XAF1 inhibited IFN-induced apoptosis; conversely, overexpression of XAF1 overcame resistance to apoptosis induction by IFN-ß. As occurred with apoptosis-resistant melanoma cells in vitro, tumor growth inhibition in the nude mouse of human A375 melanoma xenografts resulted from treatment with 5-AZA-dC in combination with IFN-ß, an effect not resulting from either single agent. The importance of epigenetic remodeling of expression of immune-modifying genes in tumor cells was further suggested by identifying reactivation of the cancer-testis antigens MAGE and RAGE in ACHN cells after DNMT1 depletion. Thus, inhibitors of DNMT1 may have clinical relevance for immune modulation by augmentation of cytokine effects and/or expression of tumor-associated antigens.
Like mutational deletions, epigenetic regulation of gene expression through methylation and acetylation are integral and common parts of the neoplastic process.1-4 Hypermethylation of DNA in promoter regions can result in heritable silencing of genes that control cell differentiation, proliferation, apoptosis, and function. Silencing of genes is maintained by DNA methyltransferases (DNMTs), which, during DNA replication copy methylation patterns to the daughter strand.1-4 Epigenetic editing of gene expression by aberrant methylation of DNA in both tumor cells and lymphocytes may influence signaling and expression of proteins important for functioning of the innate and acquired immune systems.1-6 As products of the activated immune system, interferons (IFNs) influence differentiation, function, and survival of not only immune effector cells but also cells of stroma, endothelium, and organ parenchyma.7-9 Suppression of endogenous IFNs in mice enhances tumor development.10-11 Gene expression profiling, protein expression, and cytogenetic analyses have identified decreases in the transcriptionally regulated, IFN-stimulated genes (ISGs) in immune effector and in melanoma and colon, breast, and hematologic malignancies.12-22 Epigenetic silencing by DNA methylation of genes such as STAT1, TRAIL R1, IRF-7, and DAPK that are essential for various actions of IFNs, has been identified, suggesting that hypermethylation may confer growth advantage through resistance to endogenous IFNs.23-26 Thus, silencing of ISG expression may influence tumor development or progression. Functional consequences of epigenetic silencing by hypermethylation of cytokine and IFN signaling pathways have been evaluated little. To confirm our hypothesis that inhibition of actions of IFNs could result from epigenetic silencing of ISGs, resistance to gene expression and apoptosis were assessed in melanoma and renal cell carcinoma (RCC) after an inhibitor of DNMTs, 5-AZA-2'-deoxycytidine (5-AZA-dC), and a selective antisense oligonucleotide for DNMT1. To further suggest clinical relevance, expression of tumor-associated antigens in vitro and murine tumor growth in vivo was assessed after the methylation inhibitors.
Cell Lines and IFNs ACHN, A375, A375.S2, SK-Mel-1, SK-Mel-3, SK-Mel-28, MeWo, HeLa (American Type Culture Collection, Manassas, VA), SK-RC-45, SK-RC-29 (Memorial Sloan-Kettering Cancer Center, New York, NY), MUM-2B, MUM-2C, OCM-1, C-918 (ocular melanomas provided by Mary J. Hendrix, Northwestern University, Evanston, IL), and normal kidney epithelial cells NKE39 and NKE58 (from nephrectomy specimens, Cleveland Clinic Foundation, Cleveland, OH) were cultured in 5% CO2 using Minimum Essential Medium (MEM; GIBCO, Invitrogen, Carlsbad, CA) or Dulbecco's MEM with 0.1 mmol/L nonessential amino acids (GIBCO), 1.0 mmol/L pyruvate (GIBCO), 10% fetal bovine serum, penicillin G (50 U/mL), and streptomycin (50 µg/mL). Recombinant IFN-alpha ( ) 2b (Schering-Plough, Kenilworth, NJ) and IFN-beta (ß) 1a (Serono, Rockland, MA) had specific activities of 2 x 108 U/mg protein.
5-AZA-dC, DNMT1 Antisense, and XAF-1siRNA Transfections
Western Blotting
Growth Inhibition (sulforhodamine B) and Apoptosis Assays
RNA Isolation and cDNA Synthesis
Bisulfite Modification and Methylation-Specific PCR
Tumor Xenografts Ncr nude mice (Taconic Farms, Hudson, NY) of approximately 6 weeks of age were inoculated subcutaneously in the flank with 106 A375 melanoma cells. Mice were killed humanely after 26 days of tumor growth.
