Abnormal expression of the Notch and Wnt/β-catenin signaling pathways in stem-like ALDHhiCD44+ cells correlates highly with Ki-67 expression in breast cancer

Abstract

Previous studies have reported that breast cancer stem cells may be closely associated with tumor metastasis, recurrence, and even the failure of chemotherapy and radiotherapy. The aim of the present study was to investigate whether important cell signaling pathways associated with drug resistance are activated in stem-like acetaldehyde dehydrogenase (ALDH)^hi cluster of differentiation (CD)44⁺ cells, and to analyze the association between ALDH^hiCD44⁺ cells and specific pathological features. ALDH^hiCD44⁺ cells and non-stem-like ALDH^lowCD44⁺ cells were separated from MDA-MB-231 cells by fluorescence-activated cell sorting, and the mRNA expression levels of Notch1 and β-catenin were estimated by performing quantitative polymerase chain reaction in the stem-like and non-stem-like cells. Line correlation analysis was used to evaluate the correlation between an immunohistochemical panel of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67, and ALDH^hiCD44⁺ cells from patients with invasive breast carcinoma. The mRNA levels of Notch1 and β-catenin were significantly higher in the ALDH^hiCD44⁺ cells compared with those in the ALDH^lowCD44⁺ cells (P<0.05); furthermore, the present study determined a high correlation (P<0.05) between the ALDH^hiCD44⁺ cells and Ki-67 expression (P=0.007), but no correlation (P≥0.05) with ER (P=0.065), PR (P=0.107) and HER2 (P=0.050). Overall, these data clearly indicate that ALDH^hiCD44⁺ cells may serve as novel diagnostic and prognostic factors in breast cancer.

Keywords: breast cancer; immunohistochemical; signaling pathway; stem-like cell.

Figure 1

Proportion of cluster of differentiation (CD)44⁺ and acetaldehyde dehydrogenase (ALDH)^hi cells in an MDA-MB-231 cell suspension, detected using a fluorescence microscope (magnification, ×100). (A) MDA-MB-231 cell suspension. (B) High proportion of CD44⁺ cells (red fluorescence) and (C) low proportion of ALDH^hi cells (green fluorescence).

Figure 2

Results of flow separation experiment and expressions of activated Notch1 and β-catenin genes in stem-like ALDH^hiCD44⁺ and non-stem-like ALDH^lowCD44⁺ cells. (A) ALDH+diethylaminobenzaldehyde+immunoglobulin G1-PE(Q3); (B) proportion of ALDH^hiCD44⁺(Q2) and ALDH^lowCD44⁺ cells (Q1) in the MDA-MB-231 cells. (C) The Notch1 gene; and (D) the β-catenin gene. GAPDH was used as a reference gene. (C) and (D) represent the mean cycle threshold values of five independent experiments. ALDH, acetaldehyde dehydrogenase; CD, cluster of differentiation; PE, phycoerythrin; FITC, fluorescein isothiocyanate.

Figure 3

Contents of the primary culture of breast tumor and tumor-adjacent tissues analyzed using flow cytometry, and the correlation between the abundance of stem-like cells and the immunohistochemical staining results. Microscopy of the (A) breast tumor and (B) tumor-adjacent tissues (magnification, ×100). (C) Correlation between the ALDH^hiCD44⁺ cells and Ki-67 expression levels. (D) The expression of the ALDH^hiCD44⁺ cells was significantly higher in the tumor tissues compared with the tumor-adjacent tissues (P<0.05). ALDH, acetaldehyde dehydrogenase; CD, cluster of differentiation.