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. Author manuscript; available in PMC: 2015 Mar 1.
Published in final edited form as: Mol Cancer Res. 2014 Jan 14;12(3):447–463. doi: 10.1158/1541-7786.MCR-13-0224-T

BRAFV600E Cooperates With PI3’-Kinase Signaling, Independent of AKT, To Regulate Melanoma Cell Proliferation

Jillian M Silva 1, Christina Bulman 1, Martin McMahon 1,*
PMCID: PMC3966216  NIHMSID: NIHMS556388  PMID: 24425783

Abstract

Mutationally activated BRAFV600E cooperates with PTEN silencing in the conversion of normal melanocytes to metastatic melanoma cells, but the mechanism underlying this cooperation is poorly understood. Here the consequences of pharmacological blockade of BRAFV600E or PI3'-kinase signaling were explored using pathway-targeted inhibitors and a panel of human BRAF mutated melanoma-derived cell lines. Blockade of BRAFV600E→MEK1/2→ERK1/2 or class I PI3'-kinases inhibited melanoma proliferation, whereas inhibition of AKT had only modest effects, even in cells with mutated or amplified AKT. Although single-agent inhibition of either BRAFV600E or PI3'-kinase signaling elicited anti-proliferative effects, combinatorial inhibition was more potent. Analysis of signaling downstream of BRAFV600E or PI3'-kinase revealed that these pathways cooperated to regulate protein synthesis through AKT-independent, mTORC1-dependent effects on p70S6K, ribosomal protein S6 and 4E-BP1 phosphorylation. Moreover, inhibition of mTORC1/2 inhibited cell proliferation as profoundly as single-agent inhibition of either BRAFV600E or PI3'-kinase signaling. These data reveal a mechanism by which BRAFV600E and PI3'-kinase signaling cooperate to regulate melanoma proliferation through AKT-independent effects on protein translation. Furthermore, this study provides a potential foundation for pathway-targeted combination therapy designed to enhance the therapeutic benefit to melanoma patients with combined alterations in BRAF and PI3'-kinase signaling.

Keywords: Melanoma, BRAF, PI3’-kinase, AKT, Signal Transduction

IMPLICATIONS

PI3'-kinases, but not AKT, represent potential targets for melanoma therapy.

INTRODUCTION

Melanoma is known for its rapidly increasing incidence, aggressive clinical behavior and propensity for lethal metastasis (1). Mutational activation of BRAF, detected in ≥50% of patients, is one of the earliest and most common genetic alterations in melanomagenesis (2, 3). The importance of mutationally activated BRAFV600E in melanoma maintenance was demonstrated by the clinical success of vemurafenib and dabrafenib, ATP competitive inhibitors of BRAFV600E activity (4, 5).

BRAF mutations are detected at high frequency in benign nevi, non-malignant melanocytic lesions that display hallmarks of senescence and rarely progress to melanoma (3). Malignant progression of BRAFV600E expressing melanocytes is frequently promoted by silencing of the tumor suppressor PTEN, a phosphatidylinositide (PI) 3’-lipid phosphatase that suppresses the production of PI3’-lipids in the cell (6–10). The sufficiency for these alterations in melanomagenesis was demonstrated using genetically engineered mouse (GEM) models of metastatic melanoma built upon this same foundation (11–13).

Recently, RAF→MEK1/2→ERK1/2 and PI3’-kinase→AKT signaling was demonstrated to cooperatively regulate protein translation in carcinomas through inhibitory phosphorylation of 4E-BP1, a negative regulator of the eIF4E-mRNA complex and cap-dependent translation (14). In this study, using pharmacological agents and a panel of melanoma cells, we confirm that PI3’-kinase signaling is necessary to cooperate with BRAFV600E signaling in melanoma. However, inhibition of AKT had little or no anti-proliferative effects on BRAF mutated human melanoma cell lines regardless of PTEN status. Similarly, the anti-proliferative effects of pharmacological blockade of AKT in BRAF mutated melanoma cells expressing mutated or amplified AKT1 or AKT2, respectively, was not as profound as PI3’-kinase inhibition. Although single agent inhibition of BRAFV600E→MEK1/2→ERK1/2 or PI3’-kinase often displayed substantial anti-proliferative activity, the effects of combined inhibition of both BRAFV600E plus PI3’-kinase signaling were significantly more potent. These data support the hypothesis that PI3’-kinase signaling cooperates with BRAFV600E for melanoma maintenance through the regulation of protein translation and provides a biochemical basis for the simultaneous targeting of these two pathways in patients with BRAF mutated melanoma (15–17).

MATERIALS AND METHODS

Cell Culture and Drug Treatments

Human melanoma cell lines, WM793, WM9, and A375, were kindly provided from the well-curated cell line repositories established by Dr. Meenhard Herlyn (Wistar Institute, Philadelphia, PA) and genomic sequencing of these cells was performed in the laboratory of Dr. Katherine Nathanson (University of Pennsylvania, Philadelphia, PA) (Supplementary Table S1) (18–20). The cell lines were cultured in DME-H16 media containing 3 mg/ml glucose, 0.584 mg/ml L-glutamine, 0.11 mg/ml sodium pyruvate and 3.7 mg/ml NaHCO3 supplemented with 10% FBS, 5 µg/ml of insulin, L-glutamine, penicillin/streptomycin and fungizone. M249 and M262 melanoma cells were kindly provided by Dr. Antoni Ribas (U.C. Los Angeles) and authenticated by genomic sequencing as previously described (Supplementary Table S1) (21). These cells were maintained in RPMI 1640 supplemented with 10% FBS, L-glutamine, penicillin/streptomycin and fungizone. Pathway-targeted pharmacological agents were obtained from various colleagues in the private sector or commercial sources and drug concentrations used for each treatment are listed in Supplementary Table S2.

