Oncogenic Gαq activates RhoJ through PDZ-RhoGEF.

Oncogenic Gαq causes uveal melanoma via non-canonical signaling pathways. This constitutively active mutant GTPase is also found in cutaneous melanoma, lung adenocarcinoma, and seminoma, as well as in benign vascular tumors, such as congenital hemangiomas. We recently described that PDZ-RhoGEF (also known as ARHGEF11), a canonical Gα12/13 effector, is enabled by Gαs Q227L to activate CdcIn addition, and we demonstrated that constitutively active Gαq interacts with the PDZ-RhoGEF DH-PH catalytic module, but does not affect its binding to RhoA or Cdc. This suggests that it guides this RhoGEF to gain affinity for other GTPases. Since RhoJ, a small GTPase of the Cdc42 subfamily, has been involved in tumor-induced angiogenesis and the metastatic dissemination of cancer cells, we hypothesized that it might be a target of oncogenic Gαq signaling via PDZ-RhoGEF. Consistent with this possibility, we found that Gαq Q209L drives full-length PDZ-RhoGEF and a DH-PH construct to interact with nucleotide-free RhoJ-G33A, a mutant with affinity for active RhoJ-GEFs. Gαq Q209L binding to PDZ-RhoGEF was mapped to the PH domain, which, as an isolated construct, attenuated the interaction of this mutant GTPase with PDZ-RhoGEF's catalytic module (DH-PH domains). Expression of these catalytic domains caused contraction of endothelial cells and generated fine cell sprouts that were inhibited by co-expression of dominant negative RhoJ. Using relational data mining of uveal melanoma patient TCGA datasets, we got an insight into the signaling landscape that accompanies the Gαq/PDZ-RhoGEF/RhoJ axis. We identified three transcriptional signatures statistically linked with shorter patient survival, including GPCRs and signaling effectors that are recognized as vulnerabilities in cancer cell synthetic lethality datasets. In conclusion, we demonstrated that an oncogenic Gαq mutant enables the PDZ-RhoGEF DH-PH module to recognize RhoJ, suggesting an allosteric mechanism by which this constitutively active GTPase stimulates RhoJ via PDZ-RhoGEF. These findings highlight PDZ-RhoGEF and RhoJ as potential targets in tumors driven by mutant Gαq.

Endothelial cell sprouting driven by RhoJ directly activated by a membrane-anchored Intersectin 1 (ITSN1) RhoGEF module.

Endothelial cell sprouting is a critical event in tumor-induced angiogenesis. In melanoma and lung cancer murine models, targeting RhoJ prevents endothelial sprouting, tumor growth and metastasis and enhances the effects of conventional anti-neoplastic therapy. Aiming to understand how RhoJ is activated, we used a gain of function approach to identify constitutively active Rho guanine nucleotide exchange factors (RhoGEFs) able to promote RhoJ-dependent actin-driven membrane protrusions. We demonstrate that a membrane-anchored Intersectin 1 (ITSN1) DH-PH construct promotes endothelial cell sprouting via RhoJ. Mechanistically, this is controlled by direct interaction between the catalytic ITSN1 DH-PH module and RhoJ, it is sensitive to phosphorylation by focal adhesion kinase (FAK) and to endosomal trapping of the ITSN1 construct by dominant negative RhoJ. This ITSN1/RhoJ signaling axis is independent of Cdc42, a previously characterized ITSN1 target and a RhoJ close homologue. In conclusion, our results elucidate an ITSN1/RhoJ molecular link able to promote endothelial cell sprouting and set the basis to explore this signaling pathway in the context of tumor-induced angiogenesis.

Protumoral bone marrow-derived cells migrate via Gßγ-dependent signaling pathways and exhibit a complex repertoire of RhoGEFs.

Reciprocal communication among cells of the tumor microenvironment contributes to cancer progression. Here, we show that a protumoral population of cultured bone marrow-derived cells (BMDC) containing Tie2+/CD45+/CD11b + cells responded to lung carcinoma cells and reciprocally stimulated them. These cells migrated via heterotrimeric G protein-dependent signaling pathways and strongly activated the PI3K/AKT, ERK and mTOR signaling cascades in response to conditioned media and chemotactic agonists. To get insight into the molecular machinery involved in BMDC migration, we revealed their repertoire of guanine nucleotide exchange factors for Rho GTPases (RhoGEFs) and G proteins in comparison with fresh bone marrow cells, proven that these cell populations had contrasting effects on tumor growth. BMDC exhibited a higher expression of G protein regulated RhoGEFs including P-Rex1, PDZ-RhoGEF, LARG, Trio and some less well characterized RhoGEFs such as ARHGEF5, ARHGEF17 and PLEKHG6. G proteins such as Gα12/13, Gαq, and the small GTPase RhoJ were also highly expressed in BMDC. Our results indicate that Tie2+/CD45+/CD11b + BMDC express a unique variety of chemotactic transducers and effectors potentially linked to their protumoral effect, warranting further studies to their characterization as molecular targets.

Gßγ signaling to the chemotactic effector P-REX1 and mammalian cell migration is directly regulated by Gαq and Gα13 proteins.

G protein-coupled receptors stimulate Rho guanine nucleotide exchange factors that promote mammalian cell migration. Rac and Rho GTPases exert opposing effects on cell morphology and are stimulated downstream of Gßγ and Gα12/13 or Gαq, respectively. These Gα subunits might in turn favor Rho pathways by preventing Gßγ signaling to Rac. Here, we investigated whether Gßγ signaling to phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-REX1), a key Gßγ chemotactic effector, is directly controlled by Rho-activating Gα subunits. We show that pharmacological inhibition of Gαq makes P-REX1 activation by Gq/Gi-coupled lysophosphatidic acid receptors more effective. Moreover, chemogenetic control of Gi and Gq by designer receptors exclusively activated by designer drugs (DREADDs) confirmed that Gi differentially activates P-REX1. GTPase-deficient GαqQL and Gα13QL variants formed stable complexes with Gßγ, impairing its interaction with P-REX1. The N-terminal regions of these variants were essential for stable interaction with Gßγ. Pulldown assays revealed that chimeric Gα13-i2QL interacts with Gßγ unlike to Gαi2-13QL, the reciprocal chimera, which similarly to Gαi2QL could not interact with Gßγ. Moreover, Gßγ was part of tetrameric Gßγ-GαqQL-RGS2 and Gßγ-Gα13-i2QL-RGS4 complexes, whereas Gα13QL dissociated from Gßγ to interact with the PDZ-RhoGEF-RGS domain. Consistent with an integrated response, Gßγ and AKT kinase were associated with active SDF-1/CXCL12-stimulated P-REX1. This pathway was inhibited by GαqQL and Gα13QL, which also prevented CXCR4-dependent cell migration. We conclude that a coordinated mechanism prioritizes Gαq- and Gα13-mediated signaling to Rho over a Gßγ-dependent Rac pathway, attributed to heterotrimeric Gi proteins.