The expression landscape of FOXP3 and its prognostic value in breast cancer

Abstract

Background: Forkhead Box Protein 3 (FOXP3), as an essential marker of regulatory T cell (Treg) development, is reportedly overexpressed in invasive breast carcinoma (BRCA) and could be a potential prognostic factor for BRCA. However, the biological function of FOXP3 in BRCA is still unclear. In this study, we comprehensively explored the expression landscape of FOXP3 and its prognostic value in BRCA.

Methods: FOXP3 transcriptomic expression data were mainly obtained from The Cancer Genome Atlas (TCGA). The Kaplan-Meier plotter and receiver operating characteristic (ROC) curve were used to assess the prognostic and diagnostic value of FOXP3 in BRCA. UALCAN, cBio-Portal, and MethSurv were used to evaluate the genomic variation of FOXP3. Gene set enrichment analysis (GSEA) was performed to explore the FOXP3 pathways involved in BRCA. Morover, we detected the expression of FOXP3 in 123 BRCA specimens and 5 BRCA cell lines to verify the biological value of FOXP3 in BRCA. The Kaplan-Meier method was adopted for the overall survival (OS) analysis, and a Cox proportional hazards model was used to estimate the hazard ratio (HR) for OS.

Results: FOXP3 was more highly expressed in BRCA than in normal tissues (2.808±1.020 vs. 1.409±0.656, P<0.001), and overexpressed FOXP3 was associated with a better prognosis. The ROC curve demonstrated a significant diagnostic value of FOXP3 in BRCA (area under the ROC curve, AUC: 0.877). Genomic analysis revealed that promoter hypomethylation of FOXP3 may be the underlying mechanism of FOXP3's upregulation in BRCA. GSEA found that FOXP3 coexpressed genes were mainly involved in the Toll-like receptor pathway, JAK/STAT pathway, cell cycle, and apoptosis. Moreover, high FOXP3 expression was an independent protective factor for OS in our 123 BRCA tissues (HR: 0.367; P=0.036). In vitro, we found that FOXP3 knockdown with siRNA promoted migration and invasion in MCF-7 cells.

Conclusions: This study demonstrated that FOXP3 shows prognostic and diagnostic value for BRCA. We provided evidence that promoter hypomethylation and a high expression of FOXP3 were both related to a favorable prognosis in BRCA, which maybe associated with the Toll-like receptor pathway, JAK/STAT pathway, cell cycle, and apoptosis.

Keywords: Forkhead Box Protein 3 (FOXP3); The Cancer Genome Atlas (TCGA); breast carcinoma (BRCA); prognosis.

Figure 1

FOXP3 expression in BRCA and other types of human cancer from TCGA data. (A) FOXP3 expression levels in different tumor types from TCGA database; (B) expression levels of FOXP3 mRNA in BRCA (n=1,109) and normal tissues (n=113); (C) the expression of FOXP3 in BRCA (n=112) and its paired adjacent tissues (n=112); (D) ROC curve analysis of FOXP3 in BRCA (n=1,109). *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance; BRCA, breast carcinoma; TCGA, the cancer genome atlas; ROC, receiver operating characteristic.

Figure 2

FOXP3 expression in BRCA based on different characteristics from TCGA data. (A) The association of FOXP3 expression and ER status in BRCA (n=485); (B) the association of FOXP3 expression and PR status in BRCA (n=478); (C) the association of FOXP3 expression and T classification in BRCA (n=498); (D) the association of FOXP3 expression and HER2 status in BRCA (n=486); (E) the association of FOXP3 expression and pathologic TNM stage classifications in BRCA (n=498); (F) the association of FOXP3 expression and different subtypes in BRCA (n=481). *, P<0.05, **, P<0.01, and ***, P<0.001; ns, no significance; BRCA, breast carcinoma; TCGA, the cancer genome atlas; ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2; TNBC, triple-negative breast cancer.

Figure 3

The prognostic value of FOXP3 expression in BRCA. (A) RFS survival curves of breast cancer in the Kaplan-Meier plotter database (n=2,032); (B) OS survival curves of breast cancer in the Kaplan-Meier plotter database (n=943); (C) OS survival curves of basal-like breast cancer in the Kaplan-Meier plotter database (n=278); (D) OS survival curves of breast cancer with HER2 positive status (n=157) from TCGA data; (E) OS survival curves of breast cancer with HER2 negative status from TCGA data (n=558); (F) DMFS survival curves of breast cancer from GSE 2034 data (n=286). BRCA, breast carcinoma; RFS, relapse-free survival; OS, overall survival; TCGA, the cancer genome atlas; HER2, human epidermal growth factor receptor 2; DMFS, distant metastasis-free survival.