Proliferation and Growth Assays

Melanoma cell proliferation was assessed by seeding 105 cells in 12-well plates. Cells were treated with the various pharmacological agents as described in Supplementary Table S2 for 24, 48 and 72 hours. Viable cells were enumerated using a Countess® automated cell counter (Invitrogen). Data presented is representative of three independent experiments. To complement short-term proliferation assays, replicate cultures of melanoma cells were plated in 6-well plates and cultured in the absence or presence of drug for 4–11 days with viable cells fixed and stained with Crystal Violet. Cell proliferation was quantified by solubilizing the Crystal Violet stained cells in 33% acetic acid and measuring the absorbance at 562nm using a plate reader.

Immunoblot Analysis

Cells were lysed using RIPA buffer (50mM Tris, 150mM NaCl, 0.5mM EDTA, 10mM NaF, 0.1% SDS, 0.5% Sodium Deoxycholate, 1% NP-40) containing protease and phosphatase inhibitors (Pierce/Thermo Scientific) and then centrifuged at 14,000 rpm for 5 minutes at 4°C to generate post-nuclear lysates with protein concentrations measured using the BCA assay (Pierce/Thermo Scientific) (22). 30µg of protein were separated using NuPAGE Novex Bis-Tris gels (Invitorgen) and transferred to PVDF membrane using an iBlot transfer apparatus (Invitrogen). Immunoblots were blocked in Odyssey® blocking buffer (LI-COR Biosciences) and probed with the primary antibodies as described in Supplementary Tables S3 and S4. Antigen-antibody complexes were detected using fluorescent goat anti-Rabbit IRDye 800 or goat anti-Mouse IRDye 680 secondary antibodies (LI-COR Biosciences) and visualized with a LI-COR infrared imaging system (Odyssey Classic or Fc). Immunoblot data was analyzed using either the Odyssey application software v3.0.30 or Image Studio v2.0 software (LI-COR Biosciences) (22).

Protein Synthesis Assays

Melanoma cells, plated at ~6×105 cells in 6-well plates, were treated with the indicated agents for 18 hours and then deprived of methionine for 1 hour prior to culturing in DME-H16 media containing 20µCi of [35S]-methionine for 1 hour. Cells were prepared as previously described and 30µg of cell extracts were electrophoresed and then transferred to PVDF membrane. Radiolabeled proteins were visualized by exposure to X-Ray film and quantified by densitometric analysis using ImageJ 1.45 software (NIH).

m7GTP-Sepharose Cap Binding Assays

Following the indicated drug treatments for 24 hours, melanoma cells were lysed in Buffer A (10mM Tris, 140mM KCl, 4mM MgCl2, 1mM EDTA, 1% NP-40 with protease and phosphatase inhibitors). 400µg of protein were incubated with 40µl of m7GTP-sepharose beads (GE Healthcare) in Buffer B (10mM Tris, 140mM KCl, 4mM MgCl2, 1mM EDTA with protease and phosphatase inhibitors) overnight at 4°C. eIF4E-associated complexes were washed twice with Buffer C (10mM Tris, 140mM KCl, 4mM MgCl2, 1mM EDTA, 0.5% NP-40 containing protease and phosphatase inhibitors) and then twice with cold PBS containing 5mM EDTA. m7GTP-sepharose affinity precipitated proteins were boiled in 4× LDS Sample Buffer (Invitrogen) and analyzed by immunoblotting with the indicated antibodies.

Statistical Analysis

All quantitative data is represented as means ± SEM. GraphPad Prism 6 statistical software was used to determine p values by performing either a two-way ANOVA analysis with Bonferroni’s multiple comparisons test for the proliferation graphs or paired, two-tailed t tests for the quantification of cell proliferation by Crystal Violet stained cells.

RESULTS

Pharmacological blockade of either BRAFV600E or PI3’-kinase signaling inhibits proliferation and signaling of human BRAF mutated melanoma cells

To determine the signal pathway dependencies of BRAF mutated melanoma cells, we examined the effects of pharmacological blockade of BRAFV600E→MEK1/2→ERK1/2 or PI3’-kinase→AKT signaling using human melanoma-derived cell lines that were selected based solely on their BRAF mutation status, some of which are proficient or deficient for PTEN expression (Supplementary Table S1) (18–20). Agents employed to inhibit BRAFV600E→MEK1/2→ERK1/2 or PI3’-kinase→AKT signaling were chosen because of their reported specificity and selectivity against the relevant targets (Supplementary Fig. S1 and Supplementary Table S2) (23–35). Where possible, results were verified by targeting multiple components of the same pathway using structurally unrelated, mechanistically dissimilar inhibitors of the same target.

Treatment of WM793, WM9, and A375 cells with either a MEK (1µM PD0325901/MEKi1) or an ERK1/2 (1µM SCH772984/ERKi) inhibitor for 24, 48, and 72 hours led to robust inhibition of cell proliferation (Fig. 1A, 72 hours: p<0.001), however we did not observe a significant decrease in cell number, which is consistent with the negligible pro-apoptotic effects of MEK inhibition on cultured melanoma cells (36). Similarly, inhibition of class I PI3’-kinases (5µM GDC-0941/PI3Ki1) inhibited melanoma cell proliferation without a measureable decrease in cell number at 72 hours (WM793: p<0.05; WM9 & A375: p<0.0001). Surprisingly, inhibition of AKT with either of two structurally unrelated and mechanistically dissimilar inhibitors (MK-2206/AKTi1 or GSK690693/AKTi2) was largely without effect in WM793 and A375 cells. By contrast, the proliferation of WM9 cells at 72 hours was inhibited by ~50% by both AKT inhibitors (AKTi1: p<0.01) (Fig. 1A). In addition to short-term growth assays, the effect of these agents on longer-term cell proliferation (4–6 days) was examined (Fig. 1B and Supplementary Fig. S4A). These data largely confirmed the short-term assays, in which single agent inhibition of BRAFV600E, MEK1/2, ERK1/2 or PI3’-kinase had potent anti-proliferative activity compared to modest effects of AKT inhibition.

