Abstract
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.
Highlights
Bcl-2 inhibitor of transcription 1 (Bit1) is a mitochondrial protein that is part of apoptosis pathway, which is uniquely regulated by integrin-mediated cell attachment
We have previously observed that suppression of endogenous Bit1 expression in the human cervical cancer Hela cells resulted in enhanced spindle shape-like morphology and migratory capacity and molecular changes consistent with epithelial–mesenchymal transition (EMT) [11]
To investigate the role of Bit1 in EMT, we examined the effect of altering Bit1 expression on the EMT phenotype of the human lung adenocarcinoma A549 cell line
Summary
Bit is a mitochondrial protein that is part of apoptosis pathway, which is uniquely regulated by integrin-mediated cell attachment. Following loss of cell attachment, Bit is released to the cytosol and interacts with the transcriptional regulator Amino Enhancer slip (AES) protein to induce a caspase-independent form of apoptosis [1]. In addition to integrin-mediated cell attachment, the groucho TLE1 corepressor protein which exhibits survival function in several cellular models [2,3,4], protects cells from Bit apoptosis. AES potently induces apoptosis in cells that express Bit. In line with the Bit1/AES complex as the apoptogenic factor, the integrin-mediated cell attachment and TLE1 corepressor protein block Bit apoptosis by inhibiting the formation of this complex [1]. Characterization of the TLE1 transcriptional pathway and its regulation by the Bit1/AES axis is currently under investigation
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