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. 2019 Sep 23;9(24):7122–7139. doi: 10.7150/thno.35729

Figure 7.

Figure 7

The RCS-ROS metabolic enzymes are activated by ALDH1A1 and ALDH1A1 confers resistance to erlotinib via the ROS-RCS metabolic pathway in vivo. (A-C) ALDH1A1 suppression downregulated GPX4 and SOD2 mRNA (A) and protein (B and C) expression levels by treatment with the siRNA (A and B) or the inhibitor (C). The cells were transfected with ALDH1A1 or control mock siRNA for 72 h or treated with 100 μM DSF for 6 h. (D) Conditional induction of ALDH1A1 ecto-expression in HCC827 cells upregulated GPX4 and SOD2 assayed by western blot analysis. DOX, doxycycline. (E-F) SOD2 was downregulated by knockdown or inhibition of GPX4 via siRNA (E) or inhibitor RSL3 (F), respectively, in HCC827-ER5 cells. The cells were transfected with 20 nM GPX4 or mock control siRNA for 72 h, or 100 nM RSL3 for 6 h. (G) Analysis of GPX4 protein levels after SOD2 knockdown in HCC827-ER5 cells. The cells were transfected with 20 nM SOD2 or mock control siRNA for 72 h. (H-I) Knockdown of ALDH1A1 suppressed the promoter activity of GPX4 (H) and SOD2 (I), and knockdown of GPX4 suppressed the promoter activity of SOD2 (J) as analyzed by the dual-luciferase assay for target gene promoter activity. The cells were transfected with 20 nM siRNA for 48 h. M, mock. (K) Kaplan-Meier analysis of the association between the probability of overall survival (OS) of lung adenocarcinoma patients who were received chemotherapy and their ALDH1A1, GPX4, and SOD2 gene co-expression profiles. The analysis was performed by using the online KM-plotter tool (http://kmplot.com/analysis/index.php?p=service&cancer=lung). Low or high levels of genes in tumor samples were defined as higher or lower than the median value of 36 patients. In KM-plotter tool database, the number of lung adenocarcinoma patients who were received chemotherapy is 36. The number of patients whose tumors displayed high expression of all ALDH1A1, GPX4, and SOD2 is 12 and the number of patients with low expression of all three genes is 11. (L-M) DOX-induced ALDH1A1 conferred HCC827 cell-derived xenograft tumor resistance to erlotinib, and this effect was abrogated by GSH synthesis inhibitor BSO. ALDH1A1 in the cells was induced by the Tet-On system for doxycycline-inducible gene expression as described in the Materials and Methods. The mice with subcutaneously implanted tumors were treated with erlotinib (30 mg/kg, qd, po), doxycycline (50 mg/kg, qd, po), BSO (450 mg/kg, qod, ip), or their combinations as indicated. The weight of resected tumors was measured at the end of the experiments. Tumor volumes (2 per mouse) are presented as the mean ± SEM from five mice per group. CTR, vehicle control; Erlo, erlotinib; DOX, doxycycline.