Downregulation of the transcription factor, FoxD3, is associated with lymph node metastases in invasive ductal carcinomas of the breast

Abstract

FoxD3 is a transcription factor of the forkhead gene family. We investigated its expression in invasive ductal carcinomas (IDC) of the breast and its association with metastasis. The expression of FoxD3, human epidermal growth factor receptor-2 (HER-2), estrogen receptor (ER), progesterone receptor (PR) and Ki67 was examined by immunohistochemistry in samples from 121 patients with IDC. Non-tumorous breast adenosis tissues served as controls. HER2 expression was confirmed by fluorescence in situ hybridization (FISH). The expression levels of FoxD3 in IDC tissues and the breast cancer cell lines MCF-7 and MDA-MB-231 were additionally measured by western blotting. A greater percentage of total IDC patients and patients with lymph node metastases showed reduced FoxD3 expression compared to adenosis controls (p<0.05). Overall, FoxD3 was associated with metastatic status of IDC but not with age, pathological or clinical staging, or status of HER-2, ER, or PR. In particular, FoxD3 protein expression was down-regulated in the tumor epithelia of IDC samples from patients with metastases. Furthermore, FoxD3 protein expression was decreased in the metastatic MDA-MB-231 breast cancer cell line relative to the non-metastatic cell line, MCF-7. A greater number of patients with invasive, triple-negative breast cancer were also negative for FoxD3 expression than in other, non-triple-negative tumor types. These results suggest an inverse relationship between FoxD3 expression and tumor metastasis and warrants further investigation.

Keywords: Breast cancer; FoxD3; HER-2; invasive ductal carcinomas.

Figure 1

Representative immunohistochemistry images of FoxD3 protein expression in adenosis of breast (A), IDC without lymphatic metastasis (B), IDC with lymphatic metastasis (C) and a cervical cancer positive control (D); 200 × magnification; scale bar=20 μm. (E) Semi-quantitative analysis of immunohistochemistry indicated there was significantly lower expression of FoxD3 in IDC samples (p=0.0044).

Figure 2

Semi-quantitative analysis of immunohistochemistry indicated there was significantly lower expression of FoxD3 in IDC with lymphatic metastasis than in IDC without lymphatic metastasis (A, p=0.0028). Protein levels of FoxD3 detected by western blotting in IDC samples without (left lane) or with (right lane) lymphatic metastasis, β-actin served as a loading control (B). Protein levels of FoxD3 in the metastatic breast cancer cell line, MDA-MB-231, and the non-metastatic breast cancer cell line, MCF-7, as detected by western blotting. β-actin served as a loading control (C).

Figure 3

Representative immunohistochemistry images of FoxD3 protein staining in TNBC (A) and non-TNBC invasive ductal breast cancer (B) tissues (200 × magnification, scale bar=20 μm). Semi-quantitative analysis of immunohistochemistry indicated there was significantly lower expression of FoxD3 in TNBC than in non-TNBC, invasive ductal breast cancer tissues (C, p=0.0174).

Figure 4

Semi-quantitative analysis showed that the expression of FoxD3 was not significantly correlated with Ki67 index in IDC (r=-0.108, p=0.4394).