Multiomics analysis reveals CT83 is the most specific gene for triple negative breast cancer and its hypomethylation is oncogenic in breast cancer

Abstract

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer (BrC) subtype lacking effective therapeutic targets currently. The development of multi-omics databases facilities the identification of core genes for TNBC. Using TCGA-BRCA and METABRIC datasets, we identified CT83 as the most TNBC-specific gene. By further integrating FUSCC-TNBC, CCLE, TCGA pan-cancer, Expression Atlas, and Human Protein Atlas datasets, we found CT83 is frequently activated in TNBC and many other cancers, while it is always silenced in non-TNBC, 120 types of normal non-testis tissues, and 18 types of blood cells. Notably, according to the TCGA-BRCA methylation data, hypomethylation on chromosome X 116,463,019 to 116,463,039 is significantly correlated with the abnormal activation of CT83 in BrC. Using Kaplan-Meier Plotter, we demonstrated that activated CT83 is significantly associated with unfavorably overall survival in BrC and worse outcomes in some other cancers. Furthermore, GSEA suggested that the abnormal activation of CT83 in BrC is probably oncogenic by triggering the activation of cell cycle signaling. Meanwhile, we also noticed copy number variations and mutations of CT83 are quite rare in any cancer type, and its role in immune infiltration is not significant. In summary, we highlighted the significance of CT83 for TNBC and presented a comprehensive bioinformatics strategy for single-gene analysis in cancer.

Figure 1

Screening strategies and basic annotations of CT83. Screening strategies for genes that significantly overexpressed in TNBC but not in non-TNBC subtypes. (B) The location of CT83 on chromosome X. (C) The schematic diagram of the only transcript of CT83.

Figure 2

The expression of CT83 mRNA in breast cancer. The expression of CT83 mRNA in breast cancer tissues of different subtypes according to data from the TCGA-BRCA (A,E) and METABRIC (B,F) dataset. The expression of CT83 mRNA in TNBC tissues according to data from the FUSCC-TNBC dataset (D). The expression of CT83 mRNA in breast cancer cell lines based on data from the CCLE-BRCA dataset (C,G). The expression of ESR1, PGR, and ERBB2 was plotted for distinguishing the subtype of breast cancer cell lines. The median expression of CT83 in each subtype was labeled as dashes. The positive rate of CT83 mRNA was labeled as percentage numbers in A (> 1 FPKM), 2C (> 1 RPKM), and 2D (> 1 Log2 FPKM), while the positive rate of CT83 in B was not presented because the positive expression cutoff cannot be determined based on the METABRIC raw data. Only samples with available both CT83 expression and subtype information were used for plotting. The asterisks in A–C represent the statistical difference (t-test p values) of CT83 expression between the Basal subtype and other subtypes. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 3

The expression of CT83 in normal tissues, cancer tissues, and cancer cell lines. (A) The expression of CT83 in normal adult tissues based on 8 RNA-seq datasets obtained from the Expression Atlas. (B) The expression of CT83 in pan-cancer and paired normal tissues based on RNA-seq data from the GEPIA2 database. The abbreviations of involved cancers are available on this webpage (https://gdc.cancer.gov/resources-tcga-users/tcga-code-tables/tcga-study-abbreviations). (C) The expression of CT83 in pan-cancer cell lines based on RNA-seq data from the CCLE dataset.

Figure 4

Copy number variations of CT83 in cancer. Copy number variations of CT83 in breast cancer tissues based on data from the TCGA-BRCA (A) and the METABRIC (B) dataset. Copy number variations of CT83 in breast cancer cell lines (C) and pan-cancer cell lines (D) based on data from the CCLE dataset. The median expression levels of CT83 mRNA were labeled as dashes. Copy number variations of CT83 in pan-cancer tissues (E) based on TCGA cancer data. The correlation between CT83 DNA copy number and CT83 mRNA expression levels (F).

Figure 5

Mutations of CT83 in cancer. (A) CT83 mutations in breast cancer tissues, (B) breast cancer cell lines, (C) TCGA pan-cancer tissues, and (D) CCLE pan-cancer cell lines. (E) The correlation between CT83 mRNA expression and mutations of key genes in breast cancer based on data from the TCGA-BRCA dataset. CT83 mutation points were labeled in the corresponding regions in the linear schematic diagram of its protein. Permutation test p-values were calculated by comparing CT83 expression in key-gene-mutated samples with key-gene-unmutated samples. N.A., not available; WT, wild type; MUT, mutated.

Figure 6

CT83 methylation in breast cancer. (A) The correlation between CT83 mRNA expression and its methylation status in breast cancer tissues. (B) Locations of CT83 methylation probes relative to CT83 DNA sequence. It should be noted that CT83 is located on the reverse strand of chromosome X. (C) Enlarged views for locations CT83 methylation probes. Pearson’s correlation coefficients between CT83 mRNA expression levels and methylation beta values were labeled as colorful dots. D-K) Pearson’s correlation coefficients between CT83 mRNA expression levels and methylation beta values of corresponding probes.

Figure 7

Prognostic significance of CT83 in breast cancer and pan-cancer. The hazard ratios of CT83 (high vs. low) for (A) RFS, (B) OS, and (C) DMFS in breast cancer grouped by different clinical characteristics. The hazard ratios of CT83 (high vs. low) for (D) RFS, (E) OS in different cancers. Abbreviations: RFS, relapse-free survival; OS, overall survival; DMFS, distant metastases-free survival; HR, hazard ratio; 95%CI, 95% confidence interval; P, log-rank test P-value; see https://gdc.cancer.gov/resources-tcga-users/tcga-code-tables/tcga-study-abbreviations for TCGA study abbreviations.

Figure 8

Bioinformatics prediction of CT83 biological functions in breast cancer by GSEA. GSEA of CT83 in breast cancer for (A) hallmark gene sets and (B) KEGG pathway gene sets using the TCGA-BRCA and METABRIC dataset. The red dots represent gene sets positively correlated with CT83 that are common in the TCGA-BRCA and METABRIC dataset with statistical significance, while the green dots were negatively correlated shared gene sets. Details of the three enriched gene sets associated with cell cycle signaling, namely (C) Cell Cycle, (D) G2M Checkpoint, and (E) E2F targets. Abbreviations: NES, normalized enrichment score; NOM p, normalized P-value; FDR q, false discovery rate q-value; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ^#, q < 0.25; ^##, q < 0.05; ^###, q < 0.01; ^####, q < 0.001.