p53 inhibits alpha 6 beta 4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB.

Although the interaction of matrix proteins with integrins is known to initiate signaling pathways that are essential for cell survival, a role for tumor suppressors in the regulation of these pathways has not been established. We demonstrate here that p53 can inhibit the survival function of integrins by inducing the caspase-dependent cleavage and inactivation of the serine/threonine kinase AKT/PKB. Specifically, we show that the alpha6beta4 integrin promotes the survival of p53-deficient carcinoma cells by activating AKT/PKB. In contrast, this integrin does not activate AKT/PKB in carcinoma cells that express wild-type p53 and it actually stimulates their apoptosis, in agreement with our previous findings (Bachelder, R.E., A. Marchetti, R. Falcioni, S. Soddu, and A.M. Mercurio. 1999. J. Biol. Chem. 274:20733-20737). Interestingly, we observed reduced levels of AKT/PKB protein after antibody clustering of alpha6beta4 in carcinoma cells that express wild-type p53. In contrast, alpha6beta4 clustering did not reduce the level of AKT/PKB in carcinoma cells that lack functional p53. The involvement of caspase 3 in AKT/PKB regulation was indicated by the ability of Z-DEVD-FMK, a caspase 3 inhibitor, to block the alpha6beta4-associated reduction in AKT/PKB levels in vivo, and by the ability of recombinant caspase 3 to promote the cleavage of AKT/PKB in vitro. In addition, the ability of alpha6beta4 to activate AKT/PKB could be restored in p53 wild-type carcinoma cells by inhibiting caspase 3 activity. These studies demonstrate that the p53 tumor suppressor can inhibit integrin-associated survival signaling pathways.

Vascular endothelial growth factor is an autocrine survival factor for neuropilin-expressing breast carcinoma cells.

We identify a novel function for the vascular endothelial growth factor (VEGF) in its ability to stimulate an autocrine signaling pathway in metastatic breast carcinoma cells that is essential for their survival. Suppression of VEGF expression in metastatic cells in vitro induced their apoptosis, in addition to inhibiting the constitutively elevated phosphatidylinositol 3'-kinase activity that is characteristic of these cells and important for their survival. Hypoxia enhanced the survival of metastatic cells by increasing VEGF expression. The importance of the VEGF receptor neuropilin was indicated by the ability of a neuropilin-binding VEGF isoform to enhance breast carcinoma survival. Moreover, the expression of neuropilin in neuropilin-deficient breast carcinoma cells protected them from apoptosis. The identification of this VEGF autocrine signaling pathway has important implications for tumor metastasis and therapeutic intervention.

Postbinding functions of CD4 in HIV infection.

Several studies have suggested that the CD4 molecule, in addition to serving as the HIV receptor, may also be involved in postbinding events of HIV infection. The CD4 molecule has been shown to assume an altered conformation on the T-cell surface following HIV binding, which may be involved in these postbinding events.

The metastatic odyssey: the integrin connection.

This article explores the mechanistic basis of carcinoma progression by focusing on the contribution of integrins. Integrins are essential for progression because of their ability to mediate physical interactions with extracellular matrices and their ability to regulate signaling pathways that control actin dynamics and cell movement, and for growth and survival. This article centers on a6 integrins (a6B1 and a6B4), which are receptors for the laminin family of basement membrane components. Numerous studies have implicated these integrins in cancer progression and have provided a rationale for studying the mechanistic basis of their contribution to aggressive disease.

A human recombinant Fab identifies a human immunodeficiency virus type 1-induced conformational change in cell surface-expressed CD4.

