PT Unknown
AU Quentin Dr. rer. nat., J
TI Role of the Rac1-P29S driver mutation in tumorigenesis and acquisition of BRAF inhibitor resistance
PD 07
PY 2020
DI 10.17185/duepublico/73416
LA en
AB The Rho-GTPase Rac1 is well-known to control the actin cytoskeleton. Recently, the highly active fast-cycling Rac1-P29S mutant was identified as a new driver mutation in melanoma beside the long-characterized mutations in BRAF and NRAS. Initial studies suggest that Rac1-P29S in linked to enhanced melanoma cell proliferation by activation of the MAPK protein ERK 1/2. Moreover, it is proposed that Rac1-P29S promotes early metastatic progression of melanoma and adaption of BRAF inhibitor resistance. However, little is known how this driver mutation regulates these processes. The aim of this study was therefore to characterize the role of Rac1-P29S in cell proliferation, cell survival as well as in BRAF inhibitor resistance and to identify molecular mechanisms that mediate these functions. For this, we focused on two different patient-derived melanoma cell lines. Both cell lines harbor an activating BRAF mutation but express different Rac1 variants. While Rac1-wildtype (wt) is endogenously expressed in Ma-Mel-86c cells, the Rac1-P29S mutant was found in the UKE-Mel-55b cell line. The characteristic morphology of these cells, including enhanced lamellipodia formation and dynamic membrane ruffling differs substantially from the Rac1-wt expressing Ma-Mel-86c cell line. In the first part of this study, we used RNA interference together with expression of siRNA-resistant constructs to confirm that Rac1-P29S indeed promote this phenotype. In addition, we also established that endogenous expression of Rac1-P29S in UKE-Mel-55b cells is required for accelerated early cell adhesion and spreading as well as for their migratory capacity. Next, we could show that Rac1-P29S is required for the strongly enhanced proliferation of UKE-Mel-55b cells, while Rac1-wt in Ma-Mel-86c cells does not modulate cell proliferation substantially. In agreement with such prominent role of Rac1-P29S in proliferation in UKE-Mel-55b cells, live-cell imaging revealed a significant decrease of cell division as well as a pronounced mitotic delay upon Rac1 depletion. We next studied the MAPK protein ERK1/2, the cell survival protein AKT and the actin regulated transcriptional coactivators Yap/Taz as potential downstream effectors. Data suggest that Rac1-P29S expression similarly modulates AKT activity as compared to Rac1-wt expressing cells. In contrast, Rac1-P29S strongly stimulates activities of the proliferation related proteins ERK1/2 and Yap/Taz as compared to Rac1-wt. First, Yap/Taz levels were significantly higher in UKE-Mel-55b cells as compared to Ma-Mel86c. In addition, Rac1-P29S depletion in UKE-Mel-55b cells distinctly decreased activitie of ERK1/2 and Yaz/Taz, whereas lack of Rac1-wt in Ma-Mel-86c cells did not have any marked effect on these signaling proteins. Our studies also revealed significantly elevated activities of the PAK effector family in UKE-Mel-55b cells as compared to Ma-Mel-86c cells. This implicates the PAK proteins as important effectors in enhanced cell proliferation downstream of Rac1-P29S. In the second part, we focused on the role of Rac1-P29S in the acquisition of BRAF inhibitor resistance in melanoma cells and studied potential effects on associated signaling pathways. Overall, our proliferation studies suggest that expression of Rac1-P29S in melanoma cells provides an advantage in proliferation as compared to Rac1-wt. In particular, proliferation rates during treatment with the BRAF inhibitor remained low in Rac1-wt cells, while we measured a marked recovery of proliferation in Rac1-P29S expressing cells, despite continuous inhibitor (PLX-4032) treatment. Interestingly, activity of the MAPK protein ERK1/2 in Rac1-wt cells was lost entirely after PLX-4032 treatment, while a substantial residual activity level in the Rac1-P29S cell line was measured. Depletion of Rac1-P29S significantly decrease the residual ERK1/2 activity, further indicating that endogenous expression of this Rac1 variant in melanoma stimulates ERK1/2 activation during BRAF inhibitor treatment. In addition, BRAF inhibition lead to increased nuclear Yap/Taz localization in both cell lines. Depletion of Rac1-P29S reduced this effect significantly, suggesting that the driver mutant promotes activity of these transcriptional coactivators upon BRAF inhibition. Enhanced stress fiber formation upon PLX-4032 treatment in both melanoma cell lines suggested that contractility regulatory Rho-GTPase might be also involved in acquisition of drug resistance in addition to Rac1-P29S. While stress fiber formation is typically associated with RhoA, our preliminary results with RNAi and the C3 inhibitor suggest that activities of RhoB and RhoC might be functionally more relevant in this cellular context. Taken together, our findings have established a stimulatory role of the Rac1-P29S driver mutation in tumor-related cell proliferation and advanced our understanding of how this mutation might promote BRAF inhibitor resistance in melanoma patients. Thus, the targeted inhibition of downstream signaling pathways of the Rac1-P29S mutation could therefore enable the development of new pharmacological treatment strategies for melanoma patients.
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