The binding of extracellular matrix proteins to integrins initiates survival signals that inhibit anoikis, a form of apoptosis induced upon the detachment of cells from extracellular matrix (Meredith et al. 1993
; Frisch and Francis 1994
). In the current studies, we show that the α6β4 integrin suppresses anoikis exclusively in carcinoma cells that lack functional p53. Furthermore, we demonstrate that this α6β4-associated survival function depends on the ability of this integrin to activate the serine/threonine kinase AKT/PKB in p53-deficient cells. Finally, we provide evidence that p53 inhibits the α6β4-mediated activation of AKT/PKB by promoting the caspase 3–dependent cleavage of this kinase. Collectively, our findings establish that p53 can inhibit an integrin-associated survival function, a phenomenon that has important implications for tumor cell growth.
Our results suggest that the α6β4 integrin can enhance the survival of carcinoma cells in an AKT/PKB–dependent manner. Although previous studies have shown that cell attachment to matrix proteins promotes the survival of primary epithelial cells (Khwaja et al. 1997
; Farrelly et al. 1999
), α6β4 is the first specific integrin to be implicated in the delivery of AKT/PKB–dependent survival signals to carcinoma cells. The importance of AKT/PKB in α6β4 survival signaling was indicated in our studies by the ability of a dnAKT/PKB construct containing inactivating mutations in the catalytic domain to inhibit the survival effect of α6β4 in serum-starved MDA-MB-435 cells. Although this dnAKT/PKB has been used extensively to implicate AKT/PKB in survival pathways, it is possible that it associates with phosphoinositide-dependent kinases and inhibits their activity. However, our observation that the expression of a constitutively active AKT/PKB in MDA-MBA-435 enhances their survival (data not shown) strongly suggests that α6β4 expression promotes the survival of these cells by activating AKT/PKB.
Our demonstration that p53 can inhibit AKT/PKB kinase activity is of interest in light of the recent finding that the PTEN tumor suppressor can also inhibit cell growth by inhibiting AKT/PKB in a manner that is dependent on its lipid phosphatase activity (Myers et al. 1998
; Stambolic et al. 1998
; Davies et al. 1999
; Ramaswamy et al. 1999
; Sun et al. 1999
). Together, our current findings on p53 and the previously described activities of PTEN highlight the impact of tumor suppressors on integrin-mediated functions. Moreover, our demonstration that p53 inhibits α6β4 survival signaling by promoting the caspase-dependent cleavage of AKT/PKB provides a mechanistic link between tumor suppressor function and the regulation of integrin signaling, similar to the phosphatase activities of PTEN. Although previous studies have demonstrated that caspases can be activated by p53 in both cell-free systems (Ding et al. 1998
) as well as in response to DNA damage (Fuchs et al. 1997
; Yu and Little 1998
), our findings suggest that caspases can also be activated by an integrin in a p53-dependent manner. Indeed, it will be informative to determine if other activators of p53 such as DNA damage (Siegel et al. 1995
; Komarova et al. 1997
) can promote the caspase-dependent cleavage of AKT/PKB.
The finding that AKT/PKB activity can be regulated by caspase 3 substantiates the hypothesis that caspases play an important role in many forms of apoptosis based on their ability to cleave signaling molecules that influence cell survival. For example, caspases have been shown to cleave and inactivate an inhibitor of caspase-activated deoxyribonuclease (CAD). Importantly, the cleavage of this inhibitor results in the activation of CAD, which is the enzyme responsible for the DNA fragmentation that is characteristic of apoptosis (Enari et al., 1998; Sakahira et al. 1998
). Caspase 3 has also been shown to cleave bcl-2, resulting in an inhibition of its anti-apoptotic function (Cheng et al. 1997
). While AKT/PKB has been suggested to be a target of caspase activity based on the reduced levels of this kinase observed in T cells in response to fas stimulation (Widmann et al. 1998
), our results extend this finding by establishing definitively that AKT/PKB is cleaved by caspase 3. More importantly, we provide evidence that this cleavage event results in the inhibition of AKT/PKB kinase activity, and implicate this event in the inhibition of α6β4 integrin survival function.