To explore the role of the CD4 molecule in human immunodeficiency virus (HIV) infection following initial virus-CD4 binding, we have characterized CD4-specific antibodies raised by immunizing an HIV-1-infected human with human recombinant soluble CD4 (rsCD4). Fabs were selected from a human recombinant Fab library constructed from the bone marrow of this immunized individual. Here, we describe a human rsCD4-specific recombinant Fab clone selected by panning the library over complexes of human rsCD4 and recombinant HIV-1 envelope protein. While this Fab does not bind to CD4-positive T-cell lines or to human T lymphocytes, it recognizes cell surface-expressed CD4 following the incubation of these cells with a recombinant form of HIV-1 gp120 or with HIV-1 virions. The Fab is not HIV-1 envelope specific, since it does not bind to recombinant gp120 or to native cell surface-expressed HIV-1 envelope proteins. As confirmation of its CD4 specificity, we show that this Fab immunoprecipitates a 55-kDa protein, corresponding to the molecular mass of cellular CD4, from an H9 cell lysate. The specificity of this human Fab provides evidence for a virus-induced conformational change in cell surface-expressed on CD4. The characterization of this altered CD4 conformation and its effects on the host cell will be important in defining postbinding events in HIV infection.

Activation of p53 function in carcinoma cells by the alpha6beta4 integrin.

The interaction of integrins with extracellular matrix is known to promote cell survival by inhibiting apoptotic signaling. In contrast, we demonstrate here that the alpha6beta4 integrin induces apoptosis in carcinoma cells by stimulating p53 function. Specifically, we show that expression of alpha6beta4 in carcinoma cells that lack this integrin stimulates an increase in the transactivating function of p53 as demonstrated by the ability of this integrin to up-regulate the expression of a p53-sensitive reporter gene as well as the endogenous p53 response gene, bax. In addition, we report that alpha6beta4 triggers apoptosis in carcinoma cells that express wild-type but not mutant p53 and that these alpha6beta4 functions are inhibited by a dominant negative p53 construct. Importantly, we provide a link between integrin signaling and p53 activation by demonstrating that the clustering of alpha6beta4 with a beta4 integrin-specific antibody promotes p53-dependent apoptosis in cells that express both alpha6beta4 and wild-type p53. These studies are the first to demonstrate that a specific integrin can promote apoptosis by activating p53. Moreover, given the ability of alpha6beta4 to stimulate invasion (Shaw, L. M., Rabinovitz, I., Wang, H. F., Toker, A., and Mercurio, A. M. (1997) Cell 91, 949-960), these studies suggest that the ability of alpha6beta4 to promote carcinoma progression will be enhanced in tumor cells that express mutant, inactive forms of p53.

The cleavage of Akt/protein kinase B by death receptor signaling is an important event in detachment-induced apoptosis.

Epithelial cells undergo death receptor-dependent apoptosis when detached from matrix, a process termed anoikis. Activation of Akt/protein kinase B (PKB) by matrix attachment protects cells from anoikis. In this study, we establish a link between anoikis and Akt/PKB-mediated survival by demonstrating that Akt/PKB is cleaved by caspases in matrix-detached epithelial cells by a mechanism that involves death receptors. Reduced levels of Akt/PKB protein were observed in detached Madin-Darby canine kidney cells relative to cells attached to collagen. Equivalent levels of Akt/PKB, however, were detected in matrix-adherent and detached cells after inhibition of caspase activity or expression of an Akt/PKB mutant (D108+119A) that is resistant to caspase cleavage. The contribution of death domain-containing proteins to Akt/PKB cleavage was evidenced by the ability of dominant negative Fas-associated death domain to restore normal levels of Akt/PKB in matrix-detached cells. Importantly, expression of a cleavage-resistant Akt/PKB mutant protected matrix-detached cells from apoptosis. These studies suggest that members of the death receptor family promote the caspase-mediated cleavage of Akt/PKB and that this event contributes to anoikis.

Integrin laminin receptors and breast carcinoma progression.

This review explores the mechanistic basis of breast carcinoma progression by focusing on the contribution of integrins. Integrins are essential for progression not only for their ability to mediate physical interactions with extracellular matrices but also for their ability to regulate signaling pathways that control actin dynamics and cell movement, as well as for growth and survival. Our comments center on the alpha6 integrins (alpha6beta1 and alpha6beta4), which are receptors for the laminin family of basement membrane components. Numerous studies have implicated these integrins in breast cancer progression and have provided a rationale for studying the mechanistic basis of their contribution to aggressive disease. Recent work by our group and others on mechanisms of breast carcinoma invasion and survival that are influenced by the alpha6 integrins are discussed.