Upregulation of FOXP4 in breast cancer promotes migration and invasion through facilitating EMT

Figures & data

Figure 1 FOXP4 is highly expressed in BC tissues and cell lines.

Notes: (A) FOXP4 expression in BC tissues was detected by qRT-PCR and Western blot. *P<0.05. (B) FOXP4 expression in BC cell lines, including MCF-7 and MDA-MB-231, was detected by qRT-PCR and Western blot. *P<0.05. (C) Kaplan–Meier analysis was used to identify the effect of FOXP4 expression on the prognosis of BC patients. *P<0.05.

Abbreviations: BC, breast cancer; qRT-PCR, real-time polymerase chain reaction.

Table 1 Clinicopathological variables in the 134 BC patients

Variables	Number of patients (N=134)	FOXP4 protein expression		P-value
		Low (n=51)	High (n=83)
Age (years)
≥40	77	32	45	0.332
<40	57	19	38
Tumor size
Large (≥5 cm)	74	21	53	0.010
Small (<5 cm)	60	30	30
Pathological grade
I–II	65	31	34	0.026
III–IV	69	20	49
Lymph node metastasis
Yes	62	16	46	0.007
No	72	35	37

Abbreviation: BC, breast cancer.

Figure 2 FOXP4 promotes EMT in BC cells.

Notes: (A) The mRNA level and protein level of FOXP4 were determined in MCF-7 and MDA-MB-231 cells transfected with vector, FOXP4, SCR, and siFOXP4. *P<0.05. (B) The levels of EMT markers including epithelial markers, such as E-cadherin, and mesenchymal markers, such as N-cadherin and Vimentin, were measured in indicated MCF-7 and MDA-MB-231 cells by using qRT-PCR and Western blot. *P<0.05. (C) The morphological alterations of the cells were observed by phase-contrast microscopy. MCF-7 cells were transfected with FLAG-FOXP4.

Abbreviations: BC, breast cancer; EMT, epithelial–mesenchymal transition; qRT-PCR, real-time polymerase chain reaction; siFOXP4, FOXP4 siRNA; SCR, scramble siRNA.

Figure 3 FOXP4 transcriptionally activates SNAI1 expression.

Notes: (A) The levels of EMT-associated transcription factors including Snail, Slug, and ZEB1 were measured in indicated MCF-7 and MDA-MB-231 cells by using qRT-PCR and Western blot. (B) ChIP and qChIP assays were performed using anti-FOXP4 antibody in MCF-7 and MDA-MB-231 cells. (C) The luciferase reporter assay was used to determine the luciferase activity of FOXP4 in MCF-7 and MDA-MB-231 cells transfected with 2 µg vector or 0.5 µg, 1 µg, and 2 µg FOXP4 plasmid. *P<0.05.

Abbreviations: EMT, epithelial–mesenchymal transition; qRT-PCR, real-time polymerase chain reaction; siFOXP4, FOXP4 siRNA; SCR, scramble siRNA.

Figure 4 SNAI1 reverses FOXP4-induced tumor progression in vitro.

Notes: (A) The expression of Snail in MDA-MB-231 cells transfected with scramble siRNA (SCR) and siFOXP4 was detected by qRT-PCR and Western blot. *P<0.05. (B) Wound-healing assay determined the migratory ability of MDA-MB-231 cells transfected with vector, FOXP4, SCR and FOXP4 siRNA (siFOXP4), siFOXP4, and Snail. *P<0.05. (C) Transwell invasion assay determined the invasive ability of MDA-MB-231 cells transfected with vector, FOXP4, SCR and FOXP4 siRNA (siFOXP4), siFOXP4, and Snail. *P<0.05.

Abbreviations: qRT-PCR, real-time polymerase chain reaction; siFOXP4, FOXP4 siRNA; SCR, scramble siRNA.

Figure 5 FOXP4 facilitates cell proliferation in BC.

Notes: (A) The cell proliferation was detected in MCF-7 and MDA-MB-231 cells overexpressed or knocked down FOXP4 using CCK-8 kit. *P<0.05. (B) The cell proliferation was detected in MCF-7 and MDA-MB-231 cells overexpressed or knocked down FOXP4 using colony formation assay. *P<0.05.

Abbreviations: BC, breast cancer; CCK-8, cell-counting kit 8; siFOXP4, FOXP4 siRNA; SCR, scramble siRNA.

Figure S1 Analysis of Enerly Yakhini Breast GSE19536 dataset.