This proposal tests if a melanoma-enriched, hyperactivating mutation in Rac1 can drive pro-survival signaling in melanoma cells by altering intracellular forces. This would be a powerful mechanism linking the effects of an oncogenic Rac1 mutation to alterations in physical stimuli that amplify biochemical signals and drive malignancy. Next-generation tumor sequencing has led to the discovery of a reoccurring mutation in the Rac1 GTPase gene in melanoma that results in a proline-to-serine switch at residue 29 (Rac1P29S). The Rac1P29S mutation is transforming, is associated with increased risk of metastasis, and confers resistance to chemotherapeutics like vemurafenib in melanoma. The mechanisms by which Rac1P29S drives melanoma progression are unknown. Wild type Rac1 is a key regulator of cytoskeletal organization and adhesion formation between the cell and the extracellular matrix (ECM). Adhesion proteins sense mechanical interactions between the cell and the ECM and activate pro-survival pathways in cancer. Expression of hyperactive Rac1P29S results in cells with large, elongated focal adhesions and prominent stress fibers, suggesting high contractility and tension at the cell-ECM adhesions. Additionally, Rac1P29S-mediated pro-survival signaling is abrogated upon inhibition of these adhesions. This proposal tests if the role of Rac1P29S in driving melanoma progression is dependent on mechanical cues resulting from the increased cell contractility and tension at cell-ECM adhesions. The first goal of this proposal is to test if Rac1P29S increases cell contractility and forces at cell-ECM adhesions. Traction force microscopy (TFM) will be used to measure traction forces at cell-ECM adhesions in Rac1P29S and Rac1WT cells. The second goal of this proposal is to test if Rac1P29S increases RhoA signaling by competitively binding RhoGDI, an inhibitor of both Rac1 and RhoA. It is expected that RhoA activity will be increased in Rac1P29S and Rac1WT cells, but will be abrogated upon inhibition of Rac1P29S-RhoGDI interaction. It is also expected that inhibition of traction forces and RhoA signaling will reduce Rac1P29S's pro-survival activity, resulting in increased melanoma sensitivity to a drug challenge. The final goal of this study is to determine if Rac1P29S itself is responsive to force stimuli at adhesions, establishing a positive feedback loop that would further amplify pro-survival signaling. If force stimuli at adhesions regulate Rac1P29S activity will be tested by measuring melanoma drug sensitivity following inhibition of adhesion-activated force-sensitive Rac1 regulators. Elucidating Rac1P29S's bi-directionality in amplifying mechano-responsive signaling by both stimulating and responding to force stimuli at adhesions would place Rac1P29S at the center of mechanically sensitive positive-feedback loop that drives melanoma malignancy.
Cancer cell interaction with the extracellular environment contributes significantly to the development and progression of melanoma, a highly aggressive malignancy responsible for over 75% of all skin cancer deaths. However, most chemotherapies for melanoma target intracellular signaling abnormalities. Studying the hyperactivating melanoma mutation in the Rac1 protein, which coordinates intracellular and extracellular signaling, may lead to promising strategies for melanoma drug development that target mechanisms of cancer cell interaction with the surrounding environment.
