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

Introduction

Gastric cancer (GC) is one of the most common upper gastrointestinal tumors in China, and is the 5^th most common cancer and the 4^th leading cause of cancer-related death in the world (1). In 2020, there were 479,000 new cases of GC and 374,000 deaths related to GC in China (2). As the clinical symptoms of early GC are not obvious, most patients are already in the middle and advanced stages when they are diagnosed. According to the latest data, the 5-year survival rate of GC patients in China is 35.1% (3). Thus, the molecular mechanism underlying the malignant progression of GC needs to be urgently identified.

Ferroptosis was first proposed by Dr. Stockwell of Columbia University in 2012 (4). It is an iron-dependent, new type of programmed cell death that differs to apoptosis, cell necrosis, and autophagy. The essence of ferroptosis is the depletion of glutathione (GSH), and the characteristic of ferroptosis is the accumulation of iron and lipid peroxidation (5,6). The metabolism of System Xc and GSH is an important process of ferroptosis, and solute carrier family 7 member 11 (SLC7A11) and GSH peroxidase 4 (GPX4) are important genes in this process (7). Wnt/β-catenin signaling pathway inhibits ferroptosis of GC cells by upregulating GPX4 expression to reduce lipid peroxidation levels (8). A study has shown that ferroptosis-related genes can influence the tumor microenvironment. Ferroptotic cancer cells generate some “find-me” and “eat-me” immunostimulating signals, particularly damage-associated molecular patterns (DAMPs), which robustly recruit dendritic cells (DCs), macrophages, and other immune cells properly to the site of dying tumor cells (9). Ferroptosis can impair the self-renewal ability of cancer cells and downregulate the expression of genes related to angiogenesis (10). In addition, some chemotherapeutic agents such as cisplatin, targeted agents such as sorafenib, and radiation therapy strongly induce ferroptosis (11). The latest research shows that the dysfunction of ferroptosis is closely related to tumor progression. Further, research has shown that suppressor of variegation 3-9 homolog 1 (SUV39H1) can inhibit the occurrence of ferroptosis in kidney cancer (12), and ubiquitin-specific peptidase 35 (USP35) can also regulate ferroptosis in lung cancer (13). The induction of ferroptosis has become an attractive strategy for the treatment of various types of cancer. The key genes that regulate ferroptosis (e.g., SLC7A11 and GPX4) are potential therapeutic targets for cancer treatment.

Thyroid hormone receptor interacting protein 6 (TRIP6) is a 476aa protein that belongs to the zyxin family and focal adhesion molecule. TRIP6 is involved in key cellular processes, including cell proliferation, differentiation, and survival. Studies have shown that TRIP6 is abnormal in colorectal cancer, breast cancer, and other cancers, and may play an important role in the occurrence, proliferation, and metastasis of cancer (14-16). Zhu et al. showed that high expression of TRIP6 was significantly associated with shorter survival time in patients with GC (17). Very few studies have been conducted on TRIP6 in GC.

In this study, we conducted a bioinformatics analysis of SLC7A11 and GPX4, the key factors regulating ferroptosis in GC. We identified some genes closely related to ferroptosis, and also discovered that TRIP6 may be an important factor that potentially regulates ferroptosis. Further, we found that TRIP6 inhibited the occurrence of GC ferroptosis and promoted tumor progression by binding c-Fos protein transcription to regulate the expression of GPX4 messenger RNA (mRNA). We present this article in accordance with the MDAR reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-1178/rc).

Methods

Bioinformatics analysis

Heat maps and volcano plots were generated using the heatmap package (https://cran.r-project.org/web/packages/pheatmap/index.html). Disease progression served as the study endpoint. Clinical data were retrieved from The Cancer Genome Atlas (TCGA; https://www.cancer.gov/ccg/research/genome-sequencing/tcga). The survival analysis was conducted on log₂-transformed (normalized +1) data. A univariate Cox analysis was performed using the R survival package to evaluate the impact of genes on overall survival. Using version 1.69 of the weighted gene co-expression network analysis (WGCNA) R package, co-expression networks were built based on the expression matrix of GPX4 and SLC7A11. Gene co-expression similarity was used to construct a Pearson correlation coefficient matrix. A scale-free network was established by selecting a β value when the independence degree (R²) reached 0.9. The weighted adjacency matrix was then converted into a topological overlap matrix (TOM) to assess network connectivity. A gene dendrogram (minimum module size =50) was constructed, and modules with correlations >0.7 were merged. Hierarchical clustering with mean linkage was used to group genes with similar expression profiles into modules based on TOM-based dissimilarity. Module gene expression was represented by the module signature genes.

