Combination of betulinic acid and EGFR-TKIs exerts synergistic anti-tumor effects against wild-type EGFR NSCLC by inducing autophagy-related cell death via EGFR signaling pathway

Abstract

Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of lung cancer patients with mutated EGFR. However, the efficacy of EGFR-TKIs in wild-type EGFR tumors has been shown to be marginal. Methods that can sensitize EGFR-TKIs to EGFR wild-type NSCLC remain rare. Hence, we determined whether combination treatment can maximize the therapeutic efficacy of EGFR-TKIs.

Methods: We established a focused drug screening system to investigate candidates for overcoming the intrinsic resistance of wild-type EGFR NSCLC to EGFR-TKIs. Molecular docking assays and western blotting were used to identify the binding mode and blocking effect of the candidate compounds. Proliferation assays, analyses of drug interactions, colony formation assays, flow cytometry and nude mice xenograft models were used to determine the effects and investigate the molecular mechanism of the combination treatment.

Results: Betulinic acid (BA) is effective at targeting EGFR and synergizes with EGFR-TKIs (gefitinib and osimertinib) preferentially against wild-type EGFR. BA showed inhibitory activity due to its interaction with the ATP-binding pocket of EGFR and dramatically enhanced the suppressive effects of EGFR-TKIs by blocking EGFR and modulating the EGFR-ATK-mTOR axis. Mechanistic studies revealed that the combination strategy activated EGFR-induced autophagic cell death and that the EGFR-AKT-mTOR signaling pathway was essential for completing autophagy and cell cycle arrest. Activation of the mTOR pathway or blockade of autophagy by specific chemical agents markedly attenuated the effect of cell cycle arrest. In vivo administration of the combination treatment caused marked tumor regression in the A549 xenografts.

Conclusions: BA is a potential wild-type EGFR inhibitor that plays a critical role in sensitizing EGFR-TKI activity. BA combined with an EGFR-TKI effectively suppressed the proliferation and survival of intrinsically resistant lung cancer cells via the inhibition of EGFR as well as the induction of autophagy-related cell death, indicating that BA combined with an EGFR-TKI may be a potential therapeutic strategy for overcoming the primary resistance of wild-type EGFR-positive lung cancers.

Keywords: Autophagic cell death; Betulinic acid; Cell cycle arrest; Combination therapy; EGFR-TKIs; Non-small cell lung cancer; Primary drug resistance; Wild-type EGFR.

Fig. 1

BA bound and inhibited the activation of wt-EGFR in lung cancer cells. A The BA-binding mode for wt-EGFR. BA is shown as sticks (C, yellow; O, red), and EGFR is depicted in cartoon representation with key residues indicated as green sticks and labeled. Hydrogen bonds are shown as yellow lines. B 2D diagram representation of BA in the wt-EGFR binding site using Discovery Studio Visualizer. C Docking study of BA with EGFR^WT, EGFR^L858R, and EGFR^T790M. D BA specifically inhibited the phosphorylation of tyrosine residue 1068 on wt-EGFR and its downstream signaling in the A549 and H1299 cell lines

Fig. 2

BA at a low dose synergistically enhanced the cytotoxicity of EGFR-TKIs against NSCLC cells expressing wt-EGFR. A and B Heatmaps of drug combination responses. BA combined with gefitinib (A) or osimertinib (B) acted synergistically on A549 and H1299 cells. BA and gefitinib/osimertinib at the indicated concentrations were used to treat cells for 48 h, and cell viability was assessed by a CCK-8 assay. ZIP synergy scores were calculated using SynergyFinder software. Scores > 10 were considered to indicate strong synergistic effects. The gradation of the red regions indicates the intensity of synergism. Mean ± SD, n = 3. C and D The combination of BA with gefitinib (C) or osimertinib (D) synergistically inhibited the growth of A549 and H1299 cells. The data (mean ± SD, n = 6) were analyzed using Student’s t test. **** p < 0.0001. E and F Colony formation assays showed that the combination of BA with gefitinib (E) or osimertinib (F) decreased the number of colonies. The data (mean ± SD, n = 3) were analyzed using Student’s t test. **** p < 0.0001. G and H Photographs showing morphological changes in A549 (G) and H1299 (H) cells after the indicated treatment for 24 h

