Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase

Introduction: The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. Methods: MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and beta 1-integrin, we examined activation of the beta 1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and beta 1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. Results: JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the beta 1-integrin substrate fibronectin. This was accompanied by reduced protein expression of beta 1-integrin and its binding partners alpha V- and alpha 5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and beta 1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF-6 and the Rap1 activator PDZ-GEF2 in MCF7 cells and in primary cultures from breast cancer patients. Conclusions: Our findings provide compelling evidence of a novel role for JAM-A in driving breast cancer cell migration via activation of Rap1 GTPase and beta 1-integrin. We speculate that JAM-A over-expression in some breast cancer patients may represent a novel therapeutic target to reduce the likelihood of metastasis.