Metformin accelerates the growth of BRAF^V600E-driven melanoma by upregulating VEGF-A

AMPK activation does not inhibit the growth of BRAF mutant melanoma cells

We initiated this study by investigating the effects of metformin and another AMPK activator, AICAR (5-aminoimidazole-4-carboxamide riboside) (5, 14) in BRAF and NRAS mutant melanoma cells (see Supplemental Table 1 for cells used). On standard tissue culture plastic metformin and AICAR only modestly affected the growth of these cells (Supplemental Fig 1A), but when the cells were grown in soft agar, whereas NRAS mutant cell growth was inhibited by metformin and AICAR by 68-100%, BRAF mutant cell growth was insensitive to these agents (Fig 1A, Supplemental Fig 1B, 1C). Thus, NRAS mutant melanoma cells were sensitive to the anti-growth effects of metformin and AICAR, whereas BRAF mutant cells were resistant.

To test if this difference arose because AMPK was not activated in the BRAF mutant cells, we western blotted cell extracts for AMPKα phosphorylation on T172 and ACC phosphorylation on S79. AICAR induced slow phosphorylation of AMPKα and ACC in BRAF mutant SK-Mel28 and A375 cells, with no increase apparent for the first two hours, but robust phosphorylation at 6 hours and beyond (Fig 1B, 1C). AICAR also activated AMPK in NRAS mutant D04 cells, with clear increases in AMPKα and ACC phosphorylation apparent within an hour of AICAR treatment (Fig 1D). We also show that metformin and glucose starvation activated AMPKα in A375 cells (Supplemental Figs 1D, 1E), and phosphorylation of ACC is increased in response to 12 hour metformin and AICAR treatment in 5 additional BRAF-mutant melanoma lines (Supplemental Fig 1F). Thus, AMPK was activated in BRAF mutant melanoma cells, albeit it with slow kinetics, but it did not inhibit their growth.

RSK mediates BRAF mutant melanoma cell resistance to AMPK

To investigate why BRAF mutant cells are resistant to AMPK, we examined TORC1 signaling because this pathway is a key AMPK target in cells (19). We show that 4E-BP1 and rpS6 phosphorylation (see Supplemental Fig 2A) was suppressed in metformin-treated NRAS mutant (D-04, MM-415 and WM-1366), but not BRAF mutant (A375, Mel-HO and SK-Mel 28) cells (Fig 2A). Thus TORC1 signaling was insensitive to AMPK in BRAF mutant cells. TORC1 can be activated by two pathways (5). In the canonical pathway downstream of RAS, phosphatidylinositide 3-kinase (PI3-K) activates Akt, which inhibits TSC1/2, allowing Rheb to activate TORC1 (see Supplemental Fig 2A). In the non-canonical pathway downstream of RAF, ERK activates the protein kinase RSK, which then inhibits TSC1/2 and directly activates TORC1 (Supplemental Fig 2A) and accordingly, both BRAF and MEK inhibitors block RSK phoshporylation in A375 cells (Supplemental Fig 2B).

We measured RSK activity by western blotting for phosphorylation of S380/S386 on RSK1/RSK2. We show that RSK activity was generally higher in BRAF mutant than NRAS mutant cells (Fig 2B) and that metformin and AICAR activated RSK in A375 cells (Fig 2C and Supplemental Fig 2C). Critically, metformin did not block 4E-BP1 or rpS6 phosphorylation in A375 cells, whereas the RSK inhibitor BI-D1870 suppressed phosphorylation of both (Fig 2D). Note also that metformin and BI-D1870 cooperated both to inhibit rpS6 phosphorylation and to activate AMPK in A375 cells (Fig 2D). Critically, whereas neither metformin nor BI-D1870 inhibited the growth of A375, SK-Mel28 or WM-266.4 cells, together they strongly inhibited the growth of these cells (Fig 2E).

We observed similar results with RNA interference (RNAi). Thus, whereas downregulation of RSK1 and RSK2 did not affect the growth of A375 cells, it did make these cells more sensitive to the growth inhibitory effects of metformin (Fig 2F). Note, loss of either protein alone did not sensitize A375 cells metformin (Fig 2F), demonstrating that these proteins are functionally redundant. Finally, constitutively active myristoylated RSK1 (myr-RSK); (20) increased RSK and rpS6 phosphorylation in NRAS mutant D04 cells (Fig 2G) and caused them to become resistant to the growth inhibitory effects of metformin and AICAR (Fig 2H). We conclude that RSK mediates BRAF mutant melanoma cell resistance to metformin.

