The CD9/CD81 Tetraspanin Complex and Tetraspanin CD151 Regulate alpha 3 beta 1 Integrin-Dependent Tumor Cell Behaviors by Overlapping but Distinct Mechanisms

Integrin alpha 3 beta 1 potently promotes cell motility on its ligands, laminin-332 and laminin-511, and this may help to explain why alpha 3 beta 1 has repeatedly been linked to breast carcinoma progression and metastasis. The pro-migratory functions of alpha 3 beta 1 depend strongly on lateral interactions with cell surface tetraspanin proteins. Tetraspanin CD151 interacts directly with the alpha 3 integrin subunit and links alpha 3 beta 1 integrin to other tetraspanins, including CD9 and CD81. Loss of CD151 disrupts alpha 3 beta 1 association with other tetraspanins and impairs alpha 3 beta 1-dependent motility. However, the extent to which tetraspanins other than CD151 are required for specific alpha 3 beta 1 functions is unclear. To begin to clarify which aspects of alpha 3 beta 1 function require which tetraspanins, we created breast carcinoma cells depleted of both CD9 and CD81 by RNA interference. Silencing both of these closely related tetraspanins was required to uncover their contributions to alpha 3 beta 1 function. We then directly compared our CD9/CD81-silenced cells to CD151-silenced cells. Both CD9/CD81-silenced cells and CD151-silenced cells showed delayed alpha 3 beta 1-dependent cell spreading on laminin-332. Surprisingly, however, once fully spread, CD9/CD81-silenced cells, but not CD151-silenced cells, displayed impaired alpha 3 beta 1-dependent directed motility and altered front-rear cell morphology. Also unexpectedly, the CD9/CD81 complex, but not CD151, was required to promote alpha 3 beta 1 association with PKC alpha in breast carcinoma cells, and a PKC inhibitor mimicked aspects of the CD9/CD81-silenced cell motility defect. Our data reveal overlapping, but surprisingly distinct contributions of specific tetraspanins to alpha 3 beta 1 integrin function. Importantly, some of CD9/CD81's alpha 3 beta 1 regulatory functions may not require CD9/CD81 to be physically linked to alpha 3 beta 1 by CD151.