Fig. 1. Sensitivity of human melanoma-derived cell lines to pharmacological inhibition of BRAFV600E or PI3’-kinase signaling.

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A. WM793 (BRAFV600E, PTENNull), WM9 (BRAFV600E, PTENNull), and A375 (BRAFV600E, PTENWT) cells were treated with DMSO (CTL) or inhibitors of MEK (1µM PD0325901/MEKi1), ERK1/2 (1µM SCH772984/ERKi), PI3’-kinase (5µM GDC-0941/PI3Ki1) or AKT (5µM MK-2206/AKTi1 or 5µM GSK690693/AKTi2) with cell proliferation assessed for 24, 48, and 72 hours. Results are expressed as relative cell number to the DMSO-treated controls and presented as means ± SEM (n = 3).

B. Cells were plated at a 30% confluency and cultured in the continuous presence of inhibitors of BRAFV600E (1µM GSK2118436/BRAFi1), MEK (MEKi1), ERK1/2 (ERKi), PI3’-kinase (PI3Ki1) or AKT (AKTi1 or AKTi2) for 4 (A375) or 6 (WM793 and WM9) days with viable cells fixed and stained with Crystal Violet.

C. & D. WM793, WM9, and A375 cells were treated with DMSO (CTL) or inhibitors of MEK (MEKi1), ERK1/2 (ERKi), PI3’-kinase (PI3Ki1) or AKT (AKTi1 or AKTi2) for 4 (C) or 24 (D) hours and cell lysates were immunoblotted with the indicated antibodies.

To examine the consequences of pathway-targeted inhibition on signaling downstream of BRAFV600E or PI3’-kinase, extracts were prepared from cells treated with the various agents for 4 or 24 hours and analyzed by immunoblotting (Figs. 1C & 1D). Information regarding the significance of each phosphorylation site is included in Supplementary Table S4. Control (CTL) WM793, WM9 and A375 cells displayed readily detectable phosphorylation of ERK1/2 (P-T202/Y204), AKT (P-S473), p70S6K (P-T389), ribosomal protein S6 (rpS6) (P-S235/236 & P-S240/244) and 4E-BP1 (P-T37/46 & P-S65). Inhibition of MEK (1µM PD0325901/MEKi1) or ERK1/2 (1µM SCH772984/ERKi) led to decreased P-ERK1/2 with little or no effect on P-AKT. Similarly, inhibition of class 1 PI3’-kinases (5µM GDC-0941/PI3Ki1) or AKT (5µM MK-2206/AKTi1) potently suppressed P-AKT, but had no effect on P-ERK. Thus, BRAFV600E→MEK1/2→ERK1/2 and PI3’-kinase→AKT signaling appear to be largely insulated from the inhibitory effects of the other pathway, at least over a 24 hour exposure period.

MEK or ERK1/2 inhibition resulted in decreased P-p70S6K and P-rpS6 in all three cell lines at 24 hours, whereas P-4E-BP1 was only significantly reduced in MEK-inhibited WM793 and A375 cells (Fig. 1D and Supplementary Fig. S2). Although MEK1/2 or ERK1/2 inhibition for 4 hours potently suppressed P-p70S6K, there was no effect on P-rpS6 in any of the cell lines at this time point (Fig. 1C). Interestingly, inhibition of PI3’-kinase for either 4 or 24 hours significantly reduced P-rpS6 in all cell lines, which occurred in the absence of changes in P-p70S6K (Figs. 1C & 1D & Supplementary Fig. S2). Inhibition of PI3’-kinase also led to reduced P-4E-BP1 at both 4 and 24 hours with the most profound effect at the later time point (Figs. 1C & 1D and Supplementary Fig. S2). Strikingly, AKT inhibition with either of two inhibitors had no discernible effect on either P-p70S6K or P-4E-BP1 and only reduced P-rpS6 by ~50% in WM9 cells at both time points (24 hours: p<0.05) (Figs. 1C & 1D and Supplementary Fig. S2). Taken together, these data suggest that BRAFV600E→MEK1/2→ERK1/2 and PI3’-kinase signaling regulate mTORC1-dependent phosphorylation of rpS6 and 4E-BP1 signaling events through distinct mechanisms that are largely independent of AKT activity.

Temporal regulation of rpS6 and 4E-BP1 phosphorylation by BRAFV600E→MEK1/2→ERK1/2 or PI3’-kinase

To further assess differences in the regulation of rpS6 and 4E-BP1 phosphorylation downstream of BRAFV600E and PI3’-kinase signaling, WM793 or A375 cells were treated with inhibitors of either MEK1/2 (PD0325901/MEKi1) or PI3’-kinase (GDC-0941/PI3Ki1) for 1–10 hours (Fig. 2A). Both of these agents exerted robust and selective inhibitory effects against P-ERK1/2 or P-AKT after one hour of treatment. Although MEK1/2 inhibition led to a rapid decrease of P-p70S6K, this did not result in an immediate decrease in rpS6 phosphorylation as we noted a 4–8 hour delay between effects of MEK1/2 inhibition on P-p70S6K and dephosphorylation of rpS6. As before, MEK1/2 inhibition had negligible effects on P-4E-BP1 at all time points evaluated. Although PI3’-kinase inhibition had no effect on p70S6K phosphorylation at any time point, there was a notable decrease in P-rpS6 in both cell lines within 4 hours, which was most striking in WM793 cells after 10 hours (Fig. 2A). In addition, PI3’-kinase inhibition also led to decreased P-4E-BP1 that was detected within 1 hour in both cell lines (Fig. 2A). Thus, inhibition of either MEK1/2 or PI3’-kinase resulted in decreased phosphorylation of rpS6, but elicited their effects with different kinetics and contrasting effects on the activation status (P-T389) of p70S6K.

