Purchase this article with an account.
Dallas Shujing Shi, Jae Hyuk Yoo, Weiquan Zhu, Dean Li; Oncogenic GNAQ orchestrates multiple signaling pathways via ARF6. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5313.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Activating mutations in GNAQ and GNA11, occur in over 80% of uveal melanomas. However, the molecular mechanisms governing this stimulation remain unknown. Using biochemical tools, cellular assays, a small molecule inhibitor, and animal models of uveal melanoma, we tested the hypothesis that the small GTPase, ARF6, is necessary and sufficient for oncogenic GNAQ signaling.
For in vitro experiments, Mel 92.1 and Mel 202 cells were maintained in RPMI 1640 with 10% FBS. Cells were transfected with siRNA against ARF6 or control siRNA using Lipofectamine RNAiMAX (Invitrogen). Transfected cells were then assessed for proliferation using CyQUANT (Invitrogen), anchorage-independent colony growth using CytoSelect 96-Well Cell Transformation Assay (Cell Biolabs), cell invasion using BD BioCoat Tumor Invasion Assay System (BD Bioscience), and ARF6/RhoA/Rac1/PLC levels using pull-down kits (Cells Biolabs, Millipore, Invitrogen).<br /> For in vivo experiments, nude mice (Jackson) were anesthetized with ketamine/xylazine. Then, 105 cells transfected with either ARF6 siRNA or control siRNA were injected into the posterior chamber. In a subset of mice, non-transfected cells were injected and the mice were subsequently treated with either 30mg/kg of NAV-2729 or DMSO control. All mice were euthanized after 5 weeks, and eyes were collected, fixed, embedded, sectioned, stained with H&E, and examined histologically for primary tumors by a pathologist who was blinded to the treatment regimen.
Compared to controls, uveal melanoma cells with ARF6 knockdown exhibited decreased proliferation, decreased anchorage-independent colony growth, decreased cell invasion, and decreased downstream signaling to PLC-PKC and Rac1/Rho-MAPK pathways (n=3, p<0.01). In vivo, mice with uveal melanoma xenografts using ARF6 knocked down cells exhibited lower tumor incidence and smaller tumor size compared to mice treated with control xenograft cells (n=12, p<0.05). Mice with uveal melanoma xenografts also had decreased tumors when treated with a novel ARF6 inhibitor, NAV-2729 (n=9, p<0.05).
In this study, we demonstrate that an activated GNAQ-ARF6 complex orchestrates the activity of distinct tumorigenic pathways in uveal melanoma. This suggest that targeting ARF6 may inhibit all of the currently known GNAQ-mediated oncogenic signaling pathways and presents a new strategy for treating uveal melanoma.
This PDF is available to Subscribers Only