High Potency VEGFRs/MET/FMS Triple Blockade by TAS-115 Concomitantly Suppresses Tumor Progression and Bone Destruction in Tumor-Induced Bone Disease Model with Lung Carcinoma Cells

<div><p>Approximately 25–40% of patients with lung cancer show bone metastasis. Bone modifying agents reduce skeletal-related events (SREs), but they do not significantly improve overall survival. Therefore, novel therapeutic approaches are urgently required. In this study, we investigated the anti-tumor effect of TAS-115, a VEGFRs and HGF receptor (MET)-targeted kinase inhibitor, in a tumor-induced bone disease model. A549-Luc-BM1 cells, an osteo-tropic clone of luciferase-transfected A549 human lung adenocarcinoma cells (A549-Luc), produced aggressive bone destruction associated with tumor progression after intra-tibial (IT) implantation into mice. TAS-115 significantly reduced IT tumor growth and bone destruction. Histopathological analysis showed a decrease in tumor vessels after TAS-115 treatment, which might be mediated through VEGFRs inhibition. Furthermore, the number of osteoclasts surrounding the tumor was decreased after TAS-115 treatment. <i>In vitro</i> studies demonstrated that TAS-115 inhibited HGF-, VEGF-, and macrophage-colony stimulating factor (M-CSF)-induced signaling pathways in osteoclasts. Moreover, TAS-115 inhibited Feline McDonough Sarcoma oncogene (FMS) kinase, as well as M-CSF and receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. Thus, VEGFRs/MET/FMS-triple inhibition in osteoclasts might contribute to the potent efficacy of TAS-115. The fact that concomitant dosing of sunitinib (VEGFRs/FMS inhibition) with crizotinib (MET inhibition) exerted comparable inhibitory efficacy for bone destruction to TAS-115 also supports this notion. In conclusion, TAS-115 inhibited tumor growth via VEGFR-kinase blockade, and also suppressed bone destruction possibly through VEGFRs/MET/FMS-kinase inhibition, which resulted in potent efficacy of TAS-115 in an A549-Luc-BM1 bone disease model. Thus, TAS-115 shows promise as a novel therapy for lung cancer patients with bone metastasis.</p></div

Histological examination after TAS-115 treatment in the bone disease model with A549-Luc-BM1 cells.

<p>(A) Tumor microvessel density (MVD) in the mouse bone disease model after oral administration of TAS-115 (100 mg/kg), cabozantinib (15 mg/kg), crizotinib (100 mg/kg), or sunitinib (40 mg/kg) for 2 weeks. The Y axis indicates MVD in A549-Luc-BM1-implanted tibiae. Data are expressed as means ± SE (n = 4–5). * and **, p<0.05 and p<0.01, respectively, in comparisons of the treated group with the control group, (Dunnett’s test). Scale bar indicates 100 μm. (B) Ki-67 staining of A549-Luc-BM1-implanted tibiae. TAS-115 (200 mg/kg), cabozantinib (15 mg/kg), sunitinib (40 mg/kg), crizotinib (100 mg/kg), and the combination of sunitinib (40 mg/kg) and crizotinib (100 mg/kg) were administered orally for 2 weeks. Data are expressed as means ± SE (n = 5). * and **, p<0.05 and p<0.01, respectively, in the comparison of the treated group with the control group (Dunnett’s test). Scale bar indicates 200 μm. (C) TRAP staining of A549-Luc-BM1-implanted tibiae. TAS-115 (200 mg/kg), cabozantinib (15 mg/kg), sunitinib (40 mg/kg), crizotinib (100 mg/kg), and the combination of sunitinib (40 mg/kg) and crizotinib (100 mg/kg) were administered orally for 2 weeks. The red arrows indicate TRAP-positive osteoclasts. Data are expressed as means ± SE (n = 5). B: Bone, T: Tumor (A549-Luc-BM1). **, p<0.01 in the comparison of the treated group with the control group (Dunnett’s test). Scale bar indicates 100 μm.</p

Histological examination after TAS-115 treatment in the bone disease model with A549-Luc-BM1 cells.

<p>(A) Tumor microvessel density (MVD) in the mouse bone disease model after oral administration of TAS-115 (100 mg/kg), cabozantinib (15 mg/kg), crizotinib (100 mg/kg), or sunitinib (40 mg/kg) for 2 weeks. The Y axis indicates MVD in A549-Luc-BM1-implanted tibiae. Data are expressed as means ± SE (n = 4–5). * and **, p<0.05 and p<0.01, respectively, in comparisons of the treated group with the control group, (Dunnett’s test). Scale bar indicates 100 μm. (B) Ki-67 staining of A549-Luc-BM1-implanted tibiae. TAS-115 (200 mg/kg), cabozantinib (15 mg/kg), sunitinib (40 mg/kg), crizotinib (100 mg/kg), and the combination of sunitinib (40 mg/kg) and crizotinib (100 mg/kg) were administered orally for 2 weeks. Data are expressed as means ± SE (n = 5). * and **, p<0.05 and p<0.01, respectively, in the comparison of the treated group with the control group (Dunnett’s test). Scale bar indicates 200 μm. (C) TRAP staining of A549-Luc-BM1-implanted tibiae. TAS-115 (200 mg/kg), cabozantinib (15 mg/kg), sunitinib (40 mg/kg), crizotinib (100 mg/kg), and the combination of sunitinib (40 mg/kg) and crizotinib (100 mg/kg) were administered orally for 2 weeks. The red arrows indicate TRAP-positive osteoclasts. Data are expressed as means ± SE (n = 5). B: Bone, T: Tumor (A549-Luc-BM1). **, p<0.01 in the comparison of the treated group with the control group (Dunnett’s test). Scale bar indicates 100 μm.</p

