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  • Contrary to the transient nature of signal

    2022-05-20

    Contrary to the transient nature of signal transmission through PLCβ, genome-wide RNAi screens revealed that the signaling events driven by Gαq that result in aberrant cell proliferation depends on highly specific protein-protein interactions, rather than solely on diffusible second messenger systems. Specifically, prior systems biology approaches have identified the RhoGEF TRIO as critical for activating Gαq-driven AP-1-regulated transcriptional networks independently of PLCβ to achieve sustained stimulation of proliferative pathways (Vaque et al., 2013). Further work has shown that this pathway converges in the activation of YAP and that YAP activation is critical for the oncogenic potential of UM (Feng et al., 2014a, Feng et al., 2014b, Yu et al., 2014a). The Hippo/YAP cascade is a key growth-regulating pathway in normal cellular physiology (Bhatt et al., 2010, Moroishi et al., 2015, Yu et al., 2015). Unsurprisingly, dysregulation of the Hippo pathway is seen frequently in cancer; however, its core components are rarely mutated (Martin et al., 2018, Moroishi et al., 2015). Rather, external pressures from upstream oncogenes typically drive YAP-dependent cell proliferation. Identifying the key molecular players that facilitate oncogenic signaling through Hippo/YAP pathway may also uncover potential network vulnerabilities. Interestingly, inhibition of PLCβ does not impact the activation of YAP after Gαq stimulation (Feng et al., 2014b). Together, these findings suggest that the canonical Gαq-PLCβ-MAPK signaling axis may be critical for tumor SC-10 rather than tumor maintenance, and that opportunities for intervention may lie within the distinct signaling circuitry transduced through TRIO. FAK is a non-receptor tyrosine kinase whose role as a downstream target of Gαq has been well established by biochemical studies (Gutkind and Robbins, SC-10 1992); however, the contribution of FAK as a mediator of oncogenic Gαq signaling has not been previously explored. Our finding that FAK is rapidly activated by Gαq-linked GPCRs and the oncogenic mutant Gαq through TRIO and RhoA, rather than PLCβ, prompted us to focus on the possibility that FAK may represent an integral component of the non-canonical pathway by which Gαq regulates aberrant cell growth. We found that inhibition of FAK was sufficient to reduce UM cell proliferation and, if prolonged, to trigger apoptotic cell death. This response was unanticipated as FAK inhibitors often have limited activity in most cancers as single agents but instead synergize with cytotoxic agents, as we have shown for ovarian cancer, which overexpresses FAK as a typical example (Sulzmaier et al., 2014). We hypothesized that, compared with other cancer types with FAK overexpression, the compounding impact of PTK2 copy-number gain and overexpression together with Gαq-driven FAK activity in UM creates a unique cellular state that may be highly dependent on the activity of FAK and therefore highly sensitive to FAK inhibition. This convergence of computational predictions, and biochemical and genetic information, enabled the discovery of the therapeutic potential of inhibiting FAK for UM treatment. FAK has been recently linked to YAP activity in mechanotransduction and in the coordination of cell proliferation and differentiation in mouse incisors during development (Hu et al., 2017, Lachowski et al., 2018). However, the underlying cell context-specific and developmental mechanisms are still not fully understood. We provide evidence that in UM the role of FAK converges on promoting YAP activity through the tandem inhibition of Hippo pathway signals by phosphorylation of Y26 of MOB1 and Y357 of YAP. In the case of YAP phosphorylation, these observations extend prior studies indicating the role of JAK2 and SRC in Y357 phosphorylation (Li et al., 2016, Taniguchi et al., 2015). However, downstream of FAK, we observed both tyrosine-phosphorylated YAP and a decrease in pS127 YAP, the latter a direct target of the Hippo signaling pathway. In this regard, there is increasing evidence suggesting that Hippo signaling is tightly regulated by the assembly and dissociation of key signaling complexes. Our interrogation of these complexes in response to FAK activation led to the finding that FAK phosphorylates MOB1 on Y26, resulting in the disassembly of the MOB1/LATS complex and disruption of the Hippo pathway downstream of MST1, effectively rewiring the molecular mechanisms controlling YAP activity. Mutation of Y26 of MOB1 is sufficient to abolish the effect of FAK. Whereas further work may be required to establish the structural basis for this inhibition, as well as alternative FAK-driven pathways in mechanotransduction and development, our findings support that disruption of the MOB1/LATS signaling complex by FAK is a key regulatory step resulting in YAP activation by Gαq. Ultimately, this mechanism may coordinate the Gαq-induced increase in cytosolic free YAP, which is mediated by Rho-induced actin polymerization (Feng et al., 2014b), with Hippo kinase cascade inhibition through the FAK-mediated phosphorylation of MOB1, resulting in the YAP-dependent UM cell growth.