It is important to consider the mechanism by which the α6β4-induced, caspase-dependent cleavage of AKT/PKB inhibits its kinase activity. We detected an AKT/PKB fragment (Mr
, 49 kD) after the in vitro incubation of AKT/PKB with recombinant caspase 3. This fragment was recognized by a rabbit antiserum raised against a peptide corresponding to the extreme carboxy-terminal amino acids of the molecule, suggesting that caspase 3 cleaves AKT/PKB at its amino terminus. Interestingly, the pleckstrin homology domain, which resides in the amino terminus of AKT/PKB, is important in both the translocation of this kinase to the membrane and its subsequent activation (Franke et al. 1995
; Andjelkovic et al. 1997
). It is possible that the caspase 3–dependent cleavage of AKT/PKB prevents the membrane translocation of this kinase, thus, preventing its activation. However, we were unable to identify an AKT/PKB fragment in vivo after the clustering of α6β4, despite our detection of reduced AKT/PKB levels under these conditions. This result suggests that after the initial cleavage of AKT/PKB by caspase 3, this kinase is subjected to further cleavage by other caspases, as has been shown for ICAD (Tang and Kidd 1998
). Moreover, our inability to detect AKT/PKB fragments in vivo after the clustering of α6β4 suggests that AKT/PKB cannot be detected by immunoblotting after its cleavage by multiple caspases. The ability of a caspase 3 inhibitor to restore both normal AKT/PKB levels as well as the α6β4-mediated activation of AKT/PKB suggests that the degradation of AKT/PKB observed in vivo is dependent on the initial cleavage of this kinase by caspase 3.
In contrast to our finding that p53-dependent, caspase 3 activity inhibits AKT/PKB, other studies have concluded that constitutively active AKT/PKB can delay p53-dependent apoptosis (Sabbatini and McCormick 1999
), inhibit caspases (Cardone et al. 1998
), and block caspase-dependent forms of apoptosis (Berra et al. 1998
; Gibson et al. 1999
). The demonstrated ability of AKT/PKB to inhibit p53 and caspase activity in these studies may relate to the kinetics of AKT/PKB activation. Specifically, the rapid stimulation of AKT/PKB may impede p53 or caspase activation. In contrast, the ability of α6β4 clustering to promote the caspase 3–dependent inactivation of AKT/PKB in p53 wild-type carcinoma cells may relate to the fact that α6β4 signaling stimulates caspase activity before AKT/PKB activity in these cells. Alternatively, it is possible that the ability of caspase 3 to cleave AKT/PKB was not observed in previous studies because insufficient amounts of endogenous caspase activity were present to inhibit the activity of exogenously introduced, active AKT/PKB. Nonetheless, these results suggest that an intimate crosstalk exists between AKT/PKB and caspases that contributes to the regulation of cell survival.
We have previously demonstrated that the α6β4 integrin activates p53 function (Bachelder et al. 1999
). The current studies describe an important consequence of this α6β4 activity, namely the inhibition of AKT/PKB activity and its associated cell survival function. Similar to previous results from our laboratory (Clarke et al. 1995
; Shaw et al. 1997
; O'Connor et al. 1998
) and others (Kim et al. 1997
; Sun et al. 1998
), the current studies demonstrate that the survival function of α6β4 is ligand-independent in β4-transfected, p53-deficient carcinoma cells. This ligand-independent survival function may be attributable to the ability of the β4 cytoplasmic domain to self-associate (Rezniczek et al. 1998
In addition to demonstrating that p53 inhibits α6β4-mediated survival, we observed that α6β4 increases the level of apoptosis observed in serum-starved p53 wild-type carcinoma cells. This result suggests that the apoptotic signaling pathway activated by α6β4 can augment the apoptotic signaling initiated by serum deprivation. Although p53 has been implicated in the apoptosis induced in endothelial cells upon their detachment from matrix (Ilic et al. 1998
), others have reported that epithelial cell anoikis is p53-independent (Boudreau et al. 1995
). In agreement with the results of the latter study, we observed apoptosis in p53-deficient cells, including MDA-MB-435 cells and dnp53-expressing RKO cells, upon their detachment from matrix. These results indicate that carcinoma cells are subject to a p53-independent form of anoikis. In combination with our previous observation that α6β4 apoptotic signaling requires p53 activity (Bachelder et al. 1999
), our findings suggest that the p53-independent apoptosis of carcinoma cells that occurs in response to matrix detachment can be enhanced by p53-dependent, α6β4 apoptotic signaling.
