Zinc finger and SCAN domain-containing protein 18 is a potential DNA methylation-modified tumor suppressor and biomarker in breast cancer

Abstract

Introduction: Zinc finger and SCAN domain-containing protein 18 (ZSCAN18) has been investigated as a putative biomarker of multiple human cancers. However, the expression profile, epigenetic modification, prognostic value, transcription regulation, and molecular mechanism of ZSCAN18 in breast cancer (BC) remain unknown.

Methods: In the study, we present an integrated analysis of ZSCAN18 in BC based on public omics datasets with the use of multiple bioinformatics tools. Genes potentially regulated through restoration of ZSCAN18 expression in MDA-MB-231 cells were investigated to identify pathways associated with BC.

Results: We observed that ZSCAN18 was downregulated in BC and mRNA expression was significantly correlated with clinicopathological parameters. Low expression of ZSCAN18 was found in the HER2-positive and TNBC subtypes. High expression of ZSCAN18 was associated with good prognosis. As compared to normal tissues, the extent of ZSCAN18 DNA methylation was greater with fewer genetic alterations in BC tissues. ZSCAN18 was identified as a transcription factor that might be involved in intracellular molecular and metabolic processes. Low ZSCAN18 expression was associated with the cell cycle and glycolysis signaling pathway. Overexpression of ZSCAN18 inhibited mRNA expression of genes associated with the Wnt/β-catenin and glycolysis signaling pathways, including CTNNB1, BCL9, TSC1, and PFKP. ZSCAN18 expression was negatively correlated with infiltrating B cells and dendritic cells (DCs), as determined by the TIMER web server and reference to the TISIDB. ZSCAN18 DNA methylation was positively correlated with activated B cells, activated CD8+ and CD4+ T cells, macrophages, neutrophils, and activated DCs. Moreover, five ZSCAN18-related hub genes (KDM6B, KAT6A, KMT2D, KDM1A, and HSPBP1) were identified. ZSCAN18, ZNF396, and PGBD1 were identified as components of a physical complex.

Conclusion: ZSCAN18 is a potential tumor suppressor in BC, as expression is modified by DNA methylation and associated with patient survival. In addition, ZSCAN18 plays important roles in transcription regulation, the glycolysis signaling pathway, and the tumor immune microenvironment.

Keywords: DNA methylation; ZSCAN18; breast cancer; integrated analysis; transcription regulation; tumor suppressor.

Figure 1

The expression profile of ZSCAN18 in BC. (A) The mRNA expression of ZSCAN18 in multiple human cancers by comparison of tumor and adjacent normal tissues based on TCGA datasets constructed by TIMER, which identifying genes that are upregulated or downregulated in the tumors compared to normal tissues for each cancer type, as displayed in gray columns when normal data are available; BRCA. Tumor, breast cancer; BRCA. Normal, breast normal tissues; **p < 0.01; ***p < 0.001; (B) The comparison of ZSCAN18 mRNA expression in breast primary tumor and normal tissues based on TCGA datasets constructed by UALCAN; (C) The mRNA expression of ZSCAN18 in tumor subtypes of breast invasive carcinoma; (D) The ZSCAN18 mRNA level in breast tumor, healthy, and tumor-adjacent tissues based on TCGA and GTEx datasets; (E) The protein expression of ZSCAN18 in primary tumor of BC and normal tissues and (F) in tumor subtypes of breast invasive carcinoma. Z-values represent standard deviations from the median across samples for the given cancer type. Log2 Spectral count ratio values from CPTAC were first normalized within each sample profile, and normalized across samples.

Figure 2

The relationships among ZSCAN18 expression and clinicopathological significance of BC constructed by bc-GenExMiner. (A) The mRNA level of ZSCAN18 in BC patients with different ages (≤ 51 and > 51); (B) The mRNA level of ZSCAN18 in BC patients with different ER status (ER-positive and ER-negative); (C) The mRNA level of ZSCAN18 in BC patients with different PR status (PR-positive and PR-negative); (D) The mRNA level of ZSCAN18 in BC patients with different HER2 status (HER2-negative and HER2-positive); (E) The mRNA level of ZSCAN18 in BC patients with different basal-like status (Non-basal-like and Basal-like); (F) The mRNA level of ZSCAN18 in BC patients with different TNBC status (Non-TNBC and TNBC); (G) The mRNA level of ZSCAN18 in BC patients with different NPI (NPI1, NPI2, and BPI3); (H) The mRNA level of ZSCAN18 in BC patients with different SBR (SBR1, SBR2, and SBR3); (I) The mRNA level of ZSCAN18 in BC patients with different P53 status (Wild-type and Mutated) and (J) the mRNA level of ZSCAN18 in BC patients with different tumor stages. The p value indicated statistical significance.

