The Anoikis Effector Bit1 Inhibits EMT through Attenuation of TLE1-Mediated Repression of E-Cadherin in Lung Cancer Cells

<div><p>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 <i>in vitro</i> and tumorigenicity <i>in vivo</i>. 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 <i>in vivo</i> 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.</p></div

Bit1 regulates E-cadherin expression at the transcriptional level through the transcriptional regulator AES.

<p>A. and B. Stable control shRNA and Bit1 shRNA A549 cells as well as A549 cells treated with control or Bit1 siRNAs were subjected to total RNA isolation, reverse transcription, and real time PCR analysis using specific E-cadherin primers (A) and E-cadherin promoter luciferase activity assay (B) as described in materials and methods. C and D. Stable control and Bit1 mito A549 cells as well as A549 cells transiently transfected with vector, Bit1 mito, or Bit1 cyto construct were subjected to RNA isolation and Real time PCR analysis (C) to assess for E-cadherin mRNA levels and E-cadherin promoter luciferase assay (D) to quantify E-cadherin promoter activity. E. and F. A549 cells were treated with control or AES siRNAs, and 24 hr later cells were transfected with the C-terminally myc-tagged Bit1 (Bit mito) or empty vector construct as indicated. 24h after the plasmid transfection, cells were harvested and subjected to immunoblotting with the indicated antibodies (E) and E-cadherin promoter luciferase assay (F). G and H. A549 cells were transfected with the indicated construct. The amount of plasmid transfected into cells was normalized with the vector construct. 24h post transfection, cells were harvested and subjected to immunoblotting with the indicated antibodies (G) and E-cadherin promoter luciferase assay (H). In A, B, C, D, F, and H, three independent experiments were performed in triplicates, * indicates p<0.05 by Student’s t test.</p

Knockdown of Bit1 expression promotes EMT in A549 cells.

<p>A. Stable control shRNA and Bit1 shRNA pool of cells derived from the A549 cell line were subjected to total cell lysate isolation, SDS-PAGE, and immunoblotting against specific antibodies to E-cadherin, vimentin, Bit1, and B-actin. In B, C, and D, the control shRNA and Bit1 shRNA cells were subjected to phase contrast microscopy (100x magnification) under normal culture conditions (B), wound closure (C), and Boyden chamber migration (D) assays. E and F. A549 cells were transfected with control or Bit1 specific siRNAs, and 48 h post-transfection, cells were harvested and subjected to immunoblotting against the indicated antibodies (E) and Boyden chamber migration assay (F). In D and F, results are representative of three independent experiments, *p<0.05 as compared with the control cells (Student’s t test).</p

Bit1 negatively regulates TLE1-mediated E-cadherin repression.

<p>A., B. and C. Stable control and Bit1 mito A549 cells were transfected with the GFP-tagged TLE1 or vector construct. 24h post transfection, cells were harvested and subjected to immunoblotting with the indicated antibodies (A), E-cadherin promoter luciferase analysis (B), or Boyden chamber migration assay (C). D. and E. Stable control shRNA and Bit1 shRNA A549 cells were analysed by ChIP assay. Chromatin was precipitated using antibodies against TLE1, acetyl-histone H3 (Ac-H3), and control IgG as detailed in the materials. The E-cadherin promoter sequence was amplified by PCR and subjected to agarose gel electrophoresis (D). The ChIP experiments were repeated at least three times and a representative experiment is shown. Enrichment of the E-cadherin promoter fragment in TLE1-ChIP and acetyl-histone 3-ChIP over IgG-antibody in control shRNA and Bit1 shRNA cells is shown in E. F. and G. Stable control and Bit1 mito A549 cells were subjected to ChIP assay with antibodies against TLE1, acetyl-histone H3 (Ac-H3), and control IgG. The E-cadherin promoter fragment was amplified by PCR and subjected to agarose gel electrophoresis (F). Enrichment of the E-cadherin promoter fragment in TLE1-ChIP and acetyl-histone 3-ChIP over IgG-antibody in control and Bit1 mito cells is shown in G. H., I., and J. Control and TLE1 expressing pool of A549 cells were transfected with the Bit1 mito or empty vector construct, and 24h later cells were harvested and subjected to immunoblotting with the indicated antibodies (H), E-cadherin promoter luciferase analysis (I), and Boyden chamber migration assay (J). In B, C, E, G, I, and J, three independent experiments were performed in triplicates, * indicates p<0.05 by Student’s t test.</p

A model depicting the regulation of EMT and metastasis by Bit1 through inhibition of TLE1-mediated repression of E-cadherin expression.