DNMT1 Depletion by Antisense or 5-AZA-dC Reverses Resistance to IFN-Induced Apoptosis in RCC and Melanoma Cells RCC and melanoma cell lines were treated with 5-AZA-dC followed 1 day later by IFN- 2 or IFN-ß. Synergistic growth inhibition resulted (Fig 2). Microscopic observation identified condensed nuclei and increase in floating cells suggestive of apoptosis with combined treatment (data not shown). Therefore, frequency of cells containing apoptotic fragmented DNA was quantified by TUNEL assay in RCC and melanoma cell lines after sequential 5-AZA-dC and IFN-2 or IFN-ß.
Although all cell lines in Figures 2 and 3 were completely resistant to apoptosis induction by IFNs alone (< 10% TUNEL+ cells after 4 to 5 days of IFN- 2 or IFN-ß at 50 to 500 U/mL), pretreatment with 5-AZA-dC (100 to 200 nmol/L over 4 days) resulted in at least two-fold increase of apoptotic cells by IFN-2 (Fig 3A). IFN-ß at equal dose (50 to 500 U/mL over 4 to 5 days) was more potent, resulting in 25% to 83% apoptotic cells after pretreatment with 5-AZA-dC (100 to 200 nmol/L over 4 days) (Fig 3A). Normal kidney epithelial (NKE) cells treated with 100 nmol/L 5-AZA-dC over 4 days before 100 U/mL IFN-2 or IFN-ß over 5 days responded differently, whereas 5-AZA-dC caused moderate apoptosis (13%) addition of IFN-2 or IFN-ß minimally altered frequency of apoptotic cells (6.5% to 20.5% apoptotic cells) and caused no apoptosis (< 10% TUNEL+) when administered alone (Fig 3A).
Treatment ACHN and SK-RC-45 cells with the selective DNMT1 antisense (AS) inhibitor MG98 similarly overcame resistance to IFN-induced apoptosis (Fig 3A). Western blotting for cleaved (activated) caspase 3 and PARP cleavage (Fig 3B) and caspase 3 activity assays (data not shown) confirmed apoptosis induction.
Cell-Specific Reactivation by DNMT1 Inhibitors of Genes Participating in IFN-Induced Apoptosis XAF1 was increased more than 10-fold by methylation inhibition in six of 12 cancer cell lines (between 27- and 150-fold), including one of three RCC (ACHN), two of five melanoma (A375, MeWo), and three of four ocular melanoma cell lines (MUM-2B, MUM-2C, OCM-1). RASSF1A was increased more than 100-fold in eight of 12 tumor cell lines, including all RCC cells, two of five melanoma (A375, SK-Mel-28), and three of five ocular melanoma cells (MUM-2C, OCM-1, C-918), TRAIL R1 was increased 30- to 70-fold in two melanoma cell lines (SK-Mel-1, SK-Mel-3), TRAIL R2 90- to 130-fold in two ocular melanoma cells (MUM-2C, OCM-1), TRAIL 20- to 80-fold in two ocular melanoma and one melanoma cell line (MUM-2C, OCM-1, MeWo), and STAT2 13-fold in MeWo melanoma cells. In NKE cells, no increase of 10-fold occurred after 5- AZA-dC (100 nmol/L over 4 days; Table 1). In summary, reactivation of genes was cell specific, suggesting that individual cell clones derived survival benefit from silencing specific genes while tolerating expression of other genes that may be more critical to the survival of different cell clones.
Immunoblots had confirmed similar DNMT1 depletion by AS and 5-AZA-dC in ACHN (Fig 1A) and SK-RC-45 cells (data not shown). In A375 cells, both 5-AZA-dC and the DNMT1 antisense also markedly reduced DNMT1 protein (Fig 1A). MSP determined that reactivation of XAF1 in ACHN and A375 cells by DNMT1 inhibitors was associated with demethylation of hypermethylated 5' regulatory region (Fig 1B). Cells not markedly altered by 5-AZA-dC had higher baseline XAF1 mRNA expression (data not shown); these had an unmethylated XAF1 5' regulatory region (SK-Mel-3 and SK-RC-45; Table 1; Fig 1B). To determine whether 5-AZA-dC increased XAF protein induction by IFN and whether silencing of XAF1 conferred resistance to IFN-induced apoptosis, ACHN cells were treated concurrently with 5-AZA-dC and XAF1 siRNA before IFN. 5-AZA-dC increased XAF1 protein induction by IFN (Fig 4A) and siRNA against XAF1 decreased apoptosis induction by IFN-ß (50 U/mL over 4 days) from 59% to 18% in cells pretreated with 2 days of 5-AZA-dC (200 nmol/L; Fig 4B). In A375 cells, 5-AZA-dC alone resulted in XAF1 protein expression, but induction of XAF1 by IFNs appeared to be intact despite DNA methylation, suggesting a more complex mechanism of transcriptional regulation in these cells. Nevertheless, XAF siRNA reduced IFN-induced apoptosis of 5-AZA-dC treated cells from 13.2% to 6.8%. To further confirm the role of XAF1 in IFN-induced apoptosis, A375.S2 cells, known to undergo apoptosis after IFNs, were transfected with siRNA against XAF1. This reduced apoptosis after 100 U/mL IFN-ß from 52% to 14% with associated decrease in XAF1 protein (Fig 4C). Thus, decreases in XAF1 resulted in diminished apoptosis.