Metformin accelerates the growth of BRAF tumors and induces expression of VEGF-A

Next BRAF mutant cells were grown as tumor xenografts in metformin-treated nude mice. Metformin did not affect the weight of the mice (Supplemental Fig 3A) or increase their blood lactate levels (Supplemental Fig 3B), demonstrating that it was well tolerated. Surprisingly, metformin increased the size of A375 (BRAF mutant) xenografts by 3.2 fold at 50 days (Fig 3A; p<0.0001) and Mel-HO (BRAF mutant) xenografts by 2.3 fold (Fig 3B; p<0.034), but decreased D04 (NRAS mutant) xenografts size by 42% (Supplemental Fig 3C; p< 0.0033). Thus, despite having little effect on BRAF mutant cell growth in vitro, metformin accelerated their growth in vivo.

Histological examination of the tumors revealed a dramatic increase in the size and number of CD31 positive vessels in A375 xenografts from metformin-treated mice (Fig 3C and Supplemental Fig 3D) and we confirmed that these structures were blood vessels by staining for endoglin/CD105 (Fig 3D). We used a human specific antibody to show that metformin increased VEGF-A protein production by A375 melanoma cells in vivo (Fig 3E) and that metformin, phenformin, AICAR and A-769662 all increased VEGF-A protein production in BRAF mutant, but not NRAS mutant melanoma cells (Fig 3F and Supplemental Fig 3E).

Metformin cooperates with anti-VEGF therapies to suppress the growth of BRAF^V600E mutant tumors

To investigate the importance of VEGF-A to BRAF mutant tumors responses to metformin in vivo, nude mice bearing A375 xenografts were treated with the VEGF receptor inhibitor axitinib. Metformin accelerated the growth of the tumors (2.2 fold increase in tumor size at day 50) whereas axitinib had no effect (Fig 4A). When these agents were combined they suppressed A375 tumor growth by 45% (Fig 4A). Note that no such cooperation was seen in vitro (Fig 4B) and axitinib and metformin did not cooperate to inhibit the growth of NRAS mutant cells either in vitro or in vivo (Supplemental Fig 4A, 4B).

These effects on A375 xenografts we reproduced using the VEGF neutralizing antibody bevacizumab. Metformin increased A375 tumor growth by 2.0 fold and bevacizumab reduced their growth by 34%, but together they reduced tumor growth by 64% (Fig 4C). Finally, these observations were also reproduced using short-hairpin RNA (shRNA). MDA-MB-435 cells were engineered to express two independent VEGF-A shRNA probes. These constructs blocked metformin and AICAR-induced VEGF-A protein production by MDA-MB-435 cells (Fig 4D), but did not affect their growth in vitro (Fig 4E). Critically, whereas metformin accelerated the growth of MDA-MB-435 tumors expressing control shRNA, it induced regression in tumors expressing VEGF-A shRNA (Fig 4F).

Taken together, these data show that VEGF-A drives the accelerated growth of BRAF mutant melanoma in metformin-treated mice, but when VEGF-A signaling was inhibited, metformin unexpectedly switched from a growth promoter to a growth inhibitor.

AMPK increases VEGF-A protein production in BRAF mutant cells

Next we investigated how metformin upregulated VEGF-A in BRAF mutant cells. We show that the AMPK inhibitor compound C blunted VEGF-A upregulation by AICAR in A375 cells (Fig 5A) and that AMPKα1 depletion by RNAi blocked VEGF-A upregulation in metformin and AICAR-treated A375, Mel-HO and MDA-MB-435 cells (Fig 5B and Supplemental Fig 5A/B). AMPKα2 depletion did not affect VEGF-A protein production in A375 cells (Supplemental Fig 5C).