Fig. 2. Kinetics of rpS6 and 4E-BP1 dephosphorylation following inhibition of BRAFV600E or PI3’-kinase signaling.

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A. WM793 and A375 melanoma cells were treated with MEK (1µM PD0325901/MEKi1) or PI3’-kinase (5µM GDC-0941/PI3Ki1) inhibitors for 1, 4, 8, and 10 hours and cell lysates were analyzed by immunoblotting with the indicated antibodies.

B. Cells were treated with inhibitors of mTORC1 (1µM or 5µM PP242/mTORi), p90RSK (1µM FMK/RSKi), or p70S6K (10µM DG2/S6Ki) for 24 hours and cell lysates were immunoblotted with the indicated antibodies.

C. Melanoma proliferation was assessed in WM793, WM9, and A375 cells treated with DMSO (CTL) or the mTORC1 inhibitor (mTORi) for 24, 48, and 72 hours. Results are expressed as relative cell number to the DMSO-treated controls and presented as means ± SEM (n = 3).

D. WM793, WM9, and A375 cells were also plated at a 30% confluency and cultured in the continuous presence of the mTORC1 inhibitor (mTORi) for 11 days with viable cells fixed and stained with Crystal Violet.

Evidence suggests that phosphorylation of rpS6 and 4E-BP1 is downstream of mTORC1 (37). However, BRAFV600E→MEK1/2→ERK1/2 signaling can also contribute to rpS6 phosphorylation through p90RSK (38). To determine the role of mTORC1 signaling and the S6 kinase, p70S6K or p90RSK, responsible for sustaining rpS6 phosphorylation, WM793 and A375 cells were treated with either PP242 (1µM or 5µM mTORi), a potent ATP competitive inhibitor of mTORC1/2 signaling; FMK (1µM RSKi), a covalent inhibitor of p90RSK; or DG2 (10µM S6Ki), a p70S6K inhibitor (Fig. 2B and Supplementary Table S2) (23, 24, 30).

As expected, PP242-mediated inhibition of mTORC1/2 resulted in decreased phosphorylation of P-rpS6 and P-4E-BP1 (Fig. 2B). In addition, a mechanistically distinct mTORC1 inhibitor, Rapamycin, also inhibited P-rpS6, but had no effect on 4E-BP1 phosphorylation (Supplementary Fig. S3A). Although inhibition of p90RSK had no effect on either P-rpS6 or P-4E-BP1, inhibition of p70S6K selectively reduced P-rpS6. These data are consistent with a model in which phosphorylation of both rpS6 and 4E-BP1 is mTORC1-dependent and the phosphorylation of rpS6 is p70S6K dependent but p90RSK independent (Fig. 2B).

In parallel, we assessed the effects of mTORC1/2 inhibition (PP242/mTORi) on melanoma cell proliferation using both short-term (1–3 days) and long-term (11 days) proliferation assays (Figs. 2C & 2D and Supplementary Fig. S4B). Proliferation of WM793, WM9, and A375 cells was significantly inhibited by either 1 or 5µM PP242 at all time points analyzed (24hrs: WM793 p<0.05, WM9 & A375 p<0.01; 48–72hrs: p<0.0001), however the anti-proliferative effect of 1µM PP242 was less robust against WM793 and A375 cells (Fig. 2C). These results are consistent with a longer term proliferation assay in which 5µM PP242 elicited more potent anti-proliferative effects against all three cell lines than 1µM PP242 (Fig. 2D and Supplementary Fig. S4B). Similar to previous analyses of BRAFV600E, PTENNull melanoma cells derived from a genetically engineered mouse model, Rapamycin was less potent in inhibiting the proliferation of all three human BRAF mutated melanoma cell lines (Supplementary Fig. S3B) (11). Thus, dual control of mTORC1 signaling by cooperative BRAFV600E and PI3’-kinase signaling appears important for melanoma cell proliferation.

BRAFV600E and PI3’-kinase signaling cooperate to regulate rpS6 and 4E-BP1 phosphorylation and melanoma cell proliferation

Given that BRAFV600E and PI3’-kinase signaling cooperate to regulate mTORC1 signaling output, we tested whether combined inhibition of these pathways might elicit more striking effects on intracellular signaling events leading to more profound effects on melanoma cell proliferation. Extracts of WM793, WM9 or A375 cells treated with inhibitors of BRAFV600E, MEK1/2 or PI3’-kinase, either alone or in combination, and analyzed by immunoblotting (Figs. 3A and 4A). As previously demonstrated, MEK1/2 (PD0325901/MEKi1) or BRAFV600E (PLX-4032/BRAFi2) inhibition led to decreased P-ERK, P-p70S6K and P-rpS6 with only modest inhibition of P-4E-BP1. PI3’-kinase inhibition (GDC-0941/PI3Ki1) robustly suppressed P-AKT and P-rpS6, had no effect on P-p70S6K and led to a modest decrease of P-4E-BP1 in WM793 cells with a more profound inhibition in WM9 and A375 cells. Although PI3’-kinase inhibition reproducibly inhibited P-4E-BP1, we noted some experiment-to-experiment variation in the magnitude of this effect. Most importantly, combined inhibition of either BRAFV600E or MEK1/2 plus PI3’-kinase routinely resulted in a more potent suppression of P-rpS6 and P-4E-BP1 compared to the corresponding single agents (Figs. 3A and 4A).

