Research ArticleDRUG DEVELOPMENT

Targeting nucleotide exchange to inhibit constitutively active G protein α subunits in cancer cells

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Science Signaling  04 Sep 2018:
Vol. 11, Issue 546, eaao6852
DOI: 10.1126/scisignal.aao6852
  • Fig. 1 FR traps mutant constitutively active Gαq in the inactive GDP-bound state.

    (A) Split luciferase complementation assays in human embryonic kidney (HEK) 293 cells, measured as reconstitution of CBG luciferase activity normalized to cotransfected, constitutively expressed Renilla luciferase to assess the effect of FR on the activity state of Gα subunits as determined by interaction of Gβ1γ2 with the indicated wild-type (WT) or mutant constitutively active forms of Gαq (q) or Gα13 (13) (Q209L and Q226L, respectively). Data are means ± SEM of three experiments. (B) Split luciferase complementation assays in HEK293 cells, as described in (A), to assess the potency of FR as a driver of interaction between Gβ1γ2 and wild-type or mutant constitutively active Gαq. Data are means ± SEM from three experiments. CBGC, C-terminal portion of CBG luciferase; CBGN, N-terminal portion of CBG luciferase. (C) Effect of FR treatment of HEK293 cells on copurification of endogenous Gβγ with overexpressed, affinity-tagged wild-type or constitutively active Gαq(Q209L). Affinity-tagged Gαq and Gβγ were detected by immunoblotting (IB) with FLAG and Gβ antibodies, respectively. Data shown are representative of three independent experiments. (D) Split luciferase complementation assays in HEK293 cells, as described in (A), to assess the potency of FR as an inhibitor of interaction between RGS2 and mutant constitutively active Gαq(Q209L) or between the RGS domain of LARG [LARG(RGS)] and mutant constitutively active Gα13(Q226L). Data are means ± SEM from three experiments. (E) Potency of FR as inhibitor of signal transduction by constitutively active Gαq or Gα13 detected with an SRE(L) promoter-driven transcriptional reporter in HEK293 cells. Data are means ± SEM from three experiments. Constitutively active Gαq(Q209L), constitutively active Gαq bearing the indicated FR-binding site mutations, and constitutively Gα13(Q226L) were studied. Concentration-response curves were fit by nonlinear regression to derive EC50 or IC50 values, which were compared by t test. *P < 0.05, **P < 0.01 by t test; significance was confirmed using q < 0.05 or q < 0.01 by the false discovery rate (FDR) method of Benjamini and Hochberg. n.s., not significant.

  • Fig. 2 Inhibition of Gαi1 bearing an engineered FR-binding site.

    (A) Amino acid residues in Gαi1 identical to or diverged from the FR-binding site in Gαq are indicated in blue and red, respectively. An FR-binding site was engineered in Gαi1 by introducing the eight indicated amino acid substitutions so as to match the corresponding residues of Gαq, thereby producing a chimeric Gα subunit termed Gi/q. (B) Effect of FR on guanine nucleotide exchange by Gαi/q in vitro. Nucleotide exchange was assayed by measuring increase in BODIPY-GTPγS fluorescence upon binding to the indicated purified His-tagged Gα subunits in the absence or presence of the indicated concentrations of FR. Gi/q(R54K) corresponds to a Gαq mutant that is less sensitive to inhibition by FR. Nucleotide exchange rates (kobs) of Gαi/q and Gαi/q(R54K) in the absence of FR were 0.24 and 0.12 min−1, respectively. Data are means ± SEM from three independent experiments. (C) Effect of FR on agonist-evoked signaling mediated by Gi/q. Inhibition of forskolin-induced cAMP Förster by Gi-coupled cannabinoid receptors was measured in Neuro2A cells transfected with a cAMP fluorescence resonance energy transfer (FRET) reporter and pertussis toxin (PTX)–resistant and EE epitope–tagged forms of the indicated Gα subunits and treated with PTX to inactivate endogenous Gi. Inhibition of forskolin-induced cAMP formation by a cannabinoid receptor agonist (WIN 55,212-2; WIN) was measured by FRET. Attenuation of this inhibitory effect by FR was quantified relative to vehicle controls. Data are means ± SEM from three experiments. IC50 values of FR toward cells expressing Gi/q and Gi/q(R54K) were 4.6 and 76 μM, respectively. (D) Failure of FR to correct the GTP hydrolysis defect of Gi/q(Q204L) in vitro. Hydrolysis of γ32P-GTP by the indicated Gα subunits present at 10-fold molar excess over GTP was measured over time. FR was added at the indicated time point (arrow). Data are means ± SEM from three independent experiments. (E) Effect of FR on guanine nucleotide exchange by constitutively active Gi/q(Q204L) in vitro, as determined by methods described in (B). The rate of nucleotide exchange (kobs) by Gi/q(Q204L) in the absence of FR was 0.14 min−1. Concentration-response curves were fit by nonlinear regression to derive IC50 values, which were compared by t test. *P < 0.05, **P < 0.01 by t test; significance was confirmed using q < 0.05 or q < 0.01 by the FDR method of Benjamini and Hochberg.

