DACH1 suppresses breast cancer as a negative regulator of CD44

Abstract

Dachshund homolog 1 (DACH1), a key cell fate determination factor, contributes to tumorigenesis, invasion, metastasis of human breast neoplasm. However, the exact molecular mechanisms for the anti-tumor roles of DACH1 in breast carcinoma are still lack of extensive understanding. Herein, we utilized immunohistochemistry (IHC) staining and public microarray data analysis showing that DACH1 was higher in normal breast, low-grade and luminal-type cancer in comparison with breast carcinoma, high-grade and basal-like tumors respectively. Additionally, both correlation analysis of public databases of human breast carcinoma and IHC analysis of mice xenograft tumors demonstrated that DACH1 inversely related to cancer stem cells (CSCs) markers, epithelial-mesenchymal transition (EMT) inducers and basal-enriched molecules, while cluster of differentiation 44 (CD44) behaved in an opposite manner. Furthermore, mice transplanted tumor model indicated that breast cancer cells Met-1 with up-regulation of DACH1 were endowed with remarkably reduced potential of tumorigenesis. Importantly, meta-analysis of 19 Gene Expression Omnibus (GEO) databases of breast cancer implicated that patients with higher DACH1 expression had prolonged time to death, recurrence and metastasis, while CD44 was a promising biomarker predicting worse overall survival (OS) and metastasis-free survival (MFS). Collectively, our study indicated that CD44 might be a novel target of DACH1 in breast carcinoma.

Figure 1

DACH1 and CD44 were correlated with tumorigenesis and histological grade of breast carcinoma. (a) Representative images of immunohistochemistry staining of DACH1 and CD44 in noncancerous and cancerous tissues were shown. (b) Representative images of immunohistochemistry staining of DACH1 and CD44 in low-grade and high-grade breast neoplasm tissues were shown.

Figure 2

The expression of DACH1 and CD44 correlated with molecular subtypes of breast carcinoma. (a) Representative images of immunohistochemistry staining of DACH1 and CD44 in luminal-type and basal-like breast cancer tissues were shown. (b) Expression analysis of public microarray dataset GSE20711 showed that the mRNA level of DACH1 was significantly higher in luminal-type than in basal-like breast tumor. (c) Expression analysis of GSE 20711 also displayed that CD44 mRNA expression was remarkably lower in luminal-type than in basal-like breast cancer.

Figure 3

DACH1 regulated the expression of some CSCs and EMT genes in Met-1 cells and suppressed tumor growth in vivo. (a) Stable expression of DACH1 in breast cancer Met-1 cells was achieved by retrovirus infection. (b) RNA microarray and cluster analysis showed that upregulation of DACH1 reduced the mRNA levels of CD24, CD44, KLF4, MYC, FN1 and VIM in Met-1 cells. (c) Western blot indicated that upregulation of DACH1 reduced the protein abundance of CD44, Fibronectin, Vimentin, p21 and p27 in Met-1 cells. (d) DACH1 overexpression significantly reduced the volume of tumors by ∼90% and slowed down the speed of tumor growth in nude mice xenograft tumors. (e) Overexpression of DACH1 also reduced mice transplanted tumor weight by ∼90%. (f) Mammary tumor growth in vivo was evaluated by subcutaneous implantation of DACH1-overexpressing Met-1 cells and the GFP controls in nude mice.

Figure 4

DACH1 reduced the expression of CD44, Myc, Sox2, Fibronectin, Vimentin, EGFR and Ki-67 in vivo. Both representative immunohistochemistry images and scoring results showed that overexpression of DACH1 (a) down-regulated the protein abundance of CD44 (b), Myc (c), Sox2 (d), Fibronectin (e), Vimentin (f), EGFR (g) and Ki-67 (h) in nude mice xenograft tumors.

Figure 5

The expression of DACH1 and CD44 correlated with VIM, FN1, YBX1, FOXA1, EGFR and MKI67 in breast cancer tissues. Correlation analysis of public dataset GSE 20685 showed that DACH1 was inversely correlated with cancer stem cell marker CD44 (a), mesenchymal markers FN1 (b) and VIM (c) as well as basal-like markers EGFR (e) and MKI67 (f), while positively associated with luminal marker FOXA1 (d). CD44 was parallel with FN1 (g) and VIM (h), YBX1 (i), EGFR (k) and MKI67 (l), while negatively associated with FOXA1 (j).

Figure 6

The association between the expression of DACH1 and CD44 with VIM, FN1, YBX1, FOXA1, EGFR and MKI67 in breast cancer cell lines. Correlation analysis of data with a total of 51 breast cancer cell lines showed that DACH1 was inversely correlated with CD44 (a), FN1 (b), VIM (c), EGFR (e) and MKI67 (f), while positively associated with FOXA1 (d). In contrast, CD44 was parallel with FN1 (g), VIM (h), YBX1 (i), EGFR (k) and MKI67 (l), but negatively related to FOXA1 (j).

Figure 7

DACH1 and CD44 were related to clinical outcomes of breast cancer patients. Meta-analysis of a total of 19 public databases showed that breast cancer patients with higher DACH1 mRNA level tended to have better overall survival (a), relapse-free survival (c), metastasis-free survival (e). On the contrary, patients with higher CD44 mRNA expression had shorter time to death (b) and metastasis (f), but did not acquire statistically significant worse relapse-free survival in comparison to patients with comparatively lower CD44 expression (d).