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. Author manuscript; available in PMC: 2009 Aug 17.
Published in final edited form as: Cancer Res. 2008 Jun 1;68(11):4392–4397. doi: 10.1158/0008-5472.CAN-07-5844

Human melanoma cytolysis by combined inhibition of mTOR and VEGF/VEGFR-2

Kerrington R Molhoek 1, Heinrich Griesemann 2, Jianfen Shu 3, Jeffrey E Gershenwald 4, David L Brautigan 5, Craig L Slingluff Jr 1,*
PMCID: PMC2727753  NIHMSID: NIHMS111175  PMID: 18519701

Abstract

Vascular endothelial growth factor (VEGF) plays a vital role in tumor angiogenesis. VEGF is produced by human melanomas, and the VEGF receptor 2 (VEGFR-2) is expressed by most advanced stage melanomas, suggesting the possibility of an autocrine loop. Here we show that bevacizumab, an anti-VEGF antibody, inhibits proliferation of VEGFR-2+ melanoma cell lines by an average of 41%; however, it failed to inhibit proliferation of VEGFR-2neg melanoma cell lines. The growth inhibitory effect of bevacizumab was eliminated by VEGFR-2 knockdown with siRNA, showing VEGF autocrine growth in melanoma is mediated through VEGFR-2. However, bevacizumab inhibition of autocrine signals did not completely inhibit cell proliferation, nor cause cell death. Cell survival is mediated partially through mTOR, which is inhibited by rapamycin. Combination of bevacizumab with rapamycin caused loss of half of the VEGFR-2+ melanoma cells, but no reduction in the number of VEGFR-2neg melanoma cells. The results show (1) an autocrine growth loop active in VEGFR-2+ melanoma, (2) a non-angiogenic mechanism for inhibition of melanoma by blocking autocrine VEGFR-2 activation, and (3) a possible therapeutic role for combination of inhibitors of mTOR plus VEGF in selected melanomas.

Keywords: VEGF, VEGFR-2, mTOR, melanoma, bevacizumab, rapamycin

Introduction

Malignant melanoma remains poorly responsive to systemic therapy. Treatments targeting molecular changes that underlie malignant behavior hold promise. Such approaches may target cell signaling pathways critical for cancer growth and survival or tumor angiogenesis and metastasis. However, the clinical benefit of targeted therapies as single agents has been less than desired. We are interested in enhancing antitumor effects in melanoma, by combining targeted therapies that inhibit growth and survival of melanoma cells. We previously showed melanoma proliferation was inhibited by low-dose rapamycin (1), a drug that inhibits mTOR in the PI3K pathway and is an FDA-approved agent for immunosuppression post-transplant. The Clinical Trials Evaluation Program (CTEP) of the NIH has initiated a program of clinical trials of combination therapies for selected malignancies.

Bevacizumab (Avastin®) is a humanized anti-VEGF monoclonal antibody approved for therapy of colorectal and lung cancers, based on a significant increase in survival, when administered in combination with cytotoxic chemotherapy (2). This agent was developed to block angiogenesis, a critical process for the survival of tumors as they increase in size (3, 4). Recognition of VEGF as an angiogenic factor was followed by the discovery that it is produced by both cancer cells and stromal cells, creating a microenvironment favorable for tumor growth (5–10). Production of VEGF seems to be an integral part of melanoma cancer progression because normal melanocytes do not produce it (11, 12), whereas tumor-derived melanoma cell lines express it (12–14). VEGF expression is upregulated in melanoma cells (15), and elevated serum VEGF levels directly correlate with stage of disease progression in melanoma patients (16, 17). The VEGF receptor 2 (VEGFR-2) is the major mediator of mitogenic, angiogenic, and permeability-enhancing effects of VEGF (3). VEGF receptors are not expressed on normal melanocytes (11, 15, 18), but VEGFR-2 expression is upregulated in some human melanoma cells during malignant transformation (15). These results suggest a role of VEGF in the development and progression of melanomas. Expression of VEGF and VEGFR-2 by some human melanoma cells raises the possibility that VEGF may be an autocrine growth factor for some human melanoma cells. Therefore, bevacizumab might have an effect on melanomas, independent of its antiangiogenic effects. Here we tested bevacizumab and rapamycin, singly and in combination, for their effects on proliferation of multiple tumor-derived human melanoma cell lines.

