Abstract

   Recent studies have suggested that ferroptosis, a form of
   iron-dependent regulated cell death, might play essential roles in
   tumor initiation and progression. Six-transmembrane epithelial antigen
   of prostate 3 (STEAP3) is a ferrireductase involved in the regulation
   of intracellular iron homeostasis. However, the clinical significance
   and biological function of STEAP3 in human cancers remain poorly
   understood. Through a comprehensive bioinformatics analysis, we found
   that STEAP3 mRNA and protein expression were up-regulated in GBM, LUAD,
   and UCEC, and down-regulated in LIHC. Survival analysis indicated that
   STEAP3 had prognostic significance only in glioma. Multivariate Cox
   regression analysis revealed that high STEPA3 expression was correlated
   with poor prognosis. STEAP3 expression was significantly negatively
   correlated with promoter methylation level, and patients with lower
   STEAP3 methylation level had worse prognosis than those with higher
   STEAP3 methylation level. Single-cell functional state atlas showed
   that STEAP3 regulated epithelial-to-mesenchymal transition (EMT) in
   GBM. Furthermore, the results of wound healing and transwell invasion
   assays demonstrated that knocking down STEAP3 inhibited the migration
   and invasion of T98G and U251 cells. Functional enrichment analysis
   suggested that genes co-expressed with STEAP3 mainly participated in
   inflammation and immune-related pathways. Immunological analysis
   revealed that STEAP3 expression was significantly correlated with
   immune infiltration cells, including macrophages and neutrophils,
   especially the M2 macrophages. Individuals with low STEAP3 expression
   were more likely to respond to immunotherapy than those with high
   STEAP3 expression. These results suggest that STEAP3 promotes glioma
   progression and highlight its pivotal role in regulating immune
   microenvironment.

   Keywords: Glioma, STEAP3, Metastasis, Immune infiltration, Prognosis

Introduction

   Glioma is the most common and fatal primary central nervous system
   tumor, characterized by a poor prognosis, with a 5-year overall
   survival rate of only 6.8% for high-grade glioma due to limited
   effectiveness of surgical resection and chemoradiotherapy
   ([30]Magalhaes et al., 2021; [31]Thorbinson & Kilday, 2021; [32]Zhang
   et al., 2021b). Isocitrate dehydrogenase (IDH) mutations and chromosome
   arms 1p and 19q co-deletion have been identified as molecular
   pathological markers in glioma, indicating a significant survival
   benefit ([33]Ceccarelli et al., 2016; [34]Louis et al., 2021). However,
   due to the highly invasive and infiltrative nature of glioma cells,
   current therapeutic regimes and disease monitoring means have achieved
   limited clinical success ([35]Magalhaes et al., 2021; [36]Xu et al.,
   2021). There is an urgent need to identify novel biomarkers for early
   diagnosis and prognosis prediction in glioma patients.

   Six-transmembrane epithelial antigen of prostate 3 (STEAP3) is located
   on chromosome 2q14.2 and encodes a multi-pass transmembrane protein
   that acts as an iron transporter. STEAP3 can reduce iron from Fe^3+ to
   Fe^2+ state, and plays an essential role in mediating intracellular
   iron homeostasis ([37]Ohgami et al., 2005; [38]Ohgami et al., 2006).
   The dysregulation of iron metabolism is tightly linked with
   ferroptosis, a form of regulated cell death modality induced by
   iron-dependent phospholipids peroxidation on cellular membranes
   ([39]Lei, Zhuang & Gan, 2022). [40]Liu et al. (2021) identified that
   STEAP3 knockdown blocked erastin or RSL3-induced ferroptosis.
   Accumulating evidence has implicated that ferroptosis participates in
   the development of diverse cancer types and affects the response to
   therapies ([41]Chen et al., 2021b; [42]Qu, Peng & Liu, 2022).
   Mesenchymal and dedifferentiated tumor cells, associated with
   resistance to common therapeutics, are susceptible to ferroptosis
   inducers ([43]Tsoi et al., 2018; [44]Viswanathan et al., 2017).
   Ferroptosis induction might be a promising strategy for cancer
   treatment.

   In order to explore the role of STEAP3 in glioma, we first
   comprehensively analyzed STEAP3 expression profiles, methylation, and
   its clinical implications with datasets acquired from The Cancer Genome
   Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA). Gene Ontology
   (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
   analyses were used to explore the potential molecular mechanisms of
   STEAP3 and its co-expressed genes. In addition, we analyzed the
   correlation between STEAP3 expression and immune infiltration. In
   general, our study indicated that STEAP3 might function as a potential
   prognostic biomarker in gliomas through immune regulation.

Materials and Methods

Gene expression and survival analysis

   The integrative bioinformatics analysis of STEAP3 in multiple cancer
   types was achieved with several bioinformatics databases ([45]Table 1).
   Tumor Immune Estimation Resource (TIMER2.0) is a web portal for
   systematical analysis of immune infiltration across various cancer
   types ([46]Li et al., 2020). We used the Gene_DE module of TIMER2.0 to
   explore the differential expression of STEAP3 gene between tumor
   samples and normal tissues. Gene expression levels were normalized by
   log2 (transcripts per million, TPM) prior to analysis. For certain
   tumor types without adjacent normal tissues, the Xiantao tool
   ([47]https://www.xiantao.love/products) was further applied to explore
   the differences in STEAP3 expression between TCGA cancer samples and
   matched TCGA normal tissues and data from the Genotype-Tissue
   Expression (GTEx) database. Gene expression levels were normalized by
   log2 (TPM + 1). Through the Xiantao tool, comprehensive bioinformatics
   analysis can be performed across diverse cancer types, including
   differential expression analysis, interaction network, functional
   enrichment analysis, etc. Univariate and multivariate Cox regression
   analysis were carried out to assess the effects of the independent
   variables on survival using the Xiantao tool. In addition, we also
   employed the Xiantao tool to assess the prognostic value of STEAP3 in
   different cancer types. The main outcomes included overall survival
   (OS), disease-specific survival (DSS), and progression-free survival
   (PFS). Median STEAP3 expression served as a cutoff to discriminate
   high- and low-expression groups.

Table 1. Integrative bioinformatics analyzed in the study.

   Database URL References