Abstract
BackgroundOptimising breast cancer treatment remains a challenge. Resistance to therapy is a major problem in both ER- and ER+ breast cancer. Tumour recurrence after chemotherapy and/or targeted therapy leads to more aggressive tumours with enhanced metastatic ability. Self-renewing cancer stem cells (CSCs) have been implicated in treatment resistance, recurrence and the development of metastatic disease.MethodsIn this study, we utilised in vitro, in vivo and ex vivo breast cancer models using ER+ MCF-7 and ER- MDA-MB-231 cells, as well as solid and metastatic breast cancer patient samples, to interrogate the effects of FKBPL and its peptide therapeutics on metastasis, endocrine therapy resistant CSCs and DLL4 and Notch4 expression. The effects of FKBPL overexpression or peptide treatment were assessed using a t-test or one-way ANOVA with Dunnett’s multiple comparison test.ResultsWe demonstrated that FKBPL overexpression or treatment with FKBPL-based therapeutics (AD-01, pre-clinical peptide /ALM201, clinical peptide) inhibit i) CSCs in both ER+ and ER- breast cancer, ii) cancer metastasis in a triple negative breast cancer metastasis model and iii) endocrine therapy resistant CSCs in ER+ breast cancer, via modulation of the DLL4 and Notch4 protein and/or mRNA expression. AD-01 was effective at reducing triple negative MDA-MB-231 breast cancer cell migration (n ≥ 3, p < 0.05) and invasion (n ≥ 3, p < 0.001) and this was translated in vivo where AD-01 inhibited breast cancer metastasis in MDA-MB-231-lucD3H1 in vivo model (p < 0.05). In ER+ MCF-7 cells and primary breast tumour samples, we demonstrated that ALM201 inhibits endocrine therapy resistant mammospheres, representative of CSC content (n ≥ 3, p < 0.05). Whilst an in vivo limiting dilution assay, using SCID mice, demonstrated that ALM201 alone or in combination with tamoxifen was very effective at delaying tumour recurrence by 12 (p < 0.05) or 21 days (p < 0.001), respectively, by reducing the number of CSCs. The potential mechanism of action, in addition to CD44, involves downregulation of DLL4 and Notch4.ConclusionThis study demonstrates, for the first time, the pre-clinical activity of novel systemic anti-cancer therapeutic peptides, ALM201 and AD-01, in the metastatic setting, and highlights their impact on endocrine therapy resistant CSCs; both areas of unmet clinical need.
Highlights
Optimising breast cancer treatment remains a challenge
FK506-binding protein like (FKBPL) and its therapeutic peptides target cancer stem cells (CSCs) and downregulate DLL4 and Notch4 in MDA-MB-231 and MCF-7 cells We have already demonstrated that FKBPL and its peptide derivatives potentially exert their activity by targeting the CD44 pathway [5, 24]
We investigated the impact of AD-01, as well as endogenous FKBPL, using cells stably overexpressing FKBPL (A3), on DLL4 and Notch 4 levels, which are implicated in metastasis and CSC fate [11, 14]
Summary
Optimising breast cancer treatment remains a challenge. Resistance to therapy is a major problem in both ER- and ER+ breast cancer. Tumour recurrence in endocrine-resistant breast cancer patients leads to a more aggressive type of breast cancer with enhanced metastatic ability [8]. In patients treated with neoadjuvant letrozole, CD44+/CD24− mammosphere forming cells, representative of CSCs, were increased and the remaining tumour cells appeared to have a mesenchymal phenotype consistent with the more aggressive, basal-like type of breast cancer [9]. This acquired endocrine therapy resistance has been attributed to the activation of survival pathways such as the epidermal growth factor receptor (EGFR) pathway and, more recently, the Notch pathway [10]. Tumour and plasma levels of the Notch 1, 2, or 4 receptors and DLL4 ligand were positively correlated with nodal and distant metastases in breast cancer and shorter disease-free or overall survival compared to patients with high DLL4 levels [14, 15]
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