Yifei Sanjie pill combined with gefitinib reduces the progression of EGFR-TKIs-resistant non-small cell lung cancer via YAP/ANKRD1 axis

Background: Resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) is a significant obstacle in non-small cell lung Cancer (NSCLC) treatment. Yifei Sanjie pill (YFSJ), a traditional Chinese herbal remedy, has been found to enhance gefitinib's effectiveness in NSCLC. However, the exact mechanisms by which YFSJ helps overcome EGFR-TKIs resistance are not yet fully understood.

Purpose: This study uses in vitro, in vivo, systems biology, and single-cell Sequencing approaches to explore the pharmacological pathways behind the combined outcomes of YFSJ and gefitinib in treating EGFR-TKIs-resistant NSCLC.

Methods: UPLC-MS/MS was employed to identify the active constituents of YFSJ and to perform quality control analysis. We assessed the effects of gefitinib and YFSJ, both individually and in combination, on NSCLC cell viability using the MTT assay. Colony formation and wound healing assays were conducted to assess clonogenic potential and migration. A xenograft tumor model in BALB/c nude mice was used to examine the agents' inhibitory effects, in vivo. RNA Sequencing and systems biology analysis of tumor tissues identified differentially expressed genes (DEGs) and pathways linked to the combined administration. Candidate gene expression was validated by qPCR, and the clinical relevance of Ankyrin Repeat Domain 1 (ANKRD1) in NSCLC was confirmed using data from the TCGA-NSCLC cohort. Western blotting was utilized for the purpose of validating the protein expression of EGFR, p-EGFR, ERK1/2, p-Erk1/2, Akt, p-AKT, ANKRD1, Ki67, Cleaved Caspase 3 and the Yes-associated protein1 (YAP1) signaling pathway in H1975 xenograft tumors. Single-cell Sequencing and cell-cell communication analysis revealed the association between ANKRD1 and EGFR activation in four types of EGFR-mutant NSCLC, as well as the underlying mechanisms.

Result: UPLC‑MS/MS analysis of YFSJ detected 1624 compounds in total, with 1177 confidently identified via MS/MS. Gefitinib and YFSJ as single agents inhibited the cell viability of NSCLC. YFSJ showed the least efficacy in inhibiting HCC827 cells, while it exhibited the best efficacy against H1975 cells. The IC₅₀ values of the combination therapy were lower than those of each drug used individually, and when administered together for 72 h in H1975 cells and 24 h in HCC827 cells, YFSJ and gefitinib exhibited synergistic effects. The combination therapy suppressed both the clonogenic ability and the combined processes of migration and proliferation in H1975 cells. In vivo experiments demonstrated that YFSJ (9.6 g/kg/day) in combined with gefitinib (50 mg/kg/day) markedly suppressed proliferation in H1975-derived xenografts. Integrated analysis suggested that ANKRD1 is a promising gene potentially associated with acquired EGFR-TKIs resistance, and down-regulate this gene may benefit patient survival. In vivo experiments, single-cell Sequencing and cell-cell communication analysis confirmed that the high expression of ANKRD1 might be related to EGFR exon 20 mutation-driven resistant NSCLC, potentially linked to ECM receptor-mediated signal transduction. YFSJ combined with gefitinib treatment inhibits EGFR and ERK1/2 phosphorylation, promoting YAP1 phosphorylation, inhibits Akt phosphorylation and downstream ANKRD1 expression, ultimately suppressing cell proliferation and promoting Apoptosis.

Conclusion: Co-administration of YFSJ and gefitinib suppresses the growth and migration of NSCLC. ANKRD1 may be a potential biomarker for EGFR exon 20 mutation-driven resistance. This therapy reduces resistance to first-generation EGFR-TKIs by blocking YAP/ANKRD1 axis to suppress cell proliferation and promoting Apoptosis. Based on our previous clinical investigations and the present preclinical findings, YFSJ-particularly in combination with gefitinib-may represent a novel therapeutic strategy and warrants further exploration for the treatment of NSCLC resistant to EGFR exon 20 mutations.

Ankyrin repeat domain 1; EGFR exon 20 mutation; EGFR-TKIs resistance; Gefitinib; Non-small cell lung cancer; YAP1; Yifei Sanjie pill.