Minimal contribution of ERK1/2-MAPK signalling towards the maintenance of oncogenic GNAQQ209P-driven uveal melanomas in zebrafish

Abstract

Mutations affecting Gαq proteins are pervasive in uveal melanoma (UM), suggesting they ‘drive’ UM pathogenesis. The ERK1/2-MAPK pathway is critical for cutaneous melanoma development and consequently an important therapeutic target. Defining the contribution of ERK1/2-MAPK signalling to UM development has been hampered by the lack of an informative animal model that spontaneously develops UM. Towards this end, we engineered transgenic zebrafish to express oncogenic GNAQQ209P in the melanocyte lineage. This resulted in hyperplasia of uveal melanocytes, but with no evidence of malignant progression, nor perturbation of skin melanocytes. Combining expression of oncogenic GNAQQ209P with p53 inactivation resulted in earlier onset and even more extensive hyperplasia of uveal melanocytes that progressed to UM. Immunohistochemistry revealed only weak immunoreactivity to phosphorylated (p)ERK1/2 in established uveal tumours—in contrast to strong immunoreactivity in oncogenic RAS-driven skin lesions—but ubiquitous positive staining for nuclear Yes-associated protein (YAP). Moreover, no changes were observed in pERK1/2 levels upon transient knockdown of GNAQ or phospholipase C-beta (PLC-β) inhibition in the majority of human UM cell lines we tested harbouring GNAQ mutations. In summary, our findings demonstrate a weak correlation between oncogenic GNAQQ209P mutation and sustained ERK1/2-MAPK activation, implying that ERK1/2 signalling is unlikely to be instrumental in the maintenance of GNAQQ209P-driven UMs.

Dysregulated GPCR Signaling and Therapeutic Options in Uveal Melanoma

“…Moreover, siRNA depletion of YAP or inhibition of YAP with the pharmacological inhibitor verteporfin suppressed the growth of UM cells in culture and in a mouse model (27, 28). Robust nuclear localization of YAP was also observed in uveal melanocytes in a recently described zebrafish model for UM (29). The mechanisms underlying Rho and Rac activation of YAP appear to involve both inhibition of upstream kinases, large tumor suppressor homolog 1 and 2 (LATS1/2), as well as increased sequestration to F-actin of the YAP-binding protein angiomotin (AMOT), resulting in both cases in increased levels of YAP available to translocate to the nucleus (27, 28, 30).…”

The biology of uveal melanoma

“…A weak phosphorylation of ERK1/2 was observed in transgenic UM, while staining for nuclear YAP was evident. Therefore, enhanced ERK1/2 signaling seems not a major feature of GNAQ(Q209P)-driven transgenic UM [401]. …”

Oncogenic signaling in uveal melanoma

“…Thus, the upregulation and activation of RasGRP3 in response to oncogenic Gα q/11 signaling stimulates the formation of GTP‐bound active RAS and MAPK pathway activation. It is worth noting that sustained activation of MAPK may not require mutant Gα q/11 proteins, as knockdown of GNAQ expression in GNAQ ‐mutant UM cell lines did not consistently suppress ERK activity (Mouti, Dee, Coupland, & Hurlstone, ). Moreover, only a small percentage of zebrafish uveal melanocytes expressing GNAQ Q209P displayed phosphorylated ERK1/2 (Mouti et al., ).…”

“…It is worth noting that sustained activation of MAPK may not require mutant Gα q/11 proteins, as knockdown of GNAQ expression in GNAQ ‐mutant UM cell lines did not consistently suppress ERK activity (Mouti, Dee, Coupland, & Hurlstone, ). Moreover, only a small percentage of zebrafish uveal melanocytes expressing GNAQ Q209P displayed phosphorylated ERK1/2 (Mouti et al., ). These data suggest that early acquisition of hyperactive GNAQ/GNA11 mutations may promote the transient activation of MAPK consistent with the rapid, yet transient activation of PLC (Ross, ).…”