To identify the role and to explore the mechanism of extracellular 5'-nucleotidase (CD73) in human breast cancer growth, CD73 expression was measured firstly in breast cancer tissues and cell lines, and then interfered with or over-expressed by recombinant lentivirus in cell lines. Impacts of CD73 on breast cancer cell proliferation and cell cycle were investigated with colony formation assay, CCK-8 and flow cytometry. The relationship between CD73 and AKT/GSK-3β/β-catenin pathway was assessed with adenosine, adenosine 2A receptor antagonist (SCH-58261), adenosine 2A receptor agonist (NECA), CD73 enzyme inhibitor (APCP) and Akt inhibitor (MK-2206). Moreover, the effect of CD73 on breast cancer growth in vivo was examined with human breast cancer transplanting model of nude mice. The results showed that the expression of CD73 was high in breast cancer tissues and increased with advanced tumor grades and lympho-node status. CD73 expression was higher in more malignant cells, and CD73 overexpression promoted breast cancer cell proliferation in both in vivo and in vitro. It activated AKT/GSK-3β/β-catenin/cyclinD1 signaling pathway through CD73 enzyme activity and other mechanism.
SummaryCD73, an ecto-enzyme overexpressed in breast-cancer cells, catalyzes the dephosphorylation of adenosine monophosphates into adenosine. Anti-CD73 slows breast cancer growth and its spread both in vivo and in vitro. In this study, we investigated the relation of CD73 to epidermal growth factor receptor (EGFR) expression using tissue array and breast cancer cell lines. We found that CD73 expression correlated positively to EGFR expression in vivo (n 5 80, r 5 0.425, P \ 0.01) and in vitro. EGFR expression can be decreased by suppressing CD73 with an inhibitor or small shRNA, and this effect was reversed by adenosine and NECA (adenosine A2 receptor agonist), which suggested that adenosine is involved in EGFR expression regulated by CD73 (P \ 0.01). We also showed that CD73 regulates EGFR phosphorylation by Src (P \ 0.01). By transcription factor (TF) assay, CD73 was found to regulate some associated TFs activity such as PPARc, which mediates EGFR expression, although whether PPARc mediates the effect of CD73 on EGFR expression needs further study. The KaplanMeier recurrence-free survival curves for CD73 were also plotted in www.kmplot.com. The curves show that CD73 expression separates the cases into significantly different prognostic groups among the estrogen receptor-negative cancers (P \ 0.01). Our results suggest that CD73 may be a potential prognostic biomarker associated with coexpression of EGFR in human breast cancer.2012 IUBMB IUBMB Life, 64(11): 911-920, 2012
Tumor stem cell theory may well explain a variety of malignant behaviors of tumors. Cells undergoing epithelial-mesenchymal transition (EMT) share many characteristics with tumor stem cells. Our previous studies showed that extracellular -5'- nucleotidase (CD73), one of the important surface markers of mesenchymal stem cells, may promote growth and metastasis of breast cancer cells both in vivo and in vitro. In this study, we assessed breast cancer stem cell (BCSC) markers [acetaldehyde dehydrogenase (ALDH) and CD44CD24] in various breast cancer cell lines with flow cytometry after overexpression (by lentivirus infection) or suppression (by siRNA interference) of CD73. We measured CD73 expression in breast cancer mammospheres with real-time PCR and western blots. Finally, we examined the expression of CD73 and EMT markers in different breast cancer cell lines, as well as in mammary cells (MCF10A) that underwent EMT induced by transforming growth factor beta (TGF-β). We found that CD73 positively correlated with ALDH or CD44CD24 subsets of breast cancer cells. CD73 was expressed more in breast cancer mammospheres than in adherent cells. CD73 and mesenchymal marker expression was higher in breast cancer cells with more malignant features, while CD73 was lower in low malignant breast cancer cells with higher epithelial markers. Furthermore, CD73 expression increased during the process of TGF-β-induced EMT. Our results indicate that CD73 may play an important role in BCSCs.
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