Fig. 3. Effects of single agent or combined inhibition of MEK1/2 or PI3’-kinase on melanoma cell signaling and proliferation.

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A. WM793, WM9, and A375 cells were treated with inhibitors of MEK (1µM PD0325901/MEKi1) or PI3’-kinase (5µM GDC-0941/PI3Ki1) either alone or in combination for 18 hours with cell lysates analyzed by immunoblotting with the indicated antibodies.

B. Single agent or combined inhibition of MEK (MEKi1) or PI3’-kinase (PI3Ki1) on WM793, WM9, and A375 cell proliferation for 24, 48, and 72 hours. Results are expressed as relative cell number to the DMSO-treated controls and presented as means ± SEM (n = 3).

C. Triplicate cultures of WM793, WM9 or A375 cell lines were treated with inhibitors of MEK (MEKi1) or PI3’-kinase (PI3Ki1) as single agents or in combination for 9 days with viable cells fixed and stained with Crystal Violet. DMSO-treated controls were split twice to prevent overgrowth and therefore staining of these wells is an underestimate of actual cell number 12-fold WM793, 22-fold WM9, 60-fold A375).

D. Quantification of the Crystal Violet staining of control vs. drug treated WM793, WM9, and A375 cells. Results are expressed as a fold change of the DMSO-treated control and presented as means SEM (n = 3). Paired, two-tailed t tests were used to determine p values (*p<005; **p<0.01).

Fig. 4. Effects of single agent or combined inhibition of BRAFV600E or PI3’-kinase on melanoma cell growth and signaling.

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A. WM793, WM9, and A375 cells were treated with inhibitors of BRAFV600E (5µM PLX-4032/BRAFi2) or PI3’-kinase (5µM GDC-0941/PI3Ki1) as single agents or in combination for 24 hours with cell lysates analyzed by immunoblotting with the indicated antibodies.

B. Melanoma cell proliferation was assessed by treating all three cell lines with inhibitors of BRAFV600E (BRAFi2) or PI3’-kinase (PI3Ki1) alone or in combination for 4 days with viable cells fixed and stained with Crystal Violet.

In parallel, the effects of combined MEK1/2 plus PI3’-kinase inhibition on cell proliferation were assessed in short- and long-term assays (Figs. 3B–D). In the short-term, single agent inhibition of MEK1/2 (MEKi1) or PI3’-kinase (PI3Ki1) significantly inhibited cell growth at 48 (p<0.01) and 72 (p<0.01) hours. As predicted by the single agent effects, combined inhibition of MEK1/2 plus PI3’-kinase also led to a significant decrease in melanoma cell proliferation, but this was not superior to single agent inhibition at any time point (Fig. 3B). In the longer-term assays (9 days), single agent MEK1/2 inhibition had more potent anti-proliferative activity compared to single agent PI3’-kinase inhibition (Fig. 3C & 3D). However, in all three cell lines, combined inhibition of both MEK1/2 and PI3’-kinase significantly inhibited melanoma proliferation compared to the corresponding single agents with the magnitude of the observed effects being greatest in WM9 and A375 cells (Fig. 3C & 3D). These results were confirmed using a short-term proliferation assay (4 days), in which combined inhibition of BRAFV600E (BRAF2) plus PI3’-kinase (PI3K1) more potently suppressed melanoma cell growth compared to single agent inhibition alone (Fig. 4B and Supplementary Fig. S4C). These data suggest that, at least in vitro, BRAFV600E and PI3’-kinase signaling cooperate to promote key signaling events downstream of mTORC1, which in turn promote melanoma cell proliferation.

Coordinate control of melanoma protein synthesis by BRAFV600E and PI3’-kinase signaling

In conjunction with previous experiments, we assessed the effects of combined inhibition of MEK1/2 (PD0325901/MEKi1) plus PI3’-kinase (GDC-0941/PI3Ki1) on protein synthesis in WM793, WM9 and A375 cells (Figs. 5A & 5B). Single agent inhibition of MEK1/2 or PI3’-kinase alone led to a 40–80% inhibition of [35S]-methionine incorporation (Fig. 5B). The effect of MEK1/2 inhibition on protein synthesis was most striking in WM793 cells, whereas PI3’-kinase inhibition had the equivalent effects on protein synthesis in all three cell lines. Importantly, combined inhibition of both MEK1/2 plus PI3’-kinase resulted in the most profound inhibitory effects on protein translation in all three cell lines. Consistent with previous observations, combined inhibition of MEK1/2 and PI3’-kinase also had the most striking effects on P-rpS6 and P-4E-BP1 compared to the corresponding single agents (Fig. 5A).

Fig. 5. Cooperation of BRAFV600E and PI3’-kinase signaling on the regulation of protein translation.

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A. Protein synthesis was measured by [35S]-methionine incorporation of newly synthesized proteins in WM793, WM9, and A375 cells treated with inhibitors of MEK (1µM PD0325901/MEKi1) or PI3’-kinase (5µM GDC-0941/PI3Ki1) as single agents or in combination for 18 hours (n=2). These same cell extracts were also analyzed for rpS6 and 4E-BP1 phosphorylation as indicated.

B. Densitometric quantification of [35S]-methionine incorporation of newly synthesized proteins in all three cell lines. Results are represented as a fold change compared to the DMSO-treated control.

C. WM793, WM9, and A375 cells were treated with inhibitors of MEK (MEKi1) or PI3’-kinase (PI3Ki1) alone or in combination for 24 hours with cell lysates precipitated with m7GTP sepharose beads followed by immunoblotting for 4E-BP1, eIF4E, or with the indicated antibodies.

