HNF4G increases cisplatin resistance in lung adenocarcinoma via the MAPK6/Akt pathway

Cell culture

The Chinese National Collection of Authenticated Cell Cultures (Shanghai, China) provided two human lung adenocarcinoma cell lines, A549 and HCC827, as well as human embryonic kidney cell line 293T, which were cultured in high-glucose Dulbecco’s modified Eagle’s medium (KeyGEN, Nanjing, Jiangsu, China) with 10% fetal bovine serum (Every Green, Huzhou, Zhejiang, China), 100 U/mL penicillin (Beyotime, Shanghai, China), and 0.1mg/ml streptomycin (Beyotime).

Lentiviruses and compounds

Lentivirus vectors of HNF4G-Flag, sh-HNF4G-1 (5′-GCACCAGAAGAAGCACATTTG-3′), sh-HNF4G-2 (5′-GCACATTTGATGGCAGCAACA-3′), and corresponding negative control were obtained from GENECHEM (Shanghai, China), transfected into A549, HCC827, and 293T cells, and then screened by puromycin (Beyotime).

Cisplatin and MK-2206 were both obtained from TOPSCIENCE (Shanghai, China).

Detection of cisplatin sensitivity

To detect the cisplatin sensitivity of cells, 4,000 cells were seeded into 96-well plates and then treated with 0, 1, 2, 4, 8, and 16 uM cisplatin. After 48 h, alamaBlue (YEASEN, Shanghai, China) was added, and the fluorescence was monitored at 545 nm excitation and 590 nm emission wavelengths to detect cell viability.

Quantitative real-time PCR (qRT-PCR)

As previously described (Liang et al., 2022), total RNA was extracted using TRNzol Universal (TIANGEN, Beijing, China), and cDNA was synthesized using Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (YEASEN). Hieff® qPCR SYBR Green Master Mix (Low Rox Plus) (YEASEN) was used for qPCR. The sequences of primers were as follows: HNG4F-F:5′-CAACGGTGTCAACTGTCTGTG-3′, HNG4F-R:5′- AAACGTGACTCTTACGAATGCT-3′ MAPK6-F: 5′-ACGGAAGACTTGGTGCTGAAGAT-3′, MAPK6-R: 5′-TACGCTGAGAAGCTCCTGACGAT-3′, β-actin-F: 5′-TGACGTGGACATCCGCAAAG-3′, β-actin-R: 5′-CTGGAAGGTGGACAGCGAGG-3′.

Western blot assay

RIPA Lysis Buffer (Beyotime, Shanghai, China), Protease Inhibitor Cocktail (TOPSCIENCE), and Phosphatase Inhibitor Cocktail III (TOPSCIENCE) were used to extract proteins. SDS-PAGE was used to separate protein samples, which were then transferred to polyvinylidene fluoride membranes. HNF4G (1:3000, PP-N3224-00; R&D System, Minneapolis, MN, US), MAPK6 (1:3000, abs152150; Absin, Shanghai, China), p-MAPK6 (1:1000, AF7407; Affinity, Liyang, Jiangsu, China), Akt (1:3000, CY5561; Abways, Shanghai, China), p-Akt (1:1000, AP0637; Abclonal, Wuhan, China), GAPDH (1:3000, AG019; Beyotime) antibodies were used to detect the specific protein bands.

Chromatin immunoprecipitation (ChIP) assay

ChIP was performed using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) (CST, Boston, MA, US) according to the manufacturer’s instructions. Flag-Tag Antibody (1:100; CST) was used to pull down the HNF4G’s binding chromatin in HNF4G-Flag overexpressing cells. The sequences of primers used in ChIP-qPCR were as follows: primer_1F: 5′-GCTAGGATTACAGGCGTGAACCA-3′, primer_1R: 5′-GCTAGGATTACAGGCGTGAACCA-3′, primer_2F: 5′-ACCATCTTGCTGAGAACCAGACA-3′, primer_2R: 5′- CGGAGGAGCGGATATAACCAGGA-3′, primer_3F: 5′-TGTCGCCTTCCAGCCAATCCA-3′, primer_3R: 5′-CCCAGTGCCAGCGTTTACTAAGC-3′.

Dual-luciferase reporter assay

The MAPK6 promoter sequences (NM 002748.4) and the corresponding mutated sequences on the predicted target sites of HNF4G were cloned into the firefly luciferase reporter plasmids by GENECHEM. These plasmids were then co-transfected with renilla luciferase reporter plasmids into HNF4G overexpressed or knockdown 293T cells. Dual-luciferase reporter assays were performed 48 h after transfection using the Luciferase Reporter Gene Assay Kit (Beyotime).

Bioinformatics and statistical analyses

The cisplatin sensitivity and the gene expression data of cell lines were obtained from the Cancer Therapeutics Response Portal (CTRP) website (https://portals.broadinstitute.org/ctrp/) and the Cancer Cell Line Encyclopedia (CCLE) website (https://sites.broadinstitute.org/ccle/) (Rees et al., 2016; Barretina et al., 2012). The correlation between HNF4G and MAPK6 in the lung adenocarcinoma dataset of The Cancer Genome Atlas (TCGA) was obtained from the GEPIA website (http://gepia.cancer-pku.cn/) (Tang et al., 2017). Survival analysis of HNF4G expression in lung adenocarcinoma patients who received chemotherapy was performed using the Kaplan–Meier Plotter website (https://kmplot.com/analysis/) (Gyorffy, 2021). ChIP-Seq data of HNF4G were obtained from the Encode website (https://www.encodeproject.org) (Luo et al., 2020) and the JASPAR website (https://jaspar.genereg.net) (Castro-Mondragon et al., 2022) was used to predict the binding sites of HNF4G.

