Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

Abstract The vascular endothelial growth factor receptor‐1 (VEGFR‐1) is a tyrosine kinase receptor frequently expressed in melanoma. Its activation by VEGF‐A or placental growth factor (PlGF) promotes tumour cell survival, migration and invasiveness. Moreover, VEGFR‐1 stimulation contributes to pathological angiogenesis and induces recruitment of tumour‐associated macrophages. Since melanoma acquired resistance to BRAF inhibitors (BRAFi) has been associated with activation of pro‐angiogenic pathways, we have investigated VEGFR‐1 involvement in vemurafenib resistance. Results indicate that human melanoma cells rendered resistant to vemurafenib secrete greater amounts of VEGF‐A and express higher VEGFR‐1 levels compared with their BRAFi‐sensitive counterparts. Transient VEGFR‐1 silencing in susceptible melanoma cells delays resistance development, whereas in resistant cells it increases sensitivity to the BRAFi. Consistently, enforced VEGFR‐1 expression, by stable gene transfection in receptor‐negative melanoma cells, markedly reduces sensitivity to vemurafenib. Moreover, melanoma cells expressing VEGFR‐1 are more invasive than VEGFR‐1 deficient cells and receptor blockade by a specific monoclonal antibody (D16F7 mAb) reduces extracellular matrix invasion triggered by VEGF‐A and PlGF. These data suggest that VEGFR‐1 up‐regulation might contribute to melanoma progression and spreading after acquisition of a drug‐resistant phenotype. Thus, VEGFR‐1 inhibition with D16F7 mAb might be a suitable adjunct therapy for VEGFR‐1 positive tumours with acquired resistance to vemurafenib.


| INTRODUC TI ON
The vascular endothelial growth factor receptor-1 (VEGFR-1) is a membrane tyrosine kinase receptor for VEGF-A, VEGF-B and placental growth factor (PlGF) that are all members of the VEGF family of angiogenic factors. 1 At variance with VEGF-A that also binds to VEGFR-2 even though with lower affinity, VEGF-B and PlGF are exclusive ligands for VEGFR-1. 2 This receptor is expressed in endothelial, smooth muscle cells and monocytes/macrophages, promoting chemotaxis and survival. 3 In particular, VEGFR-1 stimulation is implicated in pathological angiogenesis and induces the recruitment of tumour-associated macrophages that, in turn, favour cancer progression and dissemination. [4][5][6] In addition to the membrane receptor, soluble VEGFR-1 forms have been identified, which derive from alternative splicing of the pre-mRNA. 7 The soluble receptor is released in the extracellular matrix (ECM) and exerts anti-angiogenic effects by sequestering VEGF-A or PlGF, thus lowering their availability for membrane receptor activation, or by forming dominant negative complexes via heterodimerization with the membrane-associated receptors.
The VEGFR-1 expression has been frequently reported also in tumour cells of different tissue origin. 1 In melanoma cells, VEGFR-1 activation by autocrine or paracrine ligands promotes tumour cell survival, migration, invasiveness and chemoresistance. [8][9][10][11][12][13][14] Moreover, VEGFR-1 is involved in vasculogenic mimicry, which provides cancer cells with oxygen and nutrient supply and a route for metastatic spreading. [14][15][16] Using a recently developed monoclonal antibody (mAb), we have demonstrated that blockage of VEGFR-1 activation inhibits neovessel formation, myeloid progenitor mobilization, melanoma infiltration by monocytes/macrophages and vasculogenic mimicry. 1,14 The D16F7 mAb is characterized by an innovative mechanism of action, by which it down-modulates membrane receptor signalling without hampering VEGF-A or PlGF ligand binding. 1,14 Based on this property, the D16F7 mAb does not interfere with the decoy function of the soluble VEGFR-1 maintaining its anti-angiogenic effects.
Malignant melanoma is a highly aggressive malignancy and is regarded as the most lethal form of cutaneous cancer because of its ability to metastasize to distant organs. The approval of immune checkpoints inhibitors (ie the anti-PD-1 and anti-CTLA-4 mAbs) and, for BRAF mutated melanoma (approximately 50% of all cases), of BRAF/MEK kinase inhibitors has dramatically improved the outcome of patients with metastases. 17,18 Unfortunately, in the case of immune checkpoint inhibitors a significant proportion of patients derive no benefit from these therapies (primary resistance), whereas in the case of BRAF/MEK inhibitors most patients respond to treatment but responses are short-lived because of the development of drug resistance and tumour recurrence. Besides genetic alterations that result in reactivation of the MAPK and, less frequently, activation of the PI3K-Akt pathways, other mechanisms are involved in acquired resistance to BRAF inhibitors (BRAFi) including activation of pro-angiogenic pathways. 19,20 In this regard, the onset of treatment resistance to the BRAFi dabrafenib is associated with restored VEGF-A production by melanoma cells. [21][22][23] Moreover, by paradoxically activating the MAPK pathway in BRAF wild-type macrophages, BRAFi may induce the production of VEGF-A, which directly stimulates macrophage survival, tumour immune evasion and ultimately melanoma growth. 20,24,25 Conversely, treatment of susceptible BRAF mutated melanoma with BRAFi results in reduced tumour vascularity and increased T cell infiltration in melanoma that was attributed to loss or reduced VEGF-A expression and secretion. [26][27][28] In the present study, we have investigated whether VEGFR-1 might contribute to the acquisition of a BRAFi-resistant phenotype by melanoma and whether blockade of this receptor might reduce ECM invasion by resistant tumour cells in response to angiogenic factors. The results indicate that human melanoma cells rendered resistant to the BRAFi vemurafenib express higher levels of VEGFR-1 compared to their BRAFi-sensitive counterparts and that inhibition of VEGFR-1 with D16F7 mAb might be a suitable adjunct therapy for VEGFR-1 positive tumours with acquired resistance to vemurafenib.