Next, we examined VEGF-A upregulation in SK-Mel5 melanoma cells because although these cells express BRAF^V600E, they lack LKB1 and so cannot activate AMPK when treated with AICAR (21, 22). We confirmed that LKB1 was not expressed in SK-Mel5 cells and that AICAR did not activate AMPK in them (Fig 5C). Critically, AICAR did not upregulate VEGF-A in SK-Mel5 cells (Fig 5C). As an important control, we show that the calcium ionophore A23187 activated AMPKα and upregulated VEGF-A in SK-Mel5 cells and that the CAMKK inhibitor STO-609 blunted both responses (Fig 5C 5D). This suggests that AMPK is still activated by CAMKII in these cells and shows that the VEGF-A gene still responded to AMPK in SK-Mel5 cells, providing further evidence that AMPK upregulated VEGF-A in BRAF mutant cells.

AMPK stimulates VEGF-A protein production through ERK

To determine how AMPK upregulated VEGF-A, we show that AICAR increased VEGF-A mRNA levels in A375 cells (Fig 5E). Since VEGF-A is a hypoxia-regulated gene (23), we investigated if the hypoxia-inducible transcription factors (HIFs) regulated these responses. However, our experiments were performed in 20% oxygen and HIF1α was not expressed (Supplemental Fig 6A). Furthermore, metformin and AICAR did not induce HIF1α expression in these cells (Supplemental Fig 6B) and HIF1α siRNA did not block VEGF-A upregulation by metformin or AICAR (Supplemental Figs 6C, 6D). In addition, we were unable to detect HIF2α by western blot in these cells and HIF2α siRNA did not block metformin or AICAR mediated VEGF-A upregulation (Supplemental Figs 6C, 6D). Finally, we also show that although hypoxia upregulated the basal level of VEGF-A in A375 cells, metformin and AICAR further upregulated VEGF-A in the hypoxic cells (Supplemental Fig 6E). We conclude that metformin and AICAR upregulate VEGF-A independently of hypoxia.

Previous studies have shown that VEGF-A expression is also regulated by ERK signaling in some cells (24), so we examined if ERK regulated VEGF-A in metformin treated BRAF mutant melanoma cells. We show that the MEK inhibitor PD184352 downregulated the basal levels of VEGF-A mRNA in A375 cells (Fig 5E). We also show that PD184352 and the BRAF inhibitors PLX4720 and 885-A (25) blocked VEGF-A upregulation in AICAR treated cells (Fig 5F). Furthermore, BRAF depletion by siRNA also blocked VEGF-A upregulation by metformin and AICAR (Fig 5G). Note that BRAF depletion did not block AICAR or metformin-driven ACC phosphorylation (Fig 5G), showing that BRAF depletion did not inhibit AMPK through an unknown cryptic mechanism. Furthermore, while conducting this experiment, we noted that metformin and AICAR activated ERK in the scrambled siRNA control samples (Fig 5G, lanes 1-3), but not in the cells in which BRAF was depleted (Fig 5G lanes 4-9). Following this observation up, we show that ERK activation by AICAR occurred with slow kinetics and in the absence of increased MEK phosphorylation (Fig 5H) or BRAF activation (Supplemental Fig 6F).

AICAR induces degradation of DUSP6 protein in BRAF mutant melanoma cells

Since metformin and AICAR activated ERK without activating upstream signaling, we examined if AMPK disrupted ERK pathway negative feedback loops. The dual specificity phosphatase DUSP6 is an ERK negative regulator and a transcription target of BRAF^V600E/ERK signaling in melanoma cells (26-28). Commensurate with this, we show that PD184352 strongly suppressed (>99% inhibition) DUSP6 mRNA in A375 cells (Fig 6A) and this was accompanied by loss of the DUSP6 protein (Fig 6B, lanes 1, 2). Conversely, we show that ERK activation by AICAR was accompanied by an increase in DUSP6 mRNA (~4.2 fold increase, see Fig 6A), but unexpectedly this was accompanied by a reduction rather than increase in DUSP6 protein (Fig 6B, lanes 1, 3, 4). These data suggested that AMPK targets DUSP6 for degradation and accordingly we show that DUSP6 protein was downregulated in A375 xenografts from metformin treated mice (Fig 6C; Supplemental Fig 7A), correlating with increased ERK activation (Supplemental Fig 7B). We also show that the proteasome inhibitor MG132 prevented DUSP6 protein loss in PD184352 treated cells (Fig 6D), but increased DUSP6 protein in AICAR treated cells (Fig 6D). Finally, we show that DUSP6 depletion by two distinct siRNAs activated ERK (Fig 6E) and upregulated VEGF-A (Fig 6F) in A375 cells. We conclude that by targeting DUSP6 for degradation, AMPK activated ERK and upregulated VEGF-A.

