ARF6 Is an Actionable Node that Orchestrates Oncogenic GNAQ Signaling in Uveal Melanoma

Abstract

SUMMARY Activating mutations in Gαq proteins, which form the a subunit of certain heterotrimeric G proteins, drive uveal melanoma oncogenesis by triggering multiple downstream signaling pathways, including PLC/PKC, Rho/Rac, and YAP. Here we show that the small GTPase ARF6 acts as a proximal node of oncogenic Gαq signaling to induce all of these downstream pathways as well as β-catenin signaling. ARF6 activates these diverse pathways through a common mechanism—the trafficking of GNAQ and β-catenin from the plasma membrane to cytoplasmic vesicles and the nucleus, respectively. Blocking ARF6 with a small molecule reduces uveal melanoma cell proliferation and tumorigenesis in a mouse model, confirming the functional relevance of this pathway and suggesting a therapeutic strategy for Gα-mediated diseases.

Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy

“…GEP100 is another GEF that has been shown to activate ARF6 (16,27). Given that ARNO activates ARF6 in our assays, we assumed we reasoned that knocking down the GEF that activates ARF6 would have the same effect on VEGFR2.…”

“…We hypothesized that the small GTPase ARF6 may be involved in the enhanced vascular permeability seen in diabetic retinopathy, as ARF6 has been shown to mediate permeability in other disease states (14,15). We recently discovered the essential role of ARF6 in uveal melanoma and found upregulated protein levels of ARF6 in human tumor samples (16). Therefore, as an initial experiment, we examined ARF6 levels in postmortem whole-eye samples from patients with diabetes.…”

“…In HRMECs, VEGF stimulation did not lead to the phosphorylation of caveolin-1 (Supplemental Figure 3A), suggesting that ARF6 may affect VEGF signaling via an alternative mechanism. Previous work has shown that ARF6 functions in vesicular trafficking at the plasma membrane (16,23). To determine whether the effects of ARF6 on VEGFR2 signaling were due to trafficking events, we measured endocytosis by labeling cell-surface proteins with biotin, allowing time for endocytosis and then removing biotin from proteins remaining at the cell surface.…”

The BRAG/IQSec family of Arf GEFs

“…46,47 A recent study indicates that, like the cytohesins, BRAG2 can also interact with Gaq, although it is not yet clear that this interaction is direct. 48 Surprisingly, knockdown of BRAG2, but not cytohesin-2/ ARNO substantially attenuates Arf6 activation in 2 uveal melanoma cell lines expressing oncogenic Gaq, and inhibits their anchorage-independent growth in vitro. Knockdown of Arf6 in these cells attenuates a large number of signaling pathways downstream of Gaq, including the activation of PLC, ERK, p38 and JNK, Rac, RhoA and Hippo/YAP.…”

“…Knockdown of Arf6 in these cells attenuates a large number of signaling pathways downstream of Gaq, including the activation of PLC, ERK, p38 and JNK, Rac, RhoA and Hippo/YAP. 48 Interestingly, Arf6 depletion appears to cause retention of Gaq at the plasma membrane and its depletion from endosomes, suggesting that Gaq signaling from endosomes is important for oncogenic transformation.…”