Effects of Oncogenic Gα_q and Gα₁₁ Inhibition by FR900359 in Uveal Melanoma

Abstract

Uveal melanoma is the most common intraocular tumor in adults and often metastasizes to the liver, leaving patients with few options. Recurrent activating mutations in the G proteins, Gα_q and Gα₁₁, are observed in approximately 93% of all uveal melanomas. While therapeutic intervention of downstream Gα_q/11 targets has been unsuccessful in treating uveal melanoma, we have found that the Gα_q/11 inhibitor, FR900359 (FR), effectively inhibits oncogenic Gα_q/11 signaling in uveal melanoma cells expressing either mutant Gα_q or Gα₁₁. Inhibition of oncogenic Gα_q/11 by FR results in cell cycle arrest and induction of apoptosis. Furthermore, colony formation is prevented by FR treatment of uveal melanoma cells in 3D-cell culture, providing promise for future in vivo studies. This suggests direct inhibition of activating Gα_q/11 mutants may be a potential means of treating uveal melanoma.

Introduction

Uveal melanoma (UM) arises from melanocytes contained in the choroid, ciliary body, and iris (together known as the uvea) of the ocular cavity, and is the most common intraocular tumor in adults (1, 2). UM accounts for approximately 5% of all lethal melanoma cases, and the current treatment options for eye localized disease are either surgery or radiation. Metastases occur in approximately 50% of UM patients, predominantly to the liver. Patients with macro-metastases have an average survival of 2 to 8 months as there are no effective therapies (1). The mutations in UM are distinct from those commonly found in cutaneous melanoma, such as the driver mutations in BRAF and NRAS (3–6). UM predominately involves activating mutations in either GNAQ or GNA11 genes that encode the highly conserved Gα_q and Gα₁₁ subunits of heterotrimeric G proteins (7, 8). Point mutations occur at the Q209 and R183 residues in the GTPase domain of Gα_q/11 proteins. These residues are critical for the intrinsic GTP hydrolysis activity of the G proteins, which are rendered constitutively active in the GTP-bound state leading to aberrant downstream signaling (7–11). Gα_q/11-Q209 mutants are much more prevalent in UM than Gα_q/11-R183 mutants, and are unresponsive to GTP hydrolysis stimulation by regulator of G protein signaling (RGS) proteins (12). This results in a more significant increase in signaling and a more severe phenotype (8, 9).

The introduction of the Gα_q- or Gα₁₁-Q209L mutant into human or mouse melanocytes results in anchorage-independent growth and gives rise to heavily pigmented tumors in mice (8, 13, 14). Due to the redundancy of their signaling pathways, oncogenic mutations in either Gα_q or Gα₁₁ appear to cause similar cellular oncogenic properties leading to the pathogenesis of UM. For example, GNAQ and GNA11 mutations are responsible for the upregulation of the mitogen-activated protein kinase (MAPK) pathway in the absence of BRAF mutations in UM (15–18). Knockdown of Gα_q in cell lines derived from primary or metastatic UM results in decreased MAPK signaling (7). However, inhibiting the ERK1/2 pathway with MEK inhibitors has not been clinically effective in treating UM (19) and growth factors from the liver tumor microenvironment are able to mediate resistance (20, 21). Additional Gα_q/11 stimulated signaling pathways that promote tumorigenesis have been implicated in UM. Oncogenic Gα_q/11 leads to aberrant Akt signaling and increased activation of small GTPases RhoA and Rac1, which promote cell growth through JNK, p38, and yes-associated protein (YAP) directed transcription of growth promoting genes (13, 14, 22, 23). YAP is a co-transcriptional regulator involved in the cell growth regulating Hippo pathway, that when dephosphorylated translocates from the cytoplasm into the nucleus where it associates with TEAD4 to promote the transcription of growth promoting genes (24–26). Recent studies indicate that YAP dephosphorylation in UM occurs through the Gα_q/11 activation of a Trio, RhoA, Rac1 pathway (14). Considering the multiple avenues of disease progression as well as the multiple signaling pathways that are activated as a result of oncogenic Gα_q/11, it has been difficult to determine a suitable and successful treatment for UM. This raises the possibility that direct inhibition of oncogenic Gα_q/11 may be advantageous in preventing the constitutive activation of these multiple pathways.

