We report that the activity of glycogen synthase kinase-3 (GSK-3) is necessary for the maintenance of the epithelial architecture. Pharmacological inhibition of its activity or reducing its expression using small interfering RNAs in normal breast and skin epithelial cells results in a reduction of E-cadherin expression and a more mesenchymal morphology, both of which are features associated with an epithelial–mesenchymal transition (EMT). Importantly, GSK-3 inhibition also stimulates the transcription of Snail, a repressor of E-cadherin and an inducer of the EMT. We identify NFκB as a transcription factor inhibited by GSK-3 in epithelial cells that is relevant for Snail expression. These findings indicate that epithelial cells must sustain activation of a specific kinase to impede a mesenchymal transition.

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 α6β4 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 α6β4 in carcinoma cells that express wild-type p53. In contrast, α6β4 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 α6β4-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 α6β4 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.

We define a novel mechanism by which integrins regulate growth factor expression and the survival of carcinoma cells. Specifically, we demonstrate that the α6β4 integrin enhances vascular endothelial growth factor (VEGF) translation in breast carcinoma cells. The mechanism involves the ability of this integrin to stimulate the phosphorylation and inactivation of 4E-binding protein (4E-BP1), a translational repressor that inhibits the function of eukaryotic translation initiation factor 4E (eIF-4E). The regulation of 4E-BP1 phosphorylation by α6β4 derives from the ability of this integrin to activate the PI-3K–Akt pathway and, consequently, the rapamycin-sensitive kinase mTOR that can phosphorylate 4E-BP1. Importantly, we show that this α6β4-dependent regulation of VEGF translation plays an important role in the survival of metastatic breast carcinoma cells by sustaining a VEGF autocrine signaling pathway that involves activation of PI-3K and Akt. These findings reveal that integrin-mediated activation of PI-3K–Akt is amplified by integrin-stimulated VEGF expression and they provide a mechanism that substantiates the reported role of α6β4 in carcinoma progression.

Aberrant cell survival and resistance to apoptosis are hallmarks of tumor invasion and progression to metastatic disease, but the mechanisms involved are poorly understood. The epithelial-mesenchymal transition (EMT), a process that facilitates progression to invasive cancer, provides a superb model for studying such survival mechanisms. Here, we used a unique spheroid culture system that recapitulates the structure of the colonic epithelium and undergoes an EMT in response to cytokine stimulation to study this problem. Our data reveal that the EMT results in the increased expression of both VEGF and Flt-1, a tyrosine kinase VEGF receptor, and that the survival of these cells depends on a VEGF/Flt-1 autocrine pathway. Perturbation of Flt-1 function by either a blocking antibody or adenoviral expression of soluble Flt-1, which acts in a dominant-negative fashion, caused massive apoptosis only in cells that underwent EMT. This pathway was critical for the survival of other invasive colon carcinoma cell lines, and we observed a correlative upregulation of Flt-1 expression linked to in vivo human cancer progression. A role for Flt-1 in cell survival is unprecedented and has significant implications for Flt-1 function in tumor progression, as well as in other biological processes, including angiogenesis and development.

Although many tumors regress in response to neoadjuvant chemotherapy, residual tumor cells are detected in most cancer patients post-treatment. These residual tumor cells are thought to remain dormant for years before resuming growth, resulting in tumor recurrence. Considering that recurrent tumors are most often responsible for patient mortality, there exists an urgent need to study signaling pathways that drive tumor dormancy/recurrence. We have developed an in vitro model of tumor dormancy/recurrence. Short-term exposure of tumor cells (breast or prostate) to chemotherapy at clinically relevant doses enriches for a dormant tumor cell population. Several days after removing chemotherapy, dormant tumor cells regain proliferative ability and establish colonies, resembling tumor recurrence. Tumor cells from “recurrent” colonies exhibit increased chemotherapy resistance, similar to the therapy resistance of recurrent tumors in cancer patients. Previous studies using long-term chemotherapy selection models identified acquired mutations that drive tumor resistance. In contrast, our short term chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that can resume growth after drug removal. Studying unique signaling pathways in dormant tumor cells enriched by short-term chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence.