Figure 4

FOXP3 genomic analysis in BRCA. (A) Landscape of FOXP3 genetic alterations in pan-cancer. (B) Genetic alterations of FOXP3 in BRCA from eight studies’ data. Study: (a) proteogenomic landscape of breast cancer (CPTAC, Cell 2020); (b) invasive breast carcinoma (TCGA, Cell 2015); (c) invasive breast carcinoma (TCGA, Firehose Legacy); (d) invasive breast carcinoma (TCGA, PanCancer Atlas); (e) breast cancer (SMC 2018); (f) breast cancer (METABRIC, Nature 2012 & Nat Commun 2016); (g) the Metastatic Breast Cancer Project (Provisional, February 2020); (h) invasive breast carcinoma (TCGA, Nature 2012). (C) FOXP3 expression in different alteration groups. (D) The prognosis of FOXP3 based on its alteration condition. BRCA, breast carcinoma; TCGA, The Cancer Genome Atlas.

Figure 5

FOXP3 expression and methylation pattern in BRCA. (A) FOXP3 expression in BRCA and normal tissue from UALCAN; (B) FOXP3 promoter methylation level in BRCA and normal tissues from UALCAN; (C) correlation analysis of FOXP3 mRNA expression with FOXP3 promoter methylation status; (D) the visualization heat map of DNA methylation at CpG sites of the FOXP3 gene in BRCA from MethSurv data; (E) the Kaplan-Meier survival analysis of the promoter methylation of FOXP3 in BRCA from MethSurv data. ***, P<0.001. BRCA, breast carcinoma.

Figure 6

FOXP3 coexpression gene and pathway enrichment in BRCA. (A) Volcano plot of FOXP3-associated DEGs in TCGA BRCA database; (B) the PPI network of the top FOXP3 DEGs; (C-F) GSEA functional enrichment of FOXP3 associated DEGs in BRCA; (C) KEGG JAK-STAT signaling pathway; (D) WP apoptosis; (E) KEGG Toll-like receptor signaling pathway; (F) REACTOME Mitotic G1 phase and G1/S transition; (G-N) the correlation analyses of FOXP3 expression with related genes: (G) FOXP3 with pro-apoptotic protein BAX; (H) FOXP3 with Caspase 3; (I) FOXP3 with Caspase 7; (J) FOXP3 with Caspase 8; (K) FOXP3 with Caspase 10; (L) FOXP3 with antioncogene STAT1; (M) FOXP3 with cancer-promoting gene STAT3; (N) FOXP3 with anti-apoptotic protein BCL2. BRCA, breast carcinoma; DEGs, differentially expressed genes; TCGA, The Cancer Genome Atlas; PPI, protein-protein interaction.

Figure 7

FOXP3 protein expression and its prognostic value in our BRCA tissues. (A-D) Representative IHC staining patterns of FOXP3 in BRCA tissues: (A) positive, magnification (×200), (B) positive, magnification (×400), (C) negative, magnification (×200), (D) negative, magnification (×400). (E) OS rate of FOXP3 in total BRCA patients (n=123). (F) OS rate of FOXP3 in TNBC patients (n=25). (G) OS rate of FOXP3 in HER2+ BRCA patients (n=32). (H) OS rate of FOXP3 in HR+ BRCA patients (n=66). BRCA, breast carcinoma; IHC, immunohistochemistry; BRCA, breast carcinoma; OS, overall survival; TNBC, triple-negative breast cancer; HER2, human epidermal growth factor receptor 2; HR, hormone receptor.

Figure 8

FOXP3 expression in cell lines. (A) FOXP3 mRNA expression data in various cancer cell lines from the HPA database; (B) FOXP3 protein located in the nucleoplasm of MCF-7 cells from the HPA database (IF, ×400); (C) FOXP3 mRNA expression in MCF-7, MDA-MB-231, MDA-MB-453, BT-474, and SK-BR-3 BRCA cell lines. PBMC was regarded as the positive control; (D) FOXP3 protein expression decreased in MCF-7 transfected with FOXP3 siRNA by western blot; (E) FOXP3 mRNA expression decreased in MCF-7 transfected with FOXP3 siRNA by qRT-PCR; (F) FOXP3 knockdown enhanced the ability of MCF-7 cell migration evaluated by wound healing assay (microscope, ×100); (G) FOXP3 knockdown enhanced the invasion ability of MCF-7 cell in transwell chamber stained with crystal violet (microscope, ×200). **, P<0.01, ***, P<0.001. ns, no significance; qRT-PCR, quantitative real-time PCR; HPA, human protein atlas; IF, immunofluorescence; PBMC, peripheral blood mononuclear cell.