Intratumor Heterogeneity of Cancer/Testis Antigens Expression in Human Cutaneous Melanoma Is Methylation-Regulated and Functionally Reverted by 5-Aza-2′-deoxycytidine

Abstract

Cancer/testis antigens (CTA) are suitable targets for immunotherapy of human malignancies, and clinical trials are mainly focusing on MAGE-A3. However, the heterogeneous intratumor expression of CTA may hamper the effectiveness of CTA-directed vaccination through the emergence of CTA-negative neoplastic clones. We investigated the intratumor heterogeneity of CTA in human melanoma and the underlying molecular mechanism(s) at clonal level using 14 single cell clones generated from the melanoma lesion Mel 313. Reverse transcription-PCR revealed a highly heterogeneous expression of MAGE-A1, -A2, -A3, -A4, -A6, GAGE 1-6, SSX 1-5, and PRAME among melanoma clones. Only nine clones expressed MAGE-A3 and competitive reverse transcription-PCR identified relative differences in the number of mRNA molecules of up to 130-fold between clones 5 and 14. This clonal heterogeneity of MAGE-A3 expression correlated with the methylation status of specific CpG dinucleotides in MAGE-A3 promoter: i.e., hypomethylated CpG dinucleotides at positions ؊321, ؊151, ؊19, ؊16, ؊5, ؊2, ؉21, and ؉42 were found in clones expressing high but not low levels of MAGE-A3. Supporting the role of DNA methylation in generating the intratumor heterogeneity of CTA, the DNA hypomethylating agent 5-aza-2-deoxycytidine (5-AZA-dCyd) invariably induced their expression in all CTA-negative clones. Furthermore, 5-AZA-dCyd-treatment reduced to 6 folds the differential expression of MAGE-A3 between clones 5 and 14, which became recognized to a similar extent by T cells specific for a MAGE-A-encoded peptide. These findings identify promoter methylation as directly responsible for the intratumoral heterogeneity of therapeutic CTA in melanoma and foresee the use of 5-AZA-dCyd to overcome the limitations set by their intratumor heterogeneous expression to CTA-based vaccine therapy.

MAGED4 Expression in Glioma and Upregulation in Glioma Cell Lines with 5-Aza-2'-Deoxycytidine Treatment

“…Recent studies (Weber et al, 1994;Sigalotti et al, 2004;Wischnewski et al, 2006) have shown that epigenetic manipulation could enhance TAA expression including MAGE family, which is based on these TAA genes commonly with hypermethylated CpG sites in promoter region. Here, we report at the first time the methylation status of MAGED4.…”

Cancer‐testis antigen expression and its epigenetic modulation in acute myeloid leukemia

“…To assess the influence of treatment with epigenetic agents on CTA expression by AML blasts, cell lines were treated with 1 lM of demethylating agent 5 0 -aza-2 0 -deoxycytidine (Decitabine; Sigma-Aldrich, St. Louis, MO) and/or 5 lM of HDAC inhibitor trichostatin A (Sigma-Aldrich). These are standard concentrations for the in vitro treatment of tumor cell lines [15], and we have previously applied the same concentrations of both reagents to promote CTA expression, that is, in chronic myeloid leukemia (CML) cell lines [16]. Expression of CTA was evaluated in untreated cell lines and following 24-hr stimulation with the respective epigenetic agents.…”

“…Expression of several CTA has been linked to DNA hypomethylation [15,20,21]. We, therefore, investigated whether epigenetic treatment might increase the comparably infrequent CTA expression in AML cells.…”

Epigenetic regulation of X-linked cancer/germline antigen genes by DNMT1 and DNMT3b

“…The medical importance of CG-X antigens is supported by the observation that vaccine-induced CD8 þ T-cell responses to CG-X antigens, but not to melanoma differentiation antigens, correlates with tumor-free and overall survival in melanoma patients (Reynolds et al, 2003). Treatment with agents that increase the expression of CG-X antigens, including DAC (Karpf et al, 2004;Sigalotti et al, 2004) and zebularine (Cheng et al, 2004), constitute an intriguing therapeutic approach by which antitumor immunity to CG-X antigen-directed vaccines could be improved (Karpf and Jones, 2002;Maio et al, 2003;Guo et al, 2006). However, the toxicity and pleiotropic effects associated with nucleoside analog DNMT inhibitors could be a barrier to the successful clinical implementation of this combination strategy (Karpf and Jones, 2002).…”

“…These data prompted us to investigate the epigenetic mechanisms involved in regulating CG-X antigen gene expression. We have focused our investigations on three CG-X antigen genes identified in our microarray screen (MAGE-A1, NY-ESO-1, and XAGE-1); these genes represent three distinct CG-X gene families (Simpson et al, 2005) and MAGE-A1 and NY-ESO-1 encode medically relevant antigens under active investigation as cancer immunotherapy targets (Gillespie and Coleman, 1999;Odunsi et al, 2003;Reynolds et al, 2003;Qian et al, 2004;Sigalotti et al, 2004). These genes each contain 5 0 CpG island promoters that encompass their known transcriptional start sites (Figure 1).…”