It is likely that changes in integrin expression facilitate tumor invasion because of their combined effects on cell adhesion and signaling (Giancotti and Mainiero, 1994
; Varner and Cheresh, 1996
; Boudreau and Bissell, 1998
). The α6β4 integrin is expressed at higher levels in squamous carcinomas of the skin, larynx, cervix, and lung than in corresponding normal tissues (Kimmel and Carey, 1986
; Costantini et al., 1990
). In addition, whereas normal thyroid epithelium does not express α6β4, the integrin appears to be present at significant levels on the surface of a large proportion of thyroid carcinomas (Serini et al., 1996
). Several studies suggest that α6β4 facilitates tumor invasion (Wolf et al., 1990
; Carico et al., 1993
; Tani et al., 1996
; Shaw et al., 1997
). If this is true, a mechanism must exist to inactivate the ability of α6β4 to mediate stable adhesion to the basement membrane. Our results indicate that a fraction of EGF-R combines with α6β4 and, through the integrin-associated Src family kinase Fyn, induces tyrosine phosphorylation of the β4 cytoplasmic domain and disruption of hemidesmosomes. Dominant negative Fyn, but not Src, stabilizes hemidesmosomes and prevents their disassembly in response to EGF. Accordingly, carcinoma cells expressing dominant negative Fyn migrate and invade in vitro much less than control cells and are unable to form lung metastases upon injection in the tail vein of nude mice. These findings identify a mechanism of hemidesmosome disassembly important for both normal cell migration and carcinoma invasion.
Previous studies have indicated that cell migration and invasion require assembly of focal adhesions and stress fibers at the leading edge of the cell and their disassembly at the trailing edge (Horwitz and Parsons, 1999
). These processes are jointly regulated by β1 and αv integrins and by growth factor receptors through the focal adhesion kinase FAK (Sieg et al., 2000
) and the adaptor protein Shc (Collins et al., 1999
; Gu et al., 1999
). Upon activation, FAK combines with Src, which phosphorylates a number of focal adhesion components, including p130CAS
and paxillin (Giancotti and Ruoslahti, 1999
). Genetic evidence implies that Src signaling stimulates cell migration by promoting spreading of the leading edge and turnover of focal adhesions at the trailing edge (Kaplan et al., 1995
; Fincham and Frame, 1998
; Klinghoffer et al., 1999
). Shc is activated by Fyn and possibly other palmitoylated Src family kinases (Wary et al., 1998
) and plays a role in cell migration through activation of ERK and disassembly of focal adhesions (Collins et al., 1999
; Gu et al., 1999
). In addition to, or instead of, focal adhesions, many epithelial cells form hemidesmosomes. Our results argue that their disassembly in response to activation of the EGF-R and phosphorylation of the β4 tail is required for epithelial cell migration and invasion.
What is the mechanism by which EGF causes phosphorylation of the β4 cytoplasmic domain? Our results indicate that a fraction of EGF-R associates with α6β4 and induces phosphorylation of the β4 cytoplasmic domain through the Src family kinase Fyn (). Dominant negative studies suggest that Src can not replace Fyn in this pathway. This is most likely because Fyn combines with α6β4, but Src does so much less efficiently. By contrast, Yes combines relatively efficiently with α6β4. The NH2-terminal domains of both Fyn and Yes are known to be palmitoylated and we have observed that the association of Fyn with α6β4 requires this lipid modification. It is likely that the association of Yes with α6β4 similarly requires palmitoylation of the kinase.
Figure 9. Relationship between α6β4 signaling and assembly of hemidesmosomes. Hypothetical model of the underlying pathways. We have observed that the palmitoylated fraction of α6β4 is localized in lipid rafts and is preferentially (more ...)
Previous studies have indicated that palmitoylation targets Fyn and other signaling molecules to defined membrane microdomains enriched in cholesterol and glycosphingolipids (membrane rafts) (van't Hof and Resh, 1997
; Brown and London, 1998
). Increasing evidence suggests that targeting of signaling molecules to membrane rafts promotes compartmentalization of signaling complexes, thus ensuring signaling specificity. A prime example is provided by the T cell receptor. Upon T cell receptor activation, its ζ chains accumulate in membrane rafts, where they interact with Fyn and other signaling molecules (Baldari et al., 2000
). Palmitoylation of Fyn is required to localize the kinase to rafts, where it phosphorylates the ζ chain immunoreceptor tyrosine-based activation motifs (ITAMs). The SH2 domain of Fyn then combines with the phosphorylated ITAMs stabilizing the interaction with the ζ chain (van't Hof and Resh, 1999
). Interestingly, we have recently observed that a significant fraction of α6β4 is palmitoylated and localized to membrane rafts (unpublished data). It is possible that palmitoylation of α6β4 mediates localization to membrane rafts thus facilitating interaction with Fyn ().
