Abstract 1612: Overexpression or inducible expression of Snail, Zeb1 or TGFβ recapitulates epithelial-mesenchymal transition (EMT) in colorectal cancer cell lines in vitro

Abstract

Abstract Epithelial-mesenchymal transition (EMT) is one of the hallmarks of cancer and a critical process providing tumor cells with the ability to metastasize to distant sites. Several previous studies have revealed that EMT plays a crucial role in the progression and aggressiveness of colorectal cancer (CRC). Snail, Zeb1 and TGFβ are considered as master EMT genes and their overexpression has been linked to metastatic disease. Our study was conducted to establish an EMT platform in-house to determine the role of Snail, Zeb1 and TGF-β1 in mediating EMT in CRC cell lines in vitro and using them for studying drug sensitivity and target validation. Screening of six CRC cell lines for expression of epithelial (E) and mesenchymal (M) markers by western blot analysis suggested that DLD-1 and HT-29 cells are epithelial in nature and were used to generate stable overexpression and tet-inducible models. Overexpression of Snail or Zeb1 or TGF-β1 induced EMT in both DLD-1 and HT-29 colorectal cancer cells as indicated by downregulation of E-cadherin and upregulation of vimentin by western blot analysis and immunofluorescence staining. These cells which have undergone EMT also showed significant growth advantage in 3D growth on matrigel or soft agar. Stable ‘E’ and ‘M’ cells were also used to test the sensitivity of various inhibitors including erlotinib, salinomycin and chemotherapeutic agents such as paclitaxel, gemcitabine, cisplatin, oxaloplatin and 5-FU. Consistent with previous findings, erlotinib showed sensitivity only in ‘E’ cells and not ‘M’ cells. Chemotherapy agents have also shown higher sensitivity in ‘E’ cells than ‘M’ cells. In contrast, Salinomycin showed equipotent sensitivity in ‘E’ and ‘M’ cells. We have also generated tet-inducible models of Snail, Zeb1 and TGF-β1 and they behaved very similar to overexpression models. These inducible models were useful to determine whether any inhibitor treatment or knock down of specific gene (target) can block EMT. In our studies, testing of a Focal Adhesion Kinase (FAK) inhibitor or Dasatinib (SFK inhibitor) as examples showed blockade of EMT in these models. Overall, establishing several EMT models in-house enabled us to validate new targets involved in EMT, test new inhibitors whether it can block EMT or study drug sensitivity in ‘E’ vs. ‘M’ cells. Citation Format: Shripad V. Bhagwat, Nicole E. Hamm, Yu-Chih Hsu, Yuewei Qian, Robert Wild, Jonathan A. Lee, Sheng-Bin Peng. Overexpression or inducible expression of Snail, Zeb1 or TGFβ recapitulates epithelial-mesenchymal transition (EMT) in colorectal cancer cell lines in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1612.