Methods

Cell Culture

Melanoma cell lines used in this study were cultured from tumor-involved lymph nodes surgically resected from patients at the University of Virginia (VMM5A, VMM14, VMM15, VMM17, VMM18, and VMM39) or from patients at Duke University (DM6, DM13, DM93, DM122, and DM331) as described previously (1, 19–21). The VMM1 melanoma cell line was derived from a metastatic tumor in the brain surgically resected from a patient at the University of Virginia (21). SKMel24 and HT144 were both obtained from the American Type Culture Collection (ATCC, Manassas, VA). All of the cell lines were cultured in RPMI 1640 medium supplemented with 5% fetal bovine serum, 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37 °C in 5% CO2, unless otherwise indicated.

Reagents and Inhibitors

100 mg (25 mg/ml) of bevacizumab (Avastin; Genentech List No.: 15734) was purchased from the University of Virginia Hospital Pharmacy and used at 50 micrograms/ml in cell number assays. Rapamycin (R-5000) was purchased from LC Laboratories (Woburn, MA) and a stock solution was made in DMSO and used at 1 nM in cell number assays.

Assay of Cell Number

Melanoma cells (1,000 cells per well) were plated in triplicate in 96-well plates with 5% fetal bovine serum and allowed to adhere overnight. After 12–16 hours, the cells were washed and treated with serum alone or with inhibitors as indicated. Cell numbers were assayed 48 hours later (or 7 days later for Figure 4 C) using Cell Titer-Glo (Promega Catalog# G7571; Madison, WI), according to the instructions provided by the manufacturer. This assay uses luciferase to measure ATP; because ATP levels are kept constant in living cells, the level is proportional to the number of viable cells (22). Relative light units (RLU) measured in that assay are used to determine the number of viable cells. The assay has been validated and used extensively in lieu of other assays such as reduction of chromogenic substrates (MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) or pulse-labeling of DNA synthesis (23, 24).

Figure 4. Knockdown of VEGFR-2 prevents cell death in response to rapamycin and bevacizumab.

Figure 4

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DM6 (A) and VMM18 (B) cell lines were transfected with siRNA to VEGFR-2 (lanes 2, A and B) as well as a non-specific control (lane 1 in each) and analyzed by Western blot (insets of graphs). Separate cultures of siRNA-transfected DM6 and VMM18 cells were maintained in media plus serum and treated without drug (white), bevacizumab (dotted), rapamycin (hatched), or a combination of bevacizumab and rapamycin (black) as indicated. C, Relative cell number after 7 days of growth is plotted for VMM18 and DM6 melanoma cell lines transfected with a non-specific control siRNA (white bar) or siRNA to VEGFR-2 (black bar) . Error bars represent standard deviation from triplicate values.

Statistical Analysis

The logarithm transformation of the relative light units (RLUs; raw data), instead of cell numbers calculated from RLUs, was the outcome for analysis, and linear regression was performed. The six cell lines were grouped into either high or low VEGFR-2 expression. Within either group, several comparisons were examined, including effects of individual drugs (in serum-containing media) versus the control serum, as well as the effects of both drugs given in combination versus the control serum, each drug alone, and the expected effects of the two drugs working in combination but acting independently. These comparisons were analyzed for significant differences between the VEGFR-2 high and low groups.

Western Blot Analysis

For analysis of proteins, cells from all 14 melanoma lines (VMM18, HT144, VMM5A, DM331, DM13, DM6, SKMel24, VMM15, VMM14, VMM1, VMM17, VMM39, DM93, and DM122) were plated in 100 mm Petri dishes and incubated for 24 hours in RPMI medium plus 5% FBS. After 24 hours, the medium was aspirated and the cells were washed twice with 10 mls PBS and harvested and lysed as described previously (1). Protein yields were determined by BCA analysis. Proteins were resuspended in SDS-containing sample buffer, heated for 10 min at 100 °C, and 20 ug/lane was resolved by SDS-PAGE using 10 % gels and transferred to Immobilon-P (Millipore). Membranes were blocked in 1% BSA in 50 mM Tris-Cl (pH 7.5), 0.9% NaCl, 0.05% Tween 20, and 0.01% antifoam A. Membranes were probed with antibodies listed below. Proteins were detected with Pierce SuperSignal West Pico Chemiluminescent substrate (#34080) as recommended by the manufacturer and used to expose to Kodak BioMax film.