Cell lines

We used five GC cell lines (i.e., AGS, SGC-7901, HGC-27, BGC-823, and MGC-803) and a normal human gastric epithelial cell line (GES-1). GES-1, SGC-7901, HGC-27, BGC-823, and MGC-80 were cultured in RPMI-1640 (C11875500BT, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin) at 37 ℃ in a humidified carbon dioxide (5%) atmosphere. The AGS cells were cultured in F12K medium with 10% fetal bovine serum and 1% antibiotics. All the cell lines were purchased from the Chinese Academy of Sciences, Institute of Chemistry and Cell Biology (Shanghai, China).

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) analysis

Samples of patients undergoing GC resection at The Second Affiliated Hospital of Anhui Medical University were collected. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study protocol was approved by The Second Affiliated Hospital of Anhui Medical University (ethical batch number: YX2022-116), and the enrolled patients signed an informed consent form before participation. Total RNA was extracted by Trizol reagent (15596026, Invitrogen, Waltham, MA, USA) from the cells and human tissues. Complementary deoxyribonucleic acid was synthesized using a reverse transcription system kit (R312-01, Vazyme Biotech Co., Ltd., Nanjing, China) in accordance with the manufacturer’s instructions. qRT-PCR was performed using a SYBR Green PCR Kit (Vazyme Biotech Co., Ltd.) in accordance with the manufacturer’s instructions. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control for RNA integrity and loading normalization. The forward primer sequence of TRIP6 is 5'-GGCTGTTACAAGTGCGAGGAGT-3'. The reverse primer sequence of TRIP6 is 5'-GGATGGGAACTGGAACTGAGAAC-3'. The relative fold change was analyzed using the 2^−ΔΔC(t) method. The measurement was repeated three times.

Cell transfection and vector construction

We constructed bioactive short hairpin RNAs (shRNAs) targeting TRIP6. The sequences of sh-TRIP6 were sh1: 5'-GAAGCTGGTTCACGACATGAA-3', and sh2: 5'-GCCTGGACGC CGAGATAGA-3'. At the same time, we constructed a plasmid overexpressing TRIP6 and further obtained lentivirus through lentivirus packaging kit (Zorin, Shanghai, China). Stable TRIP6-overexpressing cell lines were obtained by lentivirus infecting and puromycin (2.5 µg/mL) screening.

Dual‑luciferase reporter assays

The wild-type and mutant promoters of GPX4 were amplified and cloned into the pGL3-basic luciferase reporter vectors (Promega, Madison, WI, USA). Next, using overexpression-Fos (oe-Fos) or the control, the reporter vectors were transfected into the HEK293T cells. After 48 h, dual-luciferase reporter assays (Vazyme Biotech Co., Ltd.) were used to measure the luciferase activity in accordance with the manufacturer’s instructions. Relative luciferase activity was normalized to Renilla luciferase.

Cell Counting Kit 8 (CCK8) assays

CCK8 assays were used to detect cell proliferation. The cells were seeded into 96-well plates (200 cells/well) in accordance with the manufacturer’s instructions (Beyotime, Shanghai, China), and 10 µL of CCK8 reagent was added to each well. After 2 h, absorbance was detected spectrophotometrically at 450 nm in accordance with the manufacturer’s instructions (Yeasen Biotechnology, Co., Ltd., Shanghai, China).

Colony formation assays

The sh-TRIP6, oe-TRIP6, or control cells were seeded into six-well plates at 1×10³/well, after being cultured for 14 days. The six-well plates were washed three times with cold phosphate-buffered solution (PBS), fixed with 75% ethanol, and stained with 0.1% crystal violet. Images of the stained tumor cell colonies were recorded with a digital camera.

C11-BODIPY staining

The sh-TRIP6, oe-TRIP6, or control cells were seeded into six-well cell plates and then pretreated with ferroptosis/erastin for 96 h. Before the flow cytometry, the cells were stained with 2 µmol/L of C11-BODIPY for 30 min. After washing three times with cold PBS, the C11-BODIPY green fluorescence (581 nm/591 nm) was measured by flow cytometry.

