Decitabine Up-regulates S100A2 Expression and Synergizes with IFN-; to Kill Uveal Melanoma Cells

Purpose: Metastatic uveal melanoma is resistant to conventional chemotherapy and immunotherapy. In this study, we investigated the responsiveness of uveal melanoma cell lines to IFNs and the hypomethylating agent decitabine. Experimental Design: The uveal melanoma cell lines 92-1, UW-1, OCM-1, and MKT-BR were exposed to varying concentrations of IFN-a, IFN-g, and decitabine, alone and in combination. The effects of decitabine on gene expression were examined using DNAmicroarray analysis. Results:We found that IFN-g and decitabine induced cell death in uveal melanoma.Whereas a high concentration of IFN-g (1,000 units/mL) was required to induce cell death, we observed a dose-related increase in cell death when decitabine was used at a range of 0.1 to 10 Amol/L. Strikingly,1 Amol/L decitabine synergized with10 to1,000 units/mL IFN-g to inducemassive cell death. In contrast, decitabine hadno effect on three cutaneous melanoma cell lines and exhibited no synergy with either IFN. In uveal melanoma, decitabine up-regulated the expression of genes involved in growth control and apoptosis and down-regulated genes that have been implicated in the malignant phenotype of cutaneous melanoma.The gene up-regulated to the greatest degree by decitabine and whose expression showed a dose-effect across the three concentrations of decitabinewas S100A2, a putative tumor suppressor.The genesmodulatedby decitabine inuveal melanomawere largely unaffected in cutaneous melanoma. Conclusions: These findings form a basis for testing the decitabine/IFN-g combination in metastatic uveal melanoma and for exploring the role of S100A2 in the susceptibility of uveal melanoma to IFN-mediated cell death. Metastatic uveal melanoma is an aggressive malignancy with a median survival of <6 months (1). Patients with locally advanced primary lesions also have a poor prognosis because up to 50% will subsequently develop disseminated metastases within 10 years (2). Although hepatic metastases can respond partially to intra-arterial chemotherapy or chemoembolization, metastatic uveal melanoma is largely resistant to systemic chemotherapy (3). Immunomodulatory drugs used to treat advanced cutaneous melanoma, including interleukin 2 (IL-2) and IFN-a, have also had very limited activity in uveal melanoma (4). The mechanism underlying the antitumor effect of IFN-a or IL-2 in select patients with advanced cutaneous melanoma remains poorly defined. The association of good outcome with the development of autoimmune breakthrough events (5, 6) suggests that overcoming tolerance to self-antigens plays a role in cytokine-mediated antitumor immunity. However, IFN-a and IFN-g can also exert direct antiproliferative and/or proapoptotic effects on cutaneous melanoma cell lines (7, 8), illustrating how certain cytokines can have antitumor effects that are independent of the induction of antigen-specific cellular or humoral immunity. The epigenetic suppression of gene expression through DNA methylation has been linked to the relative resistance of a cutaneous melanoma cell line to the direct proapoptotic effect of type I IFNs (9). In that model, the hypomethylating agent decitabine (5-aza-2¶-deoxycytidine) induced the re-expression of the apoptosis-associated IFN response gene XAF1. Decitabine has also been shown to augment the expression of other proapoptotic genes in cutaneous melanoma, including Apaf-1 (10) and RASSF1A (11). There is very little known about the direct effects of IFNs on uveal melanoma. Several papers have shown that type I and type II IFNs can have variable effects on cell proliferation and integrin/adhesion molecule expression in uveal melanoma (12, 13). Like cutaneous melanoma, the biology of uveal melanoma can also be affected through decitabine-induced gene re-expression. For example, p16 repression through promoter methylation has been associated with worse outcome in uveal melanoma, and re-expression of p16 by decitabine resulted in growth inhibition (14). In this report, we have further evaluated the effect of IFNs and decitabine on uveal melanoma cell lines and show for the Cancer Therapy: Preclinical Authors’Affiliations: Division of Medical Oncology, Department of Medicine, Duke University, and Division of Hematology and Oncology, Geriatrics Research, Education and Clinical Center, Department of Medicine, Durham VA Medical Center, Durham, North Carolina Received 4/9/07; revised 5/31/07; accepted 6/7/07. Grant support: NIH grant K23RR15541 (J.A. Gollob). 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 with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Jared A. Gollob, Duke University Medical Center, DUMC Box 3441, Durham, NC 27710. Phone: 919-668-3979; Fax: 919-668-2163; E-mail: [email protected]. F2007 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-0816 www.aacrjournals.org Clin Cancer Res 2007;13(17) September1, 2007 5219 Research. on April 13, 2017. © 2007 American Association for Cancer clincancerres.aacrjournals.org Downloaded from first time that IFN-g and decitabine synergize to induce cell death in uveal melanoma. Through DNA microarray analysis, we further show that decitabine has a striking effect on the expression of genes implicated in apoptosis and cell growth, including the tumor suppressor gene S100A2, whose upregulation may underlie the synergy between decitabine and IFN-g. Materials andMethods Cell culture. Human uveal melanoma cell lines 92-1, UW-1, OCM-1, and MKT-BR were provided by Dr. Prithvi Mruthyunjaya (Duke University Eye Center, Durham, NC). Human cutaneous melanoma cell lines DM6 and DM93 were provided by Drs. James Grichnik and Hilliard Seigler (Duke University Medical Center, Durham, NC) and 501mel by Dr. David Fisher (Dana-Farber Cancer Institute, Boston, MA). Uveal melanoma cell lines were grown in RPMI supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 Ag/mL streptomycin. 