The current studies may explain why the α6β4 integrin has been implicated in the apoptosis of some cells and the survival of others. Specifically, α6β4 has been shown to induce growth arrest and apoptosis in several carcinoma cell lines (Clarke et al. 1995
; Kim et al. 1997
, Sun et al. 1998
) as well as in endothelial cells (Miao et al. 1997
). However, this integrin has also been shown to promote the proliferation (Mainiero et al. 1997
; Murgia et al. 1998
) and survival (Dowling et al. 1996
) of keratinocytes. These apparently contradictory functions of α6β4 may relate to the fact that the functions of α6β4 are cell type–specific. The current studies establish that the p53 tumor suppressor is one critical signaling molecule that may influence α6β4 function in different cell types because this integrin promotes apoptosis only in wild-type p53-expressing cells and survival only in p53-deficient cells. Interestingly, the reported ability of α6β4 to promote keratinocyte survival (Dowling et al. 1996
) may relate to the reported deficiency of p53 activity in these cells (Nigro et al. 1997
One implication of our findings is that the α6β4 integrin is similar to a number of oncogenes that promote cell proliferation in some settings and cell death in others. The recent observation that oncogenes can deliver such death signals has led to their seemingly contradictory categorization as tumor suppressors in select environments. For example, although the stimulation of c-myc and E2F normally promotes cell proliferation, the activation of these oncogenes induces apoptosis in the presence of secondary stress signals such as p53 expression, serum starvation or hypoxia (Evan et al. 1992
; Shi et al. 1992
, Hermeking and Eick 1994
; Qin et al. 1994
; Wu and Levine 1994
). The ability of these stress signals to stimulate oncogene-dependent apoptosis is thought to be important in eliminating tumor cells that escape normal proliferation checkpoints as a result of oncogene expression. Similarly, the α6β4 integrin, which promotes the survival of p53-deficient cells, could also be classified loosely as a tumor suppressor based on its apoptotic function in carcinoma cells that express wild-type p53. The current studies demonstrate that, similar to the activity of oncogenes, integrin function and signaling can be profoundly influenced by physiological stimuli that activate other signaling pathways in a cell.
In summary, we have described the ability of the α6β4 integrin to promote the survival of the p53 mutant, but not p53 wild-type carcinoma cells. This ability of p53 to influence integrin-mediated functions so markedly derives from its ability to activate the caspase 3–dependent cleavage of AKT/PKB. The fact that AKT/PKB overexpression has been suggested to contribute to the transformed phenotype of tumor cells (Bellacosa et al. 1995
) suggests that the introduction of the α6β4 integrin into p53 wild-type tumors may inhibit their growth by inducing the cleavage of this transforming protein. The ability of α6β4 to induce the p53-dependent cleavage of AKT/PKB also suggests that the acquisition of inactivating mutations in either p53 or caspase 3 will provide a selective growth advantage for carcinoma cells by stimulating α6β4-mediated AKT/PKB–dependent survival signaling. Moreover, given our previous demonstration that α6β4 promotes carcinoma cell migration and invasion (Chao et al. 1996
, Shaw et al. 1997
; O'Connor et al. 1998
), we suggest that carcinoma cells that express α6β4 and mutant forms of p53 or caspase 3 will have a distinct advantage in their ability to disseminate and survive as metastatic lesions.