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Supplemental Figures 1-9 and Tables 1 and 2. Supplemental Table 1: Identification of potential FOXP3-targeted microRNAs in breast cancer cells. Supplemental Table 2: Primer sequences used in this study. Supplemental Figure 1: Expression profiles of IRAK1 and TRAF6 in MCF10A cells with miR-146a/b inhibitors. Supplemental Figure 2: Expression of nuclear FOXP3 in FOXP3-Tet-off MCF7 cells. Supplemental Figure 3: The expression profile of NF-κB target genes after FOXP3 induction in FOXP3-Tet-off MCF7 cells. Supplemental Figure 4: Identification of potential FOXP3-binding sites in promoter regions of miR-146a/b in the FOXP3-Tet-off MCF7 cells by whole-genome ChIP-seq. Supplemental Figure 5: Quantitative analysis of FOXP3-binding sites in the potential promoter region of human miR-146b in FOXP3-Tet-off MCF7 cells. Supplemental Figure 6: Specific binding of FOXP3 to the second forkhead-binding motif (GCAAATA) in the miR-146a promoter. Supplemental Figure 7: MiR-146a/b upregulation by FOXP3 transfection in MDA-MB231 cells. Supplemental Figure 8: The mRNA expression levels of IRAK1and TRAF6 in metastatic breast tumor cells after administration of miR-146a/b inhibitors. Supplemental Figure 9: A potential mechanism by which FOXP3 regulates the NF-κB-miR-146a/b feedback loop through IRAK1 and TRAF6 in tumor growth or regulates CXCR4/MMP9 in tumor metastasis.

FOXP3 functions not only as the master regulator in regulatory T cells, but also as an X-linked tumor suppressor. The tumor-suppressive activity of FOXP3 has been observed in tumor initiation, but its role during tumor progression remains controversial. Moreover, the mechanism of FOXP3-mediated tumor-suppressive activity remains largely unknown. Using chromatin immunoprecipitation (ChIP) sequencing, we identified a series of potential FOXP3-targeted miRNAs in MCF7 cells. Notably, FOXP3 significantly induced the expression of miR-146a/b. In vitro, FOXP3-induced miR-146a/b prevented tumor cell proliferation and enhanced apoptosis. Functional analyses in vitro and in vivo revealed that FOXP3-induced miR-146a/b negatively regulates NF-κB activation by inhibiting the expression of IRAK1 and TRAF6. In ChIP assays, FOXP3 directly bound the promoter region of miR-146a but not of miR-146b, and FOXP3 interacted directly with NF-κB p65 to regulate an miR-146–NF-κB negative feedback regulation loop in normal breast epithelial and tumor cells, as demonstrated with luciferase reporter assays. Although FOXP3 significantly inhibited breast tumor growth and migration in vitro and metastasis in vivo, FOXP3-induced miR-146a/b contributed only to the inhibition of breast tumor growth. These data suggest that miR-146a/b contributes to FOXP3-mediated tumor suppression during tumor growth by triggering apoptosis. The identification of a FOXP3–miR-146–NF-κB axis provides an underlying mechanism for disruption of miR-146 family member expression and constitutive NF-κB activation in breast cancer cells. Linking the tumor suppressor function of FOXP3 to NF-κB activation reveals a potential therapeutic approach for cancers with FOXP3 defects. Cancer Res; 75(8); 1703–13. ©2015 AACR.