While there are no current drugs that directly target Gα_q/11, a few compounds that effectively inhibit Gα_q have been identified. YM-254890, a cyclic depsipeptide purified from Chromobacterium sp. QS3666, has been shown to potently and selectively inhibit wild type Gα_q/11 and the R183 mutant, but not the Q209L mutant protein, by inhibiting GDP dissociation from GDP-bound Gα_q (27, 28). FR900359 (a.k.a. UBO-QIC), a highly related analog of YM-254890, was first isolated in 1988 from the leaves of the plant Ardisia crenata, and has since been shown to be a potent and selective inhibitor of Gα_q/11 (29–33). Interestingly, recent studies revealed an effect of FR900359 (FR) on cell cycle regulation in melanoma cell lines containing wild type or oncogenic Gα_q/11 (32). This suggests that FR might serve as a useful inhibitor in UM.

In this study, we present evidence that FR effectively inhibits Gα_q-Q209L, Gα_q-Q209P and Gα₁₁-Q209L mutant proteins resulting in decreased Gα_q/11 signaling, cell cycle arrest and eventual cell death of UM cells harboring a GNAQ/11-Q209 mutation. These data provide evidence that direct inhibition of activating Gα_q/11 mutants may be a potential strategy to treat UM.

Materials and Methods

FR900359 –

Some studies were performed using FR purchased from the Pharmazeutische Biologie at the Universität Bonn. In addition, FR was also isolated from the roots of Ardisia crenata sims using the targeted counter-current chromatography method with minor modifications (34, 35). In brief, the dry powder of the roots of Ardisia crenata sims was first extracted with 95% ethanol, then the ethanol extract was partitioned by n-butanol-water. The upper n-butanol phase was separated, concentrated and submitted to counter-current chromatography separation using an optimum two-phase solvent system of n-hexane-ethylacetate-ethanol-water (4.5:5.5:4.5:5.5, v/v), yielding more than 95% purity of FR. A positive ESI-MS spectra showed the characteristic molecular ions at m/z 1002.27 [M+H]⁺ and 1024.35 [M+Na]⁺ implying its molecular formula (C₄₉H₇₅N₇O₁₅). ¹H NMR and ¹³C NMR data were in close agreement with previous data (29). Similar results were obtained with both sources of FR.

Cell lines and culture –

OMM1.3 cells were obtained from Dr. Bruce Ksander’s lab and were confirmed to harbor the Q209P mutation in GNAQ by Sanger sequencing. 92.1 cells were obtained from Dr. Martine Jager and contained a GNAQ-Q209L mutation that was confirmed by Sanger sequencing, while OCM3 cells were obtained from Dr. Bruce Ksander and contained a BRAF-V600E mutation and wild type GNAQ/11 as confirmed by Sanger sequencing. UM002B cells were derived from a brain metastasis and are PTEN negative and contain a heterozygous Q209L mutation in GNA11 and no change in GNAQ as determined by Sanger sequencing (supplementary Fig. S1). 92.1, OMM1.3, UM002B, and OCM3 cells were cultured in RPMI 1640 containing 10% fetal bovine serum, 2 mM L-glutamine, 0.2 units/ml penicillin and 100 μg/ml streptomycin. Cells were cultured at 37°C and 5% CO₂ in a humidified incubator. Cells were not tested for mycoplasma.

Gα_q purification –

Wild type rat Gα_q was purified from High Five insect cells as previously described (36). Briefly, Gα_q and Glutathione-S-Transferase (GST)-tagged Ric-8A were co-expressed, GST-Ric-8A:Gα_q complexes were captured from the clarified lysates with glutathione Sepharose 4B, and Gα_q was eluted. The Gα_q was evaluated for activity using a Ric-8A-stimulated [³⁵S]-GTPγS binding assay (37). Mutant Gα_qΔ34-Q209L was purified from High-Five insect cells. Gα_qΔ34-Q209L was engineered for high level expression using a chimeric form of Gα_q/i that contained an N-terminal hexahistidine tag followed by residues 1–28 of rat Gα_i1, a tobacco etch virus protease site, and residues 35–359 of mouse Gα_q-Q209L. Purification of Gα_qΔ34-Q209L followed protocols published previously (38).

GTPγS Assay –

GTPγS binding of purified Gα_q and Gα_q-Q209L was measured using a filter-binding method. Spontaneous GTPγS binding to the Gα proteins was promoted in the presence of (NH₄)₂SO₄, as previously described (39). Briefly, purified Gα_q (250 nM) was pre-incubated with or without FR for 2 h at 20°C in assay buffer A (50 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 0.9 mM MgSO₄, 0.05% Lubrol). Reactions were started by the addition of 10 μM [³⁵S]GTPγS and 300 mM (NH₄)₂SO₄. Reactions were stopped by the addition of ice-cold wash buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgSO₄), and the mixture was filtered through nitrocellulose membranes. The membranes were washed four times with ice-cold wash buffer, air-dried and [³⁵S]GTPγS binding was quantitated by liquid scintillation counting.

