Differential regulation of invadopodia-mediated breast cancer metastasis by FAK family kinases

Introduction

Metastatic dissemination of cancer cells from the primary tumor and their spread to distant sites in the body is the leading cause of mortality in breast cancer patients. Invasive cancer cells penetrate through the basement membrane and into blood vessels using invadopodia, F-actin rich protrusions with matrix-degrading activity. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) define a distinct family of non-receptor tyrosine kinases that are highly expressed in invasive cancers, but the molecular mechanism by which these proteins regulate invasion of breast cancer cells to neighboring tissues and their subsequent metastatic dissemination is unclear at present.

Materials and Methods

To identify potential substrates and interactors of Pyk2 in invadopodia, we used high-throughput protein array screening followed by bioinformatic analysis. To examine the role of Pyk2 in invadopodia formation and activation, we used invadopodium precursor formation, matrix degradation, barbed end formation assays. Transwell invasion and two-dimensional motility assays were used to compare the roles of Pyk2 and FAK in cell migration and invasion. In vivo metastasis assays and intravital imaging of single tumor cells within a primary tumor in a live mouse were used to follow cancer cell behavior in its natural microenvironment. Patient database analysis was used to determine correlation of gene expression to metastasis.

Results and Discussion

Using high-throughput protein array screening and bioinformatic analysis, we identified cortactin as a novel substrate and interactor of Pyk2 in invadopodia. Pyk2 colocalizes with cortactin to invadopodia of invasive breast cancer cells, where it mediates epidermal growth factor-induced cortactin tyrosine phosphorylation both directly and indirectly via Src-mediated Abl-related gene (Arg) activation, leading to actin polymerization in invadopodia, extracellular matrix degradation, and tumor cell invasion. Further in vivo metastasis assays and high-resolution intravital imaging revealed that both Pyk2 and FAK regulate tumor cell invasion and consequent in vivo metastatic dissemination, but that they do so via distinct mechanisms. While Pyk2 regulates tumor cell invasion by controlling invadopodium-mediated functions, FAK controls invasiveness of tumor cells by regulating focal adhesion-mediated motility and functions. Further breast cancer patient database analysis revealed that increased mRNA co-expression of Pyk2, Arg and cortactin or high expression of both Pyk2 and FAK is associated with significantly lower distant metastasis free-survival.

Conclusion

Our data suggest that Pyk2 and FAK may be used to predict the probability of distant metastasis and that inhibition of either or both kinases may potentially reduce invadopodia-dependent functions and consequent breast cancer dissemination.