Examining the Role of p190RhoGAP on the Organization of the Actin Cytoskeleton in Breast Epithelial Cells

Abstract

Angiogenesis (i.e. blood vessel formation) and metastasis of tumors are two of the most dangerous aspects of malignant cancer cells. Metastasis requires spatial and temporal coordination of protein complexes known to affect cell adhesiveness and migration/proliferative properties. These protein complexes include E‐cadherin, Rho GTPases (e.g., Rac1 and RhoA), and the actin cytoskeleton. Rho GTPases perform different functions in the cell and have been shown to affect cancer progression, wound healing, and inflammation through regulation of cadherin and actin. For Rho GTPases to function, additional proteins increase or decrease their activity in the cell. These include guanine‐nucleotide‐exchange factors (GEFs) and GTPase‐activating proteins (GAPs). The current study aims to examine p190RhoGAP (GTPase activation protein (GAP) for RhoA) on cell adhesion and migration dynamics of normal Human Mammary Epithelial cells (HMECs) and the metastatic adenocarcinoma breast cancer cell line, MCF‐7. Cells were examined under endogenous conditions, as well as transfected with p190RhoGAP wild‐type and a catalytically inactive p190 RhoGAP‐R1283A mutant that locks RhoA into a constitutively active state. Overexpression of wild‐type and catalytically inactive p190RhoGAP had no major effect on actin stress fiber and cortical actin at cell‐cell contacts. However, HMEC cells overexpressing p190 RhoGAP‐R1283A showed reduced cytosolic RhoA protein, suggesting that loss of RhoA activity affects RhoA protein levels. Although no significant change in wound closure was observed, transwell cell invasion assays show a decreased rate of cell invasion in the presence of the chemo‐attractant, epidermal growth factor (EGF) in MCF‐7 cells. These data indicate a role for p190RhoGAP in the regulation of RhoA during cell migration and invasion. Loss of p190RhoGAP catalytic activity could affect the production of RhoA protein, leading to a decrease in the formation of actin structures (i.e., lamellipodia and filopodia) and a decrease in cell migration and invasion. Future work aims to determine how p190RhoGAP controls spatiotemporal regulation of protein complexes involved in metastatic cancer.Support or Funding InformationSaint Mary's School of Science Research Program and the Robert W. and Beverly J. Summers Scholarship