BMS-794833 reduces anlotinib resistance in osteosarcoma by targeting the VEGFR/Ras/CDK2 pathway

Abstract

Background: Osteosarcoma, a tumor that originates from bone cells, has a poor prognosis and a high degree of malignancy. Anlotinib, a small-molecule multi-target tyrosine kinase inhibitor (TKI), is the first-line drug in treating osteosarcoma, especially in late-stage osteosarcoma. However, patients often develop resistance after using anlotinib for a certain period, which poses a challenge to its further clinical application. Recently, several TKIs, for instance regorafenib and cabozantinib, have showed clinical interest in treating osteosarcoma and target both vascular endothelial growth factor receptor (VEGFR) and mesenchymal epithelial transition factor (c-MET). Therefore, the identification of new TKI warrants further investigation.

Methods: We performed CCK8 aasays to confirm that BMS-794833 sensitization osteosarcoma cells to anlotinib. Bioinformatics analysis and rescue experiments showed that the reduce of resistance were dependent on the VEGFR/Ras/CDK2 pathway. Cell line based xenograft model were used to demonstrate that BMS-794833 and anlotinib could synergistically treat OS.

Results: Here, we found that BMS-794833 reduced anlotinib resistance in osteosarcoma by targeting the VEGFR/Ras/CDK2 pathway. CCK8 assay showed that BMS-794833 significantly improved the resistance of osteosarcoma cells to anlotinib. The results of rescue experiments showed that the regulatory effects of BMS-794833 on the proliferation and drug resistance of osteosarcoma cells were dependent on the VEGFR/Ras/CDK2 pathway. In addition, BMS-794833 affected the resistance of osteosarcoma cells to anlotinib through epithelial-mesenchymal transition (EMT) and apoptosis pathways. More importantly, BMS-794833 and anlotinib exerted synergistic therapeutic effects against osteosarcoma in vivo.

Conclusion: Altogether, this study reveals a new (VEGFR)-targeting drug that can be combined with anlotinib for the treatment of osteosarcoma, which provides an important theoretical basis for overcoming anlotinib resistance.

Keywords: Anlotinib; CDK2; Osteosarcoma; Synergy effect; VEGFR.

Fig. 1

BMS-794833 could enhance the sensitivity of osteosarcoma to Anlotinib. (A-B) The U2OS and MG63 cells were treated with DMSO, BMS-794833, hVEGF-IN-1, K00546 and PD173074 respectively, and the viability of the cells was detected by CCK8 assay, and the IC50 value was calculated. All experiments carried out in triplicate. *P < 0.05 and **P < 0.01 comparing with control group with t-Test in GraphPad Prism 8.0 software.

Fig. 2

BMS-794833 inhibits the proliferation of osteosarcoma cells. (A) The volcano map showed the differential genes between DMSO treated group and BMS-794833 treated group. (B) A bubble plot of GSEA enrichment analysis. (C-D) U2OS and MG63 cells were treated with BMS-794833 at the shown concentrations (10 μM, 20 μM) for 24 h, and the proliferation of the cells was detected by clonal formation and CCK8 assay. In the control group, cells were treated with DMSO for the same time. All experiments carried out in triplicate. *P < 0.05 and **P < 0.01 comparing with control group with t-Test in GraphPad Prism 8.0 software.

Fig. 3

BMS-794833 inhibits osteosarcoma cell proliferation through the VEGFR/Ras/CDK2 axis. (A) U2OS and MG63 cells were treated with BMS-794833 at the shown concentrations (10 μM, 20 μM) for 24 h, and the expression levels of CDK2 protein and nucleic acid were detected by western blot assay and RT-PCR. (B) The volcano map showed the differential genes of the high and low expression CDK2 groups. (C) Venn diagram shows overlapping genes. DMS-794833 and DMSO were used to treat U2OS and MG63 cells transfected with shNC or shCDK2, and cell proliferation was measured by colony formation (D) and CCK8 (E). All experiments carried out in triplicate. *P < 0.05 and **P < 0.01 comparing with control group with t-Test in GraphPad Prism 8.0 software.

Fig. 4

BMS-794833 may affect the resistance of osteosarcoma to anlotinib through EMT and apoptosis pathways. (A) GSEA pathway enrichment map. (B) U2OS and MG63 cells were treated with BMS-794833 containing the shown concentrations (10 μM, 20 μM) for 24 h, and apoptosis was detected by flow cytometry. (C) Cell migration was detected by transwell assay. In the control group, cells were treated with DMSO for the same time. All experiments carried out in triplicate. *P < 0.05 and **P < 0.01 comparing with control group with t-Test in GraphPad Prism 8.0 software.

Fig. 5

BMS-794833 and Anlotinib synergistically inhibit the malignant progression of osteosarcoma cells in vitro. (A-B) 2D visualization of synergy between BMS-794833 and anlotinib at various concentrations in two different osteosarcoma cell cultures (24 h treatment) by combenefit2. (C-D) Clonal formation assay of MG63 and U2OS cells treated with BMS-794833 and anlotinib, either alone or in combination. (E-F) CCK8 assay of MG63 and U2OS cells treated with BMS-794833 and anlotinib, either alone or in combination. All experiments carried out in triplicate. *P < 0.05 and **P < 0.01 comparing with control group with t-Test in GraphPad Prism 8.0 software.

Fig. 6

BMS-794833 and Anlotinib synergistically inhibit the malignant progression of osteosarcoma cells in vivo. Mice were treated with DMSO, anlotinib, BMS-794833, and a combination of anlotinib and BMS-794833. (A) Visible tumor formation and photographs of representative tumors removed from mice after treatment. Tumor volume (B) and tumor weight (C) changes in mice were examined every 4 days for 28 days during treatment. (D) The expression of Ki67 in xenografted tumor tissues of nude mice was observed by immunohistochemical staining (20×, 200 × ). Data are expressed as the mean ± SD of three independent experiments.