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

Abstract

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.

TLE1 promotes EMT in A549 lung cancer cells through suppression of E-cadherin

“…The TLE1 specific siRNAs and the control, non-targeting siRNAs pools were purchased from Invitrogen (13, Carlsbad, CA). For transient transfection experiments, A549 cells (2 × 105) were transfected with 25 µM of each siRNA pool by using the Lipofectamine RNAiMAX transfection reagent (Invitrogen).…”

“…At each indicated time, the number of metabolically active cells was measured with the use of MTT assay as described previously (13). Briefly, MTT reagent (Sigma) was dissolved in sterile PBS at 5 mg/ml.…”

“…Protein preparation and western blotting were performed as described previously (13). Briefly, cells were harvested by adding ice-cold NP-40 lysis buffer (1% NP-40; 20mM Tris-HCL [pH7.4]; 150 mM NaCl; 10% glycerol, 2 mM sodium vanadate; 1 mM henylmethylsulfonyl fluoride; 10 µg/ml leupeptine; and 5 µg/ml aprotinin) and incubating at 4°C for 20 min.…”

“…In line with its anti-apoptotic function, we have found that TLE1 specifically inhibited the caspase-independent cell death induced by Bit1 (Bcl2-inhibitor of transcription 1) in several types of malignant cells (12). Recently, we showed that TLE1 effectively abrogates Bit1-induced anoikis in lung cancer cells (13). Based on the latest report indicating that TLE1 is a putative lung specific oncogene (14), it is interesting to speculate whether the anti-apoptotic function of TLE1 represents as an oncogenic stimulus.…”

Signaling Pathways Altered During the Metastatic Progression of Melanoma

“…The acquisition of anoikis-resistant phenotype is one of the critical steps during tumor progression. In melanoma, we and others [17][18][19] demonstrated that malignant transformation is associated with resistance to anoikis [16]. One of the key molecules involved in anoikis are integrins.…”