Third-generation EGFR inhibitor HS-10296 in combination with famitinib, a multi-targeted tyrosine kinase inhibitor, exerts synergistic antitumor effects through enhanced inhibition of downstream signaling in EGFR-mutant non-small cell lung cancer cells

Abstract

Background: As a highly heterogeneous disease, lung cancer has a multitude of cellular components and patterns of gene expression which are not dependent on a single mutation or signaling pathway. Thus, using combined drugs to treat lung cancer may be a practical strategy.

Methods: The combined antitumor effects of HS-10296, a third-generation EGFR inhibitor targeting EGFR T790M mutation, with the multitargeted tyrosine kinase inhibitor (TKI) famitinib in non-small cell lung cancer (NSCLC) were evaluated by in vitro methods such as cell proliferation, apoptosis, angiogenesis assays, and in vivo animal efficacy studies.

Results: Famitinib strengthened the effects of HS-10296 on inhibiting proliferation and inducing apoptosis of NSCLC cells, possibly by synergistic inhibition of AKT and ERK phosphorylation. Meanwhile, HS-10296 significantly potentiated the effects of famitinib on inhibiting the proliferation and migration of HUVEC, which may be through synergistic inhibition of ERK phosphorylation in HUVEC, suggesting that HS-10296 may improve the inhibition of angiogenesis by famitinib. Moreover, combination of HS-10296 and famitinib exerted synergistic antitumor activity in NCI-H1975 and PC-9 xenograft models, and this effect may be accomplished by synergistic inhibition of phosphorylation of AKT and ERK and tumor angiogenesis in tumor tissues.

Conclusions: Collectively, our results indicate that HS-10296 and famitinib exhibit significant synergistic antitumor activity, suggesting that the third-generation EGFR inhibitor combined with VEGFR inhibitor provides a promising strategy in the treatment of EGFR-mutant NSCLC.

Keywords: EGFR-mutant non-small cell lung cancer; HS-10296; famitinib; multi-targeted tyrosine kinase inhibitor; third-generation EGFR inhibitor.

FIGURE 1

HS‐10296 combined with famitinib synergistically inhibits cell proliferation and induces apoptosis in NSCLC cell lines. (a) NCI‐H1975 and PC‐9 cells were seeded in 96 well plates overnight and then treated with different concentrations of HS‐10296 and famitinib alone or in combination. The cell viability was detected by SRB assay after treatment for 72 h (n = 3; error bars denote SD). The combination index (CI) was calculated with CalcuSyn software. (b) NCI‐H1975 and PC‐9 cells were treated with HS‐10296 and famitinib alone, or in combination at the indicated concentrations for 48 h. Total cell lysates were subjected to immunoblotting with anti‐PARP and anti‐caspase‐3 antibodies

FIGURE 2

HS‐10296 combined with famitinib synergistically inhibits AKT and ERK phosphorylation in NSCLC cell lines. NCI‐H1975 (a) and PC‐9 (b) cells were treated with HS‐10296 and famitinib alone or in combination at indicated concentrations for 3 h. Total cell lysates were analyzed by Western blotting using the indicated antibodies

FIGURE 3

HS‐10296 combined with famitinib synergistically inhibits HUVEC proliferation and migration stimulated with VEGF and EGF. (a) HUVECs were treated with different concentrations of HS‐10296 and famitinib alone or in combination for 72 h, and cell viability was determined by CCK8 assay (n = 3; error bars denote SD). (b) HUVECs were treated with HS‐10296 (2 μM), famitinib (10 μM or 1 μM), HS‐10296 plus famitinib (2 μM + 10 μM or 2 μM +1 μM), and VEGF (50 ng/ml) and EGF (20 ng/ml) for 12 h. then, HUVEC migration was measured by transwell migration assay. The migrated cells from three random fields of vision (scale bar, 100 μm) were counted and analyzed using a two‐tailed Student's t‐test. **p < 0.01, ***p < 0.001. (c) Serum starved HUVECs were treated with HS‐10296, famitinib alone or in combination at indicated concentrations for 1.5 h. VEGF (50 ng/ml) and EGF (20 ng/ml) was added 5 min before harvest. Cells were harvested and lysed, and total cell lysates were analyzed by Western blotting using the indicated antibodies. Abbreviations: HS: HS‐10296, Fa, famitinib

FIGURE 4

Combined HS‐10296 and famitinib exerts synergistic antitumor efficacy in NSCLC xenografts. (a) NCI‐H1975 and PC‐9 tumor‐bearing mice received vehicle, HS‐10296 and famitinib alone or in combination. Tumor volumes were measured and bodyweights were determined. Tumor volumes and bodyweights are presented as means ± SD. ** p < 0.01 versus the 10 mg/kg famitinib group; ## p < 0.01 versus the 3 mg/kg HS‐10296 group; $ p < 0.05 versus the 5 mg/kg HS‐10296 group; $$ p < 0.01 versus the 1 mg/kg HS‐10296 group. (b) Tumor vascularization was significantly suppressed according to CD31 staining. MVD in the vehicle, HS‐10296 (5 mg/kg), famitinib (10 mg/kg), and HS‐10296 plus famitinib (5 + 10 mg/kg) groups were measured by immunohistochemistry. Scale bar 100 μm. *p < 0.05, **p < 0.01. (c) Mice were sacrificed 10 h after the last treatment with vehicle, 3 mg/kg HS‐10296, 10 mg/kg famitinib, or HS‐10296 plus famitinib, and tumors were removed and analyzed by Western blotting