Wnt/ERK/CDK4/6 activation in the partial EMT state coordinates mammary cancer stemness with self-renewal and inhibition of differentiation

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Abstract

Background: The hybrid EMT state is a key driver of tumour regenerative and metastatic potential; however, the mechanism whereby this programme regulates tumour stemness with respect to self-renewal and differentiation remains unclear. Methods: We isolated epithelial/mesenchymal (E/M) (CD104highCD44high) and mesenchymal (M) (CD104low CD44high) subpopulations from basal-like breast cancer cell lines. These were assayed for tumour-initiating potential and organoid-forming ability, as well as for transcriptional regulators of the hybrid EMT state by RNA and ATAC-sequencing, and their regulation by the Wnt/ERK/CDK4/6 signalling pathway. Results: E/M cells were endowed with organoid-forming ability as well as by tumour-initiating and metastatic potential relative to M cells. Interestingly, Wnt3a stimulates transient ERK/CDK4/6 activation in E/M cells, thereby upregulating FOXC2, and in turn TAp63 and ΔNp63, which support the hybrid state. In parallel, ERK/CDK4/6 activates S-phase and FOXM1, thereby promoting self-renewal. Remarkably, transient ERK activation by Wnt3a deactivates EGFR, thus preventing sustained ERK phosphorylation from causing E/M differentiation. Consistently, ERK/CDK4/6 drug perturbation in E/M cells suppressed FOXC2/p63, FOXM1, self-renewal, organoid formation and mammary tumour growth via epithelial differentiation. Conclusions: These findings unravelled a mechanism whereby the hybrid EMT state regulates stemness, self-renewal and differentiation via transient Wnt/ERK/CDK4/6 activation, which can be leveraged for cancer stem cell therapy.