Regulation of Focal Adhesion Kinase Activation, Breast Cancer Cell Motility, and Amoeboid Invasion by the RhoA Guanine Nucleotide Exchange Factor Net1

Abstract

Net1 is a RhoA guanine nucleotide exchange factor (GEF) that is overexpressed in a subset of human cancers and contributes to cancer cell motility and invasion in vitro. However, the molecular mechanism accounting for its role in cell motility and invasion has not been described. In the present work, we show that expression of both Net1 isoforms in breast cancer cells is required for efficient cell motility. Although loss of Net1 isoform expression only partially blocks RhoA activation, it inhibits lysophosphatidic acid (LPA)-stimulated migration as efficiently as knockdown of RhoA itself. However, we demonstrate that the Net1A isoform predominantly controls myosin light-chain phosphorylation and is required for trailing edge retraction during migration. Net1A interacts with focal adhesion kinase (FAK), localizes to focal adhesions, and is necessary for FAK activation and focal adhesion maturation during cell spreading. Net1A expression is also required for efficient invasion through a Matrigel matrix. Analysis of invading cells demonstrates that Net1A is required for amoeboid invasion, and loss of Net1A expression causes cells to shift to a mesenchymal phenotype characterized by high β₁-integrin activity and membrane type 1 matrix metalloproteinase (MT1-MMP) expression. These results demonstrate a previously unrecognized role for the Net1A isoform in controlling FAK activation during planar cell movement and amoeboid motility during extracellular matrix (ECM) invasion.

ACKNOWLEDGMENTS

We thank Sarita Menon for help with confocal microscopy and the Clark, Dessauer, and Denicourt lab meeting groups for critical analysis of this work.

This work was supported by Public Health Service grant CA116356 from the National Cancer Institute, by grant BCTR123806 from Susan G. Komen for the Cure, and by grant RP100502 from the Cancer Prevention and Research Institute of Texas.