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- Supporting Information
Cancer/testis antigens (CTAs) are considered to be suitable targets for the immunotherapy of human malignancies. It has been demonstrated that in a variety of tumors, the expression of certain CTAs is activated via the demethylation of their promoter CpG islands. In our study, we have shown that while the composite expression of 13 CTAs in 30 human glioma specimens and newly established cell lines from the Japanese population was nearly imperceptible, the DNA-demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR) markedly reactivated CTA expression in glioma cells but not in normal human cells. We quantified the diminished methylation status of NY-ESO-1-one of the most immunogenic CTAs-following 5-aza-CdR treatment by using a novel Pyrosequencing™ technology and methylation-specific PCR. Microarray analysis revealed that 5-aza-CdR is capable of signaling the immune system, particularly, human leukocyte antigen (HLA) class I upregulation. 51Cr-release cytotoxicity assays and cold target inhibition assays using NY-ESO-1-specific cytotoxic T lymphocyte (CTL) lines demonstrated the presentation of de novo NY-ESO-1 antigenic peptides on the cell surfaces. In an orthotopic xenograft model, the systemic administration of 5-aza-CdR resulted in a significant volume reduction of the transplanted tumors and prolonged the survival of the animals after the adoptive transfer of NY-ESO-1-specific CTLs. These results suggested that 5-aza-CdR induces the expression of epigenetically silenced CTAs in poorly immunogenic gliomas and thereby presents a new strategy for tumor immunotherapy targeting 5-aza-CdR-induced CTAs. © 2008 Wiley-Liss, Inc.
Over the last decade, there has been major progress in the identification and characterization of human tumor antigens recognized by the host immune system. A subgroup of tumor antigens, commonly referred to as cancer/testis antigens (CTAs), are expressed only in the tissues of the testis, ovary and placenta under normal conditions, but are also expressed in various types of human tumors.1, 2 Since normal CTA-expressing tissues do not express major histocompatibility complex (MHC) class I molecules, CD8 T cells cannot recognize CTAs expressed on these tissues, suggesting that CTAs are the ideal targets for tumor immunotherapy. CTAs and genes were originally identified through a variety of methods. These include T-cell epitope labeling,3 serological analysis of cDNA expression libraries (SEREX),4, 5 differential gene expression analysis6 and bioinformatics methods.7–9 In particular, NY-ESO-1 is the most immunogenic CTA discovered thus far, and it is considered to be a highly promising therapeutic target for immunotherapy.10 To date, very little is known regarding the physiological function(s) of these antigens or the mode in which the expression of their gene families is regulated.
Epigenetic alterations, including hypermethylation of promoter CpG islands, histone deacetylation of tumor suppressor and tumor-related genes,11–13 and global DNA hypomethylation,14, 15 have been recognized as important contributors to carcinogenesis in humans. Global DNA hypomethylation has been observed in various neoplasms and is considered to occur at the early stages of tumor development.16–18 However, it has been shown that the expression of certain CTA genes is reactivated in cancerous cells; this could be due to a loss of epigenetic regulation as observed when methylated chromatin regions are demethylated or when deacetylated histones are acetylated.19 Therefore, recent evidence shows that the deregulation of the DNA methylation apparatus that occurs during cancer development could provide new therapeutic targets for cancer treatment.
The DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR, decitabine) is a cytosine analogue that is incorporated into DNA during replication. It covalently binds DNA methyltransferase and inhibits its activity, leading to genome-wide demethylation.20–24 There have been several studies demonstrating the ability of 5-aza-CdR to activate the gene expression of CTAs in vitro and in vivo, which may be silenced by the hypermethylation of their promoters. This drug has been used in clinical studies for the treatment of chronic myelogenous leukaemia (CML), sickle cell anaemia and myelodysplastic syndrome (MDS).20, 25–27 Previous evidence has clearly defined the epigenetic regulatory role of DNA methylation in the constitutive expression of CTAs by cutaneous melanoma cells and renal cancer cells and has demonstrated that in vitro treatment with 5-aza-CdR upregulated their expression in neoplastic cells.28, 29
Gliomas are the most common primary tumors of the central nervous system; they account for 30% of adult primary brain tumors. Brain tumors remain difficult to cure despite recent advances in surgical, radiotherapeutic and chemotherapeutic approaches. In particular, there is currently no optimal treatment for glioblastoma multiforme, the most common malignant brain tumor in adults, and patients typically survive for a period less than a year. The poor outcome partly relates to the inability in delivering chemotherapeutic agents through the blood–brain barrier (BBB) and the low effect of radiation on the tumor. Therefore, new and more effective strategies are urgently required. Of these, the establishment of immunotherapy specifically targeting malignant cells is expected to improve tumor prognosis. It has recently been demonstrated that malignant glioma cells express certain known tumor-associated antigens such as HER-2, gp100, MAGE-1 and IL-13 receptor α2.30–37 However, CTA profiling of glioma cells, in particular, in the Asian population remains unknown.
In our study, we analyzed the expression of 13 CTA genes (MAGE-1, MAGE-3, MAGE-4, MAGE-6, MAGE-10, MAGE-3/6, LAGE-1, CT7, SCP-1, SSX-1, SSX-2, SSX-4 and NY-ESO-1) in 30 glioma tissues, 5 human glioma cell lines and 3 newly established cell lines from the Japanese population. Subsequently, the role of 5-aza-CdR in the regulation of the expression of various CTAs in glioma cells was analyzed. Finally, we demonstrated that NY-ESO-1, one of the most antigenic CTAs, is effectively induced in human glioma cells by 5-aza-CdR, and that these glioma cells forcibly expressing NY-ESO-1 show in vitro and in vivo sensitivity to NY-ESO-1-specific cytotoxic T lymphocytes (CTLs). Our present study provides the basis to establish novel immunotherapeutic approaches in glioma patients.