Kenji Ichikawa, Saori Watanabe Miyano, Yusuke Adachi, Masahiro Matsuki, Kiyoshi Okamoto and Junji Matsui*
Biology Research, Oncology Tsukuba Research Department, Discovery, Medicine Creation, Oncology Business Group, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
Received: 17 June, 2016; Accepted: 01 July, 2016; Published: 02 July, 2016
Junji Matsui, Ph, D, Biology Research, Oncology Tsukuba Research Department, Discovery, Medicine Creation, Oncology Business Group, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan, Tel: +81-29-847-5808, Fax: +81-29-847-5367 E-mail:
Ichikawa K, Miyano SW, Adachi Y, Matsuki M, Okamoto K, et al. (2016) Lenvatinib Suppresses Angiogenesis through the Inhibition of both the VEGFR and FGFR Signaling Pathways. Glob J Cancer Ther 2(1): 019-025.
© 2016 Ichikawa K, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Lenvatinib; VEGFR; FGFR; Sorafenib
Lenvatinib mesilate (lenvatinib) is an oral multiple-receptor tyrosine kinase inhibitor that selectively inhibits the kinase activities of Vascular Endothelial Growth Factor Receptor (VEGFR) 1-3, Fibroblast Growth Factor Receptor (FGFR) 1-4, Platelet-Derived Growth Factor Receptor (PDGFR) α, KIT, and RET. The VEGFR and FGFR signaling pathways are the master regulators of normal and tumor angiogenesis. Lenvatinib showed significant activity in patients with radioiodine-refractory thyroid cancer in a Phase III study and is used in the United States, the European Union, and Japan. Moreover, based on Phase II study, lenvatinib has been approved in the United States for the treatment of patients with advanced renal cell carcinoma in combination with everolimus. In addition, the efficacy of lenvatinib is being evaluated in other cancers, including hepatocellular carcinoma and endometrial cancer. The purpose of this study was to elucidate the mechanism underlying the clinical activities of lenvatinib by using in vitro and in vivo angiogenesis models.
First, we established an in vitro tube formation system, in which capillary-like structures formed on basement membrane extract in response to pro-angiogenic factors. Lenvatinib suppressed tube formation induced by bFGF alone and by bFGF plus VEGF. Furthermore, plasma levels of VEGF and FGF23, pharmacodynamic biomarkers of inhibition of the VEGFR and FGFR signaling pathways, respectively, were up-regulated after the administration of lenvatinib to mice. By contrast, the administration of another VEGFR inhibitor, sorafenib tosylate (sorafenib), up-regulated plasma levels of VEGF but not FGF23. Finally, lenvatinib suppressed bFGF-driven angiogenesis in Matrigel plug assays at low dosage (3 mg/kg), whereas sorafenib did so only at a higher dose (30 mg/kg). These results indicate that lenvatinib inhibits both VEGFR and FGFR in vitro and in vivo. This combined inhibition of both VEGFR and FGFR may lead significant clinical activities.
Angiogenesis, the formation of new blood vessels from the pre-existing vasculature, is required for embryonic development, wound repair, and tumor growth . In tumors, angiogenesis is necessary to supply oxygen and nutrients to proliferating cancer cells. Pro-angiogenic factors such as VEGF and FGF promote the migration, proliferation, differentiation, and eventually survival of endothelial cells to form tumor vessels [2-4].
The same newly formed vessels that transport growth factors, cytokines, and nutrients to cancer cells provide the route for cancer metastasis. Consequently, the activation of tumor angiogenesis correlates with both the growth of the tumor mass and cancer metastasis. The VEGF–VEGFR axis plays an integral role in tumor angiogenesis . VEGF activates VEGFR through homo- and heterodimerization by interacting with the extracellular domain of VEGFR on endothelial cells [6,7]. The VEGF–VEGFR signaling pathway also plays a critical role in promoting endothelial cell growth and migration and thus tumor vascularization. For these reasons, many inhibitors of the VEGFR signaling pathway, such as bevacizumab, sorafenib, and sunitinib, have been used as therapeutic agents in several tumor types, including renal cell carcinoma, non-small cell lung carcinoma, hepatocellular carcinoma, and thyroid cancer [8,9]. These inhibitors successfully suppress tumor angiogenesis and mass in a subset of patients.
