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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 Bcl-2 inhibitor of transcription 1 (Bit1) protein is part of an anoikis-regulating pathway that is selectively dependent on integrins. We previously demonstrated that the caspase-independent apoptotic effector Bit1 exerts tumor suppressive function in lung cancer in part by inhibiting anoikis resistance and anchorage-independent growth in vitro and tumorigenicity in vivo. Herein we show a novel function of Bit1 as an inhibitor cell migration and epithelial-mesenchymal transition (EMT) in the human lung adenocarcinoma A549 cell line. Suppression of endogenous Bit1 expression via siRNA and shRNA strategies promoted mesenchymal phenotypes, including enhanced fibroblastoid morphology and cell migratory potential with concomitant downregulation of the epithelial marker E-cadherin expression. Conversely, ectopic Bit1 expression in A549 cells promoted epithelial transition characterized by cuboidal-like epithelial cell phenotype, reduced cell motility, and upregulated E-cadherin expression. Specific downregulation of E-cadherin in Bit1-transfected cells was sufficient to block Bit1-mediated inhibition of cell motility while forced expression of E-cadherin alone attenuated the enhanced migration of Bit1 knockdown cells, indicating that E-cadherin is a downstream target of Bit1 in regulating cell motility. Furthermore, quantitative real-time PCR and reporter analyses revealed that Bit1 upregulates E-cadherin expression at the transcriptional level through the transcriptional regulator Amino-terminal Enhancer of Split (AES) protein. Importantly, the Bit1/AES pathway induction of E-cadherin expression involves inhibition of the TLE1-mediated repression of E-cadherin, by decreasing TLE1 corepressor occupancy at the E-cadherin promoter as revealed by chromatin immunoprecipitation assays. Consistent with its EMT inhibitory function, exogenous Bit1 expression significantly suppressed the formation of lung metastases of A549 cells in an in vivo experimental metastasis model. Taken together, our studies indicate Bit1 is an inhibitor of EMT and metastasis in lung cancer and hence can serve as a molecular target in curbing lung cancer aggressiveness.

In the original publication [1] the name of author Jeremy M. Aymard was spelled wrong. The original article was updated to rectify this error.

The Groucho transcriptional corepressor TLE1 protein has recently been shown to be a putative lung specific oncogene, but its underlying oncogenic activity in lung cancer has not been fully elucidated. In this report, we investigated whether TLE1 regulates lung cancer aggressiveness using the human lung adenocarcinoma cell line A549 as a model system. Through a combination of genetic approaches, we found that TLE1 potentiates epithelial-to-mesenchymal transition (EMT) in A549 cells in part through suppression of the tumor suppressor gene E-cadherin. Exogenous expression of TLE1 in A549 cells resulted in heightened EMT phenotypes (enhanced fibroblastoid morphology and increased cell migratory potential) and in molecular alterations characteristic of EMT (downregulation of the epithelial marker E-cadherin and upregulation of the mesenchymal marker Vimentin). Conversely, downregulation of endogenous TLE1 expression in these cells resulted in reversal of basal EMT characterized by a cuboidal-like epithelial cell phenotype, reduced cell motility, and upregulated E-cadherin expression. Mechanistic studies showed that TLE1 suppresses E-cadherin expression at the transcriptional level in part by recruiting histone deacetylase (HDAC) activity to the E-cadherin promoter. Consistently, the HDAC inhibitor TSA partially reversed the TLE1-induced E-cadherin downregulation and cell migration, suggesting a role for HDACs in TLE1-mediated transcriptional repression of E-cadherin and EMT function. These findings uncover a novel role of TLE1 in regulating EMT in A549 cells through its repressive effect on E-cadherin and provide a mechanism for TLE1 oncogenic activity in lung cancer.

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.