Western blot and immunoprecipitation

The cells were lysed in radioimmunoprecipitation lysis buffer (Yeasen Biotechnology, Co., Ltd.) with 1 mM phenylmethylsulfonyl fluoride (PMSF). Next, 40 µg of protein was separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to the nitrocellulose (NC) membranes. The membranes were blocked in non-fat milk and incubated with specific primary anti-human antibodies overnight. The next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. The signals were visualized by chemiluminescent detection. Immunoprecipitation was performed using a co-immunoprecipitation (CO-IP) kit (Yeasen Biotechnology, Co., Ltd.) in accordance with the manufacturer’s instructions. All the primary antibodies are listed in Table S1.

Statistics analysis

All the statistical analyses were performed using SPSS v22.0 and GraphPad Prism 8. A one-way analysis of variance and the Wilcoxon test were used for comparisons among multiple groups. The two-tailed Student’s t-test was used for comparisons between the two groups. All the experiments in our study were independently performed in triplicate. A P value <0.05 was considered statistically significant.

Results

Identification of ferroptosis-associated prognostic mRNAs

A differential analysis was conducted to identify the differentially expressed genes in GC (P<0.05; log₂|fold change| >1). A heat map and volcano plot show the 3,125 differentially expressed mRNAs in the GC and normal samples (see Figure 1A,1B). To identify the ferroptosis-associated mRNAs, we excluded samples with large differences by a clustering analysis according to the WGCNA method and ultimately identified 375 samples for subsequent analysis (see Figure 1C). During the downscaling analysis, a β value of 6 (free ratio R²=0.9) was considered the optimal soft threshold (see Figure 1D,1E), and 30 gene modules were subsequently identified by gene clustering methods (see Figure 1F). The red module (r=0.73; P<0.001) had the strongest correlation with GXP4 and SLC7A11 (see Figure 1G) due to the close correlation of the genes in this module with the module itself (r=0.91; P<0.001; see Figure 1H) and was thus selected as a target module. The univariate regression analysis identified 18 genes with significant prognostic significance (see Figure 1I).

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.

TRIP6 is highly expressed in GC and is associated with a poor prognosis

We then sought to further examine the role of TRIP6 in GC. First, the expression of TRIP6 in GC was analyzed in TCGA database. The expression of TRIP6 was significantly higher in GC tissues than normal tissues (see Figure 2A), and was associated with lymph node metastasis in GC (see Figure 2B). We also found that the expression of TRIP6 was higher in patients with Helicobacter pylori infection (see Figure 2C). Thus, these findings suggest that TRIP6 plays a significant role in the progression of GC. Kaplan-Meier curve prognostic analysis showed that patients with high expression of TRIP6 had worse prognosis than patients with low expression of TRIP6 (see Figure 2D).

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.

Next, the correlation between TRIP6 and the ferroptosis-related genes GPX4 and SLC7A11 was analyzed (see Figure 2E,2F), and we found that TRIP6 and GPX4 were significantly correlated. The expression of TRIP6 in 20 cases of GC tissues and adjacent tissues was analyzed by qRT-PCR, and we found that TRIP6 was highly expressed in the cancer tissues (see Figure 2G). In the GC cell lines, the qRT-PCR and western blot results showed that compared to the human gastric epithelial cell line GES-1, TRIP6 expression was significantly increased in the GC cell lines (see Figure 2H,2I).

High expression of TRIP6 inhibits ferroptosis in GC

We then constructed a TRIP6 knockdown cell line in the HGC-27 cell line, and a TRIP6 overexpressing cell line using the AGS cell line. The efficiency was verified by quantitative polymerase chain reaction (qPCR) and western blot. We found that sh2 knockdown worked best, and thus used sh2 for the next experiment (see Figure 3A,3B). Next, we used CCK8 assays to measure cell proliferation. We found that after knocking down TRIP6, cell proliferation ability was significantly reduced, and after TRIP6 was overexpressed, cell proliferation ability was significantly enhanced (see Figure 3C). The western blot assays showed that after knocking down or overexpressing TRIP6, GPX4 changed significantly (see Figure 3D). Additionally, the clone formation assay produced similar results (see Figure 3E,3F). When a ferroptosis inhibitor (fer-1) was added to the knockdown TRIP6 cell line, the cell proliferation ability was higher than that of the sh-TRIP6 group. When the ferroptosis activator erastin was added to the TRIP6 overexpression cell line, the cell proliferation ability was lower than that of the oe-TRIP6 group (see Figure 3G). The C11-BODIPY 581/591 lipid peroxidation fluorescent probe was used to assess changes in cell ferroptosis. We found that after knocking down TRIP6, the percentage of cell ferroptosis increased significantly, and after TRIP6 was overexpressed, the percentage of cell ferroptosis decreased (see Figure 3H).