XAF1 was overexpressed in ACHN cells to determine whether increased XAF1 protein expression alone, independent of other genes reactivated by 5-AZA-dC, could overcome resistance to apoptosis induction by IFNs. IFN-ß (50 U/mL over 24 hours) weakly induced XAF1 protein in empty vector controls, whereas stable transfection of XAF1 resulted in XAF1 protein expression that was detectable without addition of IFN. Treatment with IFN augmented expression to levels comparable to those achieved by treatment of cells with 5-AZA-dC and IFN (Figs 4 and 5). ACHN cells overexpressing XAF1 treated with IFN-ß at 50 U/mL over 5 days underwent little apoptosis (6.5% TUNEL+) suggesting that reactivation of additional genes may be necessary for apoptosis induction by low doses of IFN in DNMT1-depleted cells. However, when treated with 500 U/mL IFN-ß over 5 days, more than 80% of XAF1 overexpressing cells were apoptotic, whereas empty vector-carrying cells were resistant to even high doses of IFN-ß (<3.2% TUNEL+ after 5 days at 500 U/mL; Fig 5B). This further confirmed a role for XAF1 in apoptosis induction by IFNs.
To determine whether other genes that might enhance anticancer effect of IFNs were reactivated through DNA demethylation, cRNA array (Affymetrix [Santa Clara, CA] U133A microarray) analysis with RNA harvested from ACHN cells 24 hours after the eighth DNMT1 AS transfection was undertaken. In this assay, compared with mismatch control oligonucleotide (MM), DNMT1 AS resulted in a 94% reduction in DNMT1 expression, while not affecting expression of other DNMTs (data not shown). Among the 43 genes increased at least four-fold (P < .045) by DNMT1 AS, seven were cancer-testis antigens (CTAs), and 12 had functions related to cytokine signaling (Table 2). DNA sequences were assessed for CpG islands within 200 base pairs (bp) 5' of the transcription start using a CpG island searcher (http://www.usccancer.com/cpgislands/cpg.aspx) and a definition of length of at least 200 bp, GC% at least 50, and observed/expected CpGs at least 0.6. Most tumor antigens had CpG islands close to the transcription start, suggesting direct regulation of transcription by DNA methylation, whereas those genes involved in cytokine signaling, lacking CpG islands in close proximity to the transcription start, may have been increased through indirect mechanisms. Thus, whether through DNA demethylation or indirect mechanisms, DNMT1 depletion led to changes in gene expression that might enhance antitumor immune responses by effects on cytokine signaling and cell-surface antigen expression.
To define further the potential clinical relevance of DNMT1 suppression by 5-AZA-dC, A375 melanoma cells were inoculated into nude mice and treated with either a single dose of 5-AZA-dC, daily doses of IFN-ß, or the combination (Fig 6). Significant suppression of tumor growth resulted only from the combination and was evident throughout the growth curve. As control tumors grew larger, the combination treatment resulted in significant suppression (P < .05) in comparison to control and also in comparison to either single agent.
In normal cells, the dinucleotide CpG, particularly in promoter regions of genes, is only sparsely methylated. In contrast, in tumor cells, heavy methylation of CpG islands in promoters occurs, resulting in inhibition of binding of many critical transcription factors.1-4 This increased methylation probably results from increased activity of DNMTs, which can be many-fold higher in activity in neoplastic cells when compared with nontumor cells.1-4 Suppression of DNMT activity can be achieved by the nucleoside analogs of cytidine, which after incorporation into DNA, covalently bind DNMTs and result in re-expression of methylation silenced transcription.3,4,27,29,30 Although azacytidine analogs have broad specificity for DNMTs, removing them from their sites of enzymatic action, incorporation into DNA could also activate DNA damage pathways. Thus, although potentially with some loss of potency because of the various isoforms of DNMT, greater specificity could result from antisense oligonucleotides targeted at specific DNMTs.
