Vascular endothelial growth factor A (VEGF-A) and its receptors (VEGFR1 and R2) play important roles in the progression of malignant melanoma through tumor angiogenesis. However, it is not clear whether the VEGF-A/VEGFR1 signaling pathway is involved in the proliferation and migration of melanoma cells. Thus, the effect of VEGF-A on cell migration was investigated in human melanoma cell lines. Of several splicing variants of VEGF-A, VEGF165 is the most abundant and responsible for VEGF-A biological potency. VEGF165 facilitated the migration of melanoma cells in both a chemotactic and chemokinetic manner, but cell proliferation was not affected by VEGF165. VEGF165 also induced the phosphorylation of Akt. In addition, VEGF165-induced cell migration was inhibited significantly by VEGFR1/2 or a VEGFR1-neutralizing antibody. Furthermore, the downregulation of VEGFR1 via the transfection of VEGFR1-targeting antisense oligonucleotides suppressed VEGF165-induced cell migration. Moreover, wortmannin, an inhibitor of phosphatidylinositol-3 kinase (PI3K) in the PI3K/Akt pathway, suppressed VEGF165-induced Akt phosphorylation and VEGF165-induced cell migration. These findings suggest that the motility of melanoma cells is regulated by signals mediated through the PI3K/Akt kinase pathway with the activation of VEGFR1 tyrosine kinase by VEGF165. Thus, the downregulation of signaling via VEGF-A/VEGFR1 might be an effective therapeutic approach that could prevent the progression of malignant melanoma.
One characteristic property of malignant tumors is their ability to invade the surrounding tissues and form metastatic foci in distant organs. Metastasis involves a series of steps, including the detachment of cancer cells from the primary lesion, migration into connective tissues, intravasation into the circulation, and implantation into distant organs (Bravo-Cordero et al. 2012; Clark and Vignjevic 2015).
Tumor cells are known to produce growth factors and cytokines, such as vascular endothelial growth factor (VEGF), transforming growth factors, and basic fibroblast growth factors, which have various biological activities in tumor cells and stroma cells, including endothelial cells and fibroblasts (Hayashido et al. 1998; Guo et al. 2021; Motwani and Eccles 2021). VEGF is a potent angiogenic factor that binds to two tyrosine kinase-type receptors, VEGF receptor-1 (VEGFR1)/fms-like tyrosine kinase (Flt-1) and VEGFR2/kinase insert domain receptor (KDR)/fetal liver kinase 1, which are specifically and highly expressed in vascular endothelial cells. The interaction of VEGF and VEGFRs has a stimulatory effect on the proliferation and migration of vascular endothelial cells (Vaisman et al. 1990; Myoken et al. 1991). Importantly, VEGF is known to be upregulated in several tumors and to contribute to tumor angiogenesis.
The VEGF family consists of VEGF-A–E and placental growth factor (PlGF). VEGF-A plays a central role in tumor angiogenesis in relation to blood vessel sprouting, repair, and regeneration (Dvorak 2021). VEGF-A consists of several splice variants with different numbers of amino acids, such as VEGF121, VEGF145, VEGF165, and VEGF189. VEGF165 is the most abundant and responsible for VEGF-A biological potency (Dvorak 2021). Although VEGF-A binds to both VEGFR1 and VEGFR2, VEGF-B and PlGF bind only to VEGFR1. The affinity of VEGF-A to VEGFR1 is about tenfold higher than its affinity to VEGFR2, whereas the tyrosine kinase activity of VEGFR1 is about tenfold lower than that of VEGFR2 (Shibuya 2006, 2011; Apte et al. 2019). VEGFR1 contributes to pathological angiogenesis in tumors, rheumatoid arthritis, and cerebral ischemia, and VEGFR2 is the regulator of both physiological and pathological angiogenesis (Dvorak 2021). Although PlGF is not involved in physiological angiogenesis, it participates in pathological angiogenesis in cancer tissues via VEGFR1 (Dewerchin and Carmeliet 2012).
In general, tumor cells have the ability to produce VEGFs, whereas their expression of VEGFRs is strongly suppressed. Previous studies have shown that VEGFRs are expressed in many types of cancers, including melanoma, pancreatic, lung, and ovarian cancers, suggesting that VEGFs might regulate tumor progression through not only paracrine mechanisms but also autocrine mechanisms (Gitay-Goren et al. 1993; Frank et al. 2011; Shibuya 2011; Borsotti et al. 2015).
Melanoma is a malignant tumor derived from melanocytes in the skin and mucous membrane (Iversen and Robins 1980; Yde et al. 2018; Ahmed et al. 2020). Melanoma frequently metastasizes due to its ability to migrate effectively and form a vascular network in tumor tissues (Streit and Detmar 2003; Pasquali et al. 2018). Moreover, melanoma is known to express high levels of PlGF and VEGF-A. In vivo studies have shown that when melanoma cells are inoculated into transgenic mice that overexpress PlGF, tumor growth is increased significantly and metastatic potential is relatively higher than that in control mice inoculated with melanoma cells (Lacal et al. 2000; Graziani et al. 2016; Lacal and Graziani 2018). Furthermore, VEGFR1-expressing melanoma cells have been shown to be more invasive compared with melanoma cells that do not express VEGFR1, and the blockade of VEGFR1 using a specific monoclonal antibody reduces VEGF‐A- and PlGF-inducible extracellular matrix invasion (Hennequin et al. 1999). These results suggest that a signal mediated via VEGFR1 might regulate the invasion of melanoma cells. However, the mechanism underlying the tumor-produced VEGF-regulated invasion and metastasis of melanoma remains unclear. Thus, in the present study, we examined the expression of VEGF-A and VEGFR1 in human melanoma cells and investigated the effects of VEGF165/VEGFR on the migration and proliferation of human melanoma cells as well as the VEGF165/VEGFR-related signaling pathway.
