RAP2 Functions in Regulating Small Monomeric Proteins and Their Role in Migratory Phenotypes in Breast Cancer

Introduction Breast Cancer is known for being the most diagnosed cancer and the leading cause of cancer-related deaths among women globally, according to WHO (1). Defects in cell migration, including improper movement or dysfunction, contribute to various diseases (2). Actin cytoskeleton dynamics play a crucial role in cell mobility, with actin filaments polymerizing to form protrusions at the front and retractions at the back. Small monomeric GTPases, such as Rac1 and RhoA, regulate these processes, acting as molecular switches between active, GTP-bound, and inactive, GDP-bound states. While Rac1 controls leadingedge dynamics, RhoA is emerging as a key but less understood regulator of actin dynamics in cell migration (3). Here, we demonstrate how Rap2 influences membrane remodeling and actin cytoskeleton dynamics to regulate cell movement using the MDA MB 231 cell line (4). Aim This study aims to investigate the role of Rab and Rap GTPases, members of the Ras oncogene family, in regulating cell migration using CRISPR-Cas9-mediated knock-out (KO) models in MDA MB 231 breast cancer cells. We will assess their impact on cell migratory properties, actin cytoskeleton dynamics, and membrane remodeling. The findings will provide insights into how these GTPases influence cancer cell motility & metastatic potential. Methods To investigate Rap2’s role in cell migration, a single-cell migration assay was performed, revealing its necessity for migration through length and velocity analysis. GTP pull-down assay assessed the interaction of Rap2 with Rab40 in a GDP/GTP-dependent manner. Cortactin staining was visualized at the leading-edge dynamics, while immunocytochemistry confirmed Myosin and Phopho-Paxillin as focal adhesion markers in control and Rap2 KO cells. To assess focal adhesion dynamics, GFP-paxillin transfection, and time-lapse live imaging confirmed the role of Rap2 in enhancing focal adhesion stability during migration. Further GraphPad Prism was used for statistical analysis. Results Rap2 KO impaired cell migration, reducing length and velocity. Pull-down assays confirmed its GDP/GTP-dependent interaction. Cortactin staining showed diminished leading-edge dynamics, while immunocytochemistry revealed altered focal adhesions with reduced Myosin2B and Phospho-Paxillin. GFP-paxillin using live imaging revealed stable adhesions in impaired Rap2. Conclusions These findings demonstrate that Rap2 plays a crucial role in regulating cell migration by modulating actin cytoskeleton dynamics and focal adhesion stability. Its interaction with Rab40, influence on leading-edge formation, and impact on adhesions turnover, highlighting Rap2 as a key regulator of migratory behaviour. Understanding Rap2’s function provides new insights into the cell motility mechanism, potentially informing therapeutic strategies for migration-related diseases.