Antibodies

GAPDH Antibody (Catalog # MAB374, used at 1:500) was purchased from Chemicon International. Anti-VEGFR-2 antibody (Catalog # 2479, used at 1:1000) was purchased from Cell Signaling. Anti-VEGF antibody (Catalog # ab1316–100, used at 1: 1000) was purchased from Abcam. This is an antibody to VEGF-A. Anti-phospho MAP Kinase (ERK1/2), clone 12D4 antibody (Catalog # 05–481, used at 1:500) was purchased from Upstate. Anti-Mouse IgG, peroxidase-linked species-specific whole antibody from sheep, secondary antibody (Catalog #NA931, used at 1:5000) and anti-rabbit IgG, peroxidase-linked species-specific whole secondary antibody from donkey (Catalog # NA934, used at 1:5000) were purchased from Amersham Biosciences.

Quantitative Real-time PCR

Total RNA samples were obtained using the RNEasy mini kit according to manufacturer’s instructions (Qiagen, Valencia, CA). Reverse transcription was done with MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA) and random hexamers as per the manufacturer’s instructions. The resulting cDNA was then subjected to qRT-PCR as described previously (25). The data collected from these quantitative PCRs defined a threshold cycle (Ct) of detection for the target (VEGFR-2) or the control genes (GAPDH and HPRT1) in each cDNA sample. To convert the Ct value into a relative abundance of target and control gene per sample, a standard curve was generated for the control gene using serial dilutions of cDNA sample: an arbitrary value of template was first assigned to the highest standard and then corresponding values were assigned to the subsequent dilutions, and these relative values were plotted against the Ct value determined for each dilution, resulting in the generation of the standard curve. The relative amount of target and control genes in each sample was then determined using the comparative Ct method (Applied Biosystems). The ratios of VEGFR-2 transcript are normalized by the geometric mean of the two control genes GAPDH and HPRT1 and are plotted on the graph.

ELISA

For the quantitative determination of VEGF concentrations from each of the 14 melanoma cell cultures, 2.5 × 106 melanoma cells were plated on 100 mm dishes in 5 mls, and the medium was collected 24 hrs later. ELISA was performed with the Quantikine Immunoassay to Human VEGF kit (R&D systems, Minneapolis, MN; Catalog No. DVE00, Lot 250272), according to the instructions provided by the manufacturer, with the exception that 100 microliters of medium was used. A standard curve was generated using the supplied reagents and pg/ml of VEGF was determined for each of the melanoma cell supernatants using the average value from triplicate samples and medium with serum was used as a blank.

siRNA experiments

siRNA oligonucleotides that target VEGFR-2 and a non-targeting siRNA pool were purchased from Upstate (catalog # 60–104) and resuspended according to the manufacturer's instructions. For transfection, 1 × 106 melanoma cells (DM6 and VMM18) were used in each condition (100 nM SMARTpool or non-specific negative control) with the Amaxa nucleofector kit (Cat. No. VCA-1005) according to manufacturer’s instructions, and plated into each well of a 12-well dish and incubated for 18–24 hours in culture medium before cell number assays and Western blot analysis.

Human Subjects

All of the research involving human subjects was approved by the University of Virginia’s IRB (Human Investigation Committee, HIC 5202 and HIC 10598) in accordance with assurances filed with and approved by the Department of Health and Human Services.