The interaction of integrins with extracellular matrix is known to promote cell survival by inhibiting apoptotic signaling. In contrast, we demonstrate here that the ␣ 6 ␤ 4 integrin induces apoptosis in carcinoma cells by stimulating p53 function. Specifically, we show that expression of ␣ 6 ␤ 4 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 ␣ 6 ␤ 4 triggers apoptosis in carcinoma cells that express wild-type but not mutant p53 and that these ␣ 6 ␤ 4 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 ␣ 6 ␤ 4 with a ␤ 4 integrin-specific antibody promotes p53-dependent apoptosis in cells that express both ␣ 6 ␤ 4 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 ␣ 6 ␤ 4 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 ␣ 6 ␤ 4 to promote carcinoma progression will be enhanced in tumor cells that express mutant, inactive forms of p53.Integrins are the primary receptors used by cells to interact with extracellular matrices. Although initial studies had emphasized the functional contribution of integrins to cell adhesion and migration, a significant finding was the observation that integrins are essential for cell survival (2, 3). Specifically, epithelial cells, endothelial cells, and fibroblasts are prone to growth arrest and apoptosis when deprived of integrin-mediated contact with the extracellular matrix (4, 5). To date, several integrins including ␣ 5 ␤ 1 (6 -7), ␣ v ␤ 3 (8 -10), and ␣ 6 ␤ 1 (11) have been implicated in the promotion of cell growth and survival.Arguably, one of the most complex integrins in terms of both structure and function is ␣ 6 ␤ 4 . This integrin is distinguished structurally from other integrins on the basis of the unusually large cytoplasmic domain of its ␤ 4 subunit (12-14). Aside from its involvement in cell adhesion and migration (1,(15)(16)(17)(18)(19)(20), the ␣ 6 ␤ 4 integrin can promote growth arrest and apoptosis in some carcinoma cells. Specifically, we reported that ␣ 6 ␤ 4 expression induces the growth arrest and apoptosis of the RKO colon carcinoma cell line (21), a finding that has been substantiated in other carcinoma cell lines (22-23), as well as in endothelial cells (24). These findings, however, conflict with considerable evidence that supports a role for ␣ 6 ␤ 4 in promoting carcinoma invasion and progression (1,19). In order to understand how ␣ 6 ␤ 4 can deliver these apparently conflicting signals, we analyzed the mechanism by which ␣ 6 ␤ 4 promotes apoptosis in more detail. S...

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/PKBmediated 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 domaincontaining 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 matrixdetached cells. Importantly, expression of a cleavageresistant Akt/PKB mutant protected matrix-detached cells from apoptosis. These studies suggest that members of the death receptor family promote the caspasemediated cleavage of Akt/PKB and that this event contributes to anoikis.

The integrin A 6 B 4 has been shown to facilitate key functions of carcinoma cells, including their ability to migrate, invade, and evade apoptosis. The mechanism involved seems to be a profound effect of A 6 B 4 on specific signaling pathways, especially the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. An intimate relationship between A 6 B 4 and growth factor receptors may explain this effect of A 6 B 4 on signaling. Previously, we showed that A 6 B 4 and ErbB-2 can function synergistically to activate the PI3K/Akt pathway. Given that ErbB-2 can activate PI3K only when it heterodimerizes with other members of the epidermal growth factor receptor family, these data imply that other receptors cooperate in this process. Here, we report that A 6 B 4 can regulate the expression of ErbB-3 using several different models and that the consequent formation of an ErbB-2/ErbB-3 heterodimer promotes the A 6 B 4 -dependent activation of PI3K/Akt and the ability of this integrin to impede apoptosis of carcinoma cells. Our data also support the hypothesis that A 6 B 4 can regulate ErbB-3 expression at the translational level as evidenced by the findings that A 6 B 4 does not increase ErbB-3 mRNA significantly, and that this regulation is both rapamycin sensitive and dependent on eukaryotic translation initiation factor 4E. These findings provide one mechanism to account for the activation of PI3K by A 6 B 4 and they also provide insight into the regulation of ErbB-3 in carcinoma cells. [Cancer Res 2007;67(4):1645-52]