Cell Growth –

92.1, OMM1.3, UM002B and OCM3 cells were plated on poly-L-lysine coated 96-well white bottom plates at 5 × 10³ cells per well and allowed to sit overnight at 37°C and 5% CO₂ in a humidified incubator. Cells were then treated with DMSO or 100 nM, 300 nM or 1 μM FR for 1–4 days and harvested by removing the media and lysing the cells with 100 μl of cell-grade water for 1 h at 37°C. 100 μl of 200-fold diluted Quant-iT™ PicoGreen™ (Invitrogen) reagent in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was added to the mixture and incubated for 1 h at room temperature in the dark. DNA-bound PicoGreen™ fluorescence was detected using a Tecan Infinite F500 microplate reader.

Western Blotting –

Cell lysates were separated by SDS-PAGE and proteins were transferred to PVDF membrane. Immunoreactivity was detected using horseradish peroxidase conjugated secondary antibodies (Vector) and chemiluminescence substrate (ThermoScientific) on autoradiography film. HER3/ErbB3 (#4754), p-STAT3 (Tyr705, #4113), p-S6 (Ser235/236, #2211), p-S6 (Ser240/244, #2215), p-Rb (Ser807/811, #9308), and PLK1 (#4513) antibodies were all from Cell Signaling Technology. pCDK1 (Thr14/Tyr15, #44686G) antibody was from Invitrogen.

ERK1/2 phosphorylation –

Cell lysates were separated by SDS-PAGE and proteins were transferred to PVDF membranes and incubated with ERK2 (Santa Cruz) and pERK1/2 (Cell Signaling) antibodies followed by IRDye 800 CW and IRDye 680RD fluorescent secondary antibodies (LI-COR). Detection was with a LI-COR Odyssey and fluorescence was quantified using ImageStudioLite.

YAP localization –

Cells were grown on coverslips in RPMI 1640 containing 10% FBS. After 48 h, cells were washed in serum-free RPMI 1640, maintained overnight in serum-free RPMI 1640, and then treated for 6 h with vehicle (DMSO) or 0.1–100 nM FR900359. Cells were fixed in 3.7% formaldehyde, and processed for immunofluorescence using an anti-YAP antibody (Santa Cruz; mouse monoclonal Ab 63.7, 1:100) followed by an anti-mouse antibody conjugated to Alexa594 (Invitrogen, A11032; 1:250). Coverslips were mounted on glass slides using ProLong Diamond Antifade (Invitrogen, P36970). Subcellular localization of YAP in individual cells was scored as: C>N, indicating the majority of YAP staining in the cytoplasm; C=N, indicating that YAP staining is equally distributed in cytoplasm and nucleus; C<N, indicating that YAP staining is predominantly detected in the nucleus. Data are presented as the percentage of cells exhibiting the above criteria. 100 cells were counted in each of three separate experiments. Images were recorded with an Olympus BX-61 microscope and 60X PlanApo objective with an ORCA-ER (Hamamatsu, Bridgewater, NJ) cooled charge-coupled device camera controlled by Slidebook version 4.0 (Intelligent Imaging Innovation, Denver, CO).

Reverse Phase Protein Array (RPPA) Analysis –

92.1 and OMM1.3 cells were plated in six-well dishes at 1.5 × 10⁵ cells per well. Cells at ~80% confluency were treated with DMSO or 1 μM FR for 24 h. Cells were lysed in RPPA lysis buffer (1% Triton X-100, 50 mM HEPES, pH 7.4, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 100 mM NaF, 10 mM Na pyrophosphate, 1 mM Na₃VO₄, 10% glycerol, freshly added protease and phosphatase inhibitors from Roche Applied Science Cat. # 05056489001 and 04906837001), and proteins were prepared and analyzed by RPPA, as previously described (40). RPPA data were used to determine proteins that are significantly different in both 92.1 and OMM1.3 cell lines treated with FR or DMSO. Comparisons were performed for each cell line by the two-sample t-test method with 10,000 permutations and assumed unequal variance. Proteins with a Storey FDR < 0.05 and a log-2 ratio > 1 were considered significant. Supervised hierarchical clustering was performed based on median-centered log2-transformed expression values. Statistical calculations were performed in Matlab® (v2017a) using the mattest and mafdr functions. RPPA was performed at MD Anderson where antibodies are extensively validated before being included in the panel. Antibodies must give one major band by western blotting that is within a dynamic range as determined by drug treatment or siRNA. Full details are provided at https://www.mdanderson.org/content/dam/mdanderson/documents/core-facilities/Functional%20Proteomics%20RPPA%20Core%20Facility/RPPA_Ab%20Validation%20Example.pdf. The RPPA results were validated by Western blotting for key targets.

