Establishment of MAGEC 2‐knockout cells and functional investigation of MAGEC 2 in tumor cells

Cancer/testis antigen MAGEC 2, a member of the type I melanoma‐associated antigen family, is expressed in a wide variety of cancer types but not in normal somatic cells. MAGEC 2 has long been recognized as a tumor‐specific target, however, its functions remain largely unknown. In this study, we established MAGEC 2‐knockout A375 melanoma cell lines using the CRISPR /Cas9 system. Seven clonal cell lines were generated by using four single guide RNA s targeting the coding region of the MAGEC 2 gene, which produced indels that abolished MAGEC 2 protein expression. To identify the differentially expressed protein profiles associated with MAGEC 2 loss, isobaric tag for relative quantitation‐based comparative proteomics experiments were carried out on the MAGEC 2‐knockcout and control A375 cells. Mining of the proteomics data identified a total 224 (61.6% upregulated and 38.4% downregulated) proteins to be significantly altered in expression level in MAGEC 2‐knockcout cells. Ingenuity Pathway Analysis indicated that the significantly altered proteins were involved in critical neoplasia‐related biological functions such as cell death, proliferation, and movement. Gene ontology analysis identified “apoptosis signaling” as the top‐most upregulated pathway associated with MAGEC 2 loss. We showed that knockout or knockdown of the MAGEC 2 gene sensitized melanoma cells to tumor necrosis factor‐α‐induced apoptosis. Interestingly, actin‐based motility by Rho and RhoA signaling, known to promote cell migration, were also identified as the top downregulated pathways in MAGEC 2‐knockout A375 cells. In short, our study provides a suitable cell model for exploring the biological functions of MAGEC 2 in malignant cells, and sheds light on the molecular pathway by which MAGEC 2 promotes tumor development.