One of the most important cellular functions of integrin-mediated cell adhesion to extracellular matrix (ECM) is to promote cell survival by mediating anti-apoptotic signals from ECM to cells. Most types of cells depend on ECM attachment for survival, and, if denied ECM attachment, undergo apoptosis (anoikis). Malignant cells are less dependent on integrin-mediated survival signals than normal cells. A pathway activated by some, but not all integrins, up-regulates the anti-apoptotic protein Bcl-2 and reduces apoptosis. In screening for cDNAs that regulate this anti-apoptotic pathway, we isolated a cDNA that encodes a 179-residue mitochondrial protein, which we have named Bit1. This protein appears to be part of a previously unknown apoptosis pathway that is regulated by integrin-mediated cell attachment. Bit1 is a mitochondrial protein that is released from mitochondria when cells have detached from ECM. It forms a complex with the transcriptional regulator protein Amino-terminal Enhancer of Split (AES) in the cytoplasm. The Bitl/AES complex is the active apoptotic moiety; forced expression of cytoplasmic Bit1 causes apoptosis in cells that express AES, and AES causes apoptosis in cells that express Bit1. Plating cells onto fibronectin reduces Bitl/AES complex formation and counteracts the apoptosis-inducing effect of Bit1 and AES. In contrast, transfection with caspase inhibitors, or activated H-Ras, PI3-K or Akt, does not block apoptosis induced by Bit1 or AES. Bcl-2 also has no effect on apoptosis induced by Bit1, but partially blocks apoptosis induced by AES, presumably because it stabilizes mitochondria and prevents Bit1 release into the cytoplasm. Restoring the expression of mitochondrial Bit1 in cells that lack Bit1 enhances susceptibility to anoikis. These results suggest that the Bitl/AES pathway may be, at least in part responsible for the anti-apoptotic effect of integrin-mediated cell adhesion. This application proposes experiments to identify the integrin signaling pathways that regulate the Bitl/AES anoikis, and to determine whether the Bitl/AES pathway is dysregulated in malignant cells. The results of these studies may delineate a signaling pathway that could be of fundamental importance in the anchorage dependence of normal cells. Cells that become malignant may bypass this pathway in becoming anchorage independent and metastatic.
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