Antitumor activity of lenvatinib (e7080): an angiogenesis inhibitor that targets multiple receptor tyrosine kinases in preclinical human thyroid cancer models

Abstract

Inhibition of tumor angiogenesis by blockading the vascular endothelial growth factor (VEGF) signaling pathway is a promising therapeutic strategy for thyroid cancer. Lenvatinib mesilate (lenvatinib) is a potent inhibitor of VEGF receptors (VEGFR1-3) and other prooncogenic and prooncogenic receptor tyrosine kinases, including fibroblast growth factor receptors (FGFR1-4), platelet derived growth factor receptor α (PDGFRα), KIT, and RET. We examined the antitumor activity of lenvatinib against human thyroid cancer xenograft models in nude mice. Orally administered lenvatinib showed significant antitumor activity in 5 differentiated thyroid cancer (DTC), 5 anaplastic thyroid cancer (ATC), and 1 medullary thyroid cancer (MTC) xenograft models. Lenvatinib also showed antiangiogenesis activity against 5 DTC and 5 ATC xenografts, while lenvatinib showed in vitro antiproliferative activity against only 2 of 11 thyroid cancer cell lines: that is, RO82-W-1 and TT cells. Western blot analysis showed that cultured RO82-W-1 cells overexpressed FGFR1 and that lenvatinib inhibited the phosphorylation of FGFR1 and its downstream effector FRS2. Lenvatinib also inhibited the phosphorylation of RET with the activated mutation C634W in TT cells. These data demonstrate that lenvatinib provides antitumor activity mainly via angiogenesis inhibition but also inhibits FGFR and RET signaling pathway in preclinical human thyroid cancer models.

Figure 1

Antitumor activity of lenvatinib in human thyroid cancer xenograft models in nude mice. Nude mice bearing tumor xenografts were treated orally once daily with either vehicle or lenvatinib at the indicated doses when tumor volumes reached between 100 and 300 mm³ (day 1). Each group consisted of 5 mice. The change in tumor volume in the treated group relative to that in the control group ΔT/C (%) was calculated as (ΔT/ΔC) × 100%, where ΔT and ΔC are the change in tumor volume for the treated and vehicle control group, respectively. (a) DTC xenograft models. ΔT/C on day 15. (b) ATC xenograft models. ΔT/C on day 15. (c) TT xenograft model. ΔT/C on day 29. Data are shown as means ± SD. *P < 0.05 compared with vehicle-treated mice.

Figure 2

Antiangiogenesis activity of lenvatinib in human thyroid cancer xenograft models in nude mice. Nude mice bearing tumor xenografts were treated orally once daily with either vehicle or lenvatinib at the indicated doses when tumor volumes reached between 100 and 300 mm³ (day 1). Microvessel density (MVD) was analyzed by immunohistochemical staining of endothelial cells with an anti-mouse CD31 antibody within the resected tumor xenografts as described in Section 2. MVD is expressed as the average number of microvessels per mm² in 5 regions of interest (ROIs). Each group consisted of 5 mice. Data are shown as means ± SD. *P < 0.05 compared with vehicle-treated mice. DTC: differentiated thyroid cancer, MTC: medullary thyroid cancer, and ATC: anaplastic thyroid cancer.

Figure 3

Effect of the selective FGFR kinase inhibitor PD173074 on the FGFR signaling pathway in human DTC RO82-W-1 cells. (a) Western blot analysis of the expression of FGF receptors in vitro. Expression levels of FGF receptors in RO82-W-1 cells were compared to those in Nthy-ori 3-1 cells (normal thyroid cells). (b) Western blot analysis of the effects of PD173074 on the phosphorylation of FGFR1 and its downstream effectors. After starvation overnight, RO82-W-1 cells were treated with PD173074 at the indicated concentrations for 1 h and were then stimulated for 10 min with bFGF (20 ng/mL) and heparin before being lysed. (c) Antitumor activity of PD173074 against RO82-W-1 xenografts in nude mice. Nude mice bearing RO82-W-1 xenografts were treated orally once daily for 14 days with either vehicle or PD173074 at the indicated doses when tumor volumes reached about 300 mm³ (day 1). The tumor volume was measured on the indicated days after administrations. Each group consisted of 5 mice. Data are shown as means ± SD. *P < 0.05 compared with vehicle-treated mice.

Figure 4

Effect of lenvatinib on FGFR1 signaling pathway in human DTC RO82-W-1 cells in vitro. After starvation overnight, RO82-W-1 cells were treated with vehicle (control), lenvatinib or sorafenib at the indicated concentrations for 1 h and were then stimulated for 10 min with bFGF (20 ng/mL) and heparin before being lysed. Western blot analyses of the phosphorylation of FGFR1 and its downstream effectors in RO82-W-1 cells were then performed and representative images were shown.

Figure 5

Effect of lenvatinib on the phosphorylation of RET with an activating mutation or a rearrangement in vitro. (a) Western blot analyses of the phosphorylation of RET in human medullary thyroid TT cells. TT cells were seeded and cultured overnight. They were then treated with lenvatinib at the indicated concentrations for 1 h before being lysed. (b) Western blot analyses of the phosphorylation of KIF5B-RET fusion proteins in normal thyroid cells; Nthy-ori 3-1 transfectants overexpressing KIF5B-RET (wild-type) or KIF5B-RET (M918T). Nthy-ori 3-1 transfectants were cultured overnight and then treated with lenvatinib at the indicated concentrations for 1 h before being lysed. Western blot analyses of the phosphorylation of RET and its downstream effectors in Nthy-ori 3-1 transfectants were then performed and representative images were shown.