Regulation of epidermal growth factor receptor tyrosine kinase inhibitor resistance via Ambra1-mediated autophagy in non-small cell lung cancer.

Abstract

To explore the molecular mechanisms related to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) resistance, along with potential therapeutic targets and strategies. The autophagy and Beclin 1 regulator 1 (Ambra1) short hairpin ribonucleic acid (shRNA) lentivirus vector and Ambra1 overexpression plasmid, constructed with a plasmid cloning deoxyribonucleic acid (pcDNA) 3.1 vector, were used to down-regulate and up-regulate Ambra1 expression in the human lung adenocarcinoma erlotinib-resistant cell line (PC9/ER), respectively, as well as to screen stable transgenic cell lines. The IC50 of Erlotinib in these cell lines were measured to determine their resistance status. The real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure messenger ribonucleic acid (mRNA) expression of resistance-related genes like multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1 (MRP1), and lung drug-resistant-related protein (LRP). Western blot was performed to analyze the protein expressions of the autophagy-related genes Beclin 1, LC3II/I, and p62. Each stable transgenic line formed a tumor under the skin in nude mice; the mice with subcutaneous tumorigenesis of PC9/ER cells and shAmbra1-PC9/ER cells were subsequently treated with rapamycin (RAPA) and chloroquine (CQ), respectively. The mRNA expressions of MDR1, MRP1, and LRP in each tumor tissue sample were detected by qRT-PCR. The protein expressions of adenosine monophosphate-activated protein kinase (AMPK), phosphorylated-AMPK (p-AMPK), forkhead box O3 (FoxO3a), and phosphorylated forkhead box O3 (p-FoxO3a) in the AMPK/FoxO3a signaling pathway were analyzed via Western blot. The qRT-PCR result revealed that the level of Ambra1 in EGFR-TKI-resistant cells had increased. This was further exacerbated by the overexpression of Ambra1 and was reduced after its inhibition. Additionally, Ambra1 upregulated the mRNA expression of drug-resistant genes and the expression of autophagy-related proteins. Subcutaneous tumorigenesis of RAPA-treated shAmbra1-PC9/ER cells resulted in increased expression of drug resistance-related genes and a concomitant decrease in p-AMPK and increase in p-FoxO3a. The results revealed that Beclin-1/β-actin, p62/β-actin, and LC3II/I in the model group were all significantly increased compared to the control group, with P<0.05. Compared to the model group, Beclin-1/β-actin, p62/β-actin, and LC3II/I were all significantly higher in the pcDNA-Ambra1 group, with P<0.05. Compared to the model group, Beclin-1/β-actin, p62/β-actin, and LC3II/I were all significantly decreased in the shAmbra1 group, with P<0.05. Thus, these data suggest that Ambra1 promotes cellular autophagy. In addition, subcutaneous tumorigenesis of CQ-treated shAmbra1-PC9/ER cells resulted in reduced expression of drug resistance-related genes, and a concomitant increase in p-AMPK and decrease in p-FoxO3a. The results of this study revealed that Ambra1-mediated autophagy regulated EGFR-TKI resistance in NSCLC, most probably through the AMPK/FoxO3a signaling pathway.