Materials and methods
Chemicals and antibodies
Insulin, transferrin, 2-aminoethanol, sodium selenite, 2-mercaptoethanol, oleic acid conjugated with fatty acid–free bovine serum albumin (BSA), and PlGF were purchased from Sigma-Aldrich (St. Louis, MO). Recombinant Human VEGF165 and Human VEGF Quantikine ELISA Kits were obtained from R&D Systems Inc. (Minneapolis, MN). Type I collagen solution (Native Collagen Acidic Solution, IAC-50) was purchased from Koken (Tokyo, Japan). The VEGFR1/2 tyrosine kinase activity inhibitor [CB676475, (4-[(4′-chloro-2′-fluoro) phenylamino]-6,7-dimethoxyquinazoline)] was purchased from Calbiochem (San Diego, CA), and the VEGFR2 kinase inhibitor II [(Z)-5-bromo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl) methylene]-1,3-dihydroindol-2-one] was purchased from Merck Biosciences (Nottingham, UK). Wortmannin, a kinase inhibitor of phosphatidylinositol-3 kinase (PI3K), was obtained from Sigma-Aldrich.
Rabbit polyclonal anti-phospho-VEGFR1 antibody (Y1059; CSB-PA000747) and rabbit polyclonal anti-phospho-VEGFR2 antibody (Y1048; CSB-PA009634) were purchased from Cusabio Technology (Houston, TX). Rabbit polyclonal anti-VEGFR1 antibody (A1277) and rabbit polyclonal anti-VEGFR2 antibody (A5609) were purchased from ABclonal (Boston, MA). Rabbit monoclonal anti-phosphorylated Akt antibody (Ser473; #4060), rabbit monoclonal anti-phosphorylated extracellular signal-regulated kinase-1/2 (Erk1/2) (Thr202/Tyr204; #4370), rabbit monoclonal anti-Akt antibody (#4685), anti-Erk1/2 antibody (#4695), rabbit monoclonal anti-β-Actin (#4970), and horseradish peroxidase (HRP)–conjugated anti-rabbit IgG antibody (#7074) were purchased from Cell Signaling Technology (Danvers, MA). Anti-VEGFR1 blocking monoclonal antibody (KM1750) was kindly provided by Dr. Shibuya (Jobu University, Isesaki, Japan) and Dr. Shitara (Kyowa Hakko Kirin Co., Ltd, Tokyo, Japan).
Cells and culture
The human melanoma cell lines SK-MEL-28 (RRID:CVCL_0526) (Shiku et al. 1976), HMV-II (RRID:CVCL_1282) (Kasuga et al. 1976), G361 (RRID:CVCL_1220) (Peebles et al. 1978), and C32TG (RRID:CVCL_2324) (Jia et al. 1997) were provided by RIKEN BRC (Tsukuba, Japan) and used in this study. Malignant melanoma (MM) cells established in our laboratory from a patient with malignant melanoma of the gingiva were also used (Okamoto et al. 1996). These cell lines are free from mycoplasma contamination using e-Myco™ plus Mycoplasma PCR Detection Kit (iNtRON, Seongnam-Si, South Korea) and have been authenticated using short tandem repeat (STR) profiling (BEX Co., Ltd., Tokyo, Japan) within the last 3 mo (Supplementary Figure S1 and Table S1). The STR profiles of these cell lines, except for MM cell line, matched with the publicly available reference profiles (ICLAC Databases. 2021). As MM cell line has not yet deposited to the cell bank, the STR profile of MM cell line did not match with any other STR data in the databases.
All cells were grown in DF medium (1:1 mixture (by volume) of Dulbecco’s modified Eagle medium (DMEM) and Ham F-12 medium) supplemented with 5% fetal bovine serum in a humidified 95% air/5% CO2 atmosphere at 37 °C in a CO2 incubator (Thermo Fisher Scientific, Waltham, MA). Cell proliferation was estimated as follows. The wells of 24-well tissue culture plates were coated with 100 µg/mL of type I collagen, and cells (5 × 103) suspended in DF 6F serum–free medium supplemented with 10 µg/mL of insulin, 5 µg/mL of transferrin, 10 µM of 2-aminoethanol, 10 nM of sodium selenite, 10 µM of 2-mercaptoethanol, and 9.4 µg/mL of oleic acid conjugated with fatty acid–free BSA were seeded in each well of the culture plates (Sato et al. 1987). After 24 h, various concentrations of VEGF165 were added, and the cells were cultured in 5% CO2 for 5 d at 37 °C. Subsequently, the number of cells was counted using a Coulter counter (Beckman Coulter, Tokyo, Japan), and the measurements were collected in triplicate. All reagents used in the cell culture were free from mycoplasma and viral pathogens.