EdU and Propidium Iodide Incorporation Assays –

Cells were treated with DMSO or 1 μM FR for 6–24 h, and then incubated with 10 μM EdU. Cells were prepared according to the Click-iT™ Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen) protocol. Cells were incubated in 500 μl of Click-iT™ saponin-based permeabilization and wash reagent containing 40 μg/ml propidium iodide (PI) and 100 μg/ml RNaseA (ThermoScientific). EdU and PI fluorescence was measured and analyzed using a FACS Calibur Flow Cytometer.

Caspase 3/7 Activation Assays –

Cells were treated with DMSO or FR for 24 h. Cells were prepared following the CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit (ThermoScientific) procedure. Cells were incubated with 500 nM CellEvent Caspase-3/7 Green Detection Reagent for 30 min at 37°C, protected from light. Caspase-3/7 Green Detection Reagent fluorescence was measured and analyzed using a FACS Calibur Flow Cytometer.

3D Cultures in Matrigel –

The morphogenesis analysis was done using 3D cultures in Cultrex. Briefly, 5 × 10³ OMM1.3 cells treated or not with FR were seeded on top of a Cultrex matrix (Trevigen, Gaithersburg, MD) as described (41). Cells were cultured under these conditions for 7 to 12 days and the morphology of the colonies was monitored daily by phase contrast microscopy and by confocal microscopy at the end of the experiment to obtain optical sections to determine the effect of the treatment on cell survival and/or differentiation. The 3D cultures were processed as described (41) and mounted in Prolong gold with DAPI (Molecular Probes, Grand Island, NY) to visualize the nuclei and activated caspase 3 and Melan-A to determine induction of apoptosis and melanocyte differentiation, respectively.

Results

FR900359 inhibits GTPγS binding to Gα_q and Gα_q-Q209L proteins in vitro.

FR has recently been reported to be capable of inhibiting wild type Gα_q, Gα₁₁ and Gα₁₄ and is thought to act as a guanosine nucleotide dissociation inhibitor (GDI) by inhibiting the GDP/GTP exchange of the Gα protein (30, 32, 33). Because the Q209L mutation is unlikely to alter the structure of Gα_q/11, it is possible that FR might be able to bind to and inhibit Gα_q/11-Q209L. Indeed, FR effectively blocked spontaneous [³⁵S]GTPγS binding to both purified Gα_q and Gα_q-Q209L in a dose-dependent manner with an IC₅₀ ~75 nM (Fig. 1). This provides evidence that FR can inhibit nucleotide binding to Gα_q-Q209L, suggesting that FR can directly bind to mutant Gα_q.

Figure 1.

FR900359 inhibits GTPγS binding to Gα_q. Dose-dependent inhibition of GTPγS binding to Gα_q and Gα_q-Q209L. FR-preincubated Gα_q and Gα_q-Q209L were incubated with GTPγS for 120 min in the presence of 300 mM (NH₄)₂SO₄ and analyzed for GTPγS binding. Error bars represent the mean ± SEM from 3 experiments.

Uveal melanoma cell growth is inhibited upon treatment with FR900359.

To determine if FR can inhibit the Gα_q mutant protein in cells, we next treated UM cells containing GNAQ-Q209L/P or GNA11-Q209L mutations with varying concentrations of FR and tracked their growth over 4 days (Fig. 2A and B). The growth of primary 92.1 and metastatic OMM1.3 cells, containing GNAQ-Q209L and GNAQ-Q209P mutations, respectively, was effectively inhibited by treatment of FR at concentrations as low as 100 nM. We also observed inhibited growth of metastatic UM002B cells, containing a GNA11-Q209L mutation, suggesting FR is also able to inhibit mutant Gα₁₁ (Fig. 2C). In contrast, FR had no effect on the growth of OCM3 melanoma cells, containing a BRAF-V600E mutation (Fig. 2D). Thus, FR effectively inhibits the growth of UM cells containing activating mutations of Gα_q or Gα₁₁.

Figure 2.

FR900359 inhibits cell growth of GNAQ/11-Q209 containing uveal melanoma cells. UM cell lines were treated with DMSO or FR (100 nM, 300 nM, 1 μM) for 4 days and growth was tracked using the DNA intercalator, PicoGreen. Error bars represent the mean ± SEM from 3 experiments done in triplicate. P < 0.05 (*), P < 0.001 (***) for differences between DMSO and 100 nM FR treated cell growth, Student’s t test.

Oncogenic Gα_q inhibition in uveal melanoma cells abolishes MAPK signaling and prevents YAP localization to the nucleus.

