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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.

SUBMITTER: Zhang M

PROVIDER: S-EPMC8046080 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:OBJECTIVE:To investigate the inhibitory effect of epidermal growth factor receptor tyrosine kinase inhibitor (EGFRTKI) HS-10296 on the proliferation of triple-negative breast cancer (TNBC) MDA-MB-231 cells and explore the possible molecular mechanism. METHODS:MDA-MB-231 cells were treated with HS-10296 for 24, 48, or 72 h, and CCK-8 assay was used to assess the changes in the cell viability. The inhibitory effect of HS-10296 on cell proliferation was determined by clonogenic assay. JC-1 and flow cytometry were employed for analyzing the cell apoptosis, and the ultrastructure of the cells was observed under electron microscope. After pretreatment with autophagy inhibitor chloroquine (CQ), MDA-MB-231 cells were divided into control group, CQ treatment group, HS-10296 (4 and 6 ?mol/L) treatment groups and combined treatment groups, and the sensitivity of the treated cells to HS-10296 was determined using CCK-8 assay. The effects of HS-10296 on EGFR pathway and apoptosis- and autophagy-related proteins in MDA-MB-231 cells were investigated using Western blotting. RESULTS:HS-10296 significantly inhibited the proliferation of MDA-MB-231 cells with IC50 values at 24, 48 and 72 h of 8.393, 2.777 and 2.016 ?mol/L, respectively. JC-1 and flow cytometry showed that HS-10296 induced obvious apoptosis of MDA-MB-231 cells, which showed an apoptosis rate of (21.63 ± 2.97)% following treatment with 8 ?mol/L HS-10296. Autophagy vesicles were observed in the cells treated with HS-10296 under electron microscope. In MDA-MB-231 cells pretreated with CQ, inhibition of autophagy significantly enhanced HS-10296-induced cell death. Western blotting showed that the apoptosis-related protein caspase-3 was activated after HS-10296 treatment to cut its substrate PARP. The expression of autophagy-related protein light chain 3B (LC3B) was significantly enhanced after HS-10296 treatment (P < 0.01), which also resulted in inhibited phosphorylation of EGFR and AKT proteins in the cells. CONCLUSIONS:HS-10296 can inhibit the proliferation and induce autophagy and apoptosis of MDA-MB-231 cells by inhibiting the EGFR/PI3K/AKT signaling pathway.

Project description:BackgroundThe third generation Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor (TKI) osimertinib has been initially approved for T790M positive Non-Small Cell Lung Cancer (NSCLC) and more recently for first-line treatment of EGFR-mutant T790M negative NSCLC patients. Similarly to previous generation TKIs, despite the high response rate, disease progression eventually occurs and current clinical research is focused on novel strategies to delay the emergence of osimertinib resistance. In this study we investigated the combination of osimertinib with pemetrexed or cisplatin in EGFR-mutated NSCLC cell lines and xenografts.MethodsTumor growth was evaluated in a PC9T790M xenograft model and tissue composition was morphometrically determined. PC9, PC9T790M and HCC827 cell lines were employed to test the efficacy of osimertinib and chemotherapy combination in vitro. Cell viability and cell death were evaluated by MTT assay and fluorescence microscopy. Protein expression and gene status were analysed by Western blotting, fluorescence in situ hybridization analysis, next-generation sequencing and digital droplet PCR.ResultsIn xenograft models, osimertinib significantly inhibited tumor growth, however, as expected, in 50% of mice drug-resistance developed. A combination of osimertinib with pemetrexed or cisplatin prevented or at least delayed the onset of resistance. Interestingly, such combinations increased the fraction of fibrotic tissue and exerted a long-lasting activity after stopping therapy. In vitro studies demonstrated the stronger efficacy of the combination over the single treatments in inhibiting cell proliferation and inducing cell death in PC9T790M cells as well as in T790M negative PC9 and HCC827 cell lines, suggesting the potential role of this strategy also as first-line treatment. Finally, we demonstrated that osimertinib resistant clones, either derived from resistant tumors or generated in vitro, were less sensitive to pemetrexed prompting to use a chemotherapy regimen non-containing pemetrexed in patients after progression to osimertinib treatment.ConclusionsOur results identify a combination between osimertinib and pemetrexed or cisplatin potentially useful in the treatment of EGFR-mutated NSCLC patients, which might delay the appearance of osimertinib resistance with long-lasting effects.