At doses that alone caused minimal apoptosis, 5-AZA-dC overcame complete resistance to apoptosis induction by IFNs (Fig 3). Inhibition of the DNMT1 isoform alone by a targeted oligonucleotide antisense or siRNA has been sufficient for re-expression of silenced genes.27,30 Consistent with this, suppression of DNMT1 by an antisense oligonucleotide had similar effects on overcoming resistance to programmed cell death induction by IFNs as did 5-AZA-dC (Fig 1 To further examine the clinical potential of the combination treatment, the human A375 melanoma cell line was grown in the nude mouse. A375 cells were resistant to apoptosis induction in vitro and treatment with IFN-ß in vivo (Figs 3 and 6). A single dose of 5-AZA-dC administered 4 days before beginning treatment of the established tumors with IFN-ß resulted in significant (P < .05) suppression of growth of the melanoma otherwise resistant to IFN-ß, whereas neither treatment alone result in significant decreases in tumor size from the control. Since this dose and schedule of 5-AZA-dC was chosen semi-empirically, greater suppression may result from higher or more frequent doses or by use of the antisense to DNMT1. Counteracting cellular apoptotic processes are proteins involved in preventing unregulated cell suicide. Among these are the inhibitors of apoptosis (IAPs) such as XIAP.33 XIAP binds to caspases, thus functioning as competitive inhibitors of the autocatalytic function of caspases.33-36 Yeast two-hybrid studies have identified an XIAP-interacting protein designated XAF1.37,38 Incubation of recombinant XIAP in the presence of XAF1 blocked apoptosis inhibition by XIAP.39 Because expression was lower in tumor cell lines as compared with normal tissues including melanoma, XAF1 has been implicated as a tumor suppressor.37,39,40
Hypermethylation of a CpG island in the 5' regulatory region of XAF1 was found in ACHN and A375 cells, and DNMT1 depletion led to demethylation (Fig 1). This was associated with reactivation of mRNA expression and additional augmentation of XAF1 protein induction by IFNs (Table 1; Fig 4A). Inhibition of XAF1 by siRNA in DNMT1 depleted ACHN cells markedly reduced apoptosis to low doses of IFN-ß (Fig 4B). Conversely, on overexpression of XAF1, 10-fold higher doses of IFN-ß were required to elicit marked apoptosis (Fig 5B). IFNs induced high levels of XAF1 protein predominantly in cell lines sensitive to the proapoptotic effects of IFN- Suppression of ISGs in humans is emerging as an important contributor to development of clinical neoplasia. Expression profiling of melanoma cell and other cell lines, when compared with melanocytes, has identified a large number of IFN-stimulated genes (ISGs) that are suppressed in expression.20 Mutation of a gene in the IFN response pathway, RNase L, increases prostate cancer risk.43,44 Critical protein components of immune effector cells are regulated by IFNs.7,9,45,46 Reaching the full potential of exogenous IFNs as cancer therapeutics in humans will result from additional understanding of the mechanisms of antitumor action and more potent induction of the transcriptionally regulated ISGs such as XAF1. Hypermethylation of CpG islands in gene promoters can influence expression of a diversity of proteins that influence interaction of tumor cells with their microenvironment and the host immune response.5,6 DNMT1 depletion increased expression (10 to 100x) of cytokine signaling molecules and of CTAs (Table 2). 5-AZA-dC has increased expression of CTAs and enabled cell lysis by CTA-specific cytotoxic lymphocytes.47 Clones from a melanoma with variable CTA expression treated with 5-AZA-dC became relatively homogenous in CTA expression.48 A 7-day continuous infusion schedule of 5-AZA-dC reactivated MAGE 1 with acceptable toxicity.49 Thus, inhibitors of DNA methylation may result in "immunologic synergism" in augmenting function of the signaling pathways of not only the innate immune system, as illustrated by effects on IFN-induced apoptosis, but also acquired immunity through effects on augmentation of tumor-associated antigens.
Thus, inhibitors of DNMT1 may have clinical relevance for immune modulation by augmentation of cytokine effects and/or expression of tumor-associated antigens. We have identified a proapoptotic ISG, XAF1, that was silenced by methylation in RCC and melanoma and whose re-expression augmented apoptosis induced by IFNs. RCC and melanoma have been treated with IFN-
Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Dollar Amount Codes (A) < $10,000 (B) $10,000-$99,900 (C)
published online ahead of print at www.jco.org on June 26, 2006. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article. Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.
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Samlowski WE, Leachman SA, Wade M, et al: Evaluation of a 7-day continuous intravenous infusion of decitabine: Inhibition of promoter-specific and global genomic DNA methylation. J Clin Oncol 23:3897-3905, 2005 Submitted July 11, 2005; accepted April 5, 2006. This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
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