Silencing of Prrx1b suppresses cellular proliferation, migration, invasion and epithelial-mesenchymal transition in triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) is a highly aggressive tumour subtype associated with poor prognosis. The mechanisms involved in TNBC progression remains largely unknown. To date, there are no effective therapeutic targets for this tumour subtype. Paired-related homeobox 1b (Prrx1b), one of major isoforms of Prrx1, has been identified as a new epithelial-mesenchymal transition (EMT) inducer. However, the function of Prrx1b in TNBC has not been elucidated. In this study, we found that Prrx1b was significantly up-regulated in TNBC and associated with tumour size and vascular invasion of breast cancer. Silencing of Prrx1b suppressed the proliferation, migration and invasion of basal-like cancer cells. Moreover, silencing of Prrx1b prevented Wnt/β-catenin signaling pathway and induced the mesenchymal-epithelial transition (MET). Taken together, our data indicated that Prrx1b may be an important regulator of EMT in TNBC cells and a new therapeutic target for interventions against TNBC invasion and metastasis.

Keywords: epithelial-mesenchymal transition; invasion; paired-related homeobox 1b; proliferation; triple-negative breast cancer.

Figure 1

The Prrx1b expression levels were frequently up‐regulated in TNBC. (A) Immunohistochemistry analysis of Prrx1b protein in TNBC and adjacent non‐cancerous tissue samples. Magnification, 200×. (B) RT‐PCR analysis demonstrated the expression of Prrx1b in TNBC and matched adjacent non‐cancerous tissues. β‐actin served as loading control. (C) Statistical analysis with relative level of Prrx1b in paired TNBC tissue samples from 141 patients. N non‐malignant breast tissues, T primary breast tissues. Prrx1b, Paired‐related homeobox 1b; TNBC, Triple‐negative breast cancer.

Figure 2

Silencing of Prrx1b induces morphological alterations in breast cancer cell lines. (A) The expression of Prrx1b in cell lysates from three basal‐like breast cancer cell lines (HCC‐1937, MDA‐MB‐468, and MDA‐MB‐231) and breast epithelial cell line MCF‐10A was detected by western blotting and normalized with GAPDH expression. Silencing of Prrx1b in MDA‐MB‐468 (B) and MDA‐MB‐231 (C) cells significantly decreased Prrx1b expression, detected by western blotting and normalized with GAPDH expression. (D) Cell morphology of Prrx1b silenced MDA‐MB‐231 cells and c ontrol cells. Magnification, 200×. *P < 0.05. Prrx1b, Paired‐related homeobox 1b.

Figure 3

Silencing of Prrx1b suppresses breast cancer cell proliferation in vitro and tumour growth in vivo. The cell proliferation of control and sh1, sh2 groups in MDA‐MB‐468 (A) and MDA‐MB‐231 (B) was determined by MTT assay at 0, 24, 48, 72, 96 h, respectively. (C) Morphologic characteristics of tumours from mice inoculated with MDA‐MB‐231/Control and MDA‐MB‐231/sh2 cells. (D) Tumour volumes and weights of control and Sh2 groups from C. (E) Decreased Prrx1b expression in xenograft tumours from the Prrx1b silenced group compared with the control group, detected by immunohistochemistry staining. Each group had eight mice. Magnification, 100×. Prrx1b, Paired‐related homeobox 1b.

Figure 4

Silencing of Prrx1b suppresses breast cancer migration and invasion in vitro. (A) Representative wound healing images of MDA‐MB‐231. Quantification of wound healing rates was analysed in MDA‐MB‐468 (B) cells and MDA‐MB‐231 (C) cells. (D) Representative migration images of MDA‐MB‐468 and MDA‐MB‐231 silenced and control cells. (E) Representative invasion images of MDA‐MB‐468 and MDA‐MB‐231 silenced and control cells. (F, G) Quantifications of cells on the lower surface of the membrane. *P <0.05. Prrx1b, Paired‐related homeobox 1b.

Figure 5

Silencing of Prrx1b induces mesenchymal‐epithelial transition in MDA‐MB‐231. Silencing of Prrx1b decreased the expression of vimentin (A), but increased the expression of E‐cadherin (B) and cytokeratin (C) detected by western blotting and analysed by normalizing to GAPDH expression. (D) The level of matrix metalloproteinase (MMP)‐2 and MMP‐9 in MDA‐MB‐231 cells were determined by western blotting. *P < 0.05. Prrx1b, Paired‐related homeobox 1b.

Figure 6

Silencing of Prrx1b inhibits the Wnt/β‐catenin signaling pathway. (A) Relative mRNA expression levels of ZEB1, ZEB2, snail, twist and slug in MDA‐MB‐231 and MDA‐MB‐231 cells compared with control cells. Immunoblotting of β‐catenin in cytosolic (B) and nuclear (C) extracts of MDA‐MB‐468 and MDA‐MB‐231 cells was carried out, expression of histone H3 was used as a loading control. (D) Silencing of Prrx1b decreased TOPflash activity by TOP/FOPflash report gene assay. (E) Silencing of Prrx1b induced a reduction in cyclin‐D1 expression, one of Wnt/β‐catenin target genes. Densitometric analysis of cyclin‐D1 is shown below, normalized to GAPDH. *P < 0.05. Prrx1b, Paired‐related homeobox 1b.