Abstract 4017: DLC1 and α-catenin protein interaction enhances DLC1 antioncogenic activity by stabilizing adherens junctions and suppressing NFκB signaling

Adherens junctions (AJs), which are cell-cell adhesion complexes closely associated with actin cytoskeleton, play an important role in maintaining epithelial tissue homeostasis. Both malignant transformation, and tumor progression to metastasis are often associated with major changes in cytoskeletal organization, the cell-cell adhesion, and aberrant adhesion-mediated signaling. The tumor suppressor gene DLC1 (Deleted in Liver Cancer 1) encodes a RhoGTPase activating protein (GAP) that acts as a negative regulator of the Rho family of small GTPases, which are implicated in cytoskeleton organization and regulation of AJs. A yeast two-hybrid screening identified α-catenin, a component of AJs, as a potential DLC19s binding partner. We relied on this finding and investigated the role of DLC1 in the regulation of AJs and in adhesion-mediated signaling. Using human embryonic kidney (HEK 293) cells we demonstrated that DLC1 interacted with α-catenin and associated with E-cadherin and β-catenin, both constitutive AJs proteins. Co-immunoprecipitation using various deletion constructs of DLC1 and of α-catenin established that the N-terminal (340-435 aa) of DLC1 interacted with the N-terminal (39-161 aa) of α-catenin. Immuno-fluorescence staining and western blot of cellular membrane and cytoplasmic fractions, demonstrated that binding of DLC1 to α-catenin recruited α-catenin to the plasma membrane and required DLC1 GAP activity. In contrast, the DLC1 GAP mutant (R718E) failed to produce the same effect. Fluorescence Recovery After Photobleaching (FRAP) analysis demonstrated that DLC1-α-catenin interaction increased the GFP-E-cadherin mobility at cell-cell junctions, whereas phalloidin staining showed intact actin cable formation around the cell periphery, resulting in the stabilization of AJs. The DLC1-α-catenin interaction is instrumental for a maximal oncosuppressive effects of DLC1 (i.e., by inhibiting proliferation and abolishing colony formation of prostate carcinoma cells); DLC1 mutant defective for α-catenin binding failed to reproduce such an effect. Loss of α-catenin is usually accompanied by an increase in the activation of NFkB, which is associated with increased cell proliferation and resistance to apoptosis. We demonstrated that, by immobilizing NFkB to the AJ complex, DLC1-α-catenin interaction effectively inhibited NFkB phosphorylation and, therefore, its activation. Together, these results unravel a new mechanism through which DLC1 exerts its oncosuppressive function by stabilizing AJs and suppressing NFkB signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4017. doi:10.1158/1538-7445.AM2011-4017