Cancer-Testis Antigen MAGE-C2/CT10 Promotes Proliferation and Enhances Resistance to p53 Mediated Apoptosis in Multiple Myeloma

Cancer-testis antigens belonging to the MAGE class I family of genes are commonly expressed in Multiple Myeloma (MM). Expression of MAGE class I genes is associated with an aggressive clinical course of MM and resistance to chemotherapy, suggesting that MAGE genes may confer a survival advantage on myeloma cells. MAGE-C2/CT10 is member of the MAGE class I family of genes thought to be a good candidate for cancer immunotherapy given its very frequent expression in primary myeloma. In normal cells, MAGE-C2/CT10 seems to suppress p53 expression by promoting its polyubiquitination and degradation. However, the function of MAGE-C2/CT10 in malignancies is completely unknown. We, therefore, investigated for the first time the role of MAGE-C2/CT10 in tumor cells derived from patients with MM.

MAGE-C2/CT10 expression was analysed by real-time PCR and western blot in myeloma cell lines (N=8) and in PBMC from healthy donors (N=8). For the functional evaluation of MAGE-C2/CT10 we decided to use myeloma cell line U-266 which constitutively expresses MAGE-C2/CT10 and a missense mutant p53 (A161T) that has partially lost its transcriptional activity. The biological role of MAGE-C2/CT10 was investigated by stably silencing its expression using lentiviral short hairpin RNA (shRNA). The effects of silencing MAGE-C2/CT10 expression on myeloma cell biology were examined by determining the number of viable or apoptotic cells using a colorimetric MTT assay and annexin-V/7AAD staining followed by flow cytometry, In addition, we measured myeloma cell proliferation and the anchorage-independent growth using a BrdU incorporation assay and a colony formation assay, respectively. Finally, we investigated cell cycle phase distribution by flow cytometry and we analyzed the expression of key molecules involved in cell cycle progression and apoptosis using a real-time PCR array as well as western blot.

We found MAGE-C2/CT10 to be constitutively expressed in all myeloma cells lines but not in PBMC from healthy donors. Lentivirus-mediated silencing of MAGE-C2/CT10 inhibited significantly the proliferation and the anchorage-independent growth of myeloma cells. Cell cycle analysis demonstrated that the anti-proliferative effect of MAGE-C2/CT10 silencing in U-266 was due to a 70% decrease of cells in the S phase, a cell cycle arrest at both G0/G1 and G2/M transitions and an increase in the subG0/G1 population due to an activation of apoptotic cell death. The serine-threonine checkpoint effector kinase 2 (CHK2) and its substrate, the tumor suppressor protein p53, are essential for cell cycle control, DNA repair and apoptosis. We found that the loss of MAGE-C2/CT10 expression was associated with the activation of CHK2 through phosphorylation at Thr68 as well as the activation of p53 by phosposphorylation at Ser20. Furthermore, we observed a three-fold increase in the endogenous level of p53 protein which correlated with an up-regulation of two transcriptional targets of p53, the cyclin-dependant kinase inhibitor p21^WAF1 and the growth arrest and DNA-damage-inducible alpha protein (GADD45A), known to be essential for p53-induced G1 and G2 arrest, respectively. Finally, using the Human Apoptosis Profiler PCR array that contains a number of p53 target genes, we found that apoptosis induced by MAGE-C2/CT10 knockdown was due to a more than two-fold increase in the transcription of pro-apoptotic genes like BAX (Bcl2-associated × protein), BAD (BCL2-associated agonist of cell death), Cytochrome c, APAF1 (Apoptosis activating factors) as well as several caspases, which are the down-stream mediators of p53-dependant apoptosis in response to DNA damage.

Our collected findings support an anti-apoptotic function of MAGE-C2/CT10 in MM, likely through the regulation of key molecules involved in cell cycle progression and p53-mediated apoptosis. The central role of MAGE-C2/CT10 in the biology of myeloma strongly suggest that this cancer-testis antigen represents a promising target for myeloma-specific immunotherapies or other targeted modes of therapy for MM.

Kröger:Fresenius Biotech: Honoraria, Research Funding.