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

TLE1 inhibits Bit1 anoikis function.

<p>A. and B. A549 cells were transfected with Bit1 mito and/or with GFP-tagged TLE1 construct. 24 h post transfection, the expression of exogenous Bit1 and TLE1 in transfected cells was confirmed by western blotting using specific antibodies to myc and GFP tags. The amount of plasmid transfected into cells was normalized with the vector construct. In parallel, transfected cells were grown in suspension for 48 h and then subjected to Cell Death ELISA assay (B). C. A549 can HOP-92 cells were transfected with Bit1 cyto or GFP-TLE1 construct. 48 h post-transfection, cells were harvested and analyzed for apoptosis by Cell Death Elisa. D. A549 cells were transfected with control- or TLE1 specific siRNAs, and 48 h later, cells were subjected to immunoblotting with antibodies against TLE1 and B-actin to confirm the knockdown of TLE1 expression. E. A549 cells were transfected with vector or Bit1 mito construct, and 24 hr later, the mitochondrial Bit1 treated cells were transfected with control- or TLE1-siRNAs as indicated. Cells were then cultured in suspension for 48 hr and subjected to Cell Death Elisa. In B, C, and E, three independent experiments were performed in triplicates, * indicates p<0.05 by Student's t test.</p

Bit1 is downregulated in NSCLC tissues.

<p>A. Lung tumor tissue array slides were stained with the affinity purified rabbit anti-Bit1 antibody. Images are representative of each respective case type: normal lung (i, ii 10×), squamous cell carcinoma (iii, iv, viii 10×), adenocarcinoma (v, vi 10×) and large cell carcinoma (vii). B. The average Bit1 immunostaining intensity in normal bronchial and alveolar epithelial tissues and in various types of NSCLC cancer was determined as described in materials and methods. The normal bronchial and alveolar epithelial tissues was statistically significant from the NSCLC tumors using the ANOVA and subsequent Tukey post-hoc analysis (*, P<0.05).</p

Anoikis insensitivity of A549 cells is associated with lack of caspase activation.

<p>A. Cells were cultured being attached (A) or detached from the ECM for 24 hours (D24) and 48 hours (D48) and were harvested for apoptosis analysis using the Cell Death Elisa ELISA. In parallel, cells were also treated with 1 µM staurosporine (STS) for 24 hours and subjected to Cell Death Elisa ELISA. B. Cytosolic fractions were obtained from cells in A and analysed for the presence of cytochrome c by western blot. C. The presence of caspase-3-like activity in the cell lysates from cells treated in A was determined by the cleavage of a fluorescent substrate z-DEVD-AFC (DEVD). D. and E. Cells in A were subjected to western blotting to detect the processing of pro-caspase 3 (D) and PARP (E). In A and C, three independent experiments were performed in triplicates, * indicates p<0.05 as compared to attached conditions (Student's t test).</p

Suppression of Bit1 enhances tumorigenecity of A549 cells <i>in vivo</i>.

<p>A. A549 derived control shRNA and Bit1 shRNA cells (1.0×10⁶) were injected subcutaneously in 8 week old BALB/c nude mice. Tumors were measured periodically with a calliper on the days after injection as indicated. B. and C. Mice were anesthetized and sacrificed on the day 35 after injection. Subcutaneous tumors were surgically excised, and the tumors were then photographed (the representative tumors are shown in (B) and weighed (C). In D and E, tissue sections of control shRNA and Bit1 shRNA xenografts were subjected to TUNEL assay to detect cellular apoptosis. Representative images of TUNEL stained tumor sections are shown in (C). Arrows indicate apoptotic cells characterized by a brown staining. D. TUNEL positive tumor cells in serial sections of control shRNA and Bit1 shRNA tumors were quantified. In C and E, * indicates p<0.05 as compared to control tumors (Student's t test).</p