501mel cells were grown in F-10 medium supplemented with 10% heatinactivated FBS and penicillin/streptomycin. The other cell lines were grown in MEM supplemented with 5% FBS and penicillin/streptomycin. Cultures were maintained at 37jC with 5% CO2, and adherent cells were harvested from plastic plates using trypsin/EDTA (Invitrogen). When cells were cultured with decitabine (5-aza-2¶-deoxycytidine; Sigma), fresh decitabine in DMSO (Sigma) was added to cultures everyday, and cells were split every 48 h. When cells were exposed to decitabine and either IFN-g or IFN-a2a (R&D Systems), the IFN was added to the culture on the day the cells were split after a prior 48-h incubation with decitabine. For cells exposed to IFN-g or IFN-a alone, the IFN was added to the culture on the day the cells were split after a prior 48-h incubation with DMSO. Cell cycle analysis. Adherent and suspension cells were harvested and combined, and cells were fixed with 3 mL absolute ethanol for 1 h at 4jC. The cells were then stained with a 50 Ag/mL propidium iodide (BD Biosciences) solution containing 10 Ag/mL RNase A (Roche Applied Science) for 3 h at 4jC. The cells were analyzed by flow cytometry using the CellQuest program. Arrays. Arrays were printed at the Duke Microarray Facility using the Genomics Solutions OmniGrid 100 Arrayer. The arrays contain the Operon Human Genome Oligo Set version 4.0 (Operon) that possess 35,354 optimized 70-mers, representing 35,354 genes. Probe preparation and microarray hybridization. Total RNA (10 Ag) from each cell line and the reference (Universal Human Reference RNA, Stratagene) was hybridized to oligo-dT primers at 65jC and then incubated at 42jC for 2 h in the presence of reverse transcriptase, Cy5 or Cy3-dUTP and Cy5 or Cy3-dCTP, and a deoxynucleotide mix. NaOH was used to destroy residual RNA. Cell line and reference cDNA were pooled and mixed with 1 hybridization buffer (50% formamide, 5 SSC, and 0.1% SDS), COT-1 DNA, and poly-dA to limit nonspecific binding and heated to 95jC for 2 min. This mixture was pipetted onto a microarray slide, coverslipped, and hybridized overnight at 42j. The array was then washed at increasing stringencies and scanned on a GenePix 4000B microarray scanner (Axon Instruments). Data processing and statistical analysis. GeneSpring 6.1 program (Agilent Technologies, Silicon Genetics) was used to perform data analysis. Intensity-dependent (Lowess) normalization was done on the entire data set. Based on triplicates of each condition, a threshold of 2-fold increase or decrease in expression relative to the control and a two-way ANOVA with a P value cutoff of 0.05 were done. The expression of each gene was reported as the ratio of the value obtained for each condition relative to control conditions after normalization of the data. Western blotting. Following indicated treatment, whole cell extracts were prepared in lysis buffer [20 mmol/L Tris-HCl (pH, 8.0), 1% Igepal/NP40, 10 mmol/L EDTA, 137 mmol/L NaCl, 100 mmol/L NaF, complete protease inhibitor mixture (Roche Molecular Biochemicals), Fig. 1. IFN-g induces cell death to variable degrees in uveal melanoma.The 92-1 (A) and UW-1 (B) uveal melanoma cell lines were incubated for 48 h in mediumwith DMSO alone, following which cells were split and incubated for an additional 48 h with either DMSO or the indicated concentrations of IFN-g or IFN-a2a. Adherent and suspension cells were then harvested, stained with propidium iodide, and analyzed by flow cytometry.The number imbedded in each panel above the sub-G1region of the histogram shows the percentage of dead cells. 3 All protocols are available in greater detail on theDukeMicroarray Facility web site, http://mgm.duke.edu/genome/dna___micro/core/spotted.htm. 4 All raw data files and gene lists are found at http://data.cgt.duke.edu/Gollob3. php. Cancer Therapy: Preclinical www.aacrjournals.org Clin Cancer Res 2007;13(17) September1, 2007 5220 Research. on April 13, 2017. © 2007 American Association for Cancer clincancerres.aacrjournals.org Downloaded from and complete phosphatase inhibitor cocktail (Sigma)]. Protein in the lysates was quantitated, and equal amounts were subjected to SDSPAGE and immunoblotting. Blots were incubated with the indicated primary antibody and then incubated with horseradish peroxidase– conjugated goat anti-mouse, goat anti-rabbit, or rabbit anti-goat IgG antibody for 1 h at room temperature. Blots were then developed using the enhanced chemiluminescence system (Pierce). Primary antibodies used included antibodies to S100A2 (the kind gift of Professor C.W. Heizmann, Zurich, Switzerland), Tubulin (NeoMarkers), IL-24/mda-7 (R&D Systems), p15 INK4B, and phosphoinositide-3-kinase (PI3K) p110 g (Cell Signaling, Technology).