RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma

Abstract

Constitutive activation of Gαq signaling by mutations in GNAQ or GNA11 occurs in over 80% of uveal melanomas (UMs) and activates MAPK. Protein kinase C (PKC) has been implicated as a link, but the mechanistic details remained unclear. We identified PKC δ and ɛ as required and sufficient to activate MAPK in GNAQ mutant melanomas. MAPK activation depends on Ras and is caused by RasGRP3, which is significantly and selectively overexpressed in response to GNAQ/11 mutation in UM. RasGRP3 activation occurs via PKC δ- and ɛ-dependent phosphorylation and PKC-independent, DAG-mediated membrane recruitment, possibly explaining the limited effect of PKC inhibitors to durably suppress MAPK in UM. The findings nominate RasGRP3 as a therapeutic target for cancers driven by oncogenic GNAQ/11.

Keywords: DAG; GNA11; GNAQ; MAPK; PKC; RasGEF; RasGRP3; melanoma; uveal melanoma.

Figure 1. PKC δ and ε Are Important Downstream Effectors of Oncogenic GNAQ in Mediating MAPK Signaling

(A) Expression of PKC isoforms in melanoma cell lines with and without GNAQ or GNA11 mutations. Previously validated antibodies against various PKC isoforms (Figures S1A and S1B) were tested on a broad panel of melanoma cell lines. Suitable positive controls for isoforms found not to be expressed are shown on the rightmost lane. PC, positive control. (B) Knockdown of GNAQ and PKC isoforms δ and ε reduced levels of pMEK and pERK in the GNAQ-mutated uveal melanoma cell line 92-1, but not in the GNAQ wild-type cell line C8161. (C) Cells were transfected with respective siRNAs either singly or in combination and then lysed. −, no siRNA; NT, non-targeting siRNA. (D) PKC δ and ε synergized with GNAQ^Q209L in activating MAPK in 293FT cells. 293FT cells were transfected for 24 hr. PKC isoforms expression was detected using HA tags and GNAQ^Q209L using a Glu-Glu tag (EE). (E) Effect of knockdown of PKC α, δ, ε, and GNAQ or GNA11, on the proliferation of GNAQ mutant melanoma cells (92-1 and OMM1.3) and GNA11 mutant cell line (UPMD-2) and GNAQ/11 wild-type melanoma cells (C8161 and SK-MEL-28 from cutaneous origin and MEL285 from uveal origin). Cells were transfected with various combination of siRNAs as indicated. After transfection (48 hr), cells were counted and seeded at equal density. Cells were counted at days 1, 3, and 5 after plating. −, no siRNA control; NT, non-targeting siRNA control. Error bars represent the SEM. See also Figures S1 and S2.

Figure 2. Oncogenic GNAQ Activates Ras

(A) GNAQ^Q209L transfected alone or combined with PKC δ or ε into 293FT cells increased Ras-GTP levels. 293FT cells were transfected with indicated cDNA plasmids for 24 hr. Ras-GTP pull-down was performed and detected by western blot with a pan-Ras antibody. TPA stimulation was used a positive control. Expression levels of GNAQ^Q209L were monitored with Glu-Glu (EE) tag. PKC δ and ε were detected via HA tags. (B) Oncogenic GNAQ activates all three Ras isoforms. 293FT cells were transfected with indicated cDNA plasmids for 24 hr. Ras-GTP pull-down was performed and detected by western blot with Ras isoform-specific antibodies. (C) Depletion of Ras isoforms reduced MAPK signaling as demonstrated by decreased pMEK and pERK in GNAQ mutant melanoma cell lines. Three human uveal melanoma cell lines with GNAQ mutation (92-1, OMM1.3, and MEL202) were transfected with mock (−), non-targeting siRNAs (NT), siRNAs against GNAQ, Hras (H), Kras (K), Nras (N), or indicated combinations for 72 hr and lysed. See also Figure S2.