Results

VEGFR-2 in Melanoma Cell Lines

Levels of VEGFR-2 mRNA and protein were analyzed in fourteen melanoma cell lines (Figure 1). Quantitative real-time PCR for VEGFR-2 expression revealed mRNA in all 14 cell lines; however, the expression varied over a 1000-fold range, normalized with GAPDH and HPRT1 (Figure 1A, note a logarithmic scale). Extracts of the 14 melanoma cell lines were analyzed by immunoblotting for VEGFR-2 protein and quantitated by densitometry normalized to GAPDH, as a loading control (Figure 1B–C). The results revealed that 7 of the 8 cell lines with the highest mRNA levels also expressed VEGFR-2 protein. DM331 was the exception in this group, without detectable VEGFR-2 protein. None of the 6 melanoma cell lines with the lowest levels of mRNA exhibited any VEGFR-2 protein (Figure 1B). Among the cell lines that expressed VEGFR-2 protein, the levels of protein did not correspond to the relative levels of mRNA, suggesting that there are post-transcriptional mechanisms for regulation of VEGFR-2. From this set of 14 melanoma cell lines, we chose for further study the 3 lines with the highest levels of VEGFR-2 protein (VMM18, DM13, and DM6) and the 3 cell lines with the lowest levels of mRNA and no detectable VEGFR-2 protein (DM122, DM93, and VMM39).

Figure 1. Expression of VEGFR-2 mRNA and protein in human melanoma cell lines.

Figure 1

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A, qRT-PCR was performed as described in the materials and methods and the levels were normalized to the geometric mean of GAPDH and HPRT1 as controls. Results are plotted on a logarithmic scale to allow comparison of the 1000-fold range in mRNA levels. Error bars show standard deviation from triplicate samples. The melanoma cell lines are in rank order starting with the cell line that expressed the highest amount of VEGFR-2 mRNA. B–C, Western blot analysis of VEGFR-2 protein expression in various melanoma cell lines. Cell extracts were immunoblotted with VEGFR-2 antibody (top panels, C) and with anti-GAPDH as a loading control (bottom panels, C). Staining intensity was quantitated, normalized to the amount of GAPDH, and plotted (panel B) relative to DM13 (set as 100). The cell lines are arranged in the same order as in panel A.

VEGF in Melanoma Cell Lines

Western blotting showed that melanoma cell lines expressed VEGF, whether or not they expressed VEGFR-2 (Figure 2A). As expected, RT-PCR analysis showed VEGF mRNA was expressed in all six of these cell lines (not shown). We also analyzed, by ELISA, VEGF secreted from all 14 melanoma cell lines (Figure 2B). There was greater than a 3000-fold range in the amount of secreted VEGF in the 14 melanoma cell lines. The data for secreted VEGF are arranged in rank order based on the levels of the VEGFR-2 mRNA (Figure 1A). This shows there is no correspondence between the levels of secreted VEGF and the levels of VEGFR-2 mRNA or VEGFR-2 protein. It seems that melanoma cell lines independently produce VEGF and VEGFR-2.

Figure 2. Expression of VEGF by human melanoma cell lines.

Figure 2

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A, Western blot analysis of VEGFR-2+ and VEGFR-2neg melanoma cell lines with VEGF antibody (top panels) as described in materials and methods. GAPDH was immunoblotted as a loading control (bottom panels). B, ELISA for analysis of secreted VEGF collected from human melanoma cell lines after 24 hrs in culture is shown in pg/ml. DM331 was too high to measure. The melanoma cell lines are in the same rank order as Figure 1A.

Bevacizumab and Rapamycin Effects on Melanoma Cell Proliferation

We examined proliferation of human melanoma cell lines in vitro, and the effects of bevacizumab and rapamycin individually and in combination (Figure 3). Cells were grown with or without bevacizumab at concentrations estimated to be achieved in serum with therapeutic human dosing (26), and with or without rapamycin below levels estimated to be achieved in serum with therapeutic human dosing (27). Bevacizumab added alone significantly inhibited proliferation of the VEGFR-2+ cell lines VMM18, DM13, and DM6 (p < 0.001), but did not inhibit proliferation of cell lines without detectable VEGFR-2 protein DM122, DM93, and VMM39 (p = 0.18). We concluded that there is an autocrine loop where VEGF, which is produced by all of the melanoma cell lines, promotes proliferation only of those melanoma cell lines that express VEGFR-2.