Our mutagenesis results indicate that the association of α6β4 with Fyn requires the membrane proximal segment of the cytoplasmic domain of β4, but not the SH2 domain, the SH3 domain, or the enzymatic function of Fyn, suggesting that palmitoylation of α6β4 and Fyn may be required but not sufficient to mediate the interaction between the two molecules. It is likely that the membrane proximal portion of the cytoplasmic domain of β4 and the NH2-terminus of Fyn establish a protein–protein interaction. However, future studies will be required to identify more precisely the protein sequences involved in this interaction. In addition, although our data are most consistent with the hypothesis that Fyn interacts with and phosphorylates β4 directly, this remains to be established formally.
The cytoplasmic domain of β4 has both a signaling and a cytoskeletal function. What is the relationship between these two functions? Ligation of α6β4 causes a rapid phosphorylation of the β4 tail, followed by recruitment of Shc and activation of ERK (Mainiero et al., 1995
). In addition, there is evidence that α6β4 activates PI-3K, either through Ras or IRS-1 and 2, rapidly (Mainiero et al., 1997
; Shaw et al., 1997
; Shaw, 2001
). Finally, our results suggest that the EGF-R can activate α6β4 signaling, by acting on Fyn, with a similarly rapid kinetics. By contrast, assembly of hemidesmosomes is a much slower process, at least in cultured cells (Riddelle et al., 1991
). In addition, because exposure to EGF induces both phosphorylation of β4 and disruption of hemidesmosomes, it is likely that α6β4-dependent signaling and nucleation of hemidesmosomes are mutually exclusive processes. It is possible that phosphorylation of β4 by Fyn prevents incorporation of α6β4 in hemidesmosomes ().
Previous studies have indicated that mutation of the four tyrosine residues in β4 necessary for binding to Shc prevents disassembly of hemidesmosomes in cells exposed to the tyrosine phosphatase inhibitor pervanadate (Dans et al., 2001
). However, this mutation does not interfere with hemidesmosome disruption in response to EGF in both 804G cells (Dans et al., 2001
), and in keratinocytes (unpublished data). It is possible that EGF prevents assembly of hemidesmosomes by promoting phosphorylation of additional tyrosine residues, as suggested by the fact that mutation of the four tyrosines necessary for binding to Shc reduces but does not suppress tyrosine phosphorylation of β4 in response to EGF (unpublished data). In addition, it is known that the EGF-R activates PKC through PLC-γ (Chen et al., 1996
), and there is evidence suggesting that inhibition of PKC prevents EGF-mediated disruption of hemidesmosomes (Rabinovitz et al., 1999
). Thus, it is also possible that PKC contributes to disruption of hemidesmosomes by phosphorylating the β4 tail on serine residues (Rabinovitz et al., 1999
) or by contributing to the activation of Fyn (Shanmugam et al., 1998
). Finally, although we did not detect tyrosine phosphorylation of BPAG-1 or -2, or plectin/HD-1 in cells treated with EGF (unpublished data), activation of the EGF-R could affect the function of other hemidesmosomal components or the stability of other interactions within hemidesmosomes.
Introduction of α6β4 in breast carcinoma cells that have lost its expression promotes activation of the PI-3K–Rac pathway and invasion through Matrigel (Shaw et al., 1997
). Because it is well established that PI-3K–Rac signaling is important for tumor invasion (Keely et al., 1997
), it is possible that, in addition to promoting disruption of hemidesmosomes and thus releasing the cell's brakes, α6β4-associated Fyn promotes migration and invasion by activating the PI-3K–Rac pathway or signaling to ERK ().
In squamous carcinoma cells, the fraction of EGF-R associated with α6β4 appears to be activated even in the absence of exogenous EGF. Hence, these cells display enhanced tyrosine phosphorylation of β4 and reduced ability to assemble hemidesmosomes. These observations imply that the signaling pathway that prevents assembly of hemidesmosomes is constitutively activated in squamous carcinoma cells. Dominant negative Fyn suppresses the ability of these cells to migrate and invade both in vitro and in vivo suggesting that disruption of hemidesmosomes is required for carcinoma invasion. Because in our experimental metastasis assay the cells were injected directly into the blood stream, Fyn is probably required in one or more steps of the extravasation process, perhaps traversal of the endothelial basement membrane and/or underlying interstitial matrix. To our knowledge, our study provides the first evidence that inhibition of a Src family kinase suppresses a complex phenomenon such as metastasis. It is plausible that strategies aimed at inhibiting the kinase activity of Fyn may be useful to control squamous carcinoma progression.