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. 2021 Jul 7:11:656190.
doi: 10.3389/fonc.2021.656190. eCollection 2021.

FOXP3 Inhibits the Metastasis of Breast Cancer by Downregulating the Expression of MTA1

Chenlin Liu  1 , Jun Han  1 , Xiaoju Li  1 , Tonglie Huang  1 , Yuan Gao  1 , Baolong Wang  1 , Kuo Zhang  1 , Shuning Wang  1 , Wangqian Zhang  1 , Weina Li  1 , Qiang Hao  1 , Meng Li  1 , Yingqi Zhang  1 , Cun Zhang  1
Affiliations

FOXP3 Inhibits the Metastasis of Breast Cancer by Downregulating the Expression of MTA1

Chenlin Liu et al. Front Oncol. .

Abstract

Background: FOXP3, as a tumour suppressor gene, has a vital function in inhibiting the metastasis of breast cancer cells, but the mechanisms by which it inhibits metastasis have not been fully elucidated. This study intended to explore a new mechanism by which FOXP3 inhibits breast cancer metastasis.

Methods: Bioinformatic analysis was performed to identify potential downstream molecules of FOXP3. The function of FOXP3 in inhibiting MTA1 expression at the mRNA and protein levels was verified by real-time PCR and Western blot analysis. The interaction between FOXP3 and the MTA1 promoter was verified by transcriptomic experiments. In vitro and in vivo experiments were used to determine whether the regulation of MTA1 by FOXP3 affected the invasion and migration of breast cancer cells. Immunohistochemistry was adopted to explore the correlation between the expression levels of FOXP3 and MTA1 in breast cancer samples.

Results: Bioinformatics-based sequencing suggested that MTA1 is a potential downstream molecule of FOXP3. FOXP3 downregulated the expression of MTA1 in breast cancer cells by directly inhibiting MTA1 promoter activity. Importantly, FOXP3's regulation of MTA1 affected the ability of breast cancer cells to invade and metastasize in vitro and in vivo. Moreover, analysis of clinical specimens showed a significant negative correlation between the expression levels of FOXP3 and MTA1 in breast cancer.

Conclusion: We systematically explored a new mechanism by which FOXP3 inhibits breast cancer metastasis via the FOXP3-MTA1 pathway.

Keywords: FOXP3; MTA1; breast cancer; metastasis; transcription factor.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
MTA1 is a downstream molecule of FOXP3. (A) KEGG analysis showed that the target genes of FOXP3 might be involved in the regulation of cell movement and migration pathways. (B) GO analysis showed that the identified differentially expressed genes participate in cell adhesion and cytoskeletal reorganization. (C) Seven molecules were selected from the transcriptome sequencing results. (D) The mRNA expression levels of these 7 potential downstream molecules were determined by real-time PCR. The data are shown as the mean ± s.e.m. ns, P > 0.05; *P < 0.05; **P < 0.01; and ***P < 0.001.
Figure 2
Figure 2
FOXP3 binds to the MTA1 promoter and reduces its expression. Real-time PCR was performed to determine the mRNA levels of FOXP3 and MTA1 in MDA-MB-231 cells (A) and the mRNA levels of FOXP3 and MTA1 in MCF-7 cells (D) in the control group and FOXP3 group. Western blotting was used to determine the protein expression levels of FOXP3 and MTA1 in MDA-MB-231 cells (B) and MCF-7 cells (E) in the control group and FOXP3 group. (C, F) Grey-level analysis of the bands in (B, E) respectively. (G) Potential FOXP3 binding sites in the promoter region of the MTA1 gene. (H) The ChIP assay showed that FOXP3 had a stronger ability to bind to specific sequences in the MTA1 promoter than to the negative IgG control. The dual-luciferase reporter assay showed that transfection with the FOXP3 overexpression plasmid significantly reduced the transcriptional activity of the MTA1 gene promoter in HEK293T cells (I) and MDA-MB-231 cells (J) in a dose-dependent manner. The data are shown as the mean ± s.e.m. ns, P > 0.05; *P < 0.05; **P < 0.01; and ***P < 0.001.
Figure 3
Figure 3
The FOXP3-MTA1 pathway regulates the invasion and migration of breast cancer cells in vitro and in vivo. Transwell assays were used to examine the invasion abilities of MDA-MB-231 cells (A) and MCF-7 cells (C). (B, D) Statistical analysis of the experimental data in (A, C) respectively. (E) The mRNA expression levels of potential downstream molecules were determined. (F), In vivo imaging technology was used to demonstrate that FOXP3 can affect the ability of breast cancer cells to metastasize in vivo by regulating MTA1. (G) Statistical analysis of fluorescence values in (F). (H) H&E staining showed that FOXP3 inhibited lung metastasis from breast cancer by regulating MTA1 expression. The data are shown as the mean ± s.e.m. ns, P > 0.05; *P < 0.05; **P < 0.01; and ***P < 0.001.
Figure 4
Figure 4
FOXP3 expression and MTA1 expression were negatively correlated in clinical breast cancer samples. (A) The expression level of MTA1 was lower in breast cancer tissues with positive FOXP3 expression than in breast cancer tissues with negative FOXP3 expression. FOXP3 expression was negatively correlated with MTA1 expression. Scale bars, 250 μm (10×) and 50 μm (40×). (B) A negative correlation between nuclear FOXP3 expression and MTA1 expression was found in breast cancer specimens. Spearman rank correlation analysis. (C) Analysis of 1166 breast cancer patients in the TCGA database showed a negative correlation between FOXP3 and MTA1 expression levels in clinical breast cancer samples. Pearson correlation analysis. (D) Data from breast cancer cases were analysed on the GEPIA website, verifying that the expression levels of FOXP3 and MTA1 were negatively correlated in clinical breast cancer samples. Pearson correlation analysis.
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