| Cell lines and culture conditions
The human melanoma A375 cell line was obtained from American Type Culture Collection (ATCC). The human melanoma M14C2 clone (hereafter referred to as M14) was obtained by limiting dilution from the corresponding bulk cell population, as previously described. 12 The human melanoma GR-Mel cell line was established in the Laboratory of Molecular Oncology, IDI-IRCCS (Rome, Italy). A375 and M14 melanoma cell lines with acquired resistance to vemurafenib, hereafter referred to as A375-VR and M14-VR, respectively, were generated in our laboratories by exposing the parental cell line to increasing concentrations of vemurafenib (up to 5 µmol/L) for 3 months.
Transfection was performed using Lipofectamine 2000 (Invitrogen; ThermoFisher Scientific), as described by the manufacturer, and transfected cells were selected in blasticidine (Invitrogen) containing culture medium. Antibiotic resistant clones were isolated by ring cloning, and transfected clones maintained in the presence of 2.5 μg/mL blasticidine. VEGFR-1 expressing subclones were identified by Western blotting.

| ECM cell invasion assay
In vitro invasion assays were performed using Boyden chambers equipped with 8-µm pore diameter polycarbonate filters (Nuclepore; Whatman Incorporated, Clifton, NJ), coated with 20 µg of matrigel, as described. 29 Briefly, melanoma cells were suspended in invasion medium
After 5 days, 20 μL of MTS solution was added to each well and cells were incubated at 37°C for 1-3 hours. Absorbance was read at 490 nm (reference wavelength 655 nm) using a 3550-UV Microplate reader (Bio-Rad, Hercules, CA, USA). Chemosensitivity was measured in terms of IC 50 , that is the concentration of the drug capable of inhibiting cell growth by 50%.

| Analysis of VEGFRs transcripts
Quantification of membrane VEGFR-1 and VEGFR-2 transcripts was performed by quantitative real-time reverse transcriptasepolymerase chain reaction (qRT-PCR) according to the dual-labelled fluorigenic probe method and using an ABI Prism 7000 sequence detector (PerkinElmer, Groningen, the Netherlands) and using SYBR green master mix reagent, as previously described. 30 Expression levels were calculated by the relative standard curve method.