RNA extraction and RT-PCR for VEGF-A and VEGFR mRNAs
Total RNA was isolated from the cells using TRIzol reagent (Thermo Fisher Scientific) following the manufacturer’s protocol, and RNA quality was determined according to the following criteria: RNA concentration > 0.5 µg/µL; OD 260/280 = 1.8–2.0. Reverse transcription was performed using the Super Script First-strand Synthesis System (Life Technologies, Carlsbad, CA). PCR was performed for VEGF-A and VEGFRs with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as the internal control. Following an initial incubation at 94 °C, each PCR cycle consisted of incubation for 30 s at 94 °C, 30 s at 55 °C, and 1 min at 72 °C. After the final cycle, the samples were incubated for a further 7 min at 72 °C and then kept at 4 °C before analysis via agarose gel electrophoresis. The following primers were used: VEGF-A forward primer, 5′-CTTGCCTTGCTGCTCTACC-3′; VEGF-A reverse primer, 5′-CACACAGGATGGCTTGAAG-3′; VEGFR1 forward primer, 5′-CATGAGGATGAGAGCTCCTGAG-3′; VEGFR1 reverse primer, 5′-AGGCCAACAGAGTGCTGCTGTC-3′; VEGFR2 forward primer, 5′-CCTGTCCACTTACCTGAGGAG-3′; VEGFR2 reverse primer, 5′-CTGGCTACTGGTGATGCTGTC-3′; GAPDH forward primer, 5′-GCTCTCTGCTCCTCCTGTTC-3′; and GAPDH reverse primer, 5′-ACGACCAAATCCGTTGACTC-3′.
Cells were lysed using cell lysis buffer (50-mM Tris HCl (pH 7.4), 150-mM NaCl, 1-mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and 0.5% sodium deoxycholate) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich). The lysates were centrifuged at 15,000 × g and 4 °C for 15 min, and the supernatants were collected. Samples containing 20 μg of total protein were electrophoresed on 10% SDS–polyacrylamide gel under reducing conditions and transferred to polyvinylidene difluoride (PVDF) membrane filters (Bio-Rad Laboratories, Hercules, CA). The filters were blocked using TBS-T (20-mM Tris HCl (pH 7.5), 137-mM NaCl, and 0.1% Tween 20) containing 5% skim milk for 1 h at room temperature, after which they were incubated with primary antibodies and then with HRP-conjugated secondary antibody. Rabbit monoclonal anti-β-actin was used as a loading control antibody. Protein bands were visualized using enhanced chemiluminescence detection (Clarity ECL Substrate; Bio-Rad Laboratories).
ELISA for soluble VEGF165
To obtain conditioned media, 80% confluent melanoma cells in 6-well plates were washed twice with DF and incubated with 2 mL of DF for 24 h. The conditioned media were then centrifuged at 10,000 × g and 4 °C for 30 min to remove cells and debris. The amount of soluble VEGF165 in the conditioned media was measured using a Human VEGF Quantikine ELISA Kit according to the manufacturer’s instructions. The levels of VEGF165 detected were corrected according to the number of cells.
Cell motility assay
Cell motility was analyzed using a modified Boyden chamber assay with Transwell inserts (6.5 mm in diameter) containing 8-μm pores (Corning Costar, Cambridge, MA) as described previously (Chen 2005; Hayashido et al. 2007). The filters were coated with 100 μg/mL of type-1 collagen to enhance cell attachment. Melanoma cells (1 × 105) resuspended in DF medium containing 0.1% BSA were added to the upper compartment of each Transwell insert, and VEGF165 or PlGF (Sigma-Aldrich) was added to the upper or lower compartment. After incubation for 24 h at 37 °C, the Transwell inserts were fixed with methanol and stained with Diff-Quik (Dade Behring AG, Dudingen, Switzerland). The cells on the upper surface of the filter were wiped with a cotton swab, and the number of cells on the lower surface of the filter was counted under a low-power field (× 50) using light microscopy. Five fields were counted in each of the three different experiments, and the results were expressed as the mean number of migrating cells/mm2 ± the standard deviation (SD). To assess the chemotactic or chemokinetic response of VEGF165, checkerboard analysis was performed by adding various concentrations of VEGF165 to both the lower and upper Transwell chambers.
Melanoma cells were cultured on 6-well plates until near confluence and starved with serum-free DF overnight. The cells were then incubated with 5 ng/mL of recombinant human VEGF165 for the indicated periods, washed with ice-cold phosphate-buffered saline containing 1 mM of sodium vanadate, and lysed with cell lysis buffer supplemented with protease inhibitor cocktail and 1 mM of sodium vanadate. The samples were separated on 10% SDS–polyacrylamide gels under reducing conditions and transferred onto PVDF membrane filters. The phosphorylation of VEGFR1 and VEGFR2 was examined using immunoblotting with rabbit polyclonal anti-phospho-VEGFR1 antibody and rabbit polyclonal anti-phospho-VEGFR2 antibody, respectively. The phosphorylation of ERK1/2 and Akt was assessed using rabbit anti-phospho-ERK1/2 monoclonal antibody and rabbit anti-phospho-Akt monoclonal antibody, respectively. Total VEGFR1, VEGFR2, Akt, and MEK1/2 were detected using rabbit anti-VEGFR1 antibody, rabbit polyclonal anti-VEGFR2 antibody, rabbit anti-ERK1/2 monoclonal antibody, and rabbit anti-Akt monoclonal antibody, respectively. After incubation with the primary antibodies, the membranes were incubated with HRP-conjugated secondary antibody, and protein bands were detected using an enhanced chemiluminescence reagent.
