Abstract
IntroductionWnt signalling has been implicated in stem cell regulation however its role in breast cancer stem cell regulation remains unclear.MethodsWe used a panel of normal and breast cancer cell lines to assess Wnt pathway gene and protein expression, and for the investigation of Wnt signalling within stem cell-enriched populations, mRNA and protein expression was analysed after the selection of anoikis-resistant cells. Finally, cell lines and patient-derived samples were used to investigate Wnt pathway effects on stem cell activity in vitro.ResultsWnt pathway signalling increased in cancer compared to normal breast and in both cell lines and patient samples, expression of Wnt pathway genes correlated with estrogen receptor (ER) expression. Furthermore, specific Wnt pathway genes were predictive for recurrence within subtypes of breast cancer. Canonical Wnt pathway genes were increased in breast cancer stem cell-enriched populations in comparison to normal breast stem cell-enriched populations. Furthermore in cell lines, the ligand Wnt3a increased whilst the inhibitor DKK1 reduced mammosphere formation with the greatest inhibitory effects observed in ER+ve breast cancer cell lines. In patient-derived metastatic breast cancer samples, only ER-ve mammospheres were responsive to the ligand Wnt3a. However, the inhibitor DKK1 efficiently inhibited both ER+ve and ER-ve breast cancer but not normal mammosphere formation, suggesting that the Wnt pathway is aberrantly activated in breast cancer mammospheres.ConclusionsCollectively, these data highlight differential Wnt signalling in breast cancer subtypes and activity in patient-derived metastatic cancer stem-like cells indicating a potential for Wnt-targeted treatment in breast cancers.
Highlights
Wnt signalling has been implicated in stem cell regulation its role in breast cancer stem cell regulation remains unclear
We show that WNT pathway inhibition preferentially reduces stem-like cell activity in patient-derived metastatic breast cancer compared to normal cells
Consistent with its gene expression level, LEF1 protein was higher in five breast cancer cell lines compared to two normal cell lines (MCF10A and 226LU19), providing further evidence that WNT signalling is active in breast cancer cell lines (Figure 1B)
Summary
Wnt signalling has been implicated in stem cell regulation its role in breast cancer stem cell regulation remains unclear. Loss of negative pathway regulators such as the extracellular inhibitor of WNT signaling, secreted Frizzled-related protein 1 (sFRP1), is found in many breast tumors and is associated with poor prognosis [3,10]. Down regulation of the inhibitor Dickkopf 1 (DKK1) in a lung metastases derived MCF7-LM cell line demonstrates the importance of Wnt regulation in the metastatic process in breast cancer [11]. These data suggest that WNT pathway de-regulation within the breast contributes to cancer formation and metastasis. Transplantation experiments using immunocomprimised mice, showed that as few as 100 human breast cancer cells with the cell surface markers CD44+CD242/low were tumorigenic and could be serially passaged to generate new tumours [13]
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