  • Fig. 3 FR inhibits signaling by constitutively active Gαq in UM cells.

    (A) Inhibition of signaling by constitutively active Gαq in UM cell lines was quantified by measuring intracellular IP1, a metabolically stable product of inositol 1,4,5-trisphosphate produced by Gαq-stimulated phospholipase Cβ. Basal IP1 values in UM cell lines driven by constitutively active Gαq(Q209L) (92.1 and Mel202 cells) and BRAF(V600E) (OCM-1A cells). Data are means ± SEM from three experiments performed in triplicate. (B) Effect of FR on IP1 abundance in 92.1, Mel202, and OCM-1A cells. Data are means ± SEM of four experiments performed in triplicate. *P < 0.01 by t test; significance was confirmed using q < 0.01 by the FDR method of Benjamini and Hochberg.

  • Fig. 4 FR-sensitive growth and viability of UM cells driven by constitutively active Gαq.

    (A) Changes in viability of UM cells treated with FR. Cell viability was quantified using a water-soluble tetrazolium salt assay. The fold change in cell viability over time is shown for Gαq(Q209L)-driven 92.1 and Mel202 cells and for BRAF(V600E)-driven OCM-1A cells in response to increasing concentrations of FR. Data are means ± SEM from four experiments performed in triplicate. (B) Potency of FR as an inhibitor of UM cell viability measured in 92.1, Mel202, and OCM-1A cell lines as in (A). Data are means ± SEM from four experiments performed in triplicate. The indicated UM cell lines were treated for 3 days with the indicated concentrations of FR and analyzed for DNA content. (C) Representative histograms from flow cytometry of the Gαq(Q209L)-driven cell lines (92.1 and Mel202) and BRAF(V600E)-driven OCM-1A cells. PI, propidium iodide. (D) Potency of FR as inducer of apoptosis (sub-G1 cells) and inhibitor of cell proliferation (S and G2-M phase cells) in 92.1, Mel202, and OCM-1A cell lines; data are means ± SEM from four experiments. *P < 0.01 by t test; significance was corrected for multiple comparisons using the Holm-Sidak method.

  • Fig. 5 FR induces redifferentiation of UM cells driven by constitutively active Gαq.

    (A) Morphological changes elicited by FR in UM cell lines driven by constitutively active Gαq (92.1 and Mel202) or BRAF-V600E (OCM-1A) treated for 3 days with FR and imaged by phase-contrast microscopy. Representative fields from one of three experiments are shown. (B) Melanocytic differentiation of FR-treated UM cells indicated by pigmentation. 92.1, Mel202, and OCM-1A UM cell lines were treated for 3 days with FR. Cells were pelleted and examined macroscopically; representative images from one of three experiments performed in triplicate. (C) Induction of melanocytic markers by FR as indicated by immunofluorescence staining of tyrosinase (TYR), dopachrome tautomerase (DCT), and premelanosome protein (PMEL) of Gαq-mutant cell lines (92.1 and Mel202) but not BRAF-driven UM cells (OCM-1A). Images are representative fields from one of three experiments. Scale bars, 50 μm.

