Functional analysis of cancer/testis antigens in human cancer

The cancer/testis (CT) antigens are a family of genes that are silenced in the majority of normal human tissues, except for the testes, but are specifically re-expressed in a number of human tumours. Interestingly, their expression is frequently found in only a subpopulation of cells in malignancies, but the reasons for this heterogeneous expression pattern have not yet been investigated. Since little is known about the regulation and the biological significance of the CT antigens, it is not yet clear whether they play an active role in tumourigenesis or whether their expression is a consequence of cancer progression. To address this question, attempts were made to develop an in vitro experimental model that mimics tumours expressing CT antigens. Twenty-one melanoma, small cell lung cancer (SCLC) and multiple myeloma (MM) cell lines were screened for the expression of three CT antigens: NY-ESO-1, its binding partner MAGE-C1, and a molecule that is closely related to MAGE-C1 called MAGE-C2. Intriguingly, some of these cell lines showed heterogeneous expression patterns that resembled those observed in tumour samples. Selected cell lines were treated with MG132, an inhibitor of the proteasome protein degradation pathway, to investigate the post-translational regulation of CT antigens. Insoluble MAGE-C1 and NY-ESO-1 were stabilised upon treatment, moreover MG132 caused accumulation of these two antigens at the centrosome. Interestingly, co-transfection experiments showed that NY-ESO-1 enhances the MG132-induced stabilisation of MAGE-C1 MAGE homology domain (MHD). In addition to proteasome degradation, SUMO-1 conjugation was also identified as a post-translational regulatory mechanism for MAGE-C1. Finally, MAGE-C1 knockdown experiments demonstrated that depletion of this antigen increases cell death in response to endoplasmic reticulum (ER) stress, while reducing the stability of the pro-survival protein Mcl-1. Thus, MAGE-C1 is a novel inhibitor of ER stress-induced apoptosis. As a whole, this study provides new insights into the expression, regulation and function of these CT antigens in cancer cells.