Antisense oligonucleotides (ASOs) and transfections
To downregulate VEGFR1 or VEGFR2, morpholino antisense ASOs specific for VEGFR1 or VEGFR2 (GeneTools, Philomath, OR) were used. The sequences of the ASOs were as follows: VEGFR1, 5′-AAGCCAGGGCCGAGCCGCACATAAT-3′; VEGFR2, 5′-GCAGCACCTTGCTCTGCATCCTGCA-3′. A standard control morpholino oligonucleotide (5′-CCTCTTACCTCAGTTACAATTTATA-3′) was used as a negative control. Delivery of the oligonucleotides into the cells was performed according to the GeneTools protocol. Briefly, 80–100% confluent SK-MEL-28 cells were treated with 10 μM of the morpholino ASOs or the standard control oligonucleotide and 6 μM of Endo-Porter reagent (GeneTools). After 24 h, the cells were used in the subsequent experiments.
Statistical analysis was performed using BellCurve for Excel (Social Survey Research Information Co., Ltd., Tokyo, Japan). All data are presented as the means ± SD of at least three independent experiments. Student’s t-test was used to compare the differences between groups, which were considered significant at p < 0.05.
Expression of VEGF-A, VEGFR1, and VEGFR2 in melanoma cells
The mRNA expression of VEGF-A, VEFGR1, and VEGFR2 in melanoma cells was examined using RT-PCR. The PCR products of VEGF121, VEGF165, and VEGF189, which are splicing variants of VEGF-A, were detected in all cell lines (Fig. 1A), as was the expression of VEGFR1/Flt-1 and VEGFR2/KDR mRNAs (Fig. 1B). VEFGR1 and VEGFR2 protein expression was examined using immunoblotting. VEGFR1 protein was expressed in all melanoma cells, whereas VEGFR2 protein was not detected via immunoblotting. To investigate VEGF165 secretion by melanoma cells, the amount of VEGF165 in the conditioned media was assayed using an ELISA. The concentrations of VEGF165 in the conditioned media were as follows: 77.4 ± 10.2 pg/mL/105 cells in SK-MEL-28 cells; 45.0 ± 17.6 pg/mL/105 cells in HMV-II cells; 176.0 ± 8.4 pg/mL/105 cells in MM cells; 3.4 ± 1.0 pg/mL/105 cells in G361 cells; and 58.0 ± 10.4 pg/mL/105 cells in C32TG cells (Fig. 1C).
Effect of VEGF165 on the proliferation of melanoma cells
VEGF165 at concentrations of 0.1–100 ng/mL did not affect the proliferation of SK-MEL-28 cells (Fig. 2A) or the proliferation of the other melanoma cells (data not shown). By contrast, purified VEGF165 half-maximally stimulated human umbilical vein endothelial cell (HUVEC) proliferation at 41 pM (1.8 ng/mL) and maximally stimulated HUVEC growth at 200 pM (8.8 ng/mL) (Myoken et al. 1991).
Effect of VEGF165 on the migration of melanoma cells
The effect of VEGF165 on the migration of melanoma cells was analyzed using a modified Boyden chamber method. Checkerboard analysis indicated that VEGF165 induced chemotactic and chemokinetic migration in SK-MEL-28 cells, and 5 ng/mL of VEGF165 added to both the upper and lower chambers led to the highest enhancement of migration (Fig. 2B). In all melanoma cells, 5 ng/mL of VEGF165 added to the upper and lower chambers enhanced migration significantly (p < 0.05) compared with that in the controls (Fig. 2C).
Effect of PIGF on the proliferation and migration of melanoma cells
To clarify whether the VEGF165-induced migration of melanoma cells is regulated via VEGFR1 or VEGFR2, the effect of PlGF on the migration of melanoma cells was investigated. PlGF increased cell migration significantly (p < 0.05) in a dose-dependent manner (Fig. 2D) but did not affect cell proliferation (data not shown). These results suggested that VEGF165-induced migration of melanoma cells was mediated only by VEGFR1.
Participation of VEGFRs in the migration of melanoma cells
SK-MEL-28 cells treated with CB676475 VEGFR1/2 tyrosine kinase inhibitor (TKI), VEGFR2 kinase inhibitor II, or anti-VEGFR1 blocking monoclonal antibody for 1 h were then suspended in DMEM containing 0.1% BSA and added to an upper Transwell compartment. After incubation in the presence of 5 ng/mL of VEGF165 for 24 h, the number of cells that migrated to the lower surface of the filter was counted. Treatment with VEGFR1/2 TKI led to the suppression of VEGF165-induced cell motility in a dose-dependent manner (Fig. 3A). In contrast, VEGFR2 TKI did not alter VEGF165-induced cell motility (Fig. 3B). The treatment of SK-MEL-28 cells with KM1750, a neutralizing antibody for VEGFR1, also suppressed VEGF165-induced cell migration in a dose-dependent manner (Fig. 3C).
The suppression of VEGFR1 by a morpholino ASO targeting VEGFR1 led to a marked decrease in the migration of SK-MEL-28 cells, and the migration of SK-MEL-28 cells transfected with a VEGFR1-targeting morpholino ASO was not stimulated by VEGF165 (Fig. 3D). Conversely, the migration of SK-MEL-28 cells transfected with a VEGFR2-targeting morpholino ASO was enhanced by VEGF165. These experiments with inhibitors of VEGF receptor activities confirm the conclusion from the experiments with PIGF-treated melanomas that VEGF165 stimulated melanoma migration through VEGFR1. To assess VEGFR signaling in melanoma cells, the phosphorylation of VEGFRs was analyzed using western blot analysis following the addition of VEGF165. Treatment with VEGF165 led to phosphorylation of VEGFR-1 (Fig. 3E).
