Fig. 1: Rac1 is required for Vav1-driven BRAFi resistance in BRAF-mutant melanoma.
From: A critical role of FAK signaling in Rac1-driven melanoma cell resistance to MAPK pathway inhibition
A BRAF V600E mutant A375 subclones NT F2 and NT B7 harbor a non-targeting control shRNA for Rac1, compared to A375 subclone G3, which harbors an effective shRNA targeting Rac1. Cells were grown in 3 µM vemurafenib (VEM). Over-expression of Vav1 in the NT F2 and NT B7 subclones (NT F2 + VAV1, and NT B7 + VAV1), but not the introduction of an empty vector control (NT F2 + EV, and NT B7 + EV) promoted resistance to the BRAFi vemurafenib (blue symbols). In contrast, in A375 subclone G3, which harbors an effective Rac1 shRNA, Vav1 over-expression was unable to promote vemurafenib resistance compared to empty vector control cells (Rac KD G3 + EV vs. Rac KD G3 + VAV1; orange symbols). B In a separate experiment, similar to panel A, Vav1 over-expression in A375 subclone B7 (with a non-targeting control shRNA for Rac1) promoted vemurafenib resistance (NT B7 + EV vs NT B7 VAV1; blue symbols), while over-expression of Vav1 in a different subclone F10, which harbors an effective Rac1 shRNA, was unable to promote vemurafenib resistance, compared to Vav1 empty vector control cells (Rac KD F10 + EV vs Rac KD F10 + VAV1; orange symbols). C, D Regardless of Rac1 knockdown or Vav1 over-expression status, all the sublines showed similar high levels of growth in experiments using DMSO vehicle control instead of the BRAFi, vemurafenib. E Immunoblotting confirmed that Rac1 was knocked down in the F10 and G3 A375, compared to the F2 and B7 subclones, and that Vav1 protein was over-expressed in the Vav1 (VAV1) cell line variants compared to their respective empty vector (EV) controls. Differences in cell proliferation were analyzed via ANOVA with Sidak’s multiple comparison test, ****P < 0.0001.
