Six-Transmembrane Epithelial Antigen of Prostate 4: An Indicator of Prognosis and Tumor Immunity in Hepatocellular Carcinoma

Abstract

Purpose: The six-transmembrane epithelial antigen of prostate 4 (STEAP4) has been linked to tumor progression via its involvement in inflammatory responses, oxidative stress, and metabolism. However, STEAP4 has rarely been studied in hepatocellular carcinoma (HCC). We explored STEAP4 expression associated with tumor prognosis to understand its role in tumor biology in HCC.

Patients and methods: STEAP4 mRNA and protein expressions were primarily analyzed using bioinformatics tools based on The Cancer Genome Atlas database to understand the expression pattern, molecular mechanism, prognostic impact, and association with immune cell infiltration. We further investigated the association between STEAP4 protein expression and clinicopathological parameters and their predictive value in HCC patients using immunohistochemical staining of tissue microarrays.

Results: The expression of STEAP4 mRNA and protein in HCC tissues was significantly lower than in normal liver tissues. Reduced expression of STEAP4 was linked to advanced HCC stages, poor recurrence-free survival (RFS), and overall survival. Furthermore, reduced STEAP4 expression was a significant predictor of worse RFS in univariate and multivariate analyses in the immunohistochemical cohort. GO, KEGG, and GSEA analyses revealed that STEAP4 is related to numerous biological processes and pathways, including drug metabolism, DNA replication, RNA metabolism, and immune response. In terms of the immune system, the decreased level of STEAP4 was correlated with the immunosuppressive microenvironment.

Conclusion: Our data indicated that reduced STEAP4 expression was significantly associated with tumor aggressiveness and poor prognosis, possibly because of its link to various biological processes and induction of HCC immune evasion. Therefore, STEAP4 expression may serve as a potential prognostic biomarker for cancer progression and immunity, as well as a therapeutic target in HCC.

Keywords: Hepatocellular carcinoma; bioinformatics; immunohistochemistry; prognostic factor.

Figure 1

The expression of STEAP4 in patients with hepatocellular carcinoma (HCC) in the TCGA database. (A) STEAP4 expression levels in different cancer and normal tissues were analyzed using the TIMER database (**p < 0.01, ***p < 0.001). (B and C) Plot chart showing the mRNA and protein expression levels of STEAP4 in HCC compared with normal control using the UALCAN database. (D) The Oncoprint of the STEAP4 gene alteration showing various genetic alterations with different colors in HCC samples using cBioPortal database. The bar diagram represents STEAP4 alteration frequencies according to HCC cohorts. (E) Box plot showing the relationship between STEAP genetic alteration and expression level. (F) Scatter plot showing the relation of STEAP4 expression with methylation level.

Figure 2

The correlation of STEAP4 mRNA expression with clinical characteristics and prognosis. The expression of STEAP4 in hepatocellular carcinoma was analyzed according to (A) Histological subtype, (B) Tumor grade, (C) Pathologic tumor stage, and (D) Clinical stage (*p < 0.05, **p < 0.01, ***p < 0.001). The effect of STEAP4 mRNA expression on (E) Recurrence-free survival and (F) Overall survival was analyzed using Kaplan–Meier plotter database.

Figure 3

STEAP4 protein expression analysis of cases with hepatocellular carcinoma (HCC) in immunohistochemical analysis. (A) In the non-neoplastic tissue, STEAP4 expression showed a diffusely even pattern. (B and C) In the HCC sample, STEAP4 expression showed the clumping pattern in cytoplasm and membranous reaction; retained expression (B) and reduced expression (C) (x400). (D) A reduced expression of STEAP4 was significantly associated with shorter RFS (p = 0.034).

Figure 4

Gene enrichment analysis of STEAP4 in hepatocellular carcinoma (HCC). (A) The volcano plots of DEGs represented the upregulated and downregulated genes in red and blue and Heatmap depicted the top 50 positively and negatively correlated genes of STEAP4 in HCC. (B) The results of Kyoto Encyclopedia of Genes and Genomes pathway analysis of upregulated genes enrichment. (C)The gene ontology of STEAP4 was analyzed according to biological functional enrichment. (D and E) The results of gene set enrichment analysis based on Kyoto Encyclopedia of Genes and Genomes databases..

Figure 5

(A) The significantly somatic mutated genes in the STEAP4 low and high groups. (B) The relationship between STEAP4 expression and immune score, stromal score, and ESTIMATE score (C) Survival analysis of CD4, CD8, Treg, B cell, macrophage, and cancer-associated fibroblast. (D) Correlation between STEAP4 and 21 kinds of tumor microenvironment infiltrating immune cells analyzed using CIBERSORT.

Figure 6

(A) Correlation between STEAP4 and immune cells such as T regs, M0, M1, and M2 macrophages (B) Correlation between STEAP4 expression and Treg gene set. (C) The relationship between STEAP4 and immune check points such as PDCD1, CTLA4, and LAG3. (D) The negative correlation between CARE scores and many compounds found in the Cancer Cell Line Encyclopedia (CCLE), Genomics of Drug Sensitivity in Cancer (GDSC), and Cancer Therapeutics Response Portal (CTRP) databases.