Student’s t-test and ANOVA test were performed to compare variables between groups using R software (version 4.0.3). The p values <0.05 were considered significant.

High expression of HNF4G is significantly positively correlated with cisplatin resistance in bioinformatic analyses

When we examined the relationship between cisplatin resistance and gene expression in 542 cell lines from the CTRP and CCLE databases, we discovered that cells with high HNF4G expression were more resistant to cisplatin. As shown in Fig. 1A, the half maximal inhibitory concentrations (IC50s) of the 50 cell lines with the highest HNF4G expression against cisplatin were significantly higher than the IC50s of the 50 cell lines with the lowest HNF4G expression against cisplatin. Simultaneously, HNF4G expression in the 50 most cisplatin-resistant cell lines was significantly higher than in the 50 most cisplatin-sensitive cell lines (Fig. 1B).

Meanwhile, we examined the prognosis of lung adenocarcinoma patients receiving chemotherapy in the Kaplan–Meier Plotter database and discovered that patients with high HNF4G expression had a worse prognosis, despite the fact that the p-value was not significant, which may be due to the small sample size (Fig. 1C).

HNF4G promotes resistance to cisplatin in lung adenocarcinoma cells

We then examined the expression of HNF4G in the lung adenocarcinoma cell lines A549 and HCC827 after treatment with different concentrations of cisplatin using qRT-PCR (Fig. 2A) and western blot (Fig. 2B), and discovered that it was significantly increased after cisplatin treatment. Using lentivirus, we overexpressed and knocked down HNF4G expression in A549 and HCC827 cells, respectively (Fig. 2C). Two different shRNA sequences were used to reduce the impact of off-target effects on the experimental results. Our results showed that overexpression of HNF4G significantly enhanced the resistance of A549 and HCC827 cells to cisplatin, whereas knockdown of HNF4G had the opposite effect (Fig. 2D). According to the findings, HNF4G significantly increased the drug resistance of lung adenocarcinoma cells to cisplatin.

HNF4G promotes MAPK6 expression and MAPK6/Akt signaling pathway

HNF4G, as a transcription factor, may bind in the promoter regions of some cisplatin resistance genes to regulate their expression and resistance. We searched the Encode database for possible downstream factors of HNF4G in the ChIP-Seq data, looking for the target genes associated with cisplatin resistance. We discovered that there are several binding peaks of HNF4G in the −1000 bp∼200 bp promoter region upstream of the transcription start site (TSS) of MAPK6 (Fig. 3A), while previous studies have shown that MAPK6 phosphorylates the S473 site of Akt to activate Akt, and the activated significantly promotes the resistance of cells to cisplatin (Cai et al., 2021; Zhang et al., 2022; Zhou et al., 2022). Meanwhile, the RNA-Seq data from the TCGA database revealed that the expression of HNF4G was significantly positively correlated with the expression of MAPK6 in patients with lung adenocarcinoma, also indicating that HNF4G regulates the expression of MAPK6 (Fig. 3B).

Using qRT-PCR (Fig. 3C) and western blot (Fig. 3D), we confirmed that HNF4G overexpression promoted the expression and phosphorylation of MAPK6 in A549 and HCC827 cells, as well as Akt S473 phosphorylation, whereas HNF4G knockdown inhibited the expression of MAPK6 and the activation of MAPK6Akt signaling pathway.

We used 1uM MK-2206, an Akt-specific inhibitor, to inhibit the activity of the MAPK6/Akt signaling pathway. When we inhibited Akt activity, there was no longer a significant difference in cisplatin resistance between A549 and HCC827 cells overexpressing HNF4G and controls (Fig. 3E), indicating that the MAPK6/Akt signaling pathway is the key mechanism by which HNF4G acts on cisplatin resistance.

HNF4G binds to the promoter region of MAPK6 to promote MAPK6 expression

We obtained potential binding sites of HNF4G in the MAPK6 promoter region from the JASPAR website, and designed three pairs of primers for ChIP-qPCR to detect HNF4G binding to the MAPK6 promoter (Fig. 4A). Our ChIP-qPCR results showed that HNF4G was significantly bound to the regions around the sequences amplified by these three pairs of primers, as shown in Fig. 4B.

To conduct dual luciferase experiments, we created a firefly luciferase plasmid with the MAPK6 −1000∼200 bp region, as well as a firefly luciferase plasmid with all potential binding sites of HNF4G in this region were randomly mutated (Fig. 4C). When the plasmid contained the MAPK6 promoter, overexpression of HNF4G significantly increased the activity of the firefly luciferase (Fig. 4D). However, when all possible binding sites of HNF4G were mutated, HNF4G overexpression no longer promoted the expression of firefly luciferase (Fig. 4D).