The FGF–FGFR axis is another pro-angiogenic signaling pathway . Indeed, high levels of bFGF expression are correlated with a worse prognosis in highly vascularized tumor types, such as renal cell carcinoma and hepatocellular carcinoma [11,12]. Injection of adenovirus encoding soluble FGFR into RIP1-Tag2 mouse showed decrease of vessel density in tumor, leading to growth inhibition . Like VEGF, bFGF induces the proliferation, survival, and differentiation of endothelial cells, ultimately activating tumor angiogenesis. Accordingly, the FGF–FGFR axis is recognized as a potential therapeutic target for blocking tumor angiogenesis.
Biomarkers for molecular-targeted agents are important for assessing drug candidates in clinical trials. For example, validated biomarkers contribute to clinical studies by providing means for predicting the mechanisms of action underlying the efficacies and toxicities of candidate drugs and for determining therapeutic doses. Two biomarkers that reliably reflect the inhibition of the VEGFR and FGFR pathways are blood concentrations of VEGF and FGF23, respectively. Indeed, several VEGFR inhibitors increased plasma VEGF levels in both preclinical and clinical studies [14-16]. FGF23 belongs to the FGF family and functions as an endocrine factor. Increased plasma FGF23 levels by FGFR inhibitors are a surrogate pharmacodynamic biomarker of FGFR inhibition .
Lenvatinib is an orally administered multiple receptor tyrosine kinase inhibitor with a novel binding mode that selectively targets the kinase activities of VEGFRs (VEGFR1-3) and FGFRs (FGFR1-4) in addition to other pro-angiogenic and oncogenic pathway-related RTKs including PDGFRα, KIT and RET [18-23]. Our previous study reveals that Ki values of lenvatinib against VEGFRs are between 0.7 and 1.3 nmol/L, and FGFRs are between 8 and 22 nmol/L . Due to the results of a Phase III clinical study, lenvatinib was approved in the United States, European Union, and Japan in 2015 for the treatment of advanced, differentiated thyroid cancer that is refractory to radioactive iodine or unresectable thyroid cancer . Furthermore, lenvatinib has been approved for the treatment of patients with advanced renal cell carcinoma following one prior anti-angiogenic therapy in combination with everolimus in the United States based on Phase II study . Currently lenvatinib is being evaluated for efficacy in various other cancers, including hepatocellular carcinoma and endometrial cancer.
In this study, we show that lenvatinib inhibited angiogenesis in vitro and in vivo through the dual targeting of VEGFR and FGFR. These results suggest that this combined anti-angiogenic activity of lenvatinib contributes to its clinical activities.
Material and Methods
Cells and reagents
Human umbilical vein endothelial cells (HUVECs) were isolated from a human umbilical cord as described previously  and cultured in supplemented EBM-2 as provided in the EGM-2 BulletKit (Lonza). The human differentiated thyroid cancer cell line RO82-W-1 was obtained from the European Collection of Authenticated Cell Cultures (ECACC) and cultured in a mixture of DMEM (WAKO), Ham’s F12 (WAKO), and MCDB 105 (Sigma-Aldrich) (2:1:1) supplemented with 10% fetal bovine serum. All cells were maintained at 37°C in a 5% CO2 atmosphere.
Lenvatinib, sorafenib, and PD173074, a selective FGFR inhibitor were synthesized at Eisai Co., Ltd. (Ibaraki, Japan).