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.

TRIP6 can bind c-Fos protein

We then sought to reveal the mechanism by which TRIP6 regulates ferroptosis. We used the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://string-db.org/) to search for proteins to which TRIP6 might bind. We found that TRIP6 might bind to the c-Fos protein (see Figure 4A). TCGA database showed a significant correlation between TRIP6 and c-Fos (see Figure 4B). Further, we conducted a CO-IP experiment. The silver staining results showed that there was a differential band around the 50 kDa (see Figure 4C). The western blot verified that the differential protein was c-Fos (see Figure 4D). Next, cycloheximide chase assays were used to verify whether the combination of TRIP6 and c-Fos stabilized the expression of c-Fos. The results showed that the expression of c-Fos in the vector group gradually decreased over time, while the degradation of c-Fos in the oe-TRIP6 group was significantly slowed down (see Figure 4E). CCK8 assays were used to evaluate the effect of the overexpression of c-Fos on cell proliferation after knocking down TRIP6. The results showed that the cell proliferation ability of the sh-TRIP6 + oe-Fos group was higher than that of the sh-TRIP6 group (see Figure 4F). The C11-BODIPY 581/591 lipid peroxidation fluorescent probe was used to assess changes in cell ferroptosis, and we found that cell ferroptosis was inhibited in the sh-TRIP6 + oe-Fos group (see Figure 4G).

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.

c-Fos transcription regulates the expression of GPX4 mRNA

We also searched related research and found that c-Fos is a transcription factor (see Figure 5A). Thus, we speculated that c-Fos transcription regulates the expression of GPX4 and thus affects cell ferroptosis. The Jaspar (https://jaspar.elixir.no/) was used to predict the binding site of c-Fos to the GPX4 promoter (see Figure 5B,5C). By taking the intersection through the Wayne graph, we identified two sites that may be the binding sites of c-Fos and GPX4 (see Figure 5D). We designed three mutation sites, and the dual-luciferase reporter was used to evaluate the binding sites of the c-Fos and GPX4 promoter (see Figure 5E). We found that c-Fos may bind to CTCAGTCCTC in the promoter region of GPX4 (see Figure 5F-5H). Thus, we found that if overexpressed in GC cells, TRIP6 may bind to c-Fos, stabilize the expression of c-Fos, and promote the transcriptional regulation of GPX4 mRNA expression (see Figure 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.

Discussion

This study revealed the key role of TRIP6 in regulating ferroptosis in GC. Ferroptosis, which has been identified as a new type of cell death, plays an important role in the occurrence and progression of tumors (18,19). A review by Xu et al. summarizes the multiple mechanisms by which ferroptosis acts in GC (20). In this study, we found that TRIP6, which is highly expressed in GC, inhibits the ferroptosis of GC cells. This may occur by TRIP6 binding to and stabilizing c-Fos. c-Fos is a transcription factor that regulates the expression of GPX4, which in turn affects the ferroptosis of tumor cells.

It was recently discovered that ferroptosis is a form of regulated cell death. Ferroptosis is different from apoptosis, cell necrosis, and autophagy, and involves iron accumulation and lipid peroxidation. The metabolic pathways that rely on nicotinamide adenine dinucleotide phosphate (NADPH/H⁺), polyunsaturated fatty-acid metabolism, mevalonate, and glutamine catabolism participate in this new form of regulated necrotic cell death (21-23). GPX4 is an important regulator of ferroptosis that inhibits the formation of lipid peroxidation. The role of GPX4 as the main regulator in the ferroptosis process is based on its unique function of reducing complex hydroperoxides (including phospholipid hydroperoxide and cholesterol hydroperoxide) to their corresponding counterparts, thereby interrupting lipid peroxidation chain reaction(24).

Another key molecule related to ferroptosis is the antiporter System Xc (xCT). The antiporter System Xc exchange extracellular cystine for intracellular glutamate and serve as a source of GSH synthesis (25). Wang et al. showed that GSTZ1 enhanced sorafenib-induced ferroptosis by inhibiting the nuclear factor-erythroid factor 2-related factor 2 (NRF2)/GPX4 axis in liver cancer cells (26). Another study has shown that SMG9 is a direct binding protein of GPX4, which increases the accumulation of GPX4 in mitochondria, thereby preventing mitochondria oxidative damage and ultimately contributing to ferroptosis resistance (27). In this study, we found that TRIP6 is closely related to GPX4. After knocking down or overexpressing TRIP6, C11 probe detection revealed that TRIP6 affects ferroptosis. Additionally, using ferroptosis inhibitors or activators, we found that TRIP6 regulates the effects of inhibitors or activators on cells.

