Mutant NRAS and RAC1 signaling in melanoma
Abstract
Melanomas with mutations in the GTPases NRAS and RAC1 comprise of a particularly aggressive subset of melanoma with limited therapeutic options. Within the mutant NRAS population, targeting downstream effectors pathways, particularly the MEK-ERK1/2 pathway, remains an attractive strategy. Mutations in RAC1, particularly P29S, were recently discovered and downstream effector pathways have yet to be elucidated. We studied the atypical IκB kinase, TBK1, downstream of the RALGEF-RALB pathway in mutant NRAS melanoma and identified TBK1 as a positive regulator of cell migration and invasion. In 3D culture systems that mimic the dermal microenvironment, targeting TBK1, with either RNA interference or small molecule inhibitors, cooperated with MEK inhibition to elicit a cytotoxic effect. These cytotoxic effects were specific to melanomas with NRAS mutations. A broad examination of the response of mutant NRAS melanoma to MEK inhibition revealed an adaptive downregulation of MIG6, a negative regulator of EGFR signaling. Depletion of MIG6 with RNA interference or overexpression revealed MIG6 to be a negative regulator of EGF-stimulated AKT activation. Transwell migration and spheroid outgrowth assays revealed MIG6 to be a negative regulator of migration, particularly in the presence of MEK inhibition. These findings suggest that the downregulation of MIG6 in MEK-inhibited mutant NRAS melanomas provides an adaptive invasive phenotype. To determine the signaling pathways regulated by RAC1 P29, we utilized an unbiased protein array. Through overexpression of RAC1 P29S in melanocytes and depletion of RAC1 in a melanoma cell line with an endogenous RAC1 P29S mutation, we identified a correlation between RAC1 P29S and PD-L1, an immune ligand that binds to PD-1 receptors on T-cells to suppress effector function. Examination of RAC1 P29S melanoma patient samples in The Cancer Genome Atlas (TCGA) confirmed the positive correlation between RAC1 and PD-L1 expression. In sum, we examined signaling pathways regulated in mutant NRAS and RAC1 melanomas to identify druggable targets. In mutant NRAS melanoma, we demonstrate that components of the RALGEF-RALB-TBK1 and the EGFR-AKT pathways are promising targets for use in combination with MEK inhibitors. In RAC1 P29S melanoma, we identified a mechanism of immune evasion through upregulation of PD-L1, whose signaling axis with PD-1 receptors can be targeted with immune checkpoint inhibitors.
Subject Area
Molecular biology|Cellular biology
Recommended Citation
Vu, Ha Linh, "Mutant NRAS and RAC1 signaling in melanoma" (2017). ProQuest ETD Collection - Thomas Jefferson University. AAI10004234.
https://jdc.jefferson.edu/dissertations/AAI10004234