Fig. 3

Combination treatment induces autophagy and autophagy-related cell death via AKT-mTOR pathway. A and B A549 and H1299 cells were treated with BA, gefitinib and osimertinib alone or in combination for 48 h. Whole-cell lysates were extracted, and the LC3, p62 and Beclin1 proteins were detected by immunoblotting. GAPDH was used as internal control. C and D A549 and H1299 cells were treated with the indicated supplements for 48 h in the absence or presence of chloroquine (10 µM) or 3-MA (5 mM). LC3, p62 and Beclin1 proteins were detected by immunoblotting. GAPDH was used as an internal control. E and F A549 and H1299 cells were treated with the indicated supplements for 48 h in the absence or presence of 3-MA (5 mM), and cell apoptosis was analyzed by flow cytometry using Annexin V-FITC and PI cell apoptosis kits. Representative images are shown. Quantification of flow cytometric analysis of apoptosis. ** p < 0.01, *** p < 0.001, **** p < 0.0001. G and H Western blot analysis of the effect of mTOR on autophagy markers in A549 and H1299 cells treated with the combination strategy. Cells were treated with the indicated supplements for 48 h in the absence or presence of 3BDO (100 µM) or MHY1485 (10 µM). LC3, p62 and Beclin1 proteins were detected by immunoblotting. GAPDH was used as internal control. CQ, chloroquine. DMSO was used for the control group

Fig. 4

Combination treatment arrested cell cycle via EGFR-AKT-mTOR pathway. A and B Effect of the combination treatment on the cell cycle distribution of A549 and H1299 cells. All the data are presented as the means ± SDs (n = 3, * p < 0.05, *** p < 0.001, **** p < 0.0001). C and D Effect of the combination treatment on the protein levels of cell cycle markers. A549 and H1299 cells were treated with the indicated supplements for 48 h. Western blot assays were conducted with specific antibodies against Cyclin D1, CDK4 and Cyclin B1. GAPDH was used as internal control. E and F A549 and H1299 cells were treated with the indicated supplements for 48 h in the absence or presence of chloroquine (10 µM) or 3-MA (5 mM). Cyclin D1 and CDK4 proteins were detected by immunoblotting. GAPDH was used as internal control. G and H Western blot analysis of the effect of mTOR on autophagy markers in A549 and H1299 cells treated with the combination strategy. Cells were treated with the indicated supplements for 48 h in the absence or presence of 3BDO (100 µM) or MHY1485 (10 µM). Western blot assays were conducted with specific antibodies against Cyclin D1 and CDK4. GAPDH was used as internal control. I and J A549 and H1299 cells were treated with MHY1485 (10 µM) for 48 h in the presence of the indicated supplements. The cell cycle distribution was analyzed by flow cytometry. All the data are presented as means ± SDs (n = 3, * p < 0.05 ** p < 0.01, **** p < 0.0001)

Fig. 5

Combination treatment enhanced the suppression of EGFR and its downstream signaling and bypass pathways in wt-EGFR NSCLC cells. A549 and H1299 cells were treated with DMSO, BA, gefitinib, osimertinib or the indicated combination and then harvested for preparation of whole-cell protein lysates and subsequent western blotting to detect the indicated proteins

Fig. 6

Combination treatment suppressed A549 xenograft growth in immunodeficient mice. A The body weight was quantified in each group. B-D Combination treatment represses tumor growth, as estimated by the tumor size (B), tumor volume (C), and tumor weight (D). E H&E staining of tumors and immunohistochemical staining of Ki67 and LC3 (scale bar, 100 µm) in the tumors