  • Fig. 6 FR represses expression of differentiation genes by restoring function of the PRC2.

    (A) Gαq-mutant 92.1 UM cells were treated with FR or vehicle, and RNA was collected 1 and 3 days (1d and 3d, respectively) after treatment for RNA-seq analysis. Results of a multidimensional gene expression analysis that compares the relative patterns of expression of all genes across all samples and groups genes with similar patterns. The graph shows samples positioned by their relative gene expression values within each pattern. Dimension 1 (x axis; the most represented pattern) shows separation based on vehicle treatment (red balls) versus FR treatment (blue balls), whereas dimension 2 (y axis; the second most represented pattern) shows separation based on time in culture (indicated by 1d or 3d on balls). (B) MA plot (M, log ratio; A, mean average) comparing gene expression between FR- and vehicle-treated 92.1 samples identifies a group of significantly reduced genes (circled; fold change, >2; FDR, q < 0.01) associated with FR treatment. (C) GO analysis of the FR-repressed gene set [circled in (B) with arrow]. (D) FR-repressed genes [circled in (B) with arrow] identified as targets of the polycomb repressive complex 2 (PRC2) by GSEA. EGF, epidermal growth factor; BMP2, bone morphogenetic protein; hESC, human embryonic stem cells. (E) Effect of the EZH1/2 inhibitor GSK503 on morphological differentiation elicited by FR. Representative fields are shown from one of three experiments of 92.1 UM cells treated for 7 days with GSK503 and for 3 days with FR and then imaged by phase-contrast microscopy. Scale bar, 100 μm. (F) Effect of GSK503 on pigmentation of FR-treated cells, visualized by macroscopic inspection. 92.1 cells were treated for 7 days with GSK503 and for 3 days with FR and pelleted; representative images from one of three experiments. (G) PRC2 inhibition by GSK503. Immunoblots of 92.1 cells treated for 7 days with GSK503 show reduced histone H3K27 trimethylation. Plot shows relative fraction of trimethyl-histone H3K27 compared to dimethyl sulfoxide (DMSO) control and normalized to total histone H3 from densitometry data from three independent experiments. *P < 0.01 by t test; significance was confirmed using q < 0.01 by the FDR method of Benjamini and Hochberg.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/546/eaao6852/DC1

    Fig. S1. FR inhibition of Gαi1 bearing an engineered FR-binding site.

    Fig. S2. Heatmaps of cell cycle and apoptosis gene expression in response to FR.

    Fig. S3. Validation of selected FR target genes.

    Data file S1. Gene sets with positive correlation to FR treatment in GSEA.

    Data file S2. Gene sets with negative correlation to FR treatment in GSEA.

    Data file S3. Genes within the FR-responsive, reduced-expression cluster.

    Data file S4. GO analysis results for the FR-responsive gene cluster.

    Data file S5. GSEA results for the FR-responsive gene cluster.

    Data file S6. Primers used for quantitative PCR gene expression analysis.

  • The PDF file includes:

    • Fig. S1. FR inhibition of Gαi1 bearing an engineered FR-binding site.
    • Fig. S2. Heatmaps of cell cycle and apoptosis gene expression in response to FR.
    • Fig. S3. Validation of selected FR target genes.
    • Legends for data files S1 to S6

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Gene sets with positive correlation to FR treatment in GSEA.
    • Data file S2 (Microsoft Excel format). Gene sets with negative correlation to FR treatment in GSEA.
    • Data file S3 (Microsoft Excel format). Genes within the FR-responsive, reduced-expression cluster.
    • Data file S4 (Microsoft Excel format). GO analysis results for the FR-responsive gene cluster.
    • Data file S5 (Microsoft Excel format). GSEA results for the FR-responsive gene cluster.
    • Data file S6 (Microsoft Excel format). Primers used for quantitative PCR gene expression analysis.

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