Integrin β4 Signaling in Cancer

The α6β4 integrin is a laminin-5 receptor expressed in many epithelial cells, in Schwann cells, and in endothelial cells. The cytoplasmic portion of β4 is much larger than that of other integrin β subunits and confers unique cytoskeletal associations and signaling functions to α6β4. Integrin α6β4 signaling proceeds through Src Family kinase (SFK)-mediated phosphorylation of the C-terminal portion of the cytoplasmic domain of α4, recruitment of Shc and activation of Ras and PI-3K. Upon dephosphorylation, the β4 tail associates with the keratin cytoskeleton, causing assembly of hemidesmosomes and, hence, strengthening adhesion to basement membranes containing laminin-5.

Integrin β4 signaling (adapted from Giancotti and Tarone. 2003. Annu. Rev. Cell Dev. Biol. 19:173-206).

A team effort in our lab led to the elucidation of the mechanism by which α6β4 mediates signal transduction. Fabrizio Mainiero discovered that ligand binding to α6β4 elicits tyrosine phosphorylation of the β4 cytoplasmic domain, recruitment of Shc, and activation of Ras to ERK signaling. Agnese Mariotti and Laurent Gagnoux-Palacios identified the SFKs Fyn and Yes as integrin-associated tyrosine kinases. Mike Dans mapped the four major tyrosine phosphorylation sites and defined the mechanism of recruitment of Shc. Fabrizio Mainiero and Agnese Mariotti demonstrated that the EGF-R combines with α6β4 and - through the integrin-associated SFK - induces phosphorylation of β4, causing disruption of hemidesmosomes and increased epithelial cell migration and proliferation. These results indicate that tyrosine phosphorylation of β4 promotes signaling but antagonizes assembly of hemidesmosomes, suggesting that these two functions may be mutually exclusive. Chiara Murgia and Pamela Blaikie generated mice carrying two distinct targeted deletions of the cytoplasmic domain of β4 and demonstrated that the β4 cytoplasmic tail is necessary for stable epidermal adhesion and proliferation in vivo. These mouse models have improved our understanding of the physiological function of α6β4 and the pathogenesis of a lethal form of skin blistering, PA-JEB (junctional epidermolysis bullosa with pyloric atresia), which is caused by mutations in the genes encoding α6, β4, or laminin-5.

Sotiris Nikolopoulos has examined the phenotype of mice lacking the signaling portion of the cytoplasmic domain of β4. These mice are viable and fertile but exhibit defects in wound healing and tumor angiogenesis. These result indicate that β4 promotes the acquisition of an invasive phenotype by keratinocytes responding to wound signals and by endothelial cells participating in angiogenesis.

The β4 integrin is upregulated in many carcinomas, including those arising in the mammary gland. We and others have shown that activation of the EGF-R, ErbB2 and Met RTKs enhances phosphorylation of the β4 cytotail, causing disruption of hemidesmosomes and increased epithelial cell migration, suggesting that these RTKs decrease the ability of β4 to mediate stable adhesion but increase its signaling function. In order to examine the potential roles of β4 signaling in mammary tumorigenesis in vivo, Wenjun Guo crossed mice carrying a targeted deletion of the β4 signaling domain to MMTV-Neu mice. The results provided clear evidence that β4 signaling is required for ErbB2-induced mammary tumor progression. As compared to control mice, the β4 mutant mice displayed delayed tumor onset and greatly decreased tumor growth. In addition, they developed fewer lung metastases. Notably, loss of β4 signaling greatly sensitized the MMTV-Neu tumors to ErbB2 kinase inhibition. Through a combination of molecular cytology on tissue sections and biochemistry on cell lines isolated from the tumors, Wenjun obtained evidence that β4 cooperates with ErbB2 to promote tumor cell proliferation through activation of c-Jun, thereby facilitating tumor initiation and growth, and to disrupt cell polarity through activation of STAT3, thus promoting tumor invasion. Importantly, Wenjun discovered that β4 forms a physical complex with ErbB2 and potentiates and expands its signaling capability.

Immunoblotting shows that the activation of STAT3 and c-Jun is defective in ErbB2-transformed mammary epithelial cells carrying a knock-in mutation that deletes the c-terminal, signaling domain of the β4 integrin (left). The model illustrates the signaling mechanisms that enable β4 to amplify oncogenic ErbB2 signaling (right). See Guo et al. Cell 10:93-95, 2006.

