SOX9 indirectly regulates CEACAM1 expression and immune resistance in melanoma cells

// Shira Ashkenazi 1,2 , Rona Ortenberg 1 , Michal Besser 1,2 , Jacob Schachter 1,* and Gal Markel 1,2,3,* 1 Ella Lemelbaum Institute of Melanoma, Sheba Medical Center, Ramat Gan, Israel 2 Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Israel 3 Talpiot Medical Leadership program, Sheba Medical Center, Ramat Gan, Israel * These authors have contributed equally to this work Correspondence to: Gal Markel, email: // Keywords : CEACAM1, melanoma, SOX9, T cells, Immune checkpoint, Immunology and Microbiology Section, Immune response, Immunity Received : November 01, 2015 Accepted : February 05, 2016 Published : February 14, 2016 Abstract As melanoma cells are immunogenic, they instigate an adaptive immune response and production of anti-tumor T-cells. A central factor in this interaction is CEACAM1 (carcinoembryonic antigen cell adhesion molecule 1), a transmembrane glycoprotein previously shown in our lab to protect melanoma cells from T cell-mediated killing. In this study, we examine the role of transcription factor SOX9 in the regulation of CEACAM1 expression and immune resistance in melanoma cells. Knockdown of endogenous SOX9 results in CEACAM1 up-regulation, while its overexpression leads to the opposite effect. We show that SOX9 controls CEACAM1 expression at a transcriptional level, but in an indirect manner, as regulation of the CEACAM1 promoter remains intact even when all eight potential SOX9-binding sites are abolished. A series of promoter truncations localizes the SOX9-controlled area to the proximal 200bp of the promoter. Point mutations in putative Sp1 and ETS1 binding sites identify these transcription factors as the primary SOX9-controlled mediators. Co-immunoprecipitation studies show that SOX9 and Sp1 physically interact in melanoma cells, while silencing of SOX9 down-regulates ETS1, but not Sp1, in the same cells. Finally, knockdown of SOX9 indeed renders melanoma cells resistant to T cell-mediated killing, in line with the increased CEACAM1 expression. In conclusion, we show that SOX9 regulates CEACAM1 expression in melanoma cells, and thereby their immune resistance. As CEACAM1 is a pivotal protein in melanoma biology and immune crosstalk, further understanding of its regulation can provide new insights and contribute to the development of novel approaches to therapy.