Increased MAPK and YAP signaling is observed in cells expressing oncogenic Gα_q (13, 17, 42). To test if Gα_q inhibition affects MAPK signaling in UM cells, we immunoblotted for pERK1/2 after treating 92.1, OMM1.3, and UM002B cells with FR (Fig. 3). ERK1/2 activation in 92.1 and OMM1.3 cells was completely abolished while it was decreased by ~65% in UM002B cells after 24 h-treatment with 1 μM FR (Fig. 3A & B). In contrast, ERK1/2 phosphorylation was unchanged by FR treatment of OCM3 cells (Fig. 3A & B). ERK1/2 activation slowly decreased in 92.1 cells over the course of 5 h with a half-life of ≤30 min, the earliest time point tested, while it decreased more rapidly in OMM1.3 cells and was almost completely abolished after 30 min treatment (Fig. 3C). We also detected YAP localization by fluorescence microscopy after treating OMM1.3 cells with FR for 6 h. FR significantly decreased the amount of YAP found in the nucleus, while cytoplasmic YAP levels were increased (Fig. 4A). FR concentrations as low as 0.1 nM were sufficient to observe a significant change in YAP localization (Fig. 4B). Significant displacement of YAP from the nucleus to the cytoplasm was also observed in 92.1 and UM002B cells, but was not observed in OCM3 cells when treated with FR for 6 h (Fig. 4C). These data suggest direct inhibition of activated Gα_q/11 in UM cells results in decreased activation of MAPK and YAP pathways.

Figure 3.

ERK activation is inhibited by FR900359 treatment of uveal melanoma cells. A) OCM3, 92.1, OMM1.3, and UM002B cells were treated with 1 μM FR or DMSO for 24 h and cell lysates were immunoblotted for ERK2 and pERK1/2. B) Quantitation of ERK activation after 24 h treatment. C) 92.1 and OMM1.3 cells were treated with 1 μM FR or DMSO for 0–5 h and cell lysates were immunoblotted for ERK2 and pERK1/2. Error bars represent the mean ± SEM from 3 experiments. P > 0.05 (ns), P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) for differences between untreated (0 h) and 1 μM FR treated cells, Student’s t test.

Figure 4.

FR900359 treatment prevents YAP localization to the nucleus in uveal melanoma cells. A) OMM1.3 cells were serum starved overnight and then treated with 100 nM FR or DMSO for 6 h. YAP was observed by fluorescence microscopy. B) Quantitation (by counting and scoring cells) of nuclear (N) or cytoplasmic localization of YAP in OMM1.3 cells. YAP was detected by immunofluorescence stain. C) Quantitation (by counting and scoring cells) of nuclear (N) or cytoplasmic (C) localization of YAP in OCM3, 92.1, and UM002B cells. YAP was detected by immunofluorescence stain. P < 0.01 (**), P < 0.001 (***) for differences between DMSO (N > C) and FR (N > C).

Oncogenic Gα_q inhibition results in decreased levels of proteins involved in cell cycle and cell proliferation.

Because Gα_q activation is responsible for initiating many different signaling cascades, we wanted to take a more global approach to determine pathways that are affected in UM cells when oncogenic Gα_q is inhibited by FR. This was done using a high-throughput antibody-based reverse phase protein array analysis that utilized validated antibodies. Cell lysates from DMSO or FR treated UM cells were probed for 218 different proteins and phosphoproteins and analyzed for changes in protein levels (Fig. 5). The 20 proteins that were most significantly up- or down-regulated upon Gα_q inhibition by FR in 92.1 and OMM1.3 cells are shown in Fig. 5A. Many of the same proteins were significantly altered in both cell lines tested, while a few were specific to one or the other. In both cell lines, proteins that promote cell cycle progression, such as CDK1, cyclin-B, PLK1, and p-Rb, were significantly decreased by FR treatment, while the observed decreased phosphorylation of the S6 ribosomal subunit and decreased p-90RSK are indicative of cell growth arrest (43). Interestingly, we also observed a number of proteins that increased in expression following FR treatment. These included Pdcd4, a programmed cell death protein that suppresses tumor progression (44), HER3 and pSTAT3 (Fig. 5A). The changes in expression or phosphorylation for many of these proteins was validated by immunoblotting of control and FR treated UM cell lysates (Fig. 5B). In contrast, OCM3 cells showed no changes in expression or phosphorylation of those proteins when treated with FR for 24 h (Fig. 5B). These data are in line with the observed effects of FR on UM cell growth (Fig. 2).

Figure 5.

Reverse Phase Protein Array analysis of FR900359 treated UM cells. A) A heat map showing supervised hierarchical clustering of samples using median-centered log2-transformed RPPA expression value data for proteins deemed significant in at least one cell line comparison. Log2-transformed fold change ratios for each cell line are compared (FR/DMSO) in the next to last set of lanes on the right as is the index of significant proteins for each cell line in the far right set of lanes. Proteins with a Storey FDR < 0.05 and a log-2 ratio > 1 were considered significant. B) Lysates from 92.1, OMM1.3 and OCM3 cells were immunoblotted for proteins and phosphoproteins that were most significantly up- or downregulated by FR treatment.

