Loss of TGF-β Adaptor β2SP Activates Notch Signaling and SOX9 Expression in Esophageal Adenocarcinoma

TGF-beta and Notch signaling pathways play important roles in regulating self-renewal of stem cells and gastrointestinal carcinogenesis. Loss of TGF-beta signaling components activates Notch signaling in esophageal adenocarcinoma, but the basis for this effect has been unclear. Here we report that loss of TGF-beta adapter beta 2SP (SPNB2) activates Notch signaling and its target SOX9 in primary fibroblasts or esophageal adenocarcinoma cells. Expression of the stem cell marker SOX9 was markedly higher in esophageal adenocarcinoma tumor tissues than normal tissues, and its higher nuclear staining in tumors correlated with poorer survival and lymph node invasion in esophageal adenocarcinoma patients. Downregulation of beta 2SP 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 beta 2SP 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 beta 2SP-silenced esophageal adenocarcinoma cells. Conversely, SOX9 silencing rescued the phenotype of esophageal adenocarcinoma cells with loss of beta 2SP. Interaction between Smad3 and ICN1 via Smad3 MH1 domain was also observed, with loss of beta 2SP increasing the binding between these proteins, inducing expression of Notch targets SOX9 and C-MYC, and decreasing expression of TGF-beta targets p21(CDKN1A), p27 (CDKN1B), and E-cadherin. Taken together, our findings suggest that loss of beta 2SP switches TGF-beta signaling from tumor suppression to tumor promotion by engaging Notch signaling and activating SOX9. Cancer Res; 73(7); 2159-69. (C)2013 AACR.