Hypo-phosphorylated 4E-BP1 binds to the eIF4E initiation complex at the 5’ cap of mRNAs to inhibit cap-dependent translation by preventing eIF4G from binding to this complex (39). Phosphorylation of 4E-BP1 by mTORC1 releases it from eIF4E allowing eIF4G to bind to the eIF4E-mRNA complex and initiate cap-dependent translation (40). Thus, hypo-phosphorylated 4E-BP1 and eIF4G directly compete for the same binding site on eIF4E, whereas hyper-phosphorylated 4E-BP1 is not a strong competitor for this site (40). Since activation of BRAFV600E and PI3’-kinase signaling promotes hyper-phosphorylation of 4E-BP1, we examined the effects of MEK1/2 or PI3’-kinase inhibition on cap-dependent protein translation, specifically the association between 4E-BP1 and the eIF4E-complex. Extracts of melanoma cells treated with inhibitors of MEK1/2 (MEKi1) or PI3’-kinase (PI3Ki1), either alone or in combination, were used in a cap pull-down assay. In this assay, eIF4E present in melanoma lysates binds to sepharose beads coupled to the cap analog 7-methyl GTP (m7GTP) in order to determine if there is an increase or decrease in 4E-BP1 bound to the eIF4E-m7GTP cap complex.

As observed previously, combined inhibition of MEK1/2 plus PI3’-kinase more potently suppressed P-rpS6 and P-4E-BP1 than the corresponding single agent treatments in the crude cell extracts used for the cap pull-down assays (Fig. 5C). However, despite the effects of MEK1/2 inhibition on P-4E-BP1, we only observed increased association of 4E-BP1 with the eIF4E-m7GTP cap complex in WM793 cells. In all three cell lines, PI3’-kinase inhibition led to increased association of 4E-BP1 with the eIF4E-m7GTP cap complex. Interestingly, and contrary to expectation, combined inhibition of MEK1/2 plus PI3’-kinase did not lead to a further increase in 4E-BP1 association with eIF4E-m7GTP cap complexes. These data suggest that the regulation of melanoma protein synthesis downstream of BRAFV600E and PI3’-kinase activation is more complex than the simple regulation of the stoichiometry of 4E-BP1 phosphorylation detected in cell lysates and its capacity for interaction with eIF4E.

Inhibitors of MEK1/2 and AKT display little or no cooperative effects against melanoma proliferation or signaling

The ineffectiveness of the AKT inhibitors in suppressing melanoma cell proliferation was surprising given the prominence ascribed to AKT as an effector of PI3’-kinase signaling in cancer (14, 31, 41). Since AKT inhibition had modest effects on melanoma cells, we assessed whether combined inhibition of MEK1/2 plus AKT might provide a more robust inhibition of melanoma cell proliferation and signaling. To test the effects of single agent versus combined MEK1/2 or AKT inhibition melanoma cells were treated with MEK1/2 (1µM GSK1120212/MEKi2) or AKT (5µM GSK690693/AKTi2) inhibitors, either alone or in combination, for 5–6 days with viable cells stained with Crystal Violet (Fig. 6A and Supplementary Fig. S4D). Similar to our previous results, single agent MEK1/2 inhibition profoundly inhibited the proliferation of all three melanoma cell lines, however the anti-proliferative effects of AKT inhibition were modest (~35–70% inhibition) and substantially less than that observed in response to single agent PI3’-kinase inhibition (Figs. 1A & 1B). Moreover, inhibition of AKT did not substantially enhance the anti-proliferative effect of MEK1/2 inhibition in these cells (Fig. 6A and Supplementary Fig. S4D).

Fig. 6. Sensitivity of melanoma cell proliferation and signaling to either single agent or combined inhibition of MEK or AKT.

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A. Proliferation of melanoma cells treated with MEK (1µM GSK1120212/MEKi2) or AKT (5µM GSK690693/AKTi2) inhibitors either as single agents or in combination was assessed for 5 (WM9 and A375) or 6 (WM793) days with viable cells fixed and stained with Crystal Violet.

B. WM793, WM9, and A375 cells were treated with inhibitors of MEK (MEKi2) or AKT (AKTi2) either alone or in combination for 24 hours and cell lysates were analyzed by immunoblotting with the indicated antibodies.

To assess the effect of combined inhibition of MEK1/2 and AKT on melanoma signaling, melanoma cells were treated with MEK1/2 (GSK1120212/MEKi2) or AKT (GSK690693/AKTi2) inhibitors, either alone or in combination, for 24 hours with extracts analyzed by immunoblotting (Fig. 6B). As expected, MEK1/2 inhibition led to decreased P-ERK, P-rpS6, and P-4E-BP1 with no effect on P-AKT. AKT inhibition greatly enhanced AKT phosphorylation, as predicted by this agent’s mechanism of action, and reduced P-PRAS40 (T246), a direct downstream substrate of AKT. Consistent with previous data, AKT inhibition had no effect on P-4E-BP1 in any of the cell lines, but moderately decreased P-rpS6 in WM9 cells with little or no discernible inhibitory effect in WM793 or A375 cells. Combined MEK1/2 plus AKT inhibition had only a modest effect on P-rpS6 in WM9 and A375 cells, whereas P-rpS6 in WM793 cells was not further decreased compared to single agent inhibition with MEK. Likewise, the combination of MEK1/2 and AKT inhibition was also ineffective in further reducing P-4E-BP1 compared to MEK1/2 inhibition alone. Hence, although AKT may modestly contribute to rpS6 phosphorylation, its contribution is substantially less than what is observed following inhibition of BRAFV600E→MEK1/2→ERK1/2 or PI3’-kinase signaling. Taken together, these data emphasize the importance of BRAFV600E and PI3’-kinase signaling in the regulation of protein translation leading to melanoma cell proliferation in a substantially AKT-independent manner.

