TRIP6/c-Fos regulating GPX4 modulates gastric cancer growth by inhibiting ferroptosis

Abstract

Background: Gastric cancer (GC) is a digestive system tumor with the highest incidence in China. In recent years, the role of ferroptosis in tumor occurrence and development has received more and more attention. In this study, we sought to identify the genes related to ferroptosis in GC and explain the mechanism of these related genes.

Methods: The Cancer Genome Atlas (TCGA) database was analyzed using bioinformatics technology to identify the gene closely related to ferroptosis [i.e., thyroid hormone receptor interacting protein 6 (TRIP6)]. We then constructed the knockdown and overexpression of TRIP6 cell lines to verify the effects of TRIP6 on cell proliferation and ferroptosis using quantitative polymerase chain reaction (qPCR), western blot, co-immunoprecipitation (CO-IP), flow analysis, dual-luciferase report, and other technologies. We also validated the results by ferroptosis inducers and inhibitors.

Results: The bioinformatics analysis showed that ferroptosis is closely related to TRIP6. TRIP6 is highly expressed in GC cells and tissues and is associated with a poor prognosis. The knockdown or overexpression of TRIP6 affects cell proliferation and ferroptosis, which we verified via recovery experiments with ferroptosis activators and inhibitors. TRIP6 also affected the expression of glutathione (GSH) peroxidase 4 (GPX4). The CO-IP and silver staining experiments verified that TRIP6 can bind to the transcription factor c-Fos and stabilize its expression. The dual-luciferase experiment confirmed that the transcription factor c-Fos regulated the expression of GPX4.

Conclusions: TRIP6, which is highly expressed in GC, binds to and stabilizes the expression of c-Fos, which acts as a transcription factor to regulate the expression of GPX4 and thereby inhibit cell ferroptosis.

Keywords: Ferroptosis; c-Fos; gastric cancer (GC); thyroid hormone receptor interacting protein 6 (TRIP6).

Figure 1

Identification of ferroptosis-associated genes. (A) Heat map showing the expression clustering of differential genes between the tumor and normal samples. (B) Volcano plot showing upregulated and downregulated genes in the differential expression analysis; the green dots represent the downregulated genes, and the red dots represent the upregulated genes. (C) Cluster analysis excluding models with large differences. (D,E) Optimal soft threshold settings. (F) Clustering dendrogram of samples: the different colors correspond to the dynamic module, which corresponds to the height above. (G) Correlation index of each module with GPX4 and SLC7A11. (H) Correlation index of genes in the red module with the overall genes. (I) Univariate regression analysis of differential genes. CI, confidence interval; FC, fold change; FDR, false discovery rate; GPX4, glutathione peroxidase 4; N, normal tissue; SLC7A11, solute carrier family 7 member 11; T, tumor tissue.

Figure 2

TRIP6 is highly expressed in GC. (A) TRIP6 is highly expressed in tumors in TCGA database. (B) TRIP6 expression is related to the distant metastasis of tumors. (C) TRIP6 expression is higher in patients with Helicobacter pylori infection. (D) TRIP6 high expression is related to poor prognosis. (E,F) The expression of TRIP6 is correlated with GPX4 and SLC7A11. (G) The expression of TRIP6 mRNA in cancerous and adjacent GC patients in 20 cases of GC. (H,I) Western blot and qPCR verified the expression of TRIP6 in GC cell lines compared to a normal human gastric epithelial cell line. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, gastric cancer; GPX4, glutathione peroxidase 4; H. pylori, Helicobacter pylori; mRNA, messenger RNA; qPCR, quantitative polymerase chain reaction; SLC7A11, solute carrier family 7 member 11; STAD, stomach adenocarcinomas; TCGA, The Cancer Genome Atlas; TPM, transcripts per million; TRIP6, thyroid hormone receptor interacting protein 6.

Figure 3

Revised TRIP6 promotes the proliferation of GC and inhibits ferroptosis. (A,B) Western blot and qPCR verified the efficiency of the knockdown and overexpression of TRIP6. (C) CCK8 assays verified the effects of TRIP6 knockdown and overexpression on the proliferation of GC cells. (D) Western blot verified the effects of TRIP6 knockdown and overexpression on the GC cells, and the effect of the ferroptosis marker GPX4. (E,F) Clone formation validated the effect of TRIP6 knockdown and overexpression on the proliferation of GC cells staining method: crystal violet staining. (G) The effect of ferroptosis inhibitors and ferroptosis activators on cell viability. (H) C11 probes verified the effects of TRIP6 knockdown and overexpression on the ferroptosis of GC cells via a flow cytometry analysis. *, P≤0.05; **, P≤0.01. CCK8, Cell Counting Kit 8; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, gastric cancer; GPX4, glutathione peroxidase 4; mRNA, messenger RNA; NC, negative control; oe, overexpression; qPCR, quantitative polymerase chain reaction; sh, short hairpin; TRIP6, thyroid hormone receptor interacting protein 6.

Figure 4

TRIP6 binds to c-Fos and stabilizes its expression. (A) STRING database predicts the possible binding sites of TRIP6, and predicts that c-Fos may bind to TRIP6. (B) The correlation between c-Fos and TRIP6 in TCGA GC database (P<0.001). (C) Protein electrophoresis and silver staining found that there are different proteins at 50 kDa after the CO-IP experiment. Red arrow, Fos. (D) Western blot verified that TRIP6 binds to c-Fos after the CO-IP experiment. (E) The cycloheximide chase assay confirmed that c-Fos degradation slowed down after TRIP6 was overexpressed. (F) CCK8 confirmed that the overexpression of c-Fos in the sh-TRIP6 cell line promoted cell proliferation. (G) Flow cytometry confirmed that the overexpression of c-Fos in the sh-TRIP6 cell line promoted cell ferroptosis. *, P≤0.05. CCK8, Cell Counting Kit 8; CHX, cycloheximide; CO-IP, co-immunoprecipitation; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, gastric cancer; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation; NC, negative control; oe, overexpression; sh, short hairpin; STRING, Search Tool for the Retrieval of Interacting Genes/Proteins; TCGA, The Cancer Genome Atlas; TPM, transcripts per million; TRIP6, thyroid hormone receptor interacting protein 6.

Figure 5

Specific binding sites of c-Fos and GPX4. (A) Transcription factor c-Fos motif. (B,C) JASPAR database: c-Fos and GPX4 promoter possible binding sites. (D) A Venn diagram of the two most likely binding sites. (E) Three mutant sequences were designed, and (F-H) verified by a dual-luciferase experiment; the GPX4 promoter 1,212–1,221 position was found to be the binding site of c-Fos. **, P≤0.01; NS, no significance. GPX4, glutathione peroxidase 4; MUT, mutant; NC, negative control; oe, overexpression; RE, random expectation; WT, wild type.

Figure 6

Illustration of TRIP6 regulating GPX4 expression by stabilizing c-Fos to promote cell ferroptosis in GC cells. GC, gastric cancer; GPX4, glutathione peroxidase 4; TRIP6, thyroid hormone receptor interacting protein 6.