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// Xin Yao 1 , Tri Pham 1 , Brandi Temple 1 , Selena Gray 1 , Cornita Cannon 1 , Camry Hardy 1 , Kamari Fletcher 1 , Shubha Kale Ireland 1 , Ahamed Hossain 1 , Renwei Chen 2 , Asim B. Abdel-Mageed 3 and Hector Biliran 1 1 Department of Biological and Public Health Sciences, Xavier University of Louisiana, New Orleans, LA 70125, USA 2 Center for Bioengineering, University of California, Santa Barbara, CA 93106, USA 3 Tulane Cancer Center, Tulane University School of Medicine, New Orleans, LA 70112, USA Correspondence to: Hector Biliran, email: hbiliran@xula.edu Keywords: TLE1, anoikis, E-cadherin, tumorigenecity, ZEB1 Received: April 27, 2017 Accepted: June 26, 2017 Published: July 31, 2017 ABSTRACT The Transducin-like enhancer of split 1 (TLE1) corepressor protein is overexpressed in human lung tumors and is a putative lung-specific oncogene. However, the molecular mechanism underlying its oncogenic function remains to be delineated. Here, we report an important role of TLE1 in promoting lung tumorigenesis by a mechanism involving induction of anoikis resistance. Using the human lung adenocarcinoma A549 and immortalized bronchial epithelial BEAS-2B cell lines, we observed that TLE1 inhibits anoikis through transcriptional repression of E-cadherin gene. In support of E-cadherin as a downstream target of TLE1 to block anoikis, forced expression of E-cadherin attenuated TLE1-induced anoikis resistance while E-cadherin downregulation decreased the anoikis sensitivity of TLE1 knockdown cells. Furthermore, we determined that E-cadherin expression is transcriptionally induced upon loss of cell attachment and functions as an effector of anoikis. Loss of E-cadherin via the siRNA strategy or exogenous TLE1 expression was sufficient to attenuate anoikis in A549 and BEAS-2B cells. Importantly, we demonstrated that the ZEB1 transcriptional factor is required for TLE1-mediated E-cadherin repression and anoikis resistance. ZEB1 interacted with and recruited the TLE1 to the E-cadherin promoter to impose histone deacetylation and gene silencing. In vivo , TLE1 strongly promoted tumorigenicity of A549 cells in a ZEB1-dependent manner. Underscoring its role in anoikis insensitivity of lung cancer cells, the TLE1-mediated E-cadherin repression was negatively regulated by the tumor suppressor Bcl-2 inhibitor of transcription 1 (Bit1) to effect anoikis. These findings identify the ZEB1/TLE1/E-cadherin transcriptional mechanism as a novel pathway that promotes anoikis resistance and oncogenicity of lung cancer cells.

The mitochondrial Bit1 (Bcl-2 inhibitor of transcription 1) protein is a part of an apoptotic pathway that is uniquely regulated by integrin-mediated attachment. As an anoikis effector, Bit1 is released into the cytoplasm following loss of cell attachment and induces a caspase-independent form of apoptosis. Considering that anoikis resistance is a critical determinant of transformation, we hypothesized that cancer cells may circumvent the Bit1 apoptotic pathway to attain anchorage-independence and tumorigenic potential. Here, we provide the first evidence of the tumor suppressive effect of Bit1 through a mechanism involving anoikis induction in human lung adenocarcinoma derived A549 cells. Restitution of Bit1 in anoikis resistant A549 cells is sufficient to induce detachment induced-apoptosis despite defect in caspase activation and impairs their anchorage-independent growth. Conversely, stable downregulation of Bit1 in these cells significantly enhances their anoikis resistance and anchorage-independent growth. The Bit1 knockdown cells exhibit significantly enhanced tumorigenecity in vivo. It has been previously shown that the nuclear TLE1 corepressor is a putative oncogene in lung cancer, and we show here that TLE1 blocks Bit1 mediated anoikis in part by sequestering the pro-apoptotic partner of Bit1, the Amino-terminal Enhancer of Split (AES) protein, in the nucleus. Taken together, these findings suggest a tumor suppressive role of the caspase-independent anoikis effector Bit1 in lung cancer. Consistent with its role as a tumor suppressor, we have found that Bit1 is downregulated in human non-small cell lung cancer (NSCLC) tissues.