| ELISA quantification of VEGF-A and PlGF levels
Conditioned media from melanoma cell lines were obtained by semi-

| Transient siRNA transfection
Melanoma cells were plated in complete medium, the day after Plates were incubated at 37°C for 5 days, and cell growth was evaluated by the MTS assay. Three replica wells were used for each group.
To analyse the influence of VEGFR-1 on the development of resis-

| Immunoblot analysis
Proteins were run in 10% SDS-polyacrylamide gels and transferred to supported nitrocellulose membranes by standard techniques.
Immunodetection was performed using antimouse or anti-rabbit Ig/ Horseradish peroxidase secondary antibodies and ECL Western blotting detection reagents from GE Healthcare (Milan, Italy).

| Statistical analysis
Statistical analysis of the differences between pairs of groups was performed by two-sided Student's t test. For multiple comparisons, the non-parametric Kruskal-Wallis followed by Dunn's post hoc test was used. P values below 0.05 were considered statistically significant.

| Generation and characterization of A375 and M14 sublines with acquired resistance to vemurafenib
The  Figure 1).

| VEGFR-1 silencing counteracts the emergence of resistance in sensitive cells and increases sensitivity to vemurafenib in resistant melanoma cells
To investigate the role of VEGFR-1 in the acquisition of a vemurafenib-resistant phenotype, A375 cells, which express basal  Figure 4B).

| Blockade of VEGFR-1 inhibits ECM invasion by vemurafenib-resistant melanoma cells
According to the phenotype switching model, metastasis formation is the result of tumour transition from a proliferative to an invasive phenotype. 32 An online gene expression-based tool developed for predicting melanoma cell phenotype (ie Heuristic Online Phenotype Prediction, HOPP) is available and has identified a set of genes that characterizes these two different melanoma phenotypes. 33  VEGFR-1 expression, to PlGF or VEGF-A failed to induce matrigel invasion and treatment with D16F7 had no effect ( Figure 5D).

| D ISCUSS I ON
The antitumour effects of vemurafenib are short-lived and the majority of patients undergoing therapy present tumour relapse within few months after the beginning of treatment. Therefore, the characterization of the mechanisms contributing to vemurafenib resistance is essential in order to improve its long-term efficacy and to identify next generation therapeutic strategies.
Adaptive tumour responses to BRAF-targeted drugs are favoured by melanoma heterogeneity and lead to treatment failure. Acquired resistance mechanisms include the increased expression of several receptor tyrosine kinases, such as platelet-derived growth factor receptor beta (PDGFRβ), insulin-like growth factor-1 receptor (IGF1R) and epidermal growth factor receptor (EGFR), 34

BSA P lGF
Anti-VEGFR-1 mAb P lGF + anti-VEGFR-1 mAb B * * * ### correlated to cell survival and chemoresistance. 3,10,11,44 Moreover, increased VEGFR-1 expression and/or up-regulation of its specific ligand PlGF are considered mechanisms of tumour resistance to VEGF-A targeting anti-angiogenic therapies. [45][46][47][48] Overall, our results strongly support the hypothesis that VEGFR-1 expression might contribute to the aggressive phenotype of melanoma cells resistant to vemurafenib. Actually, the recently described mAb D16F7, produced in our laboratories and that specifically inhibits VEGFR-1, drastically reduces invasiveness of resistant cells. Interestingly, VEGFR-1 blockade by D16F7 mAb reduces ECM invasion triggered by VEGF-A and PlGF, supporting the hypothesis that up-regulation of VEGFR-1 might contribute to tumour progression and spreading of melanoma after acquisition of a drug-resistant phenotype. Besides this activity, D16F7 mAb has also the ability to modify the tumour microenvironment at least at two different levels: hampering tumour-associated angiogenesis and reducing melanoma infiltration by pro-tumour macrophages. These D16F7 properties support its use in combination with BRAFi. It should also be noted that, since VEGFR-1 does not play a relevant role in physiological angiogenesis in the adult, this combination is likely to result in increased therapeutic efficacy without causing additive toxicity.
In conclusion, our results strongly suggest that the selective VEGFR-1 inhibition by D16F7 mAb might potentiate the effects of vemurafenib-based therapies for melanoma treatment and counteract resistance development to this BRAFi.

ACK N OWLED G EM ENTS
The research leading to these results has received funding from the

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest. The final version of the paper was read and approved by all authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.