We recently demonstrated that actively promoting the formation of cell surface complexes by engaging the tetraspanin CD151 with a monoclonal antibody (mAb 1A5) inhibits metastasis by immobilizing the tumor cells. Tetraspanins influence cellular behavior via the regulation of associated partner molecules such as integrins, cell adhesion molecules, and members of the immunoglobulin superfamily. Although CD151 is able to associate with and regulate the activity of laminin-binding integrins we demonstrate that CD151 does not associate with α3α1 in the presence of mAb 1A5. Using a 2-color flow cytometric approach we mapped the domains of CD151 required for antigen recognition by mAb 1A5 and determined that 1A5 maps to the integrin 194QRD196 motif of CD151, using this information we further performed CD151 co-immunoprecipitations and demonstrated that 1A5 recognizes CD151 not associated with α3α1.To determine the identity of the protein(s) associated with the antibody-engaged CD151 and also possibly responsible for the anti-migratory, anti-metastatic phenotype we used a proteomic approach. Using tandem mass spectrometry on CD151-associated proteins co-immunoprecipitated by mAb 1A5, we detected in excess of 200 proteins. This strategy identified several putative partners of CD151. Further analysis of the CD151 associated proteins led to our discovery of the novel CD151-associated protein ALCAM/CD166, a member of the immunoglobulin superfamily. Using an RNAi-mediated knockdown approach we demonstrate that ALCAM/CD166 is required for the CD151 complex to regulate tumor cell mobility in vitro. Conversely, CD151 is required for ALCAM to control motility. Spontaneous metastasis assays using the avian chorioallantoic membrane and subcutaneous murine xenografts demonstrate thatALCAM/CD166 is indeed required for the engaged tetraspanin to inhibit tumor cell dissemination in vivo. Further mechanistic analysis of this complex revealed that the CD151/ALCAM protein complex promotes activation of the small GTPase Rap1 via activated protein kinase C. Collectively, we present data, which demonstrate that, the ALCAM-containing CD151 complex can control metastasis. This novel complex reveals how cell-cell interactions can control migration through the regulation of cell-matrix adhesions. These results also demonstrate mechanistically that tetraspanins can integrate signaling between physically distinct molecular functions and therefore behave as a molecular clutch that controls matrix adhesion in response to cell-cell interactions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4217. doi:1538-7445.AM2012-4217