TRIP6 is an aptamer protein that interacts with many different proteins via its LIN-ISL-MEC (LIM) domain and acts as an intracellular signal protein, transcription aptamer, or auxiliary activator (28). TRIP6 binds to many molecules from the cell surface to the nucleus and serves as a platform for recruiting many molecules involved in actin assembly, cell movement, anti-apoptotic signaling, and transcriptional regulation. A previous study has shown that TRIP6 is an important auxiliary transcription activator (29). In this study, we found that TRIP6 is highly expressed in GC and is associated with a poor prognosis, which indicates that TRIP6 may play an important role in the occurrence and development of tumors. A research has shown that TRIP6 promotes the proliferation of hepatocellular carcinoma by increasing the activity of protein kinase B (AKT) and inhibiting the activity of forkhead box class O 3a (FOXO3a) (30). Zhao et al. also showed that TRIP6 maintains the stem cell-like characteristics of breast cancer cells by activating the Wnt/β-catenin pathway (31). Overexpression of TRIP6 promotes migration and invasion of colorectal cancer cells in a phosphoinositide 3-kinase (PI3K) and nuclear factor-κB (NF-κB)-dependent manner (15). Another study found that TRIP6 could reduce cell migration by interfering with Cas/Crk signaling (32). TRIP6 also regulates pro-survival signaling by activating NF-κB, extracellular signal-regulated kinase (ERK), and PI3K/AKT, and nuclear TRIP6 acts as a transcriptional co-modulator of activator protein-1 (AP-1) and NF-κB, suggesting that TRIP6 plays an important role at the confluence of multiple signaling pathways that are critical for tumor progression (33). However, the mechanism of TRIP6 in GC is still unclear. Thus, we analyzed the genes related to ferroptosis in GC in TCGA database and found that TRIP6 is a key gene that affects ferroptosis. It may be that TRIP6 binds to and stabilizes the transcription factor c-Fos, thus affecting cell ferroptosis. These results provide novel insights into the role of TRIP6 in GC.

c-Fos is encoded by the Fos gene and is the main member of the transcription factor AP-1. c-Fos enters the nucleus to form an AP-1 complex that is involved in in transcriptional regulation (34). c-Fos plays an important role in maintaining the normal physiological functions of cells, including cell proliferation, migration, and differentiation. Recent research has shown that c-Fos also has multiple functions in relation to tumor cell proliferation, differentiation, invasion, and metastasis (35). The AP-1 transcription factor complex activates the expression of downstream genes to promote the occurrence and development of tumors. Among them, c-Fos and c-Jun have a synergistic effect, which leads to changes in certain biological behaviors of cells and promotes cell development, mass proliferation, epithelial-mesenchymal transition, etc. (36). The expression of c-Fos in ovarian cancer and cervical cancer tissues is higher than that in adjacent normal tissues. The expression level of c-Fos also varies significantly with the degree of cancer differentiation (37,38). Compared with cervical cancer tissues without lymph node metastasis, c-Fos protein is highly expressed in cervical cancer tissues with lymph node metastasis (39). The c-Fos protein has also been found to be overexpressed in other types of tumors, such as esophageal cancer and lung cancer; thus, the c-Fos protein may be related to the occurrence, development, and metastasis of cancers (34,40). In this study, we found that c-Fos regulates the transcription of GPX4, thereby inhibiting cell ferroptosis. Further, we predicted the specific binding sites of c-Fos and GPX4 promoter via the dual-luciferase report experiment using database predictions.

Conclusions

In conclusion, our bioinformatics analysis revealed the TRIP6 gene is closely related to ferroptosis. TRIP6 is overexpressed in GC cells and tissues and is associated with the poor prognosis of GC patients. Mechanism studies found that TRIP6 binds to c-Fos and stabilizes it, preventing it from degradation. As a transcription factor, c-Fos regulates and promotes the expression of downstream GPX4, inhibits cell ferroptosis, and thus promotes the occurrence and development of GC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-1178/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-1178/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-1178/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-1178/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study protocol was approved by The Second Affiliated Hospital of Anhui Medical University (ethical batch number: YX2022-116), and the enrolled patients signed an informed consent form before participation.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: L. Huleatt)