FR treatment of uveal melanoma cells induces cell growth arrest and apoptosis.

We next investigated the effect of FR treatment on cell cycle, as measured by propidium iodide staining and EdU incorporation. FR reduced S phase entry in 92.1 cells (Fig. 6A). After 24 h-treatment with FR, ~96% of cells were in the G1 phase while ~0.3% were in S phase (Fig. 6B). This compared with ~61% of untreated cells in G1 phase and 30% in S phase. 24 h-treatment with FR also resulted in a decrease in cells in G2/M phase from ~8.5% in untreated cells to ~3.7% in FR treated cells. Similar results were observed in FR treated OMM1.3 and UM002B cells, while OCM3 cells were not affected by FR (Fig. 6C). We also assessed whether FR induced apoptosis of UM cells using an activated caspase-3/7 detection reagent and FACS analysis. We found that by 24 h there were significantly more 92.1 cells undergoing apoptosis when treated with 1 μM FR compared to untreated cells (Fig. 7A & B). The same effect was observed in FR treated OMM1.3 and UM002B cells, but not in OCM3 cells (Fig. 7C). By comparison, treatment with 1 μM staurosporine, which induces apoptosis through caspase-3 activation (45), for 3–5 h induced significant apoptosis in all cell lines. Therefore, FR induces G1 cell growth arrest and leads to apoptosis of UM cells containing activating mutations of Gα_q/11.

Figure 6.

Uveal melanoma cells undergo G1 cell cycle arrest upon FR900359 treatment. A) 92.1 cells were treated with 1 μM FR or DMSO over the course of 24 h. Cells were incubated with Click-iT™ Plus EdU for 30 min, harvested, then fixed and permeabilized. Click-iT™ Plus EdU was detected using Alex Fluor™ 647 picolyl azide. Cells were then stained with propidium iodide and analyzed by flow cytometry. Cell populations in G1, S, and G2/M phases were observed. B) Quantitation of percent of 92.1 cells in G1 and S phase. C) Quantitation of percent of OCM3, OMM1.3, UM002B cells in G1 and S phase after treatment with 1 μM FR or DMSO for 24 h. Error bars represent the mean ± SEM from 2 (92.1 cells - 6, 12, 18 h) or 3 (92.1, OCM3, OMM1.3, UM002B cells - 24 h) experiments. P > 0.05 (ns), P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) for differences in DMSO and 1 μM FR treated cells, Student’s t test.

Figure 7.

FR900359 treatment promotes apoptosis of uveal melanoma cells. 92.1 cells were treated with DMSO (A) or 1 μM FR (B) for 24 h. Cells were harvested and incubated with CellEvent™ Caspase-3/7 Green Detection Reagent to detect cleaved caspase 3/7. Fluorescence was detected and analyzed by flow cytometry. The area under the “Apoptotic” box represents cells positive for activated caspase-3/7. C) OCM3, OMM1.3, and UM002B cells were similarly treated, harvested and analyzed, and the percent of cells undergoing apoptosis was determined. 3 – 5 h staurosporine (STS) treatment promotes apoptosis through caspase 3. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) for differences between DMSO and 1 μM FR treated cells.

FR treatment prevents uveal melanoma colony formation and forces cellular differentiation in 3D culture.

In order to obtain a better idea of how UM cells might be affected by FR-treatment in vivo, we utilized 3D-Matrigel-cell culture, which allows cells to grow in contact with extracellular matrix and provides positional information. OMM1.3 cells were seeded in the Matrigel culture and allowed to grow and form 3D cellular colonies of at least 20 cells in size, analogous to how they would initiate colonization in the liver. Untreated cells formed colonies within 12 days, while cells that were treated with 30 nM FR immediately after being seeded were completely incapable of forming colonies during that same timespan (Fig. 8A). FR treatment also led to the disassembly of cells that had already formed OMM1.3 colonies, which continued to be unable to re-form colonies after 35 days of no additional treatment with FR (Fig. 8B). Lower doses of FR appear to induce OMM1.3 cell differentiation as evidenced by an increase in Melan-A expression with 1 and 10 nM FR-treatment. However, at higher doses we observed little Melan-A expression and an increase in the proportion of cells undergoing apoptosis (Fig. 8C). These data convey that FR prevents colony formation and, depending on the degree of inhibition, disrupts colonies through the induction of cell differentiation or apoptosis to ultimately elicit a long-lasting effect.

Figure 8.

