Figure 3. Rab25 facilitates cell migration in luminal breast cancer cells but opposes migration of claudin low cells.

(A) Shows MCF7 cells with stable Rab25 overexpression migrating to fill an artificially created scratch wound over 48 hrs. The 0 hr represents initial wounding point. The distance between cell fronts measured at time 0 is initial distance. The distance remaining after wound closure is subtracted from initial distance to calculate distance moved. Data and graph are representative of 5 independent experiments. (B) Shows MDA231 cells with stable Rab25 overexpression migrating to fill an artificially created scratch wound over 10 hrs. The 0hr represents initial wounding point. The distance between cell fronts measured at time 0 is initial distance. The distance remaining after wound closure is subtracted from initial distance to calculate distance moved. Data and graph are representative of 5 independent experiments. (C) Top panel shows immune-blot of lysates from human mammary epithelial (HMLE) cells expressing Rab25 protein that is lost with exogenous overexpression of Snail. The lower panel is an immune-blot of lysates from HMLE Snail cells where Rab25 was expressed using lenti-viral constructs. The actin panel validates equal loading. These blot are representative of multiple similar expression analysis. The side panel diagram shows HMLE as an epithelial cell before Snail is overexpressed. HMLE Snail then undergoes EMT and assumes mesenchymal morphology. With addition of exogenous Rab25 it may be possible to reverse EMT (MET) and restore epithelial features. (D) HMLE, HMLE Snail and HMLE Snail +Rab25 cells were assayed in a Matrigel based invasion assay using modified Boyden Chambers. Equal number of cells from each isogenic line was plated in 4 inserts. After 8 hours, the invaded cells were harvested, stained and counted. Introduction of exogenous Rab25 into mesenchymal cells (HMLE Snail) rescues cells partially from Snail-driven invasive phenotype. The data represents of 4 independent experiments. (E) Data from RNA Sequencing analysis of LPA mouse model showing slow growing SG Cdh1+ carcinomas in white open circles and fast growing (FGM) Cdh1-ve sarcoma-like (non epithelial) tumors. Each of the four sub-plots show that Rab25 levels correlating with Cldn7, Cldn4, and Cdh1 levels between the carcinoma and sarcoma group with non epithelial sarcoma showing undetectable levels of Rab25. Rpm stands for Reads per minute.