Participation of VEGF165 in the ERK signaling pathway
To examine the effect of VEGF165 on Erk phosphorylation in melanoma cells, the expression of phosphorylated Erk in SK-MEL-28 cells cultivated with VEGF165 was analyzed using immunoblotting. Phosphorylated Erk was expressed constitutively in SK-MEL-28 cells, and VEGF165 did not alter the expression of phosphorylated Erk (Fig. 4A).
Participation of the PI3K/AKT signaling pathway in VEGF165-induced cell migration
The participation of PI3K in Akt phosphorylation was investigated using wortmannin, a PI3K inhibitor, which suppressed VEGF165-induced Akt phosphorylation in SK-MEL-28 cells in a dose-dependent manner (Fig. 4B). Similarly, wortmannin suppressed VEGF165-induced Akt phosphorylation in the other melanoma cells (Fig. 4C). Wortmannin also suppressed VEGF165-induced SK-MEL-28 cell migration in a dose-dependent manner (Fig. 4D), and it suppressed VEGF165-induced cell migration significantly (p < 0.05) in the other melanoma cells (Fig. 4E).
Activation of the PI3K/AKT signaling pathway by VEGFR1
The effect of VEGF165 on the phosphorylation of Akt in SK-MEL-28 cells was analyzed using immunoblotting. Phosphorylated Akt was observed 1 h after the VEGF165 treatment was applied (Fig. 5A). Furthermore, the VEGF165 treatment led to phosphorylation of Akt in other melanoma cells (Fig. 5B). To determine whether VEGF165-induced Akt phosphorylation is regulated via VEGFR1 or VEGFR2, the effects of VEGFR1- or VEGFR2-targeting TKIs on VEGF165-induced Akt phosphorylation were investigated. VEGFR1/2 TKI suppressed the VEGF165-induced phosphorylation of Akt (Fig. 6A), whereas VEGFR2 TKI did not alter the expression of phosphorylated Akt (Fig. 6B). These results suggest that the PI3-kinase pathway in melanomas is activated by VEGFR1 but not VEGFR2.
The expression of VEGF-A in malignant tumors is closely related to tumor progression and prognosis (Aoyagi et al. 2010; Martins et al. 2013). The biological functions of VEGF-A are exerted through its binding to two tyrosine kinase receptors, VEGFR1 and VEGFR2, expressed in vascular endothelial cells. VEGF-A plays an important role in tumor angiogenesis by enhancing the proliferation and motility of endothelial cells (Motwani and Eccles 2021). Several studies have shown that various cancer cells produce VEGF-A and express its receptors, VEGFR1 and/or VEGFR2 (von Marschall et al. 2000; Carrillo de Santa Pau et al. 2009; Hlobilkova et al. 2009; Sopo et al. 2019). Thus, VEGF-A could promote tumor development and progression by regulating the proliferation and motility of tumor cells in an autocrine manner as well as angiogenesis induction in a paracrine manner.
In the present study, we first examined the expression of VEGF-A, VEGFR1, and VEGFR2 in melanoma cells as well as their participation in the motility and proliferation of these cells. All melanoma cells tested secreted VEGF165 into the culture media and expressed VEGFR1 and VEGFR2, suggesting that the VEGF165 produced by melanoma cells might regulate the proliferation and motility of these cells in an autocrine manner. In addition, radio-receptor assay using [125 I]-labeled VEGF165 confirmed that SK-MEL-28 cell line expressed high-affinity binding sites with a dissociation constant of 130 pM with 1300 binding sites per cell while the low-affinity sites with a dissociation constant of 4.1 nM with 20,000 binding sites per cell (data not shown). VEGF165 also facilitated the motility of melanoma cells in both a chemotactic and chemokinetic manner, although it did not alter the proliferation of melanoma cells.
PlGF, which is about 40% homologous to VEGF-A at the amino acid level, binds specifically to VEGFR1 and induces various signaling pathways (Tammela et al. 2005; Shibuya 2006). In the current study, both PlGF and VEGF165 enhanced the migration of melanoma cells, suggesting that the VEGF165-induced migration of these cells is regulated via VEGFR1.
VEGFR1 and VEGFR2 belong to the receptor tyrosine kinase (RTK) subfamily and are known to induce the activation of several intracellular signaling molecules, including PI3K, Akt, Erk1/2, and p38 mitogen-activated protein kinase (MAPK), when they bind VEGF-A (Zhang et al. 2010; Szabo et al. 2016; Roskoski 2017). To determine whether the motility of melanoma cells is regulated by VEGFR1 or VEGFR2, the effects of inhibiting the tyrosine kinase activity of VEGFR1 or VEGFR2 on VEGF165-induced cell motility were investigated. TKIs of both VEGFR1 and VEGFR2 suppressed the migration of melanoma cells induced by VEGF165, although VEGFR2 TKI did not affect VEGF165-induced cell migration. The neutralizing antibody against VEGFR1 also suppressed VEGF165-induced cell migration. Furthermore, the transfection of an ASO targeting VEGFR1 markedly reduced the migration of melanoma cells, and the addition of VEGF165 did not increase the migration of melanoma cells transfected with this VEGFR1-targeting ASO. However, the migration of melanoma cells transfected with a VEGFR2-targeting ASO was not suppressed. Additionally, VEGF165 enhanced the migration of melanoma cells transfected with this VEGFR2-targeting ASO. Collectively, these findings suggest that the VEGF165-induced migration of melanoma cells is mediated though signaling involving VEGFR1.
