DLC-3 suppresses cellular proliferation, migration, and invasion in triple-negative breast cancer by the Wnt/β-catenin pathway

Abstract

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Our study investigated the functional role of DLC-3 in TNBC. The expression of DLC-3 was assessed by immunohistochemistry in TNBC to evaluate the clinicopathologic significance of DLC-3. Recombinant lentiviral vectors encoding the DLC-3 gene were constructed for transfection into MDA-MB-231. Real-time qPCR and western blot analysis were employed to evaluate the expression of DLC-3, β-catenin, GSK-3β and c-myc in DLC-3-transfected cells. Moreover, cell proliferation assays, cell colony formation assays, and cell migration and invasion assays were performed to elucidate the role of DLC-3 in TNBC development and progression. Our data revealed that DLC-3 was downregulated in TNBC, and its expression level was associated with lymph node status and differentiation grade in breast cancer. Both real-time qPCR and western blot analyses showed that the DLC-3 gene and protein were overexpressed in the DLC-3-transfected MDA-MB-231 cells. In addition, the expression of GSK-3β was upregulated and the expression of β-catenin and c-myc gene was downregulated in the DLC-3-transfected cells. Furthermore, DLC-3 overexpression inhibited cell proliferation, colony formation, migration, and invasion in vitro. DLC-3, functioning as a tumor-suppressor gene, inhibits cell growth and invasion in TNBC, possibly through regulation of the Wnt/β-catenin signaling pathway.

Keywords: DLC-3; Triple-negative breast cancer; invasion; proliferation.

Figure 1

DLC-3 expression was frequently down-regulated in TNBC. (A) IHC results of DLC-3 expression in adjacent noncancerous and breast cancer tissue samples. DLC-3 was expressed in the nucleus of normal breast epithelial cells (Adjacent noncancerous) (a). Loss of expression of DLC-3 in poorly differentiated TNBC (b) and weak staining in well-differentiated TNBC (c). (B) Compared with normal breast tissue, DLC-3 expression was significantly decreased in TNBC (*P < 0.05). (C) IHC results of Ki-67 expression in breast cancer samples. (a) represents the low expression level group, (b) represents the high expression level group (× 200).

Figure 2

Effects of pcDNA3.1(+)-DLC-3 on DLC-3 expression in MDA-MB-231. A. The expression of DLC-3 in HCC-1937, MDA-MB-468, MDA-MB-231 and MCF-10A was detected by western blotting and normalized to GAPDH expression. B. DLC-3 protein expression in the pcDNA3.1(+)-DLC-3 cells was higher than that in the pcDNA3.1(+) group and the untransfected MDA-MB-231 group (*P < 0.05). No difference was observed between the pcDNA3.1(+) group and the untransfected MDA-MB-231 group.

Figure 3

DLC-3 overexpression inhibits proliferation and colony formation of MDA-MB-231 cells. A. Cell proliferation of the pcDNA3.1(+)-DLC-3 cells was decreased compared with the pcDNA3.1(+) group and the untransfected MDA-MB-231 group. B. The colony formation of pcDNA3.1(+)-DLC-3 cells was decreased compared with the pcDNA3.1(+) group and the untransfected MDA-MB-231 group (*P < 0.05).

Figure 4

Upregulation of DLC-3 suppresses breast cancer migration and invasion in vitro. A. Compared to the control group, upregulation of DLC-3 significantly inhibited MDA-MB-231 cells migration by wound healing assay. B and C. The number of pcDNA3.1(+)-DLC-3 cells that passed through the membrane was lower than the number in the pcDNA3.1(+) group and the untransfected MDA-MB-231 group (*P < 0.05). No difference was observed between the pcDNA3.1(+) group and the untransfected MDA-MB-231 group.

Figure 5

DLC-3 overexpression regulates the Wnt/β-catenin signaling pathway. A. GSK-3β mRNA expression was higher in the pcDNA3.1(+)-DLC-3 group, while β-catenin and c-myc mRNA expression was lower when compared to these levels in the pcDNA3.1(+) group and the untransfected MDA-MB-231 cells. B, C. GSK-3β protein expression was higher in the pcDNA3.1(+)-DLC-3 group, while β-catenin and c-myc protein expression were lower, when compared to these levels in the pcDNA3.1(+) group and the untransfected MDA-MB-231 cells (*P < 0.05).