Poster 11: Exploring the Relationship Between Integrin αvβ6 and αβ1 in Oral Squamous Cell Carcinoma

Abstract

The overall 5-year survival rate for oral squamous cell carcinoma (OSCC) remains among the lowest for major cancers, probably due to the ability of these tumors to metastasize. In OSCC, upregulation of integrin αvβ6 expression is recognized as a hallmark for metastases and poor prognosis. Integrins are a large family of αβ heterodimeric transmembrane receptors that link the cellular cytoskeleton to the extracellular matrix (ECM). Integrin expression dysregulation has been implicated in tumor formation and metastasis since successful invasion requires continuous remodeling of the ECM by integrins. Previous work from our lab suggests there might be an inverse relationship between αβ1 and αvβ6 expression in epithelial tumors; however, correlation between expression levels of these integrins and malignant phenotypes remains to be elucidated. Three OSCC cell lines, SCC9, SCC9β6 and SCC9β6β1, were used to study the effects of increased expression of αvβ6 in oral cancer. SCC9β6 cells were generated by co-culturing SCC9 cells with retroviral producer cell lines that expressed full-length β6. The SCC9β6 cells were then infected with β1-myc lentiviruses to generate the SCC9β6β1 cell line. Flow cytometry and Western blot analysis were used to evaluate levels of β6. SCC9β6 and SCC9β6β1 cells were FACS sorted to obtain cell populations with high β6 and high β6/β1 expression, respectively. The effect of differential integrin expression on growth of OSCC cells was evaluated using cell proliferation assays and scratch wound assays were used to access migratory abilities of the cells. Furthermore, the various cell lines were grown on coverslips coated with fibronectin (FN) and immunofluoresence was used to assess the ability of these cells to organize an FN matrix in vitro. Proliferation assays were also utilized to evaluate whether 0.01 mM zoledronate influenced the growth of SCC9β6 cells. Finally, FACS analysis was used to assess surface expression of αvβ6 in SCC9β6 cells and SCC9β6 cells grown in 0.01 mM zoledronate. Flow cytometry and Western blot analysis indicated there was a 10-fold increase in the expression of β6 in SCC9β6 compared to SCC9 cells. Cell proliferation assays demonstrated that SCC9β6 cells were more proliferative than the SCC9 cells, which showed more growth than the SCC9β6β1 cells. Scratch wound assays indicated that SCC9β6 cells were the most migratory, followed by SCC9β6β1 and finally by SCC9 cells. The SCC9 and SCC9β6β1 cells were able to organize the FN into a fibrillar pattern in association with peripheral focal adhesion whereas the SCC9β6 cells were not able to do so. Proliferation of SCC9β6 cells in the presence of zoledronate was greatly decreased compared to SCC9β6 cells grown in isolation and these cells showed a decrease in surface expression of αvβ6 by approximately 40%. This data suggest upregulation of integrin β6 increases the proliferative and migratory capacities of SCC9 cells while decreasing the matrix organizing ability of these cells. Furthermore, increased expression of β1 in SCC9β6 cells appears to reverse these malignant phenotypes. Therefore, the tipping of the balance of these integrins, upregulation of αvβ6 and downregulation of αβ1, may contribute to cell invasion and migration in OSCC. The ability of zoledronate to inhibit proliferation of SCC9β6 cells is of great interest since OSCC cells with increased αvβ6 expression are associated with a poor prognosis and there is currently no tumor cell specific therapy for the treatment of OSCC. Downregulation of αvβ6 in SCC9β6 cells grown in the presence of zoledronate suggests a possible mechanism by which zoledronate inhibits cell proliferation. The overall 5-year survival rate for oral squamous cell carcinoma (OSCC) remains among the lowest for major cancers, probably due to the ability of these tumors to metastasize. In OSCC, upregulation of integrin αvβ6 expression is recognized as a hallmark for metastases and poor prognosis. Integrins are a large family of αβ heterodimeric transmembrane receptors that link the cellular cytoskeleton to the extracellular matrix (ECM). Integrin expression dysregulation has been implicated in tumor formation and metastasis since successful invasion requires continuous remodeling of the ECM by integrins. Previous work from our lab suggests there might be an inverse relationship between αβ1 and αvβ6 expression in epithelial tumors; however, correlation between expression levels of these integrins and malignant phenotypes remains to be elucidated. Three OSCC cell lines, SCC9, SCC9β6 and SCC9β6β1, were used to study the effects of increased expression of αvβ6 in oral cancer. SCC9β6 cells were generated by co-culturing SCC9 cells with retroviral producer cell lines that expressed full-length β6. The SCC9β6 cells were then infected with β1-myc lentiviruses to generate the SCC9β6β1 cell line. Flow cytometry and Western blot analysis were used to evaluate levels of β6. SCC9β6 and SCC9β6β1 cells were FACS sorted to obtain cell populations with high β6 and high β6/β1 expression, respectively. The effect of differential integrin expression on growth of OSCC cells was evaluated using cell proliferation assays and scratch wound assays were used to access migratory abilities of the cells. Furthermore, the various cell lines were grown on coverslips coated with fibronectin (FN) and immunofluoresence was used to assess the ability of these cells to organize an FN matrix in vitro. Proliferation assays were also utilized to evaluate whether 0.01 mM zoledronate influenced the growth of SCC9β6 cells. Finally, FACS analysis was used to assess surface expression of αvβ6 in SCC9β6 cells and SCC9β6 cells grown in 0.01 mM zoledronate. Flow cytometry and Western blot analysis indicated there was a 10-fold increase in the expression of β6 in SCC9β6 compared to SCC9 cells. Cell proliferation assays demonstrated that SCC9β6 cells were more proliferative than the SCC9 cells, which showed more growth than the SCC9β6β1 cells. Scratch wound assays indicated that SCC9β6 cells were the most migratory, followed by SCC9β6β1 and finally by SCC9 cells. The SCC9 and SCC9β6β1 cells were able to organize the FN into a fibrillar pattern in association with peripheral focal adhesion whereas the SCC9β6 cells were not able to do so. Proliferation of SCC9β6 cells in the presence of zoledronate was greatly decreased compared to SCC9β6 cells grown in isolation and these cells showed a decrease in surface expression of αvβ6 by approximately 40%. This data suggest upregulation of integrin β6 increases the proliferative and migratory capacities of SCC9 cells while decreasing the matrix organizing ability of these cells. Furthermore, increased expression of β1 in SCC9β6 cells appears to reverse these malignant phenotypes. Therefore, the tipping of the balance of these integrins, upregulation of αvβ6 and downregulation of αβ1, may contribute to cell invasion and migration in OSCC. The ability of zoledronate to inhibit proliferation of SCC9β6 cells is of great interest since OSCC cells with increased αvβ6 expression are associated with a poor prognosis and there is currently no tumor cell specific therapy for the treatment of OSCC. Downregulation of αvβ6 in SCC9β6 cells grown in the presence of zoledronate suggests a possible mechanism by which zoledronate inhibits cell proliferation.