Molecular and Cellular Pathobiology Loss of TGF-b Adaptor b2SP Activates Notch Signaling and SOX9 Expression in Esophageal Adenocarcinoma

TGF-b and Notch signaling pathways play important roles in regulating self-renewal of stem cells and gastrointestinal carcinogenesis. Loss of TGF-b signaling components activates Notch signaling in esophageal adenocarcinoma, but the basis for this effect has been unclear. Here we report that loss of TGF-b adapter b2SP (SPNB2) activates Notch signaling and its target SOX9 in primary fibroblasts or esophageal adenocarcinoma cells. Expression of the stem cell marker SOX9 wasmarkedly higher in esophageal adenocarcinoma tumor tissues than normal tissues, and its higher nuclear staining in tumors correlatedwith poorer survival and lymph node invasion in esophageal adenocarcinoma patients. Downregulation of b2SP by lentivirus short hairpin RNA increased SOX9 transcription and expression, enhancing nuclear localization for both active Notch1 (intracellular Notch1, ICN1) and SOX9. In contrast, reintroduction into esophageal adenocarcinoma cells of b2SP and a dominant-negative mutant of the Notch coactivator mastermind-like (dnMAN) decreased SOX9 promoter activity. Tumor sphere formation and invasive capacity in vitro and tumor growth in vivo were increased in b2SP-silenced esophageal adenocarcinoma cells. Conversely, SOX9 silencing rescued the phenotype of esophageal adenocarcinoma cells with loss of b2SP. Interaction between Smad3 and ICN1 via Smad3 MH1 domain was also observed, with loss of b2SP increasing the binding between these proteins, inducing expression of Notch targets SOX9 and C-MYC, and decreasing expression of TGF-b targets p21(CDKN1A), p27 (CDKN1B), and E-cadherin. Taken together, our findings suggest that loss of b2SP switches TGF-b signaling from tumor suppression to tumor promotion by engaging Notch signaling and activating SOX9. Cancer Res; 73(7); 2159–69. 2013 AACR.