Figure 3. RasGRP3 Is Upregulated in Uveal Melanoma Cells with GNAQ/11 Mutations

(A) Scatterplot of top 487 genes (left panel) and heatmap of top 30 genes (right panel) of differentially expressed genes between 5 GNAQ/11 mutant (mt) and 5 GNAQ/11 wild-type (WT). The black arrow indicates RasGRP3. (B) qRT-PCR shows markedly elevated RasGRP3 mRNA levels in 6 GNAQ mutant cells (92-1, MEL202, OMM1.3, MEL270, UPMM2, and UPMM3) and 3 GNA11 mutant cells (OMM-GN11, UPMD-1, and UPMD-2), compared with three types of human melanocytes (IHM, immortalized human melanocytes; NHM, normal human melanocytes) and seven human melanoma cell lines without GNAQ/11 mutations. Three (MUM2C, MEL285, and MEL290) melanoma cell lines without GNAQ/11 mutations are from uveal origin. (C) RasGRP3 mRNA expression across different human cancers from TCGA RNA sequencing data. RasGRP3 expression is highest in uveal melanoma. (D) Cutaneous melanomas with GNAQ^Q209P or GNA11^Q209L mutations in the TCGA have increased RasGRP3 mRNA expression, comparable with uveal melanomas. (E) Western blot shows increased RasGRP3 protein levels in human melanoma cells with GNAQ/11 mutations compared with cell lines with other mutations (MEL285, MUM2C, and MEL290 are of uveal and the other GNAQ/11 WT cell lines are of cutaneous origin). RasGRP1, RasGRP2, and RasGRP4 were not detectable in melanoma cells with GNAQ/11 mutations. 293FT lysates transfected with RasGRPs cDNA were used as positive control. PC, positive control. (F) RasGRP3 immunohistochemistry in human uveal melanoma cell lines. RasGRP3 immunohistochemistry of pellets of formalin-fixed paraffin-embedded GNAQ mutant uveal melanoma cell lines (92-1 and MEL270) and GNAQ/11 wild-type lines (CRMM1 and CRMM2). Scale bars, 100 μm. (G) RasGRP3 immunohistochemistry in four uveal melanoma tissues with GNAQ mutation and four with GNA11 mutations. Scale bars, 100 μm. See also Figure S3.

Figure 4. Oncogenic GNAQ Signaling Regulates RasGRP3 Expression and Phosphorylation in Uveal Melanoma Cells

(A) Depletion of GNAQ with siRNAs decreased RasGRP3 expression and T133 phosphorylation in three GNAQ mutant uveal melanoma cells (OMM1.3, 92-1, and MEL202). Cells were transfected with mock (−), non-targeting siRNAs (NT), or GNAQ siRNAs for 72 hr and lysed. (B) GNAQ^Q209L or GNA11^Q209L, but not wild-type GNAQ, GNA11, or Braf^V600E, induced increased RasGRP3 expression and phosphorylation in immortalized human melanocytes. Melanocytes were infected with lentivirus expressing indicated proteins for 48 hr and analyzed by immunoblot. Expression levels of GNAQ and GNA11 were monitored using Glu-Glu tags. (C) The PKC inhibitor AEB071 decreased RasGRP3 expression and phosphorylation in OMM1.3 cells in a time-dependent manner. OMM1.3 cells were incubated with DMSO or 500 nM AEB071 for the times indicated. (D) The PKC inhibitors AEB071 and AHT956 decreased RasGRP3 expression and phosphorylation in OMM1.3 cells in a dose-dependent manner. OMM1.3 cells were incubated with DMSO or AEB071 or AHT956 at indicated doses for 24 hr. (E) TPA increased RasGRP3 expression and phosphorylation in human melanocytes. Immortalized human melanocytes were incubated with or without 200 nM TPA for the indicated times. (F) PKC δ and ε increased T133 phosphorylation of RasGRP3 in 293FT cells. 293FT cells were transfected with indicated cDNAs for 24 hr. (G) Knockdown of PKC δ and ε decreased RasGRP3 expression and phosphorylation in GNAQ mutant cells. Western blot of 92-1 cells transfected for 72 hr with mock (−), non-targeting siRNAs (NT), PKC α, δ, or ε siRNAs, singly or in indicated combination. (H) The MEK inhibitor PD0325901 has no effect on RasGRP3 expression and phosphorylation in GNAQ mutant cells. MEL202 and 92-1 cells were incubated with PD0325901 at different doses for 24 hr. See also Figures S4S6.