The MAPK pathway is a canonical signaling pathway triggered by several RTKs (McKay and Morrison 2007; Tarcic and Yarden 2010) including VEGF receptors (Yu and Sato 1999). Therefore, we investigated whether VEGF165 induces the activation of the MAPK cascade in melanoma cells. We found that Erk is constitutively phosphorylated in SK-MEL-28 cells and VEGF165 did not affect the phosphorylation of Erk, indicating that VEGF165 is not involved in the MAPK pathway of melanoma cells. In addition to the MAPK cascade, the PI3K/Akt pathway is activated through RTKs (Matsuoka and Yashiro 2014; Mayer and Arteaga 2016; Nozhat and Hedayati 2016) including VEGF receptors (Yu and Sato 1999). In the melanoma cells tested in the present study, Akt was phosphorylated by VEGF165. To clarify whether VEGF165-induced Akt phosphorylation is regulated via VEGFR1 or VEGFR2, we investigated the effects of VEGFR1 and VEGFR2 TKIs on VEGF165-induced Akt phosphorylation. VEGFR1/2 TKIs suppressed the induction of Akt phosphorylation by VEGF165 in SK-MEL-28 cells, but the VEGFR2 TKI did not affect VEGF165-induced Akt phosphorylation. These findings show that VEGF165 induces phosphorylation of Akt via VEGFR1 in melanoma cells. We also examined the participation of PI3K in the VEGF165-induced Akt phosphorylation of melanoma cells, finding that the PI3K inhibitor wortmannin suppressed VEGF165-induced Akt phosphorylation in melanoma cells. Wortmannin also suppressed the VEGF165-induced migration of melanoma cells. These findings indicate that VEGF165 promotes the migration of melanoma cells through the activation of PI3K/Akt signaling via VEGFR1. Using recombinant human VEGFR1 shows that PI3-kinase binds directly to phosphorylated tyrosine residue 1213, which resulted from an autophosphorylation event (Yu et al. 2001).
In conclusion, the melanoma cells examined in this study produced VEGF165 and expressed RNAs encoding its receptors VEGFR1 and VEGFR2. However, these melanoma cell lines expressed VEGFR1 protein but not VEGFR2 protein. We found that VEGF165 enhanced cell motility via VEGFR1 but not VEGFR2. Thus, the motility of melanoma cells may be regulated by a VEGF165/VEGFR1-mediated autocrine signaling pathway. Moreover, we found that VEGF165-induced melanoma cell motility is mediated by the PI3K/Akt pathway via VEGFR1. A survey of 167 melanoma specimens found that less than 10% of the tumors expressed VEGFR2, and they suggested that anti-VEGF proliferation therapy would not be an effective strategy for melanomas (Molhoek et al. 2011). Our results suggest that VEGF-A/VEGFR1 signaling could serve as a therapeutic target to prevent the invasion and metastasis of melanoma with inhibition of the associated signaling pathway being a therapeutic strategy to treat melanoma.
The data presented in this study are available on request from the corresponding author. Publicly available datasets were analyzed in this study.
Ahmed B, Qadir MI, Ghafoor S (2020) Malignant melanoma: skin cancer-diagnosis, prevention, and treatment. Crit Rev Eukaryot Gene Expr 30:291–297
Aoyagi Y, Iinuma H, Horiuchi A, Shimada R, Watanabe T (2010) Association of plasma VEGF-A, soluble VEGFR-1 and VEGFR-2 levels and clinical response and survival in advanced colorectal cancer patients receiving bevacizumab with modified FOLFOX6. Oncol Lett 1:253–259
Apte RS, Chen DS, Ferrara N (2019) VEGF in signaling and disease: beyond discovery and development. Cell 176:1248–1264
Borsotti P, Ghilardi C, Ostano P, Silini A, Dossi R, Pinessi D, Foglieni C, Scatolini M, Lacal PM, Ferrari R, Moscatelli D, Sangalli F, D’Atri S, Giavazzi R, Bani MR, Chiorino G, Taraboletti G (2015) Thrombospondin-1 is part of a Slug-independent motility and metastatic program in cutaneous melanoma, in association with VEGFR-1 and FGF-2. Pigment Cell Melanoma Res 28:73–81
Bravo-Cordero JJ, Hodgson L, Condeelis J (2012) Directed cell invasion and migration during metastasis. Curr Opin Cell Biol 24:277–283
de Santa Carrillo, Pau E, Arias FC, Caso Peláez E, Muñoz Molina GM, Sánchez Hernández I, Muguruza Trueba I, Moreno Balsalobre R, Sacristán López S, Gómez Pinillos A, del Val Toledo Lobo M (2009) Prognostic significance of the expression of vascular endothelial growth factors A, B, C, and D and their receptors R1, R2, and R3 in patients with nonsmall cell lung cancer. Cancer 115:1701–1712
Chen HC (2005) Boyden chamber assay. Methods Mol Biol 294:15–22
Clark AG, Vignjevic DM (2015) Modes of cancer cell invasion and the role of the microenvironment. Curr Opin Cell Biol 36:13–22
