1Valentina Folgiero, 1Giulia Bon, 1Ada Sacchi, 2Arthur M Mercurio, 1Rita Falcioni
1Regina Elena Cancer Institute – Department of Experimental Oncology - Molecular Oncogenesis Laboratory, 00158 Rome, Italy
2Department of Cancer Biology - University of Massachusetts Medical School, Worcester, MA 0605
The integrin α6β4 is a cell surface receptor whose major function in normal epithelial tissues is to promote the adhesion and to stabilize the connection between epiderma and derma. In epithelial tumours, expression of the β4 subunit is often mainatined and it facilitates key functions of carcinoma cells including their ability to migrate, invade and evade apoptosis. The mechanism involved appears to be a profound effect of α6β4 on specific signaling pathways, especially the PI3-K/Akt pathway. An intimate relationship between α6β4 and growth factor receptors may explain this effect of α6β4 on signaling.
Here, we report that α6β4 can regulate the expression of ErbB-3 at the protein level, and that the consequent formation of an ErbB-2/ErbB-3 heterodimer promotes the α6β4-dependent activation of PI3-K/Akt and the ability of this integrin to impede apoptosis and promote the survival of carcinoma cells. These findings provide one mechanism to account for the activation of PI3-K by α6β4 and they also provide insight into the regulation of ErbB-3 in carcinoma cells.
The α6β4 integrin was identified initially as an epithelial-specific integrin and it was thought that expression of the β4 subunit was limited to cells of epithelial origin (1). A tumor antigen associated with metastasis, identified as (TS180) (2), was later shown to be identical to the β4 integrin subunit (3). This work provided the first indication that α6β4 may be linked to tumor progression. Subsequently, other studies revealed that expression of α6β4 persists in some aggressive carcinomas and that its expression may be linked to the behavior of these tumors (4).
Although α6β4 has been reported to activate several key signaling molecules in carcinoma cells, its ability to activate PI3-K survival pathway has attracted the most attention, especially with regard to its influence on function. A critical, unresolved issue, however, is the mechanism by which α6β4 activates PI3-K (5). This mechanism is probably not a direct activation of PI3-K by α6β4 because the β4 cytoplasmic domain lacks a consensus sequence for binding the PI3-K. In this direction, studies by our laboratory provided the first evidence that α6β4 may associate with ErbB-2 (6), an orphan receptor of the EGFR family, on the surface of some breast carcinoma cell lines. Subsequent studies using a 3T3 fibroblast cell model system demonstrated that both α6β4 and erbB2 are required for PI3-K activation and the stimulation of invasion (7). Given that ErbB-2 is thought to function only when it heterodimerizes with other members of the EGF receptor family, these data imply that other receptors cooperate in this process.
In the current study, we investigated the relationship between α6β4 and ErbB signaling especially in the context of PI3-K activation. Specifically, we focused on ErbB-3, which possesses, in its cytoplasmic tail, six consensus binding sites for the regulatory subunit of PI3K (8). Among the EGFR family, the ErbB-2/ErbB-3 heterodimer is the strongest stimulator of the PI3K activity (9) and the over-expression of both proteins promotes breast carcinoma proliferation and survival (10-11). Our data reveal that α6β4 can regulate the expression of ErB-3 at the level of protein translation resulting in a significant induction of ErbB-2/ErbB-3 heterodimerization and consequent activation of PI3-K.
It is widely assumed that the distinct signaling properties of the α6β4 integrin account for its ability to enhance functions associated with carcinoma progression such as invasion and resistance to apoptotic stimuli (12). In this study, we highlight a novel mechanism by which α6β4 can impact activation of the PI3-K/Akt pathway. This mechanism involves the ability of α6β4 to regulate the expression of ErbB-3, and we provide evidence that this regulation occurs at the level of protein translation. The increase in ErbB-2/ErbB-3 heterodimer formation that occurs as a consequence stimulates Akt activity. Indeed, the ability of heregulin, the ErbB-3 ligand, to stimulate PI3K/Akt activity is enhanced markedly in cells that express α6β4. Although previous studies implicated α6β4 in the facilitation of growth factor receptor signaling (4), ours is the first study to demonstrate that this integrin can promote such signaling by regulating the expression of a specific growth factor receptor.
The data reported here are a direct extension of our previous work that demonstrated an interaction between α6β4 and ErbB-2 in breast carcinoma cell lines and revealed the necessity of ErbB-2 for the α6β4-dependent activation of the PI3-K/Akt pathway and for α6β4-mediated functions such as invasion and survival (5-8). Given that ErbB-2 is thought to function only when it heterodimerizes with other members of the EGF receptor family (13), the 'missing link' in our previous studies was the identification of an ErbB-2 heterodimer that facilitates α6β4-dependent functions. Clearly, ErbB-3 is one EGF receptor family member that can serve in this capacity, a finding that is highly relevant because both the ErbB-2/ErbB-3 heterodimer and α6β4 have been implicated in breast cancer progression and metastasis.
It is also worth noting that ours is the first study to implicate ErbB-3 directly in the survival of breast carcinoma cells.
Our conclusion that α6β4 can regulate ErbB-3 expression and promote ErbB-2/ErbB-3 heterodimer formation and signaling is based on evidence obtained from carcinoma cells that express endogenous α6β4 and ErbB-2/ ErbB-3, carcinoma cells that lack expression of either α6β4 or ErbB-3, as well as a 3T3 cell model system. The inference could be made from these results that all breast carcinoma cells that express α6β4 also express ErbB-3 and, conversely, that all breast carcinoma cells that express ErbB-3 also express α6β4. In a recent test of this hypothesis, Giulia Bon demonstrated that ablation of α6β4 expression by interference in an aggressive and metastatic breast carcinoma cell line significantly increase the ability of the cells to respond to hormone treatment by Tamoxifen and impaired the ability of these cells to form xenograft tumors (12). In these mammary tumor cells she also found that the ablation of β4 subunit inhibits the activity of PI3K. Subsequently, Valentina Folgiero found that the ablation of β4 down regulates the expression of ErbB-3 protein (14). These results suggested for the first time that α6β4-depedent PI3K activity and ErbB-3 expression could be one of the mechanisms responsible for Tamoxifen resistance in mammary tumors.
In summary, the data we report substantiate the hypothesis that the function and signaling properties of the α6β4 integrin in breast carcinoma cells is intimately associated with the EGF receptor family.
These data also support the possibility that the impact of α6β4 on the functions of carcinoma cells is mediated, in part, by the ability of this integrin to enhance the translation of key growth factor receptors.
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