Research ArticleG Protein Signaling

Direct targeting of Gαq and Gα11 oncoproteins in cancer cells

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Science Signaling  19 Mar 2019:
Vol. 12, Issue 573, eaau5948
DOI: 10.1126/scisignal.aau5948
  • Fig. 1 FR suppresses hallmark features downstream of Gα11oncoproteins in CM cells.

    (A) Quantification of the effect of the indicated concentrations of FR on the number of adherent HCmel12 melanoma cells cultured for 72 hours. Data are means + SEM of three experiments. (B) Viable cell metabolism was analyzed by XTT assay after treatment of HCmel12 cells with vehicle or the indicated concentration of FR for 48 hours. Data are means + SEM of three experiments. Data in (A) and (B) were normalized to those of vehicle-treated control cells. (C) Flow cytometric histograms (left) and quantitative analysis (right) of cell cycle progression in HCmel12 cells treated with vehicle or the indicated concentrations of FR using the fluorescent DNA-intercalating agent PI. (D) Flow cytometric analysis (left) and quantitative analysis (right) of annexin V– and PI-stained HCmel12 cells that were left untreated or were treated with the indicated concentrations of FR for 72 hours. Plots in (C) and (D) are representative of three biological replicates. ns, not significant. (E and F) Cell pellets (E) and bright-field microscopic images (F) of HCmel12 cells after 72 hours of treatment with vehicle or FR. Scale bars, 5 μm. Four experiments are represented by the data in (E) and (F). (G) Representative Western blotting analysis (top) and quantification (bottom) of the relative abundance of the differentiation marker gp100 in HCmel12 cells after treatment with vehicle or 10 nM FR for 72 hours. Data are means ± SEM of four experiments. (H) Basal migratory capacity of HCmel12 cells after treatment with vehicle or the indicated concentrations of FR as determined in Boyden chamber transwell assays. Graph: Data are means + SEM of three experiments. Scale bar, 200 μm. Statistical significance was determined by one-sample t test for (A), (B), and (G) or by two-way analysis of variance (ANOVA) for (D).

  • Fig. 2 FR preferentially silences mitogenic pERK signaling over canonical PLCβ signaling in cells with mutationally activated GαqQ209L through a direct interaction with its protein target.

    (A to D) Analysis of the effects of the indicated concentrations of FR on the amounts of pERK1/2 and IP1 in HCmel12 cells (A), B16 melanoma cells (B), CCh-treated HEK293 cells expressing endogenous Gq-coupled muscarinic M3 receptors (C), and propionic acid (C3)–stimulated HEK293 cells exogenously expressing free fatty acid FFA2 receptors (D). Data are from n = 7 [pERK1/2, (A)], n = 6 [IP1, (A)], n = 4 [pERK1/2, (B)], n = 3 [IP1, (B)], n = 6 [pERK1/2, (C) and (D)], and n = 4 [IP1, (C) and (D)] independent experiments. (E and F) Analysis of the effects of the indicated concentrations of FR on intrinsic IP1 accumulation (E) and SRE reporter activity (F) in Gαq/11 KO cells transiently expressing Gαqwt or GαqQ209L. Data are means ± SEM of three experiments. (G) Analysis of the effects of the indicated concentrations of FR on SRE reporter assays, with HEK293 Gαq/11 KO cells expressing WT or Q209L mutant proteins together with key FR-binding site deletions. (H) Illustration of the FR-binding site on Gαq and the sites of the individual loss-of-function mutations from (G). (I and J) Illustration of the overall structure (I) and a magnified view (J) of an AlF4-dependent complex of Gαq and PLCβ3 (PDB, 3ohm) in the absence (left) and presence (right, dark green) of FR, which was accommodated at the domain interface of the active Gαq-PLC-β3 complex. (K) Scheme for the kiss-and-run activation of PLCβ. GPCR activation promotes the exchange of GDP for GTP on the Gα subunit. Gα-GTP separates from Gβγ to bind to and activate its effector PLCβ (kiss). Deactivation of signaling occurs when PLCβ converts Gα-GTP into Gα-GDP with its intrinsic GTPase activity, causing the complex to dissociate (run) and to reset the inactive GDP-bound state. FR disrupts this cycle and attenuates PLCβ signaling by preserving Gαq in the inactive, GDP-bound state, thereby diminishing the probability of “kissing.” (L and M) Single-cell FRET imaging of HEK293T cells expressing the muscarinic M3 receptor, Gβ1γ2, YFP-PLCβ3, and CFP-Gαqwt. (L) Cells were super-fused with buffer and buffer containing 10 μM CCh consecutively as indicated, normalized to basal. Cells were (or not) additionally exposed to 1 μM FR during the period of CCh treatment (WT, n = 17 cells, four experiments; Q209L, n = 24 cells, three experiments). (M) FRET signals shown as absolute FYFP/FCFP ratio of experiments with and without 10 μM CCh (WT, n = 35 cells, four experiments; Q209L, n = 40 cells, six experiments). Data are means + SEM of the indicated number of independent experiments. Statistical significance was calculated by unpaired t test (A, B, F, L, and M) or one-way ANOVA (E). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 FR effectively dampens signaling of non-PLCβ effectors downstream of Gαq/11oncoproteins.