In vitro tube formation assay
Geltrex (45 µL/well; Thermo Fisher Scientific) was added to each well of 96-well plates and incubated at 37°C in a 5% CO2 atmosphere for 30 minutes to allow the gel to solidify. Medium 200PRF (Thermo Fisher Scientific) containing bFGF only (80 ng/mL; Invitrogen) or both bFGF (80 ng/mL) and VEGF (80 ng/mL; R&D Systems) was incubated with anti-bFGF antibody (1000 ng/mL; 05-117, MILLIPORE), anti-VEGF antibody (1000 ng/mL; MAB293, R&D Systems), or both anti-bFGF antibody and anti-VEGF antibody at 4°C overnight. HUVECs were diluted to 1.6×105 cells/mL in Medium 200PRF, and 75 µL of this cell suspension was dispensed into each well containing the solidified gel in 96-well plates. Pre-incubated antibody–bFGF or antibody–bFGF–VEGF mixture, lenvatinib solution (0.610−4000 nmol/L) and 80 ng/mL bFGF, lenvatinib solution (0.610−4000 nmol/L) and 80 ng/mL bFGF–VEGF, or vehicle only was then added to each well (25 µL; final concentration of each ligand, 20 ng/mL). The plates were cultured for 20 hours at 4°C, after which 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (100 µL; 3.3 mg/mL in phosphate-buffered saline; Sigma) was added to each well; plates were incubated for an additional 4 hours for the formation of capillary-like structures. Images of tubes were obtained by using a GelCount device (Oxford Optronix), and the length of the tubes (capillaries) was measured by using the In Cell Developer Toolbox (version 1.9.2, GE Healthcare). This experiment was performed three times in triplicate.
Measurement of VEGF in mouse plasma
Cultured RO82-W-1 cells were suspended in Hank’s Balanced Salt Solution (Thermo Fisher Scientific) and mixed with an equal volume of Matrigel (Corning) to yield a suspension containing 5×107 cells/mL. A 0.1-mL aliquot of this cell suspension was transplanted subcutaneously into the right flank region of each BALB/c nude mouse (CAnN.Cg-Foxn1nu/CrlCrlj, female, Charles River Japan). When tumor volume reached approximately 350 mm3, mice were grouped according to tumor volume and shape and randomly allocated to receive vehicle, lenvatinib, or sorafenib (n = 8 per group). Lenvatinib (doses, 3 and 10 mg/kg), sorafenib (dose, 30 mg/kg), or vehicle only (distilled water) were administered orally once daily for 12 days at 0.1 mL/10 g body weight. At 9 hours after the last administration, blood was collected from the abdominal aorta of isoflurane-anesthetized mice. Blood was then centrifuged at 20,000 g for 5 minutes at 4°C, and the plasma was collected. The amount of VEGF in the mouse plasma was measured by using the Mouse VEGF Quantikine ELISA Kit (R&D Systems); this assay was performed in duplicate.
Measurement of FGF23 in mouse plasma
Lenvatinib (doses, 3 and 10 mg/kg), sorafenib (doses, 9 and 30 mg/kg), or vehicle only (distilled water) was orally administered to BALB/c nude mice (CAnNCrlCrlj, female, Charles River Japan) at 0.1 mL/10 g body weight (n = 8 per group). At 24 hours after this single administration, blood was collected from the abdominal aorta of isoflurane-anesthetized mice, centrifuged at 9,000 g for 10 minutes at 4°C, and the plasma collected. The amount of plasma FGF23 was measured by using the FGF-23 ELISA kit (Kainos Laboratories) according to the manufacturer’s procedure. The assay was performed in duplicate.
Matrigel plug assay
BALB/c nude mice (CAnN.Cg-Foxn1nu/CrlCrlj, female, Charles River Japan) were each injected subcutaneously in the abdominal region with 300 µL of Matrigel (Corning) containing 1 µg/mL bFGF (Invitrogen) or without bFGF. Mice injected with bFGF-containing Matrigel were allocated into 6 groups (n=8 per group, Day 1). Lenvatinib and sorafenib were orally administered at 0.1 mL/10 g body weight and PD173074 was orally administered at 0.2 mL/10 g body weight once daily for 7 days (Days 1–7). On Day 8, mice were euthanized, and the Matrigel plug isolated from each mouse was minced in 400 µL of distilled water and placed in the dark at 4°C for 2 days to release any hemoglobin in the plugs into the water. To quantify the formation of neovasculature, the hemoglobin content in the Matrigel plug was measured by using Drabkin’s solution (Sigma). Briefly, the supernatant was isolated by centrifugation and plated in 96-well microtiter plates (100 µL/well), Drabkin’s solution was added at 100 µL/well, and the plate was placed in the dark for 2 hours at room temperature. The optical density of each well was measured at 540 nm (reference wavelength: 660nm) by using a microplate reader (Spectra Max250, Molecular Devices). The hemoglobin content of each plug was calculated by using the value of the hemoglobin standard.
