TEA Domain Transcription Factor 1 Inhibits Ferroptosis and Sorafenib Sensitivity of Hepatocellular Carcinoma Cells

Abstract

Background: Ferroptosis, as a unique form of cell death, plays crucial negative roles in tumorigenesis and progression. This study aimed to investigate the role and molecular mechanism of TEA domain transcription factor 1 (TEAD1) in HCC and its effect on sorafenib-induced ferroptosis.

Methods: TEAD1 expression was analyzed in HCC tissues using quantitative PCR, and western blot. The effects on cell proliferation, migration and invasion were determined by CCK-8, wound healing and Transwell assays. Intracellular iron, reactive oxygen species (ROS), malondialdehyde (MDA) and GSH measurement was used to assess ferroptosis. Chromatin immunoprecipitation and luciferase reporter gene assays were performed to verify the relationship between TEAD1 and solute carrier family 3 member 2 (SLC3A2). Expression of mTOR, ribosomal protein S6, glutathione peroxidase 4 (GPX4) and SLC3A2 was analyzed by western blot. Tumor xenografts were used assess the effect of TEAD1 on tumor growth in vivo.

Results: TEAD1 was more abundant in HCC compared with normal tissues. Overexpression of TEAD1 enhanced the proliferation, migration, and invasion of HCC cells, while knockdown of TEAD1 inhibited these cell behaviors. Further, TEAD1 inhibited ferroptosis, which was demonstrated by decreased intracellular Fe²⁺ content, ROS, and MDA levels, and increased GSH activity. Mechnistically, TEAD1 promotes the transcription of SLC3A2 and activates the mTOR signaling. Additionally, silenced TEAD1 restrained tumor growth and enhance sorafenib-induced antitumor activity in vivo.

Conclusions: TEAD1 confers resistance of HCC cells to ferroptosis, thereby promoting the progression of HCC, suggesting the potential value of TEAD1 in the diagnosis and treatment of HCC.

Keywords: Ferroptosis; Hepatocellular carcinoma; TEA domain transcription factor 1; Transcriptional regulation.

Fig. 1

TEAD1 expression in HCC tumor and matched adjacent normal tissues. A The levels of TEAD1 in HCC and normal tissues from UALCAN website. B RT-PCR analysis of TEAD1 mRNA in HCC tissues and matched normal tissues (n = 30). C Western blot results of TEAD1 protein in tissues and matched normal tissues. The representative images of Western blot (left). The plot of TEAD1 expression (n = 30). D The expression of TEAD1 mRNA in HCC cells, by RT-PCR. E The expression of TEAD1 protein in HCC cells, by Western blot. N normal tissues. T Tumor tissues

Fig. 2

TEAD1 promotes proliferation, migration, and invasion in HCC Cells. After Hep-3B cells were transfected with si-TEAD1 or scrambled control (si-NC), A CCK8 assay was performed for cell viability, B wound healing was performed for cell migration, C Transwell invasion assay was conducted used for cell invasion. After BEL-7405 cells were transfected with TEAD1-overexpressing plasmids (TEAD1_OE) or empty vector (Vector), D CCK8 assay, E wound healing, and F Transwell invasion assays were conducted for cell viability, migration and invasion. **p < 0.01; ***p < 0.001

Fig. 3

TEAD1 regulates ferroptosis. A The growth inhibition of Hep-3B/si-TEAD1 and Hep-3B/si-NC cells treated with various concentrations of Sorafenib was determined by CCK8 assay. B The growth inhibition of BEL-7405/TEAD1_OE and BEL-7405/vector cells treated with various concentrations of sorafenib was determined by CCK8 assay. C Western blot was performed to examine the expression of TEAD1 in nucleus and cytoplasm in HCC cells after treatment with 10 μM Sorafenib. D Immunofluorescent staining was used to observe the distribution of TEAD1 in HCC cells after treatment with 10 μM Sorafenib. E After BEL-7405/TEAD1_OE and BEL-7405/vector cells were treated with Sorafenib, ferrous iron, lipid ROS, MDA and GSH were analyzed. F After Hep-3B/si-TEAD1 and Hep-3B/si-NC cells were treated with Sorafenib, ferrous iron, lipid ROS, MDA and GSH were analyzed. G The cell viability of Hep-3B/si-TEAD1 and Hep-3B/si-NC cells treated with Fer-1 was determined by CCK8 assay. H After Hep-3B/si-TEAD1 and Hep-3B/si-NC cells were treated with Fer-1, ferrous iron, lipid ROS, MDA and GSH were analyzed. **p < 0.01; ***p < 0.001 verse the Vector or si-NC groups. ##p < 0.01; ###p < 0.001 verse the Vector or si-NC + SOR groups

Fig. 4

TEAD1 regulates the transcription of SLC3A2 in HCC cells. A Venn diagram reveals four ferroptosis-related genes, which were.potential target genes of TEAD1 predicted by ChIPBase database. B The mRNA expression levels of SLC3A2 in the LIHC samples was analyzed through UALCAN database. C The correlation between the mRNA expression levels of TEAD1 and SLC3A2 in the LIHC samples was analyzed through UALCAN database. D RT-PCR was used to measure the expression levels of TEAD1 protein in HCC cells after knockdowning or overexpressing TEAD1. E Western blot was used to measure the expression levels of TEAD1 protein in HCC cells after knockdowning or overexpressing TEAD1. F The schematic drawing shows the TEAD1 binding motifs on the SLC3A2 promoter. G The potential binding site between TEAD1 and SLC3A2 promoter was predicted by JASPAR bioinformatics website (http://jaspar.genereg.net/). H The dual-luciferase reporter assay verified the binding of TEAD1 in SLC3A2 promoter region in Hep-3B and BEL-7405 cells. I ChIP assay was performed to detect the enrichment of TEAD1 in SLC3A2 promoter region after overexpressing TEAD1 in BEL-7405 cells. J After BEL-7405 cells were co-treated with si-SLC3A2 and TEAD1_OE or alone, ferrous iron, lipid ROS, MDA and GSH were analyzed. *p < 0.05; **p < 0.01; ***p < 0.001 verse the control groups. #p < 0.05;##p < 0.01 verse the TEAD1_OE groups

Fig. 5

TEAD1 activates mTORC1 signaling and increases GPX4 expression. A The expression of mTOR, RPS6, and GPX4 proteins in TEAD1-overexpressing BEL-7405 cells, examined by Western blot. B The expression of mTOR, RPS6, and GPX4 proteins in TEAD1-knockdown Hep-2B cells, examined by Western blot. After TEAD1-overexpressing BEL-7405 cells were co-treated with Sorafenib and Torin or Sorafenib alone, C Cell viability of CCK8. D Ferrous iron levels, E the level of lipid ROS, measured by C11-BODIPY staining coupled with flow cytometry, and F the levels of MDA, were analyzed. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 6

TEAD1 knockdown inhibits growth of xenograft in vivo. A Hep-3B cells with TEAD1 knockdown or control were injected subcutaneously into the flank of nude mice and then treated with sorafenib or PBS (six per group). B The growth curves of tumor volume in each group. Tumor volume: 0.5 × a × b². C The images of tumors. D The tumor weight in each groups (n = 6). E The expression of TEAD1, mTOR, RPS6 and GPX4 in tumor tissues of each group. *p < 0.05; **p < 0.01; ***p < 0.001