Effects of AKT inhibition on melanoma cells with mutated or amplified AKT1/2

A small percentage of human melanoma cells are reported to express either amplified or mutated AKT in addition to mutated BRAF (21). In addition, AKT1 mutation is associated with BRAFV600E inhibitor resistance in melanoma (42, 43). Consequently, we tested the effects of AKT inhibition on the proliferation of melanoma cells with alterations in AKT1 or 2 (Supplementary Table S1) (21). M249 (BRAFV600E, PTENNull, AKT2Amp) or M262 (BRAFV600E, PTENWT, AKT1E17K/Amp) cells were treated with inhibitors of BRAFV600E (GSK2118436/BRAFi1), MEK1/2 (PD0325901/MEKi1), PI3’-kinase (GDC-0941/PI3Ki1) or AKT (MK-2206/AKTi1 or GSK690693/AKTi2) with proliferation measured at 24, 48 or 72 hours (Fig. 7A). In short-term cell proliferation assays, neither of the AKT inhibitors was as effective as single agent inhibition of MEK1/2 or PI3’-kinase at inhibiting cell proliferation (Fig. 7A). In the longer-term assays, the allosteric AKT inhibtor MK-2206 inhibited the growth of M249 cells as effectively as single agent inhibition of BRAFV600E, MEK1/2 or PI3’-kinase (Fig. 7B and Supplementary Fig. S4E). By contrast, the ATP competitive AKT inhibitor, GSK690693, was substantially less effective in these same cells. Both AKT inhibitors inhibited cell growth by 60–65% in M262 cells (AKT1E17K), but were not as effective as single agent blockade of BRAFV600E, MEK1/2 or PI3’-kinase (Figs. 7B and Supplementary Fig. S4E). Although these data suggest that AKT inhibition in melanoma cells with mutated or amplified AKT1/2 may predict for more robust anti-proliferative effects on cell growth, these effects generally fall short of what is observed with inhibitors of BRAFV600E→MEK1/2→ERK1/2 or PI3’-kinase signaling.

Fig. 7. Pharmacological inhibition of BRAFV600E or PI3’-kinase signaling in melanoma cells with mutated or amplified AKT.

Fig. 7

Fig. 7

Fig. 7

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A. M249 (BRAFV600E, PTENNull, AKT2Amp) and M262 (BRAFV600E, PTENWT, AKT1E17K/Amp) melanoma cells were treated with DMSO (CTL) or inhibitors of BRAFV600E (1µM GSK2118436/BRAFi1), MEK (1µM PD0325901/MEKi1), PI3’-kinase (5µM GDC-0941/PI3Ki1) or AKT (5µM MK-2206/AKTi1 or 5µM GSK690693/AKTi2) with cell proliferation assessed for 24, 48, and 72 hours. Results are expressed as relative cell number to the DMSO-treated controls and presented as means ± SEM (n = 3).

B. In parallel, these cells were plated at a 30% confluency and cultured in the continuous presence of DMSO or inhibitors of BRAFV600E (BRAFi1), MEK (MEKi1), PI3’-kinase (PI3Ki1) or AKT (AKTi1 or AKTi2) for 4 (M249) or 6 (M262) days with viable cells fixed and stained with Crystal Violet.

C. M249 and M262 cells were treated with DMSO (CTL) or inhibitors of BRAFV600E (BRAFi1), MEK (MEKi1), ERK1/2 (ERKi), PI3’-kinase (PI3Ki1 or 5µM BKM-120/PI3Ki2) or AKT (AKTi1 or AKTi2) for 24 hours and cell lysates were analyzed by immunoblotting with the indicated antibodies.

We next assessed the effects of the various agents on signaling downstream of BRAFV600E→MEK1/2→ERK1/2 or PI3’-kinase→AKT (Fig. 7C). As previously observed with other melanoma cells, inhibition of BRAFV600E→MEK1/2→ERK1/2 signaling in either M249 or M262 cells led to substantially decreased P-ERK, P-p70S6K and P-rpS6 with only modest effects on P-4E-BP1. Blockade of PI3’-kinase with two different inhibitors (GDC-0941/PI3Ki1 or BKM-120/PI3Ki2) suppressed P-AKT and P-rpS6, modestly reduced P-4E-BP1, but did not alter P-p70S6K. Although both AKT inhibitors had their predicted effects on P-AKT (decreased with MK-2206/AKTi1, increased with GSK690693/AKTi2), these agents had only modest effects on P-rpS6 and negligible effects on either P-p70S6K or P-4E-BP1 compared to single agent blockade of BRAFV600E, MEK1/2, ERK or class 1 PI3’-kinases.

DISCUSSION

The conversion of melanocytes to metastatic melanoma cells is accompanied by alterations in key signaling pathways that cooperate in melanomagenesis (Supplementary Fig. S1) (1, 7, 11, 44–46). The most common alterations in the PI3’-kinase→AKT pathway are silencing of the PTEN, INPP4B or PIB5PA lipid phosphatases and, less commonly, point mutation/amplification of PIK3CA or AKT1-3 (21, 47–51). Thus, cooperating alterations in PI3’-kinase signaling appear to be essential for progression of BRAFV600E initiated melanoma. Here we show that melanoma cells, regardless of their PTEN or AKT mutational status, were sensitive to single agent inhibition of BRAFV600E or PI3’-kinase signaling and even more susceptible to combined pathway inhibition. This suggests that melanoma cells, even those lacking genetically defined alterations in PI3’-lipid signaling (such as A375 cells), have a requirement for PI3’-kinase signaling for their maintenance (52, 53).