Colony formation of uveal melanoma cells in 3D-culture is prevented by FR900359 treatment. A) OMM1.3 cells were seeded and treated with FR for 7 days, followed by 5 days of no treatment (washout). Treatment started 4 h after seeding. B) OMM1.3 cells were seeded and allowed to form colonies. Colonies were then treated with FR for 7 days, followed by no treatment for 35 days. C) Confocal microscopy showing activated caspase-3 (green) and melan-A (purple) of cultures as described, treated with FR for 7 days at different concentrations.

Discussion

Uveal melanoma is the most common intraocular tumor in adults and has a high rate of metastasis, leaving patients with a short survival time (1). UM is predominantly driven by activating mutations in Gα_q/11 proteins, which promotes increased signaling in various cell proliferative pathways, such as the ERK1/2-MAPK and Hippo/YAP pathways (7, 8, 14, 22). To date, targeting proteins downstream of Gα_q/11 has yielded unsuccessful results in clinical trials (19, 46). Therefore, it may be advantageous to inhibit oncogenic Gα_q/11 directly. Here, we provide evidence that shows that a depsipeptide, that has previously been shown to inhibit wild-type Gα_q/11, is also capable of inhibiting oncogenic Gα_q and Gα₁₁ in UM cells, promoting G1 cell cycle arrest and either cell death or differentiation.

Previous studies have shown that the related compounds YM-254890 and FR900359 act as GDIs to inhibit GDP dissociation from Gα_q and thereby inhibit GTP binding and activation of Gα_q (27, 28, 31). FR has been shown to bind specifically to Gα_q, Gα₁₁ and Gα₁₄ and inhibit activity in a pseudo-irreversible manner (32). These studies also demonstrated that FR can inhibit basal signaling mediated by Gα_q-R183C and Gα_q-Q209L expressed in HEK293 cells as well as signaling in some melanoma cell lines expressing mutant Gα_q (32). Moreover, a very recent study has shown that FR is capable of inhibiting oncogenic Gα_q/11-Q209L in UM cells (47). FR appears to function by trapping mutant Gα_q in the inactive GDP-bound form by inhibiting nucleotide exchange and promoting Gα_q association with Gβγ (47). In the present study, we show that FR effectively inhibits signaling in UM cells expressing Gα_q-Q209L, Gα_q-Q209P and Gα₁₁-Q209L. We show in vitro that FR inhibits GTPγS-binding to Gα_q-Q209L with an IC₅₀ ~75 nM (Fig. 1). The IC₅₀ determined in these studies is likely driven by the high concentration of Gα_q that was used in these assays and thus doesn’t reflect the 1–10 nM IC₅₀s previously reported for FR binding to Gα_q (32, 47, 48). In addition, the high IC₅₀ may also reflect the fact that we used Gα_q in these studies while YM-254890 interacts with both the Gα_q and Gβγ subunits in the heterotrimer (presumably FR does as well) (28).

Since mutationally activated Gα subunits are thought to be constitutively associated with GTP, it is unclear how FR might inhibit the activity of activated Gα_q/11. It might bind to GTP loaded Gα_q/11-Q209L and promote GTP dissociation, or there may be a slow exchange of GDP/GTP on Gα_q/11-Q209L such that FR can bind in the GDP state. This latter possibility seems likely given the recent report on FR inhibition of Gα_q-Q209L in UM cells (47), although this inhibition occurs within minutes in some cells (Fig. 3C). It is also possible that FR binds to newly synthesized GDP-bound Gα_q/11 with the degradation of GTP-bound Gα_q/11 being required to observe an effect. However, this seems unlikely since we see rapid inhibition of ERK activity (Fig. 3C) while the half-life of Gα_q/11 is reported to be about 5 h (49). While further characterization of the mechanism of FR inhibition of mutant Gα_q/11 is warranted, the effects of FR treatment on UM cells are striking.

Gα_q/11 inhibition in UM cells containing oncogenic GNAQ/11-Q209 mutations leads to cell growth arrest within 24 h (Figs. 2 & 6). FR-mediated G1 cell growth arrest is also detected in melanoma cells with (Hcmel12) or without (B16) a GNAQ-Q209L mutation (32) as well as in primary UM cell lines containing a GNAQ-Q209L mutation (47). Additionally, we show that FR treatment is capable of producing similar results in metastatic UM cells containing GNAQ-Q209P or GNA11-Q209L mutations. Given the prevalence of Q209L and Q209P mutations in UM, it is worth noting that Gα_q-Q209P has been shown to have altered binding to various effectors and Gβγ compared to Gα_q-Q209L, although it still effectively activates downstream signaling (50). FR treatment likely does not affect the growth of mutant BRAF cell lines such as OCM3 due to the lack of dependency on GNAQ/11 for cell growth (Fig. 2 & 6). Thus, OCM3 cells have been a valuable control in our studies, providing support for the specificity and non-cytotoxic nature of FR. These results are similar to recent studies in which OCM-1A cells were also used as a control for FR treatment (47).