Characterization of the mouse bone disease model with A549-Luc-BM1 cells.

<p>(A) Representative pictures of metastatic site difference between parental A549-Luc and A549-Luc-BM1 cells after left cardiac-ventricle implantation into mice. (B) Representative optical changes after intra-tibial implantation of A549-Luc-BM1 cells. (C) Micro-CT images of A549-Luc-BM1 implanted mouse tibia. (D) Hematoxylin and Eosin staining of A549-Luc-BM1 implanted mouse tibia. Each picture was taken from different mice that were selected based on average data at each time point. A549-Luc-BM1 cells caused tumor growth in bone and aberrant bone remodeling after their implantation. Hematoxylin and Eosin staining demonstrated that the tumor protruded from the tibia at 5 weeks after implantation. Scale bar indicates 3 mm.</p

Anti-tumor efficacy of TAS-115 in the bone disease model of A549-Luc-BM1 in mice.

<p>(A) The changes in bioluminescence signals after oral administration of each of TAS-115 (200 mg/kg), cabozantinib (15 mg/kg), sunitinib (40 mg/kg), crizotinib (100 mg/kg), or the combination of sunitinib and crizotinib, or subcutaneous administration of zoledronic acid (0.2 mg/kg) for 4 weeks following intra-tibial implantation of A549-Luc-BM1 cells in mice. Relative total photon flux (RTP) was calculated according to the following formula: RTPn = (TP on each measurement day) / (TP _{day 0}) ×100. Data are expressed as means ± SE (n = 8–9). **, p<0.01 in the comparison of the treated group with the control group (Dunnett’s test). (B) Body weight changes over the 4 weeks of treatment. Data are expressed as means ± SE (n = 8–9).</p

Inhibitory activity of TAS-115 against osteoclast formation and signal transduction in osteoclasts.

<p>(A) Effect of TAS-115, cabozantinib, sunitinib, or crizotinib against M-CSF and RANKL induced osteoclast formation. The Y axis indicates absorbance at 405 nm (A405) that reflects TRAP activity in cells, which was used as an indicator of osteoclast formation. Data are expressed as means ± SD (n = 4). ##, p<0.01 in the comparison of the control group (the cultures treated with RANKL and M-CSF) with the non-treated group (the cultures without RANKL and M-CSF treatment) (Student’s <i>t</i>-test). **, p<0.01 in the comparison of the treated group with the control group (Dunnett’s test). (B) Representative photographs of TRAP-positive cells in the non-treated, control and TAS-115 treated cultures. M-CSF dependent bone marrow macrophages (MDBMs) were cultured for 4 days without RANKL, M-CSF or TAS-115 as the non-treated group. MDBMs were maintained in M-CSF and RANKL without TAS-115 for 4 days as the control group. For TAS-115 treatment, MDBMs were maintained in M-CSF, RANKL and the indicated concentration of TAS-115 for 4 days.</p

Inhibitory activity of TAS-115 against FMS, VEGFR2 and MET expressed in MDBMs.

<p>(A) Effect of TAS-115, cabozantinib, sunitinib, or crizotinib on FMS kinase mediated phosphorylation in MDBMs. MDBMs were cultured for 4 days with M-CSF in plastic dishes. Test compounds were then added at the indicated concentration to the culture media and incubated for 1 hr. Following incubation of MDBMs with hM-CSF and the indicated drugs, cell lysates were then prepared, and analyzed by subsequent Western blotting with the indicated antibodies. (B) Effect of TAS-115 and cabozantinib on ligand induced VEGFR2 and MET phosphorylation in MDBMs. Cell culture and drug treatments were performed as for the experiments with M-CSF. rhHGF (100 ng/mL) or rhVEGF (100 ng/mL) was then added to the culture media, and, following incubation for 10 or 5 min, respectively, cell lysates were prepared. VEGFR2 and its phosphorylated form were detected in MDBMs by Western blotting following immunoprecipitation with an anti-VEGFR antibody. MET and its phosphorylated form were directly detected by Western blotting. Sunitinib and crizotinib were used as positive control agents for VEGFR2 inhibition and MET inhibition, respectively.</p

Kinase inhibitory activity of TAS-115 for FMS, MET and VEGFR2.

<p>Kinase inhibitory activity of TAS-115 for FMS, MET and VEGFR2.</p