<p>In normal bronchial epithelial cells, Bit1 upregulates E-cadherin expression and maintains epithelial phenotype by relieving E-cadherin repression through removal of TLE1 corepressor and its associated repressive chromatin remodelling enzymes such as HDAC1 at the E-cadherin promoter. During lung cancer progression, loss of Bit1 expression results in suppression of E-cadherin expression as a consequence of enhanced TLE1 occupancy, HDAC1 recruitment, and histone deacetylation at the E-cadherin promoter. The suppression of E-cadherin upon downregulation of Bit1 may promote EMT, migration, and metastasis. While our current data indicate that the Zeb1 transcription factor may in part underlie TLE1 recruitment to the E-cadherin promoter to repress transcription, additional components of the TLE1 corepressor machinery including other known E-cadherin repressors and chromatin remodelling Polycomb repressive complexes (Prc) remain to be identified.</p

Suppression of Bit1 expression attenuates the epithelial phenotype of BEAS-2B cells.

<p>Stable control shRNA and Bit1 shRNA pool of BEAS-2B cells were subjected to immunoblotting with the indicated antibodies (A), phase contrast microscopy (B), and Boyden chamber migration assay (C). D and E. BEAS-2B cells were treated with control or Bit1 specific siRNAs, and 48h later, cells were subjected to immunoblotting against the indicated antibodies (D) and Boyden chamber migration assay (E). In C and E, results are representative of three independent experiments, *p<0.05 as compared with the control cells (Student’s t test).</p

Bit1 induces E-cadherin expression and inhibits EMT.

<p>A. Stable control and mitochondrial Bit1 expressing (Bit1 mito) pool of A549 cells were subjected to immunoblotting with the indicated antibodies. B and C. Stable control and Bit1 mito pool of cells were subjected to phase contrast microscopy under normal culture conditions (B) and in suspension (C). D. Stable control and Bit1 mito pool of cells were subjected to Boyden chamber migration assay. E, F, and G. A549 cells were transfected with the C-terminally myc-tagged mitochondrial localized Bit1 (Bit1 mito), N-terminally tagged cytoplasmic localized Bit1 (Bit1 cyto) or empty vector construct. 24 h post-transfection, cells were subjected to immunoblotting against the indicated antibodies (E), phase contrast microscopy in suspension culture (F), and Boyden chamber assay (G). In D and G, three independent experiments were performed in triplicates, * indicates p<0.05 by Student’s t test as compared to control cells.</p

Bit1 inhibits tumor metastasis <i>in vivo</i>.

<p>A., B., C. D., and E. Stable control and Bit1 mito A549 cells were injected into the tail vein of nude mice. After seven weeks post-injection, the mice were sacrificed and the lungs were harvested and photographed (the representative lungs are shown in A) and the number of surface tumor nodules was counted (B). The lung samples were subjected to histological analysis. Representative H & E staining of lung tissue sections is shown in C. The number of microscopic tumor nodules was quantified in D. The total lung weight from the control and Bit1 mito group of mice was measured (E). F. and G. The control shRNA and Bit1 shRNA A549 cells were injected into the tail vein, and six weeks after injection mice were sacrificed and subjected to histological analysis. The representative H & E staining of the lung sections is shown in F. The number of microscopic tumor nodules in the lung sections was quantified (G). In C and F, boxed regions are shown at higher magnification showing inflammatory (inf) cells surrounding the tumor nodules. The arrows denote tumor nodules. In B, D, E, and G, * indicates p<0.05 by Student’s t test.</p

The Anoikis Effector Bit1 Displays Tumor Suppressive Function in Lung Cancer Cells

<div><p>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 <i>in vivo</i>. 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.</p></div

Induction of E-cadherin expression contributes to Bit1-mediated inhibition of cell motility.

<p>A. A549 cells were transfected with C-terminally tagged mitochondrial Bit1 (Bit1 mito) alone or together with FLAG-tagged AES construct. The amount of plasmid transfected into cells was normalized with the vector construct. 24h post transfection, cells were harvested and subjected to Boyden chamber migration assay. B. A549 cells were treated with control- or AES-siRNAs, and 24 h later cells were transfected with the vector or Bit1 mito construct as indicated. 24h after the plasmid transfection, cells were subjected to Boyden chamber migration assay. C. and D. A549 cells were treated with control- or E-cadherin-siRNAs, and 24 h later cells were transfected with the vector or Bit1 mito construct as indicated. 24h after the plasmid transfection, cells were harvested and subjected to immunoblotting with the indicated antibodies (C) and Boyden chamber migration assay (D). E. and F. Stable control shRNA and Bit1 shRNA A549 cells were transfected with the vector or E-cadherin construct as indicated. 24h post-transfection, cells were harvested and subjected to immunoblotting with the indicated antibodies (E) and Boyden chamber migration assay (F). G. and H. Stable control shRNA and Bit1 shRNA BEAS-2B cells were transfected with the vector or E-cadherin construct as indicated. 24h post-transfection, cells were harvested and subjected to immunoblotting with the indicated antibodies (G) and Boyden chamber migration assay (H). In A, B, D, F, and H, three independent experiments were performed in triplicates, * indicates p<0.05 by Student’s t test.</p