Antitumor activity of lenvatinib and sorafenib in K1 or RO82-W1 xenografts in mice
K1 and RO82-W1 cells were washed with Dulbecco’s phosphate buffered saline, harvested with 0.25% trypsin–EDTA, and suspended with 50% BD Matrigel™ (BD biosciences) in the mixture medium at a density of 5.0 × 107 cells/ml for K1 xenografts model and 2.5 × 107 cells/mL for RO82-W1 xenografts. The cell suspension was inoculated subcutaneously into the right flank region of each mouse. Mice were selected based on their tumor volumes, shapes of tumors, physical condition, and body weights, and were randomly divided into 12 groups (Day 1). Lenvatinib mesilate (1, 3, 10, 30, and 100 mg/kg), sorafenib tosylate (3, 10, 30, 100, and 300 mg/kg), or vehicle was orally administered at 0.2 and 0.1 mL/10 g body weight, respectively, once daily. The tumor volumes were measured on day 15 for K1 xenografts model and on day 22 for RO82-W1 xenografts.
The tumor volume was calculated according to the following formula;
Tumor volume (mm3) = length (mm) × width2 (mm2) × 1/2
Length: largest diameter of tumor
Width: diameter perpendicular to length
The %TGI was calculated according to the following formula;
%TGI = (1 – dT / dC) × 100
dT = Tumor volume on last day – Tumor volume on Day 1
dC = Mean tumor volume of the vehicle-treated group on last day – Mean tumor volume of the vehicle-treated group on Day 1
All procedures using laboratory animals were conducted in accordance with the Institutional Animal Care and Use Committee guidelines of Eisai Co., Ltd.
All data are presented as mean ± SD. The differences between the means of groups were analyzed by unpaired t test or one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. A value of P < 0.05 (two sided) was considered statistically significant. Statistical analyses were performed using GraphPad Prism version 6.02.
Establishment of bFGF or bFGF plus VEGF-induced tube formation assay
To evaluate the inhibitory activity of lenvatinib against VEGFR and FGFR in angiogenesis in vitro, we established and validated a tube formation assay system in which HUVECs form capillary-like structures on Geltrex (Invitrogen), a basement membrane extract with reduced concentrations of growth factors. First, we tested whether VEGF, bFGF, and the combination of VEGF plus bFGF-induced tube formation on Geltrex. Tube formation was potently induced in response to bFGF and the combination of VEGF plus bFGF (Figure 1A, B) but not to VEGF only (data not shown). To examine ligand specificity in this system, anti-bFGF and anti-VEGF antibodies were used to disrupt the function of the bFGF and VEGF ligands, respectively. Anti-bFGF antibody significantly decreased bFGF-induced tube formation, whereas anti-VEGF antibody did not (Figure 1C, Figure S1A). Furthermore, both anti-bFGF antibody alone and a mixture of anti-bFGF and anti-VEGF antibodies significantly inhibited bFGF- plus VEGF-induced tube formation compared with that of the control, and the inhibitory activity of the antibody mixture was greater than that of anti-bFGF antibody alone (Figure 1D, Figure S1B). Treatment with anti-VEGF antibody alone failed to inhibit the tube formation induced by bFGF plus VEGF (Figure 1D). These results suggest that bFGF acts as strong pro-angiogenic factor in this assay system and that VEGF contributes to tube formation in the presence of bFGF. Additional activity of VEGF in the VEGF-bFGF combination setting did not observed (Figure 1B), probably because 20 ng/ml bFGF achieved complete effect on tube formation.