Inhibition of Gα_q/11 in UM cells harboring GNAQ-Q209L/P or GNA11-Q209L, but not in melanoma cells containing wild type GNAQ/11, abolishes MAPK signaling (Fig. 3) and inhibits YAP translocation from the cytoplasm to the nucleus (Fig. 4), thereby hindering YAP-mediated transcription of cell proliferative genes. In general, these results are similar to studies of mutant GNAQ-Q209L melanoma and UM cells treated with GNAQ siRNA or FR (13, 14, 32, 47, 51). However, we see observable differences in YAP localization amongst the different cell lines. In OCM3 cells, YAP is largely equally distributed between the nucleus and cytoplasm and is unchanged by FR treatment. In both the OMM1.3 and 92.1 cells, YAP is primarily nuclear before treatment and moves to the cytoplasm with FR treatment. The effects of FR on YAP localization in UM002B cells are not as dramatic since YAP is not highly localized in the nucleus in UM002B cells, although FR does significantly decrease the YAP levels in the nucleus. The inhibition of the MAPK and YAP pathways likely contributes to the changes in cell cycle and cell growth associated proteins we detected by RPPA.

Upon FR treatment, we observed a significant decrease in the expression of proteins involved in regulating cell cycle, such as CDK1, cyclin B, PLK1, pRb, and Wee1 (Fig. 5), as further evidence of a disruption in cell cycle. We also observed a significant decrease in the phosphorylation of the S6 ribosomal subunit and of its activating kinase, p90RSK, which is characteristic of cell growth arrest (Fig. 5) (43).

Utilizing a Matrigel 3D culture, UM cells were allowed to grow in a 3D manner as they would in vivo. FR treatment prevents the colonization of single UM cells, and disrupts colonies that were allowed to form before treatment (Fig. 8). Of note, UM cells remain unable to form colonies even after 35 days of no treatment/washout (Fig. 8B). This suggests that Gα_q/11 inhibition causes long-term effects in UM cell behavior consistent with a reprogramming into a more differentiated phenotype. Further, if used as a treatment option, FR might not need to be continuously administered to patients. Upon treatment with lower doses of FR, UM cells show an increased expression of melan-A, a protein expressed on the surface of melanocytes (52), but lose that expression and begin dying at higher doses of the molecule (Fig. 8C). Therefore, it appears that lower doses of FR have a correcting effect on aberrant Gα_q/11 signaling that causes the cells to differentiate into normal melanocytes. The differentiation of B16 melanoma cells by FR has previously been reported (32), while recent studies suggest FR promotes differentiation of primary 92.1 and Mel202 UM cells containing Gα_q-Q209L by promoting polycomb-mediated gene repression (47). UM cells treated with higher doses of FR may succumb to cell death (Figs. 7 & 8C), although there appears to be some compensatory mechanisms in play. For example, a significant increase in HER3 expression was observed upon FR treatment (Fig. 5). This is thought to be a compensatory mechanism for cell survival as a result of decreased ERK signaling, which has previously been observed in melanoma cells harboring a BRAF^V600E mutation when treated with a RAF inhibitor, effectively suppressing ERK activation (53, 54). In the presence of its ligand (neuregulin 1), HER3 heterodimerizes with HER2 to recruit PI3 kinase and activate Akt, promoting cell survival (54, 55). An increase in activated STAT3 was also detected upon FR treatment (Fig. 5). STAT3 also activates Akt and may be enhanced by HER3 signaling (56).

In summary, the data presented here show that FR inhibits oncogenic Gα_q/11-Q209 protein in vitro and in UM cells, promoting cell cycle arrest and eventual cell death, while also preventing the colonization of UM cells in 3D culture. Our work highlights the potential use of Gα_q/11 inhibitors such as FR in the treatment of uveal melanoma.

Implications:

Oncogenic Gα_q/11 inhibition by FR900359 may be a potential treatment option for those with uveal melanoma.

The abbreviations used are:

C

cytoplasmic

DMSO

dimethylsulfoxide

FR

FR900359

GDI

guanosine nucleotide dissociation inhibitor

G_q, Gα_qβγ

heterotrimer

GST

glutathione S-transferase

GTPγS

guanosine 5’-O-[gamma-thio]triphosphate

MAPK

mitogen activated protein kinase

N

nuclear

RPPA

reverse phase protein array

UM

uveal melanoma

YAP

yes associated protein