The expression of integrin alpha(v)beta6 promotes the epithelial cell morphology and suppresses invasive behavior in transformed oral keratinocytes.

The expression of the integrin alpha(v)beta6 has been correlated with oral SCC invasion. We evaluated its expression in three 4NQO transformed murine oral keratinocyte cell lines (B7E3, B7E11 and B4B8). The B7E3 cells were negative for beta6, whereas the B7E11 and the B4B8 cells were both positive. The beta6 negative B7E3 cells were fibroblast-like in appearance, whereas the B7E11 cells were more epithelial-like. The B4B8 cells were a mixture of the two cell types. Using immunofluorescent microscopy, we found that vimentin was highly expressed in the B7E3 cells, whereas the B7E11 cells keratin positive. The B4B8 cells expressed both filaments. The B7E3 cells formed large tumors when injected into nude mice, whereas the B4B8 cells formed small tumors and the B7E11 cells formed none. These results suggest that the expression of the alpha(v)beta6 integrin suppresses tumor formation and may promote the epithelial phenotype in 4NQO-transformed murine oral keratinocytes.

Matrix metalloproteinases and TGFbeta1 modulate oral tumor cell matrix.

The integrin beta6 has been shown to promote invasion and experimental metastasis by oral squamous cell carcinoma (SCC). In this study, we demonstrate that the expression of beta6 by oral SCC9 cells increased activation of the UPA --> MMP3 --> MMP9 pathway. We also demonstrate that the deposition of fibronectin and tenascin-C matrices by SCC9beta6 cells and peritumor fibroblast cocultures is counter-regulated by the UPA --> MMP3 --> MMP9 pathway. Suppression of individual components of this pathway increased the deposition of fibronectin, but decreased tenascin-C matrix assembly by the cocultures. When the SCC9beta6/PTF cocultures were incubated with TGFbeta1, the deposition of fibronectin and tenascin-C as well as the activation of MMP3 and MMP9 was increased. These results indicate that MMP3, MMP9, and TGFbeta1 are important for the modulation, composition, and maintenance of the ECM in oral SCC.

Specific alpha v integrin receptors modulate K1735 murine melanoma cell behavior.

Expression of beta 3 integrins is increased in invasive melanoma. In this study we show that K1735 cell proliferation is enhanced by the expression of either beta 3 or a constitutively active Src. We investigated possible modulators of FN matrix assembly and found that matrix metalloproteinase 2 (MMP2) was activated by alpha v beta 3. alpha v beta 3 integrin was localized to focal contacts whereas alpha v beta 5 was peripherally distributed. MMP2 was also activated by expression of CASrc. MMP2 activation inversely correlated with FN matrix assembly, in that it dramatically reduced the organization of a FN matrix. K1735 cell migration on VN and invasion through a reconstituted basement membrane were decreased in the presence of anti-MMP2 antibodies. These results demonstrate that the expression of the alpha v beta 3 complex modulates melanoma cell behavior including activation of Src, organization of the cytoskeleton, assembly of the extracellular matrix, cell motility, and activation of MMP2.

Alphavbeta6-Fyn signaling promotes oral cancer progression.

We have previously shown that the integrin beta6 is neo-expressed in invasive oral squamous cell carcinoma (SCC) and is correlated with oral tumor progression. However, the mechanism by which the integrin beta6 promotes oral tumor progression is not well understood. The purpose of the present study was to determine whether integrin beta6 signaling activates Fyn and thus promotes oral squamous cell carcinoma progression. We analyzed the integrin beta6 signaling complex and investigated the function of these signaling molecules in oral SCC cells. We found that, upon ligation of the integrin beta6 with fibronectin, beta6 complexed with Fyn and activated it. The activation of Fyn recruited and activated focal adhesion kinase to this complex. This complex was necessary to activate Shc and to couple beta6 signaling to the Raf-ERK/MAPK pathway. This pathway transcriptionally activated the matrix metalloproteinase-3 gene and promoted oral SCC cell proliferation and experimental metastasis in vivo. These findings indicate that integrin beta6 signaling activates Fyn and thus promotes oral cancer progression.

Expression of integrin beta 6 enhances invasive behavior in oral squamous cell carcinoma.

Oral squamous cell carcinoma (SCC) is characterized by invasive growth and the propensity for distant metastasis. The expression of specific adhesion receptors promotes defined interactions with the specific components found within the extracellular matrix (ECM). We previously showed that the alpha v beta 6 fibronectin receptor is highly expressed in oral SCC. Here we forced expression of the beta 6 subunit into poorly invasive SCC9 cells to establish the SCC9 beta 6 cell line and compared these two cell lines in several independent assays. Whereas adhesion to fibronectin was unaffected by the expression of beta 6, migration on fibronectin and invasion through a reconstituted basement membrane (RBM) were both increased. Function-blocking antibodies to alpha v beta 6 (10D5) reduced both migration on fibronectin and invasion through an RBM, whereas anti-alpha 5 antibodies were effective only in suppressing migration on fibronectin, not invasion. Expression of beta 6 also promoted tumor growth and invasion in vivo and modulated fibronectin matrix deposition. When grown as a co-culture with SCC9 cells, peritumor fibroblasts (PTF) organized a dense fibronectin matrix. However, fibronectin matrix assembly was decreased in co-cultures of SCC9 beta 6 cells and PTF and this decrease was reversed by the addition of function-blocking anti-alpha v beta 6 antibodies. The expression of beta 6 also resulted in increased levels of matrix metalloproteinase 3. Addition of the general MMP inhibitor GM6001 to SCC9 beta 6/PTF co-cultures dramatically increased fibronectin matrix assembly in a similar fashion as incubation with anti-alpha v beta 6 antibodies. These results demonstrate that expression of beta 6 (1) increases oral SCC cell motility and growth in vitro and in vivo; (2) negatively affects fibronectin matrix assembly; and (3) stimulates the expression and activation of MMP3. We suggest that the integrin alpha v beta 6 is a key component of oral SCC invasion and metastasis through modulation of MMP-3 activity.