207 Cancer testis antigen melanoma-associated antigen A3 (MAGE-A3) promotes cutaneous squamous cell carcinoma growth in vivo

To identify novel targets for cancer immunotherapy, our lab has profiled the expression of cancer testis antigens (CTAs) in primary human cutaneous squamous cell carcinoma (cSCC). CTAs are immunogenic and may be suitable as targets for immunotherapy, or as biomarkers for aggressive biological behavior. We recently showed that MAGE-A3 is highly expressed in aggressive cSCC and is associated with poor prognosis. We hypothesized that MAGE-A3 may be a novel target for immunotherapy and/or a biomarker for metastatic cSCCs. In this study, we identified the expression of MAGE-A3 in human and murine cSCC cell lines in vitro. Furthermore, MAGE-A3 antibody treatment was demonstrated to inhibit the growth and proliferation of human (A431) cSCC cells using MTT and crystal violet assays. In addition, we observed a 2.4-fold block in S-phase progression of A431 cells by EDU assay (P ≤ 0.001). Immunoblot analysis showed an increase in p21 and p53 cell cycle proteins after MAGE-A3 antibody treatment, with no change in RNA transcript levels. We next used Pam 212 cells to examine the role of MAGE-A3 expression in cSCC growth in vivo. Pam 212 cells develop locally invasive cSCC in immune competent BALB/c mice. Characterization of Pam 212 primary tumors revealed MAGE-A3 expression within the tumor tissue. We then utilized a CRISPR/Cas9 knockout system to demonstrate that Pam 212 tumor maintenance is impaired upon functional ablation of MAGE-A3 in vivo, with a 3.5-fold decrease in tumor volume at endpoint (n = 6/group; P ≤ 0.001). Taken together, these results suggest that cSCC may rely on signaling events downstream of MAGE-A3 to support tumor proliferation. Using the PAM 212 model in immunocompetent BALB/c mice, we will further explore immunotherapeutic options directed against MAGE A-3 in cSCC.