Increased expression of GPX4 promotes the tumorigenesis of thyroid cancer by inhibiting ferroptosis and predicts poor clinical outcomes

Abstract

Background: Ferroptosis plays a critical role in suppressing cancer progression, and its essential regulator is glutathione peroxidase 4 (GPX4). High GPX4 expression can inhibit accumulation of iron, thus suppressing ferroptosis. However, its function in thyroid cancer has not been fully illuminated. Here, we explore the effect of GPX4 on thyroid cancer tumorigenesis and prognosis.

Methods: Based on The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, GPX4 expression was investigated in cancer tissues and adjacent tissues. We determined the biological functions of GPX4-associated differentially expressed genes (DEGs) by using the "clusterProfiler" R package. In addition, the predictive value of GPX4 in thyroid cancer was assessed by using Cox regression analysis and nomograms. Finally, we conducted several in vitro experiments to determine the influence of GPX4 expression on proliferation and ferroptosis in thyroid cancer cells.

Results: GPX4 expression was obviously elevated in thyroid cancer tissues compared with normal tissues. Biological function analysis indicated enrichment in muscle contraction, contractile fiber, metal ion transmembrane transporter activity, and complement and coagulation cascades. GPX4 overexpression was associated with stage T3-T4 and pathologic stage III-IV in thyroid cancer patients. Cox regression analysis indicated that GPX4 may be a risk factor for the overall survival of thyroid cancer patients. In vitro research showed that knockdown of GPX4 suppressed proliferation and induced ferroptosis in thyroid cancer cells.

Conclusions: GPX4 overexpression in thyroid cancer might play an essential role in tumorigenesis and may have prognostic value for thyroid cancer patients.

Keywords: GPX4; clinicopathological features; ferroptosis; prognostic biomarker; thyroid cancer.

Figure 1

Differential mRNA expression profiles in thyroid cancer patients stratified by GPX4 level. (A) Comparison of GPX4 expression between different types of cancers and adjacent tissues based on TCGA. ns indicates p≥0.05, *p<0.05, **p<0.01, ***p<0.001. GPX4 expression is higher in thyroid cancer tissue than in adjacent normal tissue according to TCGA (B) as well as GSE27155 (C) and GSE33630 (D) from GEO. Differentially expressed mRNAs between the two groups are displayed by volcano plots (E) and heatmaps (F).

Figure 2

GPX4 expression in thyroid cancer tissues and paracancerous tissues. High expression of GPX4 in thyroid cancer tissues (A) and low expression of GPX4 in paracancerous tissues (B) based on immunohistochemistry (Original magnification×40); scale bars: 50 μm. Expression of GPX4 at the mRNA level (C) and protein level (D) in different tissues (T, thyroid cancer tissues; P, paracancerous tissues, N=16). ****p<0.0001.

Figure 3

Functional enrichment analysis results. Significant Gene Ontology terms of the GPX4-associated DEGs, including BP (A), CC (B), and MF (C). (D) Significant KEGG pathways of the GPX4-associated DEGs. Significant GSEA results of GPX4-associated DEGs, including Gpcr ligand binding (E), G alpha I signaling events (F), PI3KAKT signaling pathway (G), and pathways in cancer (H).

Figure 4

GPX4 expression and clinicopathological features in thyroid cancer patients. The Wilcoxon rank sum test was applied to analyze associations of clinical T stage (A) and pathologic stage (B) with GPX4 expression. (C) ROC curve analysis evaluating the diagnostic performance of GPX4 in thyroid cancer. (D) Nomogram was built to examine 1-, 3-, and 5-year overall survival based on the risk score model of GPX4 expression. (E) Calibration plot verifying the efficiency of the nomogram for overall survival. (F) Nomogram was built to examine 1-, 3-, and 5-year progression-free interval based on the risk score model of GPX4 expression. (G) Calibration plots verifying the efficiency of the nomogram for progression-free interval.

Figure 5

Knockdown of GPX4 inhibits proliferation in thyroid cancer cells. (A) GPX4 expression levels in FTC133, K1 and TPC-1 cells by western blot analysis. (B) GPX4 expression in FTC133 cells transfected with control, NC siRNA, or siGPX4 was confirmed by western blot analysis. (C) A CCK-8 assay was used to evaluate the viability of FTC133 cells transfected with NC siRNA/siGPX4. (D) Colony formation assay demonstrated the proliferation ability of FTC133 cells transfected with NC siRNA/siGPX4. (E) Ki-67 and PCNA protein expression in FTC133 cells transfected with NC siRNA/siGPX4 under erastin treatment.

Figure 6

Knockdown of GPX4 activates ferroptosis in thyroid cancer cells. Relative iron (A), GSH (B) and MDA (C) contents in FTC133 cells transfected with NC siRNA/siGPX4 after treatment with erastin. (D) Detection of lipid ROS levels in FTC133 cells transfected with NC siRNA/siGPX4 after treatment with erastin; scale bars: 50 μm. *p<0.05, **p<0.01.