Originally published as JCO Early Release 10.1200/JCO.2005.03.4074 on June 26 2006

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Overcoming Resistance to Interferon-Induced Apoptosis of Renal Carcinoma and Melanoma Cells by DNA Demethylation

Frederic J. Reu, Soo In Bae, Leonid Cherkassky, Douglas W. Leaman, Daniel Lindner, Normand Beaulieu, A. Robert MacLeod, Ernest C. Borden

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.

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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