Dewerchin M, Carmeliet P (2012) PlGF: a multitasking cytokine with disease-restricted activity. Cold Spring Harb Perspect Med 2: a011056
Dvorak HF (2021) Reconciling VEGF with VPF: the importance of increased vascular permeability for stroma formation in tumors, healing wounds, and chronic inflammation. Front Cell Dev Biol 9:660609
Frank NY, Schatton T, Kim S, Zhan Q, Wilson BJ, Ma J, Saab KR, Osherov V, Widlund HR, Gasser M, Waaga-Gasser AM, Kupper TS, Murphy GF, Frank MH (2011) VEGFR-1 expressed by malignant melanoma-initiating cells is required for tumor growth. Cancer Res 71:1474–1485
Gitay-Goren H, Halaban R, Neufeld G (1993) Human melanoma cells but not normal melanocytes express vascular endothelial growth factor receptors. Biochem Biophys Res Commun 190:702–708
Graziani G, Ruffini F, Tentori L, Scimeca M, Dorio AS, Atzori MG, Failla CM, Morea V, Bonanno E, D’Atri S, Lacal PM (2016) Antitumor activity of a novel anti-vascular endothelial growth factor receptor-1 monoclonal antibody that does not interfere with ligand binding. Oncotarget 7:72868–72885
Guo X, Chen M, Cao L, Hu Y, Li X, Zhang Q, Ren Y, Wu X, Meng Z, Xu K (2021) Cancer-associated fibroblasts promote migration and invasion of non-small cell lung cancer cells via miR-101-3p mediated VEGFA secretion and AKT/eNOS pathway. Front Cell Dev Biol 9:764151
Hayashido Y, Hamana T, Ishida Y, Shintani T, Koizumi K, Okamoto T (2007) Induction of alpha2-antiplasmin inhibits E-cadherin processing mediated by the plasminogen activator/plasmin system, leading to suppression of progression of oral squamous cell carcinoma via upregulation of cell-cell adhesion. Oncol Rep 17:417–423
Hayashido Y, Lucas A, Rougeot C, Godyna S, Argraves WS, Rochefort H (1998) Estradiol and fibulin-1 inhibit motility of human ovarian- and breast-cancer cells induced by fibronectin. Int J Cancer 75:654–658
Hennequin LF, Thomas AP, Johnstone C, Stokes ES, Plé PA, Lohmann JJ, Ogilvie DJ, Dukes M, Wedge SR, Curwen JO, Kendrew J, Lambert-van der Brempt C (1999) Design and structure-activity relationship of a new class of potent VEGF receptor tyrosine kinase inhibitors. J Med Chem 42:5369–5389
Hlobilkova A, Ehrmann J, Knizetova P, Krejci V, Kalita O, Kolar Z (2009) Analysis of VEGF, Flt-1, Flk-1, nestin and MMP-9 in relation to astrocytoma pathogenesis and progression. Neoplasma 56:284–290
ICLAC Databases (2021) International Cell Line Authentification Committee. https://iclac.org/databases/
Iversen K, Robins RE (1980) Mucosal malignant melanomas. Am J Surg 139:660–664
Jia LQ, Osada M, Ishioka C, Gamo M, Ikawa S, Suzuki T, Shimodaira H, Niitani T, Kudo T, Akiyama M, Kimura N, Matsuo M, Mizusawa H, Tanaka N, Koyama H, Namba M, Kanamaru R, Kuroki T (1997) Screening the p53 status of human cell lines using a yeast functional assay. Mol Carcinog 19:243–253
Kasuga T, Nojiri I, Furuse K, Kubo E, Noda Y, Sugano H, Sakamoto G (1976) Establishment of human melanoma cell like (HMV-II). Proceedings of the Japanese Cancer Association 35th annual meeting
Lacal PM, Failla CM, Pagani E, Odorisio T, Schietroma C, Falcinelli S, Zambruno G, D’Atri S (2000) Human melanoma cells secrete and respond to placenta growth factor and vascular endothelial growth factor. J Invest Dermatol 115:1000–1007
Lacal PM, Graziani G (2018) Therapeutic implication of vascular endothelial growth factor receptor-1 (VEGFR-1) targeting in cancer cells and tumor microenvironment by competitive and non-competitive inhibitors. Pharmacol Res 136:97–107
Martins SF, Garcia EA, Luz MA, Pardal F, Rodrigues M, Filho AL (2013) Clinicopathological correlation and prognostic significance of VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression in colorectal cancer. Cancer Genomics Proteomics 10:55–67
Matsuoka T, Yashiro M (2014) The role of PI3K/Akt/mTOR signaling in gastric carcinoma. Cancers (basel) 6:1441–1463
Mayer IA, Arteaga CL (2016) The PI3K/AKT pathway as a target for cancer treatment. Annu Rev Med 67:11–28
McKay MM, Morrison DK (2007) Integrating signals from RTKs to ERK/MAPK. Oncogene 26:3113–3121
Molhoek KR, Erdag G, Rasamny JK, Murphy C, Deacon D, Patterson JW, Slingluff CL Jr, Brautigan DL (2011) VEGFR-2 expression in human melanoma: revised assessment. Int J Cancer 129:2807–2815
Motwani J, Eccles MR (2021) Genetic and genomic pathways of melanoma development, invasion and metastasis. Genes (Basel) 12:1543
Myoken Y, Kayada Y, Okamoto T, Kan M, Sato GH, Sato JD (1991) Vascular endothelial cell growth factor (VEGF) produced by A-431 human epidermoid carcinoma cells and identification of VEGF membrane binding sites. Proc Natl Acad Sci U S A 88:5819–5823
