Expression of Cancer-Testis Antigens MAGE-C1/CT7 and MAGE-A3 Is Central to the Survival of Myeloma Cells and Their Resistance to Chemotherapy

Abstract

The cancer-testis (CT) class of tumor antigens is a group of proteins, the expression of which is characteristically restricted to cancer and the human germline. Based on their immunogenicity and restricted tissue expression, CT antigens seem ideal targets for immunotherapeutic approaches. This is particularly the case in multiple myeloma (MM) where these tumor antigens are frequently and strongly expressed. Unfortunately, very little is known about the biological function of CT antigens which seems surprising given the strong impact of CT antigen expression on the prognosis of a variety of malignancies including MM. We examined for the first time the impact of the expression of two CT antigens frequently found in MM, MAGE-C1/CT7 and MAGE-A3, on the biology of the disease conducting knock-down experiments using RNA interference (RNAi) technology. As read-out assays, Western Blots as well as a large variety of tests measuring proliferation, cytotoxicity, cell adhesion, and clonogenic growth were performed. Transfecting myeloma cell line MOLP-8 with RNAi specific for MAGE-C1/CT7 and MAGE-A3 we observed a down-regulation of CT antigen protein expression by 80% and 70%, respectively. Importantly, transfection with MAGE-A3 RNA stealth led to a specific decrease in MAGE-A3 expression while the protein expression of other MAGE genes (MAGE-A4, MAGE-C1/CT7, MAGE-C2/CT10) and non-MAGE CT antigens such as Ropporin-1 was not affected. The same was true for knock-down experiments targeting MAGE-C1/CT7 with the exception of a slight decrease in expression of the highly homologous gene MAGE-C2/CT10. Comparing myeloma cell lines treated with gene-specific versus nonsense RNA stealth, we found that knocking down MAGE-C1/CT7 and MAGE-A3 led to the induction of apoptosis in in about 70% of cells at 72h post transfection as indicated by MTT assays, LDH release assays and Annexin V expression analyzed by flow cytometry. No such effect was observed when transfection was performed with nonsense RNAi (p<0.01). Importantly, CD138-negative myeloma precursors, which also expressed the CT antigens investigated, were also affected by downregulation of CT antigen expression as indicated by an 48% decrease in clonogenic growth (p<0.01). Applying chemotherapy with melphalan to the cell cultures we observed that knock-down of MAGE-C1/CT7 or MAGE-A3 increased the chemosensitivity of MM cell lines by 67% (p<0.01). Importantly, knock-down of MAGE-C1/CT7 or MAGE-A3 specifically affected cell survival and did not alter the proliferative capacity of myeloma cells or cell adhesion. In conclusion, we show here for the first time that CT antigens MAGE-C1/CT7 and MAGE-A3, which are also expressed in the majority of patient myeloma samples, are important for the survival of myeloma cell lines and clonogenic myeloma precursors. In addition, the expression of these CT antigens most likely contributes to promoting resistance to chemotherapy in multiple myeloma. Targeting CT antigens expressed by myeloma cells, either by applying CT antigen-specifric immunotherapy or other targeted therapies, might significantly improve myeloma therapy, i.e. by eliminating chemotherapy-resistant clones.