Figure 3. Effects of rapamycin and bevacizumab on proliferation of human melanoma cell lines.

Figure 3

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Numbers of human melanoma cells (left axis) and the relative change in cell number (right axis) are shown in a cell proliferation assay using Cell Titer-Glo starting with 1000 cells/well. Cells were cultured in media plus serum, plus no treatment (white), bevacizumab (dotted), rapamycin (hatched), or a combination of bevacizumab and rapamycin (black) as indicated and described in materials and methods. The three cell lines on the left are labeled VEGFR-2 positive because of detection of the protein by Western blotting (Figure 1). The three cell lines to the right are VEGFR-2 negative with no detectable VEGFR-2 protein.

Rapamycin added alone inhibited proliferation of all six melanoma cell lines with a mean inhibition of 72%, compared to control (p < 0.001). Combination of bevacizumab plus rapamycin did not just inhibit cell proliferation, but caused a net loss of VEGFR-2+ cells, consistent with an induction of cell death. In contrast, bevacizumab plus rapamycin had no additional effect compared to rapamycin alone (77% vs. 73%) in the VEGFR-2-negative cell lines. Thus, bevacizumab does not have a significant additive effect on melanoma cells without VEGFR-2, whereas bevacizumab kills VEGFR-2+ melanoma cells in combination with rapamycin.

Knockdown of VEGFR-2 by siRNA prevents cell death from combination of bevacizumab plus rapamycin

Bevacizumab only had effects on VEGFR-2+ melanoma cells, and we wanted to test whether the observed effects were dependent on VEGFR-2. Using an siRNA SMARTpool for VEGFR-2 and a non-specific siRNA control, we knocked down VEGFR-2 in both the DM6 and VMM18 cell lines (Figure 4A and B). Immunoblotting showed that the VEGFR-2 siRNA resulted in the loss of detectable protein. Cell proliferation assays showed that bevacizumab alone did not inhibit the VEGFR-2 knocked down cell lines, compared to the same cell lines transfected with a control siRNA (Figure 4A and B). Combination treatment with bevacizumab plus rapamycin in the VEGFR-2 knocked down cell lines did not produce cell death and was no different than treatment with rapamycin alone. These results demonstrate that VEGFR-2 is necessary for the negative effects of bevacizumab on melanoma cell proliferation in vitro, and for net cell death when used in combination with rapamycin.

If VEGF was promoting melanoma cell growth with VEGFR-2, then knockdown of the receptor would be expected to inhibit cell proliferation. We did not detect an effect on proliferation at 48 hours; so to test this hypothesis, VMM18 and DM6 melanoma cell lines were transfected in parallel with control siRNA or VEGFR-2 siRNA and evaluated at 7 days. Knockdown of VEGFR-2 reduced cell proliferation in both VMM18 and DM6 by about 50% (Figure 4C).

Bevacizumab inhibited proliferation of VEGFR-2+ melanoma cells, however this did not involve significant reduction in ERK activation. Immunoblotting for phospho-ERK was not reduced in VMM18 cells treated with bevacizumab compared to untreated controls (data not shown). Therefore, ERK activation did not depend on VEGFR-2 and the VEGFR-2 receptor must have other intracellular effectors that contribute to proliferation.

Discussion

Our results demonstrate that VEGFR-2 renders melanomas especially susceptible to combination therapy of bevacizumab plus rapamycin. The combination therapy is lethal to cells with VEGFR-2, and knockdown of VEGFR-2 by siRNA renders them insensitive. Thus, melanoma-derived VEGF can cause not only angiogenesis, but also autocrine activation of tumor cells that express VEGFR-2. This discovery may be useful to guide therapy for individual patients.