    (A) Representative real-time DMR recordings of the cellular responses induced by FR and the indicated stimuli [CCh, 30 μM; forskolin (Fsk), 30 μM; EGF, 50 nM] in Gαq/11 KO cells transiently transfected with empty plasmid or with plasmid encoding Gαqwt or GαqQ209L. (B) Quantification of the effects of FR on Gαqwt and GαqQ209L from the experiments shown in (A). Data are means ± SEM of three experiments. (C and D) Representative DMR profile of CCh-induced cell responses and inhibition with 100 μM atropine (C) or FR (D) in Gαq/11 KO cells transiently expressing the indicated Gα proteins. (E) Quantification of the effects of FR on the positive DMR deflections from the experiments shown in (D). Data are means ± SEM of three experiments. (F and G) Thin-layer chromatography (TLC) of lysates of [32P]orthophosphate-labeled HEK293T cells transfected with empty vector or with plasmid encoding the indicated HA-tagged Gα subunit. Subunits were isolated either by immunoprecipitation (IP) with anti-HA antibody (F) or by their interaction with His6-Gβγ isolated by pulldown (PD:His) with TALON resin (G). The detection of Gα subunits by Western blotting analysis (G, left) confirmed their expression in total cell lysates and successful pulldown with His6-Gβγ. (H) Analysis of the effects of the indicated concentrations of FR on YAP phosphorylation (by comparing total YAP abundance to pYAP abundance) in Gαq/11 KO cells transiently transfected with empty vector or with plasmid encoding Gαqwt or GαqQ209L. (I) Analysis of the effects of the indicated concentrations of FR on pYAP abundance in Gαq/11 KO cells expressing WT Gαq or the Q209L mutant Gαq with or without the I190W mutation within the FR-binding site. For the I190W Q209L mutant, a plateau was not reached; therefore, the effect of 10 μM FR was arbitrarily set to 50%. Statistical significance was calculated using the Wilcoxon test (B) or by one-sample t test (H). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 4 FR suppresses prosurvival signaling downstream of Gαq/11 oncoproteins in UM cell lines.

    (A and B) Quantification of cell-intrinsic IP1 production in the indicated UM cell lines (A) and analysis of their sensitivity to treatment with the indicated concentrations of FR (B). Gray and blue bars in (A) indicate experiments with 10,000 cells per well and 50,000 cells per well, respectively. In (B), 10,000 cells per well were used. Data are from four biological replicates. (C) Comparison of the relative amounts of pERK1/2 in the indicated UM cell lines in the absence (Basal) and presence of 1 μM trametinib (Tram). For Mel290, Mel285, and 92.1 cells, data are from four independent experiments; for Mel270, Mel202, and OMM1.3 cells, data are from three independent experiments. (D) Analysis of the effects of the indicated concentrations of FR on pERK1/2 amounts in the indicated UM cell lines. Data are means ± SEM of three experiments. (E) Comparison of the relative amounts of pAKTS473 in the indicated UM cell lines in the absence and presence of 30 μM LY294002. Data are from four independent experiments. (F) Analysis of the effects of the indicated concentrations of FR on pAKTS473 abundance in the indicated UM cell lines expressing WT (n = 3 experiments) or mutant Gαq/11 (n = 4 experiments). Statistical significance in (B) was calculated by a one-sample t test; for (C) and (E), unpaired t tests were used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 5 FR inhibits YAP signaling in Gq mutant UM cell lines.