Reagents

Antibodies for phospho-ACC, phospho-AMPKα, total AMPKα, phospho-MEK, phospho-S6, phospho-S6K1, phospho-4EBP1, phospho-RSK, RSK2, HIF-1α and LKB1 were from Cell Signaling Technology (Cambridge-Biosciences). Antibodies for BRAF, ERK2 and AMPKα1 were from Santa Cruz Biotechnology (Santa Cruz, USA). The DUSP6 antibody was from Abcam (Cambridge, USA). Phospho-ERK1/2 and tubulin antibodies were from Sigma. Anti-RSK1 was from Millipore (Billerica, USA). A-77652 and BI-D1870 were from the Medical Research Council Protein Phosphorylation Unit, University of Dundee (UK). Metformin, phenformin, AICAR, A23157, STO-609 and rapamycin were from Sigma. Axitinib was purchased from Selleck Chemicals. 885-A was synthesized on contract by Evotec AG (Abingdon, UK). PD184352 and PLX4720 were synthesized in-house.

Preparation of cell lysates and Western blotting

The details are described in the Supplemental Material

Cell culture techniques

Details regarding cell lines, their growth conditions, mutation status and source are found in Supplemental Table 1. Cell viability was by SRB assay (40) and growth in soft agar as described (41), with macroscopic (>0.1-mm) colonies scored (10 fields per sample; triplicate determinations) after 2 weeks. The number of colonies formed/total number of cells plated (%) was calculated and is expressed relative to appropriate controls. Statistical analysis was by Student’s t-test. VEGF-A protein levels were measured by human-specific sandwich ELISA (R&D Systems). Gene expression measurements by qRT-PCR and siRNA mediated gene depletion was as described (25). Cells transfected with shRNA vectors (SA Biosciences) were selected in puromycin (1μg/mL). Further details can be found in Supplemental Material.

Xenograft studies

All animal procedures were approved by the Animal Ethics Committees of the Institute of Cancer Research in accordance with National Home Office regulations under the Animals (Scientific Procedures) Act 1986 and according to the guidelines of the Committee of the National Cancer Research Institute (Workman et al., 2010). 2.5 × 10⁶ A375, 2.5 × 10⁶ Mel-HO, 5 × 10⁶ D-04, 4 × 10⁶ MDA-MB-435/NS or 4 × 10⁶ MDA-MB-435/shV.3 cells were injected into the flanks of female (5-8 mice per group) nude mice (Charles River; UK) as described (42). Metformin (300 mg/kg) was administered in drinking water one day prior to injection of cells assuming an average water consumption of 5ml per day per mouse. Axitinib was dissolved in a vehicle of 0.5% w/v carboxymethyl cellulose, and administered daily by oral gavage at a dose of 10 mg/kg, beginning 14 days after injection of cells. Bevacizumab was injected intraperitoneally twice weekly. Tumor length and width were measured using calipers and tumor volume calculated using the formula: Volume = 0.5236 × length × width².

Immunohistochemistry

Tumor vessel density was analyzed in ~600mm³ formalin fixed tumors. 3 μm sections were stained for endoglin (CD105, dilution 1:60; Novocastra) detection with VECTOR® M.O.M.™ Immunodetection Basic Kit (Vector Laboratories). The number of endoglin positive vessels in 5 randomly selected high-powered fields (HPF) for control tumors (n=6) or metformin (n=9) treated mice was determined. The average vessel area was calculated as the sum of the vessel area (∑A) in 5 HPF per tumor with area expressed as maximum length*maximum width (A=a*b), with the average taken for all water treated and metformin treated animals.

Statistical Analysis

The Student’s t test was performed for mRNA expression, tumor xenografts, soft agar, and VEGF-A ELISA assays, the Mann Whitney ranks test was performed for the blood vessel number and area.