The melanoma cell lines examined fell into two groups, VEGFR-2+ and VEGFR-2neg. All 14 melanoma cell lines expressed VEGFR-2 transcript. However, only about half had detectable levels of the VEGFR-2 protein, and these had high levels of mRNA. However, some cell lines with high levels of mRNA had no detectable VEGFR-2 protein; therefore we suspect that there may be other modes of regulation, such as microRNAs, that limit accumulation of the protein, even when the mRNA is expressed. Regardless of the VEGFR-2 protein expression levels, we observed that every melanoma cell line tested produced VEGF in secreted and cell-bound forms. This is consistent with reports of VEGF in melanoma patient biopsies, based on immunohistochemistry (12, 14, 28–31). It also is consistent with prior reports with epithelial cancers of a growth protective effect of VEGF induced by radiation exposure (32–34).

VEGF has three receptors: VEGFR-1 is thought to be a less active “decoy receptor” (35); VEGFR-2 is a protein tyrosine kinase that activates a variety of cell signaling pathways to promote cell proliferation (36); and VEGFR-3 is closely related to VEGFR-2, but its expression is primarily in lymphatic endothelial cells (35, 37). VEGFR-2 is believed to be the dominant effector of VEGF function on cells other than lymphatic endothelial cells (38). VEGFR-2 has been found in 50–80% of melanomas by immunohistochemistry, with incidence increasing with advanced disease stage (11, 12, 16, 17, 31, 39). Using a receptor tyrosine kinase antibody array, we detected selective activation of VEGFR-2 in VMM18 (a VEGFR-2+ cell line), but not in DM122 (a VEGFR-2neg cell line; data not shown). Our hypothesis was that melanoma cells expressing VEGFR-2 depended on VEGF for proliferation. We found that bevacizumab inhibited the proliferation of 3 out of 3 VEGFR-2+ melanoma cell lines tested. Knock down of VEGFR-2 by siRNA prevented this inhibition. This demonstrated that the response to VEGF was due to VEGFR-2. Furthermore, knockdown of VEGFR-2 reduced the proliferation of VEGFR-2+ melanoma cell lines. Overall, these findings demonstrate that melanoma-derived VEGF interacts with cell-surface VEGFR-2 on melanoma cells providing an autocrine growth signal (15, 39–41).

Melanoma cell proliferation is thought to be mediated in part through activation of the mTOR pathway. mTOR is an enzyme that regulates translation and transcription during cell growth; thus, it is a logical therapeutic target, and its inhibitors, rapamycin (sirolimus) and CCI-779 (temsirolimus) may have therapeutic potential in cancer. However, in clinical trials to date, clinical activity of CCI-779 was low when it was used as a single agent in melanoma patients (42). Clinical efficacy of mTOR inhibition likely will require combination therapy that inhibits other growth pathways as well.

VEGFR-2 is believed to signal cell proliferation and survival through MAP kinase and AKT/mTOR pathways, but our prior results recently confirmed in other cancer cell lines, showed crosstalk between these pathways (1, 43). We did not find evidence of decreased activation of ERK with inhibition of VEGF, which suggests that the effect of VEGFR-2 on melanoma cell proliferation is mediated through other pathways. It is also possible that VEGFR-2 signaling may differ in melanomas with or without BRAF or RAS mutations. Additional studies are planned for elucidating the downstream effects of blocking the VEGF/VEGFR-2 autocrine loop. Regardless, the combination of bevacizumab plus rapamycin causes not only synergistic inhibition of proliferation, but also death of human melanoma cells expressing VEGFR-2. These data provide rationale for combination therapy with inhibitors of VEGF and mTOR in patients with VEGFR-2+ melanomas.

Acknowledgements

We thank the members of the Slingluff laboratory, Mark Smolkin, and Gina Petroni for helpful discussions. We thank Dr. Yongde Bao and the University of Virginia Biomolecular Core Facility for their assistance with the real-time PCR experiments. This work was partly supported by grant CA77584 to Dr. David L. Brautigan from USPHS NCI and partly supported by funds from the Farrow Fellowship and the UVA Cancer Center Support Grant, P30 CA44570 to Dr. Kerrington R. Molhoek and the Harrison Foundation to the University of Virginia Cancer Center and Dr. Craig L. Slingluff, Jr. Support was also provided by the NIH/NCI grant R01 CA57653 (to CLS) and the University of Virginia Cancer Center Support Grant (NIH/NCI P30 CA44579). The authors declare that there are no conflicts of interest or financial interests.

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