Figure 5. RasGRP3 Is Important for GNAQ-Mediated MAP-Kinase Pathway Activation and Proliferation

(A) siRNA-mediated knockdown of RasGRP3 decreases pMEK and pERK levels in three GNAQ mutant melanoma cells (92-1, omm1.3, and mel270), but not in three GNAQ/11 wild-type melanoma cells. (B) MUM2C is from uveal origin. Cells were transfected with mock(−), non-targeting, or RasGRP3 siRNAs for 72 hr and lysed for western blot analysis. (C) RasGRP3 knockdown reduces proliferation of melanoma cells with GNAQ mutations (92-1, MEL270, and OMM1.3), but not of cell lines with other mutations (SK-MEL-5 and UACC257). Cells were transfected with non-targeting or RasGRP3 siRNAs; 48 hr after transfection, cells were counted and plated at equal density. Cells were counted at days 1, 3, and 5 after plating. Data represent the mean ± SEM. (D) Western blot of stable knockdown of RasGRP3 using shRNA. 92-1 cells were infected with lentivirus expressing two different shRNAs for RasGRP3 and selected with puromycin. NT, non-targeting shRNA. (E) shRNA-mediated knockdown of RasGRP3 in 92-1 cells from (D) decreased the number and size of colonies. Cells were stained with crystal violet after 18 days of culture. (F) Nude mice were injected subcutaneously with 2 × 10⁶ 92-1 cells expressing RasGRP3 shRNA-1 (n = 5) or non-targeting shRNA (n = 5). Tumors sizes and weights for both experimental arms 13 weeks after injection. Data represent the mean ± SD. (G) The effect of co-transfection of GNAQ^Q209L with four different RasGRP isoforms on MAPK signaling. 293FT cells were transfected with mock (−), GFP, GNAQ^Q209L, RasGRP1, RasGRP2, RasGRP3, or RasGRP4 plasmids either singly or in combination as indicated. After 24 hr, cells were lysed and blotted. Arrows indicate that pRasGRP1 antibodies recognized both RasGRP1 T184 phosphorylation and RasGRP3 T133 phosphorylation. See also Figure S7.

Figure 6. Both PKC-Mediated Phosphorylation and C1 Domain Are Required for RasGRP3-Mediated MAPK Activation

(A) Mutating the PKC phosphorylation site of RasGRP3 (T133A) partially attenuated RasGRP3-mediated MAPK signaling in 293FT cells. 293FT cells were transfected with mock (−), GFP, GNAQ^Q209L, RasGRP3^wt (GRP3^wt), or RasGRP3^T133A (GRP3^T133A) either singly or in the indicated combination. After 24 hr, cells were lysed and subjected to western blot. RasGRP3 wild-type and mutant expression were detected with Myc tag antibody. GLU-GLU tag (EE) was used to detect GNAQ^Q209L. The top panel shows a schematic diagram of the RasGRP3 T133A construct. (B) PKC inhibition fails to completely abrogate RasGRP3-mediated MAPK signaling. 293FT cells were transfected with GFP, GNAQ^Q209L, RasGRP3^wt, or RasGRP3^T133A, either singly or in the indicated combinations. After 24 hr, transfected cells were treated with 1 μM AEB071 (AEB) or vehicle for another 24 hr and then lysed. (C) The MEK inhibitor PD0325901 (PD) completely suppressed RasGRP3-mediated MAPK signaling. 293 FT cells were co-transfected with GNAQ^Q209L and RasGRP3^T133A or with GNAQ^Q209L and RasGRP3^wt. After 24 hr, cells were treated for another 24 hr with DMSO, 1 μM AEB071 (AEB), 1 μm AHT956 (AHT), 100 nM PD0325901 (PD), 1 μM AEB + 100 nM PD, or 1 μM AHT956 + 100 nM PD, respectively. (D) Both T133 phosphorylation and the C1 domain of RasGRP3 contribute to oncogenic GNAQ-mediated MAPK signaling. 293 FT cells were transfected with indicated constructs for 24 hr and lysed. The left panel shows a schematic diagram of the RasGRP3 C1 domain deletion constructs used. (E) Restoration of MAPK activation by expression of membrane-targeted forms of RasGRP3 in 293FT cells. RasGRP3 constructs tagged with the membrane-targeting motifs of Fyn and Src protein as shown in the left panel. 293FT cells transfected with indicated RasGRP3 mutant constructs for 24 hr. See also Figure S7.

Figure 7. Model of Oncogenic GNAQ Activates MAPK Signaling in UM

GTP-bound GNAQ directly activates PLC β, which generates the second messenger DAG. DAG activates PKC δ and ε by binding their C1 domains. The Ras activator RasGRP3 represents a critical signaling node linking oncogenic GNAQ to the RAS/RAF/MEK/ERK signaling pathway by three independent mechanisms: binding of membrane recruitment by DAG, phosphorylation and upregulation of expression by PKC δ and ε.