We are currently studying the molecular mechanisms by which the β4 integrin expands ErbB2 signaling and the signaling pathways that mediate disruption of epithelial adhesion and polarity and suppression of anoikis in ErbB2-transformed breast tumor cells. In addition, we are examining the role of β4 signaling in prostate tumorigenesis.

Spinardi L, Ren Y-L, Sanders R, Giancotti FG. The β4 subunit cytoplasmic domain mediates the interaction of the α6β4 integrin with the cytoskeleton of hemidesmosomes. Mol. Biol. Cell 1993; 4:871-884.

Einheber S, Milner TA, Giancotti FG, Salzer JL. Axonal regulation of Schwann cell integrin expression suggests a role for α6β4 in myelination. J. Cell Biol. 1993; 123:1223-1236.

Spinardi L, Einheber S, Cullen T, Milner T, Giancotti FG. A recombinant tail-less integrin β4 subunit disrupts hemidesmosomes, but does not suppress α6β4-mediated cell adhesion to laminins. J. Cell Biol. 1995; 129:473-487.

Mainiero F, Pepe A, Spinardi L, Wary KK, Ammad M, Schlessinger J, Giancotti FG. Signal transduction by the α6β4 integrin: distinct β4 sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes. E.M.B.O. J. 1995; 14:4470-4481.

Mainiero F, Pepe A, Yeon M, Ren Y-L, Giancotti FG. The intracellular functions of α6β4 integrin are regulated by EGF. J. Cell Biol. 1996; 134:241-253.

Mainiero F, Murgia C, Wary KK, Curatola AM, Pepe A, Blumemberg M, Westwick JK, Der C, Giancotti FG. The coupling of α6β4 integrin to the Ras-MAP Kinase pathways mediated by Shc controls keratinocyte proliferation. E.M.B.O. J. 1997; 16:2365-2375.

Murgia C, Blaikie P, Dans M, Kim N, Petrie H, Giancotti FG. Cell cycle and adhesion defects in mice carrying a targeted deletion of the integrin β4 subunit cytoplasmic domain. E.M.B.O. J. 1998; 17:3940-3951.

Dans M, Gagnoux-Palacios L, Blaikie P, Klein S, Mariotti A, Giancotti FG. Tyrosine phosphorylation of the β4 integrin cytoplasmic domain mediates mitogenic signaling and antagonizes formation of hemidesmosomes. J. Biol. Chem. 2001, 276:1494-1502.

Mariotti A, Kedeshian PA, Dans M, Curatola AM, Gagnoux-Palacios L, Giancotti FG. EGF-R signaling through Fyn kinase disrupts the function of integrin α6β4 at hemidesmosomes: role in epithelial cell migration and carcinoma invasion. J Cell Biol. 2001, 155(3):447-58.

Weaver VM, Lelièvre S, Lakins JN, Chrenek MA, Jones JC, Giancotti F, Werb Z, Bissell MJ. β4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium. Cancer Cell. 2002, 2(3):205-16 [Preview by Jacks and Weinberg in Cell 11:923-925, 2002; News and Views by Yamada and Clark in Nature 419:790-791, 2002; Must Read F1000].

Gagnoux-Palacios L, Dans M, van’t Hof W, Mariotti A, Pepe A, Meneguzzi G, Resh MD, Giancotti FG. Compartmentalization of integrin α6β4 signaling in lipid rafts. J. Cell Biol. 2003, 162:1189-1196.

Nikolopoulos SN, Blaikie P, Yoshioka T, Guo W, Giancotti FG.Integrin β4 signaling promotes tumor angiogenesis. Cancer Cell. 2004 Nov;6(5):471-83.

Nikolopoulos SN, Blaikie P, Yoshioka T, Guo W, Puri C, Tacchetti C, Giancotti FG. Targeted deletion of the integrin β4 signaling domain suppresses laminin-5-dependent nuclear entry of mitogen-activated protein kinases and NF-kappaB, causing defects in epidermal growth and migration. Mol Cell Biol. 2005 Jul;25(14):6090-102.

Guo W, Pylayeva Y, Pepe A, Yoshioka T, Muller WJ, Inghirami G, Giancotti FG. β4 integrin amplifies ErbB2 signaling to promote mammary tumorigenesis. Cell. 2006, 126(3):489-502 [Preview by Muthuswamy, S.K. in Cell 126:443-445, 2006; Preview by Carraway, K.L. III, and Sweeney, C. in Cancer Cell 10(2):93-95, 2006; News and Views by Walker and Brugge in Nat Cell Biol 8:1220-1222, 2006; Exceptional F1000].