FAK Family Kinases: The Yin and Yang of Breast Cancer Metastasis

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 was unclear until recently.

Materials and Methods

To identify potential substrates and interactors of Pyk2 in invadopodia, we used high-throughput protein array screening followed by bioinformatic analysis. The role of Pyk2 in invadopodia formation and activation was examined by invadopodium precursor formation, matrix degradation, and barbed end formation assays. 2D and 3D 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 the primary tumor of a live mouse were used to follow the behavior of cancer cells in their natural microenvironment, which was validated by transcriptomics and network analysis. Patient database analysis was used to determine correlation of gene expression to metastasis.

Results and discussion

Using high-throughput screening, we identified cortactin as a substrate and interactor of Pyk2 in invadopodia. Pyk2 colocalizes with cortactin to invadopodia of invasive breast cancer cells, where it mediates EGF-induced cortactin tyrosine phosphorylation both directly and indirectly via Src-mediated Abl-related gene (Arg) activation, leading to actin polymerization in invadopodia, ECM degradation, and tumor cell invasion. 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. These observations were validated by tumor transcriptome and network analysis, revealing the in vivo molecular mechanisms and signaling pathways by which FAK family proteins coordinate the regulation of breast cancer metastasis. 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.