    (A) Quantification of cytosolic pYAP amounts in the indicated UM cell lines expressing WT or mutant Gαq/11. Dots depict single experiments; horizontal lines show total averaged data ± SEM (vertical bars). (B) Effects of FR and forskolin on pYAP abundance in the indicated UM cell lines. For Mel202 and 92.1 cells, n = 7 experiments; for OMM1.3 cells, n = 5; for all others, n = 6. Data are expressed relative to vehicle-treated cells. (C and D) Western blotting analysis (C) and quantification (D) of relative pYAP abundance in the indicated cells after treatment for 1 hour with 10 μM FR or vehicle. Data are from four independent experiments. (E) Analysis of the subcellular distribution of YAP in the indicated UM cell lines after treatment with 10 μM FR or vehicle for 1 hour. Cells were analyzed by immunofluorescence microscopy after treatment with anti-YAP antibody (green) and DAPI (blue) to stain nuclei. Images are representative of three experiments. Statistical significance was calculated with an unpaired t test for (A) or a one-sample t test for (B) and (D). Data in (B) and (D) are means + SEM of the indicated number of independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 6 Mutation-specific inhibition of proliferation and tumor growth by FR as a strategy to treat Gαq-driven cancer.

    (A) Effect of the indicated concentrations of FR on the number of the indicated UM cell lines expressing WT or mutant Gαq/11 after 72 hours of treatment. As a control, cells were treated with 1 μM trametinib. The numbers of treated cells are expressed as a percentage of the numbers of vehicle-treated controls (n = 3 biological replicates each performed in duplicate). (B) Representative bright-field images (n = 3 experiments) of UM cell lines expressing WT or mutant Gαq/11 treated with 100 nM FR or 1 μM trametinib for 72 hours. Scale bars, 100 μm. (C) Left: Effects of FR and vehicle on the growth of Mel270 (GαqQ209P) UM cells in a xenograft mouse model in vivo (n = 5 animals per control- and FR-treated group). Right: Representative histological section of a Mel270 tumor originating from xenograft experiments and stained with hematoxylin and eosin after 14 days of treatment with FR or vehicle. Tumor size was monitored every 3 to 4 days after the initiation of treatment. ***P < 0.001. (D) An experiment comparable to that shown in (C) was performed with CM A375 (B-RafV600E) xenografts (n = 6 animals per control- and FR-treated group). Data in (A), (C), and (D) are means ± SEM of the indicated numbers of experiments.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/573/eaau5948/DC1

    Fig. S1. Exemplary gating strategy used in flow cytometry analysis to detect and quantify apoptotic and necrotic cells.

    Fig. S2. HCmel12 cells display intrinsic Gq activity.

    Fig. S3. Mouse B16 melanoma cells exhibit increased levels of Gq activity.

    Fig. S4. FR does not affect total ERK protein abundance in HCmel12, B16, or HEK293 cells.

    Fig. S5. Functional expression of WT and mutant Gαq proteins in an SRE reporter assay.

    Fig. S6. Authentication of UM cell lines by Sanger sequencing at position Q209 of Gαq.

    Fig. S7. Effects of AlF4 on IP1 production in UM cell lines expressing WT or mutant Gαq.

    Fig. S8. FR inhibits AlF4-mediated IP1 accumulation in Mel290 cells.

    Fig. S9. FR and trametinib do not alter the abundance of total ERK1/2 proteins in UM cell lines.

    Fig. S10. Time course analysis of the amounts of total AKT and pAKT in UM cell lines after treatment with FR.

    Fig. S11. Neither FR nor forskolin alters the abundance of total cellular YAP protein.

  • This PDF file includes:

    • Fig. S1. Exemplary gating strategy used in flow cytometry analysis to detect and quantify apoptotic and necrotic cells.
    • Fig. S2. HCmel12 cells display intrinsic Gq activity.
    • Fig. S3. Mouse B16 melanoma cells exhibit increased levels of Gq activity.
    • Fig. S4. FR does not affect total ERK protein abundance in HCmel12, B16, or HEK293 cells.
    • Fig. S5. Functional expression of WT and mutant Gαq proteins in an SRE reporter assay.
    • Fig. S6. Authentication of UM cell lines by Sanger sequencing at position Q209 of Gαq.
    • Fig. S7. Effects of AlF4 on IP1 production in UM cell lines expressing WT or mutant Gαq.
    • Fig. S8. FR inhibits AlF4-mediated IP1 accumulation in Mel290 cells.
    • Fig. S9. FR and trametinib do not alter the abundance of total ERK1/2 proteins in UM cell lines.
    • Fig. S10. Time course analysis of the amounts of total AKT and pAKT in UM cell lines after treatment with FR.
    • Fig. S11. Neither FR nor forskolin alters the abundance of total cellular YAP protein.

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