Figure 3

The prognostic significance of ZSCAN18 in BC. (A) High ZSCAN18 mRNA expression shows a better OS for BC; (B) High ZSCAN18 mRNA expression shows a better RFS for BC; (C) High ZSCAN18 mRNA expression shows a better DMFS for BC; (D) High ZSCAN18 mRNA expression shows a better PPS for BC; (E) High ZSCAN18 mRNA expression shows a better OS for ER negative (ER-) BC; (F) High ZSCAN18 mRNA expression shows a better OS for HER2 negative (HER2-) BC; (G) High ZSCAN18 mRNA expression shows a better OS for lymph node positive (LN+) BC; (H) High ZSCAN18 mRNA expression shows a better OS for lymph node negative (LN-) BC. HR, 95% CI, and log-rank p were displayed.

Figure 4

DNA methylation and genetic alteration of ZSCAN18 in BC. (A) Promoter methylation, gene mutation, and genetic alteration profile of ZSCAN18 are constructed by the cBioPortal; Tumor samples are shown in columns. General promoter methylation but no gene mutation is found in the ZSCAN18 nucleotide sequence; Genomic alterations of ZSCAN18 are mutually exclusive. (B) The “Putative CpG Islands” in nucleotide sequence of ZSCAN18, and which are marked by red arrow. “Observed vs Expected”, the minimum average observed to expected ratio of C plus G to CpG in a set of 10 windows are required before a CpG island is reported (default value: 0.6); “Percentage”, the minimum average percentage of G plus C in a set of 10 windows are required before a CpG island is reported (default value: 50). (C) The promoter methylation level of ZSCAN18 in primary tumor and normal tissues, in which the beta value indicates level of DNA methylation ranging from 0 (unmethylated) to 1 (fully methylated). Different beta value cut-off has been considered to indicate hypermethylation (beta value: 0.7 - 0.5) or hypomethylation (beta-value: 0.3 - 0.25); (D) The promoter methylation level of ZSCAN18 in tumor subtypes of breast invasive carcinoma and normal tissues. The p value indicated statistical significance.

Figure 5

Genes transcription regulated by ZSCAN18 and its potential signaling pathways in BC. (A) The top 14 pathway enrichment clusters were found based on ZSCAN18 regulated genes and carried out with GO Biological Processes, KEGG pathway, and Reactome Gene Sets through Metascape. (B) The top-level GO biological processes were found based on ZSCAN18 regulated genes through Metascape. Length of bars represent log₁₀ (p-value) determined by the best-scoring term within each cluster. Cell cycle (C) and glycolysis signaling pathway (D) differentially enriched in ZSCAN18-low expression phenotype constructed by GSEA. Gene sets with FDR q-val<0.05 are considered as significant. NOM p-value: normalized p-value; FDR q-value: false discovery rate q-value. (E–H) The mRNA expressions of CTNNB1, BCL9, TSC, and PFKP in ZSCAN18 overexpressed MDA-MB-231 cell line. *p < 0.05.

Figure 6

Correlation of ZSCAN18 expression/DNA methylation with immune infiltration in BC. (A) ZSCAN18 expression negatively correlated with infiltration levels of B cells and DCs in TIMER database. (B) ZSCAN18 expression showed negative association with infiltration levels of activated B cells, activated CD8 T cells, activated CD4 T cells, macrophages, and activated DCs in TISIDB database (n=1100). (C) ZSCAN18 DNA methylation positively associated with activated B cells, activated CD8 T cells, activated CD4 T cells, macrophages, neutrophils, and activated DCs in TISIDB database (n=785). The correlation coefficient (cor. and/or rho.) and p value were displayed.

Figure 7

ZSCAN18-related hub genes and its PPI physical network. (A) Five ZSCAN18-related hub genes were identified by PPI network analysis using CytoHubba plugin of Cytoscape. The top five hub genes are shown in different colors except blue. (B) The mRNA expression of the top five hub genes in BC was observed based on bc-GenExMiner. The p value was displayed. (C) The correlation between the top five hub genes and OS of BC patients was investigated based on KM plotter. KDM1A: also called KDM1. The HR, 95% CI, and log-rank p were displayed. (D) A PPI physical network of ZSCAN18 was built by STRING database based on experimental evidence. The confidence score was defined as 0.7 in this analysis.