Intratumor heterogeneity of cancer/testis antigens expression in human cutaneous melanoma is methylation-regulated and functionally reverted by 5-aza-2′-deoxycytidine
Intratumor heterogeneity of cancer/testis antigens expression in human cutaneous melanoma is methylation-regulated and functionally reverted by 5-aza-2′-deoxycytidine
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DOI：
10.1158/0008-5472.can-04-1442
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发表时间：
2004-12-15
期刊：
影响因子：
11.2
通讯作者：
Maio, M
中科院分区：
文献类型：
--
作者：
Sigalotti, L;Fratta, E;Maio, M
肿瘤/睾丸抗原（CTA）是人类恶性肿瘤免疫治疗的合适靶点，临床试验主要集中在MAGE-A3上。然而，CTA的异质性肿瘤内表达可能会阻碍CTA-导向疫苗的有效性，通过CTA阴性肿瘤克隆的出现。我们研究了CTA在人黑色素瘤中的瘤内异质性和克隆水平上的潜在分子机制，使用从黑色素瘤病变Mel 313产生的14个单细胞克隆。逆转录-PCR显示MADE-A1、-A2、-A3、-A4、-A6、GAGE 1-6、SSX 1-5和PRAME在黑色素瘤克隆中的高度异质性表达。只有9个克隆表达MAGE-A3和竞争性逆转录-PCR鉴定的mRNA分子的数量的相对差异高达130倍克隆5和14之间。MAGE-A3表达的这种克隆异质性与MAGE-A3启动子中特定CpG二核苷酸的甲基化状态相关：即，在表达高水平而非低水平MAGE-A3克隆中，位置-321、-151、-19、-16、-5、-2、+21和+42的低甲基化CpG二核苷酸是圆形的。DNA低甲基化剂5-氮杂-2 '-脱氧胞苷（5-AZA-dCyd）在所有CTA阴性克隆中始终诱导其表达，这支持了DNA甲基化在产生CTA肿瘤内异质性中的作用。此外，5-AZA-dCyd-处理将克隆5和14之间MAGE-A3的差异表达降低至6倍，其被对MAGE-A编码肽特异性的T细胞以相似的程度识别。这些发现将启动子甲基化鉴定为直接导致黑色素瘤中治疗性CTA的瘤内异质性的原因，并预见使用5-AZA-dCyd来克服其瘤内异质性表达对基于CTA的疫苗治疗的限制。
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 MADE-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 round 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.