Abstract 69: Integrin α6β4 associates with plectin and vimentin in focal adhesions to enhance breast cancer cell invasion
Authors: Lei Qi, Teresa Knifley, Min Chen, Kathleen L. O’Connor
Journal: Cancer Research (2019)

Abstract Integrin α6β4 binds plectin to associate with intermediate filaments such as vimentin. While vimentin and integrin α6β4 are associated with the aggressiveness of triple negative breast cancer (TNBC), the biological importance of their association is not clear. Here, we utilized naturally occurring Epidermolysis Bullosa mutations in the integrin β4 subunit that are deficient in plectin binding to investigate the association between integrin α6β4 and vimentin. Using integrin β4-deficient BT549 cells, we generated cell lines that expressed integrin β4 wild type (β4-wt), plectin binding mutants (β4-R1225H or β4-R1281W), 1355-1662 deletion mutant (β4-Δ1355-1662; which retains plectin binding but deletes the signaling domain) or an empty control vector (EV). Migration assay and three-dimensional (3D) culture were performed to test whether integrin α6β4 signaling and its association with plectin contribute to the migratory and invasive phenotypes. Compared to EV cells, β4-wt increases cell migration and 3D invasive growth, but the β4-R1225H and β4-R1281W could not. CRISPR knockout of plectin also blocked β4-wt 3D cell growth. Utilizing total internal reflection fluorescence (TIRF) microscopy, we found that integrin β4-wt and β4-Δ1355-1662 were recruited to focal adhesions (FAs, indicated by paxillin co-staining) on laminin-1 plus fibronectin matrices, but not when plated on laminin-1 or fibronectin alone. Integrin β4-R1225H and β4-R1281W did not localize to FAs. Similarly, integrin β4-wt in plectin knockout cells could not aggregate in FAs. Furthermore, we observed that integrin αv and β1 colocalized with integrin β4 in FAs. Proximity ligation assay (PLA) indicated that the close interaction of integrin β1 and plectin. Interestingly, depletion of integrin β1 significantly reduced integrin β4 accumulation in FAs. Further immunostaining showed that integrin β4, plectin, and vimentin aggregated in FAs in the central area of the cell where FAs predominantly formed. Depletion of vimentin did not impact the recruitment of integrin β4 in FAs or cell proliferation, but significantly suppressed cell migration and 3D growth in β4-wt cells. Integrin β4-plectin-vimentin complexes were also found in MDA-MB-231 cells where they co-localized with paxillin and connected with stress fibers. In summary, our study demonstrates the essential roles of plectin binding and vimentin in promoting the migratory and invasive phenotypes by integrin α6β4 signaling in TNBC. Our study further highlights a novel function of plectin binding and integrin β1 in enhancing integrin β4 dynamics. Citation Format: Lei Qi, Teresa Knifley, Min Chen, Kathleen L. O’Connor. Integrin α6β4 associates with plectin and vimentin in focal adhesions to enhance breast cancer cell invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 69.