Nozhat Z, Hedayati M (2016) PI3K/AKT pathway and its mediators in thyroid carcinomas. Mol Diagn Ther 20:13–26
Okamoto T, Tanaka Y, Kan M, Sakamoto A, Takada K, Sato JD (1996) Expression of fibroblast growth factor binding protein HBp17 in normal and tumor cells. In Vitro Cell Dev Biol Anim 32:69–71
Pasquali S, Hadjinicolaou AV, ChiarionSileni V, Rossi CR, Mocellin S (2018) Systemic treatments for metastatic cutaneous melanoma. Cochrane Database Syst Rev 2:Cd011123
Peebles PT, Trisch T, Papageorge AG (1978) Isolation of four unusual pediatric solid tumor cell lines. Pediatr Res 12:485
Roskoski R Jr (2017) Vascular endothelial growth factor (VEGF) and VEGF receptor inhibitors in the treatment of renal cell carcinomas. Pharmacol Res 120:116–132
Sato JD, Kawamoto T, Okamoto T (1987) Cholesterol requirement of P3–X63-Ag8 and X63-Ag8.653 mouse myeloma cells for growth in vitro. J Exp Med 165:1761–1766
Shibuya M (2006) Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1): a dual regulator for angiogenesis. Angiogenesis 9:225–230
Shibuya M (2011) Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) signaling in angiogenesis: a crucial target for anti- and pro-angiogenic therapies. Genes Cancer 2:1097–1105
Shiku H, Takahashi T, Oettgen HF (1976) Cell surface antigens of human malignant melanoma. II. Serological typing with immune adherence assays and definition of two new surface antigens. J Exp Med 144:873–881
Sopo M, Anttila M, Hämäläinen K, Kivelä A, Ylä-Herttuala S, Kosma VM, Keski-Nisula L, Sallinen H (2019) Expression profiles of VEGF-A, VEGF-D and VEGFR1 are higher in distant metastases than in matched primary high grade epithelial ovarian cancer. BMC Cancer 19:584
Streit M, Detmar M (2003) Angiogenesis, lymphangiogenesis, and melanoma metastasis. Oncogene 22:3172–3179
Szabo E, Schneider H, Seystahl K, Rushing EJ, Herting F, Weidner KM, Weller M (2016) Autocrine VEGFR1 and VEGFR2 signaling promotes survival in human glioblastoma models in vitro and in vivo. Neuro Oncol 18:1242–1252
Tammela T, Enholm B, Alitalo K, Paavonen K (2005) The biology of vascular endothelial growth factors. Cardiovasc Res 65:550–563
Tarcic G, Yarden Y (2010) MAP Kinase activation by receptor tyrosine kinases: in control of cell migration. Methods Mol Biol 661:125–135
Vaisman N, Gospodarowicz D, Neufeld G (1990) Characterization of the receptors for vascular endothelial growth factor. J Biol Chem 265:19461–19466
von Marschall Z, Cramer T, Höcker M, Burde R, Plath T, Schirner M, Heidenreich R, Breier G, Riecken EO, Wiedenmann B, Rosewicz S (2000) De novo expression of vascular endothelial growth factor in human pancreatic cancer: evidence for an autocrine mitogenic loop. Gastroenterology 119:1358–1372
Yde SS, Sjoegren P, Heje M, Stolle LB (2018) Mucosal melanoma: a literature review. Curr Oncol Rep 20:28
Yu Y, Sato JD (1999) MAP kinases, phosphatidylinositol 3-kinase, and p70 S6 kinase mediate the mitogenic response of human endothelial cells to vascular endothelial growth factor. J Cell Physiol 178:235–246
Yu Y, Hulmes JD, Herley MT, Whitney RG, Crabb JW, Sato JD (2001) Direct identification of a major autophosphorylation site on vascular endothelial growth factor receptor Flt-1 that mediates phosphatidylinositol 3’-kinase binding. Biochem J 358:465–472
Zhang Z, Neiva KG, Lingen MW, Ellis LM, Nör JE (2010) VEGF-dependent tumor angiogenesis requires inverse and reciprocal regulation of VEGFR1 and VEGFR2. Cell Death Differ 17:499–512
We would like to thank Drs. Masabumi Shibuya (Jobu University) and Kenya Shitara (Kyowa Hakko Kirin Co., Ltd.) for providing KM1570, a neutralizing antibody to VEGFR1.
Grants-in-Aid for Scientific Research (C) from the Japanese Ministry of Education, Culture, Sports, Science and Technology to K.K. (grant number: 24593033) and Grants-in-Aid for Scientific Research (B) to T.O. (grant number: 18H03000).
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The authors declare no competing interests.
Editor: J. Denry Sato
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Supplementary file1 Supplementary Figure S1 Confirmation of Mycoplasma-free culture. It was confirmed that all cell lines were free of mycoplasma contamination by PCR method. (PDF 291 KB)
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Koizumi, K., Shintani, T., Hayashido, Y. et al. VEGF-A promotes the motility of human melanoma cells through the VEGFR1–PI3K/Akt signaling pathway. In Vitro Cell.Dev.Biol.-Animal 58, 758–770 (2022). https://doi.org/10.1007/s11626-022-00717-3