Role of ERalpha/NOTCH4 axis in sustaining stemness in breast cancer cells

Abstract

Early detection and new therapeutic strategies have improved breast cancer patient outcome and survival rates in the last years. However, breast cancer still remains the second leading cause of cancer-related deaths among women worldwide, and approximately 30% of patients eventually experience a tumor relapse. Treatment failure is mainly due to metastatic process and resistance to conventional therapy. Over the past decade it has been established the existence of a subpopulation of cancer stem cell (CSC) within breast cancers that is responsible for tumor initiation, progression and resistance to endocrine therapies. It is well known the “driving role” of oestrogens and its receptor alpha (ERα), in development and progression of breast cancer disease, but still unknown their role in regulating breast CSCs (BCSCs). In the past few years, several studies revealed the presence of gain-of-function mutation in ESR1, gene encoding for ERα, in metastatic breast cancer patients after long-term endocrine therapies treatment. Particularly, Y537N, Y537S and D538G are the most frequent “hot spot” mutations within ERα hormone-binding domain (HBD) that lead to ligand-independent ERα activity and consequently, resistance to endocrine therapy. Here, we studied how HBD-ESR1 mutations might account for a mechanism of metastatic process and endocrine resistance, sustaining stem cell-like phenotype. As experimental model, we used breast cancer cell lines expressing wild-type and HBDESR1 mutations. Our results, using in vivo and in vitro experiment (mammosphereforming assay and CD44+/CD24- phenotype analysis) have suggested an enrichment of BCSCs activity by HBD-ESR1 mutations, that seems to be sustained by Notch4 signaling through constitutive hyper phosphorylation of Serine 118 residue of ERα that has been demonstrated related to stem cell phenotype and tumor initiation, in mutant-expressing cells. Experiments conducted using CRISPR-Cas9 knock-in of Y537S-ERα mutation confirmed the role of this mutation in tumor initiation and progression as obtained using HBD-ESR1 stable clones. We propose a potential novel role of HBD-ESR1 mutations in sustaining BCSCs activity, that could have clinical relevance, suggesting new molecular biomarker and target to aim better therapeutic strategies for ERα-positive breast cancer metastatic patients.