Hepatic nuclear factor 4 alpha promotes the ferroptosis of lung adenocarcinoma via transcriptional activation of cytochrome P450 oxidoreductase

Abstract

Background: Lung adenocarcinoma is one of the most prevalent cancers while ferroptosis is crucial for cancer therapies. This study aims to investigate the function and mechanism of hepatic nuclear factor 4 alpha (HNF4A) in lung adenocarcinomas' ferroptosis.

Materials and methods: HNF4A expression in ferroptotic A549 cells was detected. Then HNF4A was knocked down in A549 cells while overexpressed in H23 cells. Cells with changed HNF4A expression were tested for cytotoxicity and the level of cellular lipid peroxidation. The expression of cytochrome P450 oxidoreductase (POR) expression was examined after HNF4A was knocked down or overexpressed. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) and dual-luciferase assays were performed to validate the regulation of HNF4A on POR. Finally, POR was restored in HNF4A-altered cells to check whether it restores the effect of HNF4A on ferroptosis.

Results: We found that HNF4A expression significantly decreased in the ferroptosis of A549 cells, and this change can be blocked by deferoxamine, an inhibitor of ferroptosis. Knockdown of HNF4A inhibited ferroptosis in A549 cells while overexpression of HNF4A promoted ferroptosis in H23 cells. We identified a key ferroptosis-related gene, POR serves as a potential target gene of HNF4A, whose expression was significantly changed in lung adenocarcinoma cells knocking down or overexpressing HNF4A. We demonstrated that HNF4A was bound to the POR's promoter to enhance POR expression, and identified the binding sites via ChIP-qPCR and luciferase assays. Restoration of POR expression blocked the promoting effect of HNF4A on ferroptosis in lung adenocarcinoma.

Conclusion: HNF4A promotes POR expression through binding to the POR's promoter, and subsequently promotes the ferroptosis of lung adenocarcinoma.

Keywords: Ferroptosis; HNF4A; Lung adenocarcinoma; POR; Transcript factor.

Figure 1. HNF4A expression significantly decreased in ferroptotic A549 cells.

(A) The HNF4A expression in A549 cells treated with RSL3 and IKE. (B) qRT-PCR and Western blot (C) results of HNF4A expression in A549 cells after the treatment of RSL3 and IKE, with or without DFO. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2. HNF4A promoters ferroptosis and POR’s expression in lung adenocarcinoma.

(A) The expression of HNF4A in different lung adenocarcinoma cell lines. (B) qRT-PCR and (C) Western blot results of HNF4A and POR expression in HNF4A knocked down A549 and HNF4A overexpressed H23 cells. (D) Cytotoxicity of RSL3 and IKE, and (E) the level of cellular lipid peroxidation in cells with knocked down or overexpressed HNF4A. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. HNF4A promoted POR expression via binding to POR’s promoter.

(A) HNF4A binding peaks in POR’s promoter according to the ChIP-seq data in the Encode database. (B) The locations of HNF4A’s potential binding sites and primers in POR’s promoter. (C) The ChIP-qPCR results. (D) the sequences of HNF4A’s binding sites and corresponding mutated sequences in the luciferase assays. (E) The results of dual-luciferase assays. ns, not significant; **p < 0.01, ***p < 0.001.

Figure 4. POR restoration blocked HNF4A’s promotion on ferroptosis in lung adenocarcinoma.

(A) qRT-PCR and (B) western blot results of POR restoration in cells with knocked down or overexpressed HNF4A. (C) The cytotoxicity of ferroptosis inducers and (D) the level of cellular lipid peroxidation in HNF4A-altered cells after POR was restored. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.