Abstract 3243: Dysfunctional of TGF-β signaling via loss of β2SP activates SOX9 and C-MYC via Notch signaling in Barrett's esophageal adenocarcinoma

Abstract

Abstract Background: Esophageal adenocarcinoma (EA) is often considered to arise from a clonal stem like population of cells, potentially responsible for its poor prognosis. TGF-β and Notch signaling pathways play important roles in regulating self-renewal of stem cells and gastrointestinal carcinogenesis. We have demonstrated loss of TGF-β signaling components and activation of Notch signaling in Barrett's esophageal adenocarcinoma; however, their contributions to EA and the mechanisms of their action remain unclear. Methods: Immunoblotting and immunofluorescence were used to evaluate protein expression and localization. Notch targets Hes-1, SOX9 and C-MYC transcription were assayed using their luciferase reporters and Q-PCR. Functional studies were performed in β2SP wild type and mutant MEFs cells and in EA cell lines genetically engineered to express high or low levels of β2SP. Results: Increased levels of Notch signaling Hes1 and Jagged1 occurred in EA tissues and cell lines, compared to normal tissues. Loss of β2SP in MEFs cells increased Hes1 expression by 40 fold. In addition, SOX9, a target gene of Notch signaling and a documented stem cell marker was highly up-regulated in EA tumor cells and tissues. Expression of SOX9 was increased in the absence of β2SP in MEFs cells by 9 fold. Down-regulation of β2SP in SKTG-4, FLO-1 and BE3 EA cells by lentivirus shRNA led to increased SOX9 expression and enhanced nuclear localization of both active intracellular Notch1 domain (ICN1) and SOX9. Reintroducing β2SP into EA cells with knock down β2SP decreased SOX9 promoter activity by 10 fold. Concomitantly, an increased proportion of stem cells in β2SP knock down cells were identified using stem cell marker OCT3/4, indicating expansion of putative cancer stem cells in the absence of β2SP. Most interestingly, we observed a direct interaction between Smad3 and ICN1 via Smad3 MH1 domain by GST-pull down assays and that loss of β2SP increases the binding of Smad3 with ICN1 and induces Notch targets SOX9 and C-MYC transcription. Conclusions: Our findings suggests that loss of TGF-β signaling adaptor β2SP may switch Smad3 function from tumor suppression to tumor promotion by binding intracellular Notch1 domain (ICN1) and activating Notch signaling. Thereby, a potential therapeutic value for targeted therapy in EA is in the setting of loss of β2SP/TGF-β with active Notch signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3243. doi:1538-7445.AM2012-3243