Regulation of protein translation is a process critically required for the aberrant behavior of cancer cells (54). Considerable attention has focused on the mechanisms by which PI3’-kinase signaling regulates mTORC1 activity to initiate cap-dependent protein translation. Here we demonstrate that, in BRAF mutated melanoma cells, these signaling events are under the coordinate control of both BRAFV600E and PI3’-kinase signaling in a manner that is appears largely AKT independent. Others have previously reported dual control of these signaling events in tumor cells with coincident KRAS and PIK3CA mutations, however in that case AKT was reported to be a key effector of PI3’-lipid signaling (14). Moreover, in these cells, single agent blockade of MEK1/2 or AKT had little effect on proliferation, rpS6 or 4E-BP1 phosphorylation or cap-dependent translation and combined inhibition of both pathways was required to suppress these processes (14). By contrast, we show that single agent inhibition of either BRAFV600E or PI3’-kinase signaling was sufficient to inhibit both protein synthesis and melanoma cell proliferation, but that combined pathway blockade led to more robust suppression of proliferation. Interestingly, single agent inhibition of mTORC1/2 with PP242 inhibited cell proliferation as potently as the blockade of either BRAFV600E or PI3’-kinase signaling, indicating the potential importance of mTORC1/2 signaling as an effector of cooperating BRAFV600E and PI3’-kinase activity for melanoma cell proliferation.

Mechanistically, it remains unclear how BRAFV600E or PI3’-kinase influence the phosphorylation of rpS6 and 4E-BP1. It is possible that BRAFV600E signaling influences mTORC1 signaling through ERK1/2 regulation on the TSC1/2 complex as previously proposed (55). By contrast, PI3’-kinase blockade also led to diminished rpS6 phosphorylation, but this occurred in a largely AKT-independent manner and without any change in the key activating phosphorylation of p70S6K (P-T389). One possibility is that PI3’-lipid signaling may regulate rpS6 phosphorylation through effects on PP1, the protein phosphatase that dephosphorylates rpS6 (56). Although we propose that PI3’-kinase cooperates with BRAFV600E to regulate melanoma protein synthesis through cooperative effects on the translation initiation, there are likely additional regulatory events in protein synthesis under the control of these pathways as suggested by the results of the m7GTP cap binding experiments (57).

Despite effective suppression of AKT phosphorylation by either of the two structurally unrelated and mechanistically dissimilar agents, we observed only modest effects on melanoma cell proliferation and failed to detect the expected effects on downstream signaling events. The effects of these agents on cell proliferation or signaling in cells expressing either amplified or mutated AKT1/2 was less robust than expected based on current literature (21). Moreover, the combined blockade of MEK1/2 and AKT had little or no effect on the inhibition on rpS6 and 4E-BP1 phosphorylation, which stood in contrast to the substantive cooperative effects of PI3’-kinase and BRAFV600E or MEK1/2 inhibition. Consequently, the lack of potent single agent activity and evidence of strong cooperation with the BRAFV600E pathway inhibitors, suggests that AKT is not an attractive target for melanoma therapy. This is consistent with the inability of the allosteric AKT inhibitor (MK-2206) to prevent the growth of BRAFV600E, PTENNull melanomas in a GEM model (58). However, since evidence suggests that AKT1 mutation can promote resistance of BRAF mutated melanomas to vemurafenib, AKT inhibitors might forestall the onset of drug resistance in patients (42, 43). Although alternative effectors of PI3’-lipid signaling in melanomagenesis remain to be elucidated, there are numerous proteins with PH, PX or FYVE PI3’-lipid binding domains that are potential candidates (59). Indeed, parsing out which of these effectors is important for the cooperation of oncogenic BRAFV600E and PI3’-kinase signaling will help to decipher the underlying biochemical and signaling mechanisms that exist in the melanoma cell and could lead to the identification of new potential biomarkers and molecular targets for the treatment of melanoma patients.

Supplementary Material

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ACKNOWLEDGMENTS

We thank all the members of the McMahon lab for advice and guidance on this project with special thanks to Victoria Marsh Durban and Marian Deuker. We are indebted to our colleagues Craig Stumpf and Davide Ruggero (UCSF) for assistance with protein synthesis and m7GTP cap binding assays and advice on the regulation of protein translation. We thank Kevan Shokat (UCSF) and Jack Taunton (UCSF) for DG2, PP242 and FMK as well as Dr. Meenhard Herlyn (Wistar Institute) and Dr. Antoni Ribas (UCLA) for providing melanoma cell lines. We also thank many colleagues in the private sector for providing compounds and information on their use: Leisa Johnson and Lori Friedman (Genentech) for GDC-0941; Tona Gilmer and Kirin Patel (Glaxo-Smith-Kline) for GSK2118436 and GSK1120212; Steven Townson, Heike Keilhack, and Ahmed Samatar (Merck & Co.) for MK-2206 and SCH772984; Janet Lyle, Darrin Stuart, and Emmanuelle di Tomaso (Novartis) for BKM-120; and Fei Su (formerly of Roche) for PLX-4032.

GRANT SUPPORT

This research was supported by grants from the Melanoma Research Alliance, a NIH/NCI R01 CA176839 (to MMcM), and an Institutional Research and Academic Career Development Award (IRACDA) (to JS).

Footnotes

Conflict of interest disclosure:

Dr. McMahon discloses receipt of major research support (>$10,000) from Novartis, Plexxicon and Glaxo-Smith-Kline.

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Supplementary Materials

1
NIHMS556388-supplement-1.docx (22.9KB, docx)
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NIHMS556388-supplement-2.pptx (70.9KB, pptx)
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NIHMS556388-supplement-3.pptx (170.4KB, pptx)
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NIHMS556388-supplement-4.pptx (147.9KB, pptx)
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NIHMS556388-supplement-5.pptx (226.1KB, pptx)
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NIHMS556388-supplement-6.docx (20.2KB, docx)
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NIHMS556388-supplement-7.docx (18.2KB, docx)
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NIHMS556388-supplement-9.docx (16.9KB, docx)

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