Nicastrin and Notch4 drive endocrine therapy resistance and epithelial to mesenchymal transition in MCF7 breast cancer cells.

INTRODUCTION: Resistance to anti-estrogen therapies is a major cause of disease relapse and mortality in estrogen receptor alpha (ERα)-positive breast cancers. Tamoxifen or estrogen withdrawal increases the dependence of breast cancer cells on Notch signalling. Here, we investigated the contribution of Nicastrin and Notch signalling in endocrine-resistant breast cancer cells. METHODS: We used two models of endocrine therapies resistant (ETR) breast cancer: tamoxifen-resistant (TamR) and long-term estrogen-deprived (LTED) MCF7 cells. We evaluated the migratory and invasive capacity of these cells by Transwell assays. Expression of epithelial to mesenchymal transition (EMT) regulators as well as Notch receptors and targets were evaluated by real-time PCR and western blot analysis. Moreover, we tested in vitro anti-Nicastrin monoclonal antibodies (mAbs) and gamma secretase inhibitors (GSIs) as potential EMT reversal therapeutic agents. Finally, we generated stable Nicastrin overexpessing MCF7 cells and evaluated their EMT features and response to tamoxifen. RESULTS: We found that ETR cells acquired an epithelial to mesenchymal transition (EMT) phenotype and displayed increased levels of Nicastrin and Notch targets. Interestingly, we detected higher level of Notch4 but lower levels of Notch1 and Notch2 suggesting a switch to signalling through different Notch receptors after acquisition of resistance. Anti-Nicastrin monoclonal antibodies and the GSI PF03084014 were effective in blocking the Nicastrin/Notch4 axis and partially inhibiting the EMT process. As a result of this, cell migration and invasion were attenuated and the stem cell-like population was significantly reduced. Genetic silencing of Nicastrin and Notch4 led to equivalent effects. Finally, stable overexpression of Nicastrin was sufficient to make MCF7 unresponsive to tamoxifen by Notch4 activation. CONCLUSIONS: ETR cells express high levels of Nicastrin and Notch4, whose activation ultimately drives invasive behaviour. Anti-Nicastrin mAbs and GSI PF03084014 attenuate expression of EMT molecules reducing cellular invasiveness. Nicastrin overexpression per se induces tamoxifen resistance linked to acquisition of EMT phenotype. Our finding suggest that targeting Nicastrin and/or Notch4 warrants further clinical evaluation as valid therapeutic strategies in endocrine-resistant breast cancer.

Author Correction: Differential epigenetic reprogramming in response to specific endocrine therapies promotes cholesterol biosynthesis and cellular invasion.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Activation of Farnesoid X Receptor impairs the tumor-promoting function of breast cancer-associated fibroblasts.

Cancer-associated Fibroblasts (CAFs), the principal components of tumor microenvironment, play multiple role in breast cancer progression. We have previously shown an oncosuppressive role of the nuclear Farnesoid X Receptor (FXR) in mammary epithelial cancer cells, here we assessed whether FXR activation may affect CAF tumor-promoting features. We showed that FXR is expressed in human CAFs isolated from four patients and treatment with the selective FXR agonist GW4064 decreased CAF migration, stress-fiber formation and contractility. RNA-sequencing highlighted cell movement and pathways known to govern cell cytoskeleton organization and migration among the most down-regulated functions and ingenuity canonical pathways upon GW4064 treatment. FXR activation reduced expression of different secreted factors. Coculture experiments revealed a reduced growth and motility of breast cancer cells treated with conditioned-media derived from GW4064-treated CAFs. Increased FXR levels in bulk tumors correlated with a longer patient survival. Our results evidence that FXR activation inhibits tumor-stimulatory activities of CAFs by impacting their mechanical properties and their paracrine signaling repertoire, suggesting that nuclear FXR ligands, by targeting both neoplastic cells and supportive stroma, may represent a promising avenue for the future management of breast cancer.

Acquired CYP19A1 amplification is an early specific mechanism of aromatase inhibitor resistance in ERα metastatic breast cancer.

Tumor evolution is shaped by many variables, potentially involving external selective pressures induced by therapies. After surgery, patients with estrogen receptor (ERα)-positive breast cancer are treated with adjuvant endocrine therapy, including selective estrogen receptor modulators (SERMs) and/or aromatase inhibitors (AIs). However, more than 20% of patients relapse within 10 years and eventually progress to incurable metastatic disease. Here we demonstrate that the choice of therapy has a fundamental influence on the genetic landscape of relapsed diseases. We found that 21.5% of AI-treated, relapsed patients had acquired CYP19A1 (encoding aromatase) amplification (CYP19A1amp). Relapsed patients also developed numerous mutations targeting key breast cancer-associated genes, including ESR1 and CYP19A1. Notably, CYP19A1amp cells also emerged in vitro, but only in AI-resistant models. CYP19A1 amplification caused increased aromatase activity and estrogen-independent ERα binding to target genes, resulting in CYP19A1amp cells showing decreased sensitivity to AI treatment. These data suggest that AI treatment itself selects for acquired CYP19A1amp and promotes local autocrine estrogen signaling in AI-resistant metastatic patients.

Identification of novel 2-(1H-indol-1-yl)-benzohydrazides CXCR4 ligands impairing breast cancer growth and motility.

BACKGROUND: Stromal-derived-factor-1 (SDF-1) and the G-protein-coupled receptor CXCR4 are involved in several physiological and pathological processes including breast cancer spread and progression. Several CXCR4 antagonists have currently reached advanced development stages as potential therapeutic agents for different diseases. RESULTS: A small series of novel CXCR4 ligands, based on a 2-(1H-indol-1-yl)-benzohydrazide scaffold, has been designed and synthesized. The interaction with CXCR4-active site was predicted by molecular docking and confirmed by whole cell-based [(125)I]-SDF-1 ligand competition binding assays. One of the synthesized compounds was particularly active in blocking SDF-1-induced breast cancer cell motility, proliferation and downstream signaling activation in different breast cancer cell models and coculture systems. CONCLUSION: The newly synthesized compounds represent suitable leads for the development of innovative therapeutic agents targeting CXCR4.

Activated FXR Inhibits Leptin Signaling and Counteracts Tumor-promoting Activities of Cancer-Associated Fibroblasts in Breast Malignancy.

Cancer-associated fibroblasts (CAFs), the principal components of the tumor stroma, play a central role in cancer development and progression. As an important regulator of the crosstalk between breast cancer cells and CAFs, the cytokine leptin has been associated to breast carcinogenesis. The nuclear Farnesoid X Receptor-(FXR) seems to exert an oncosuppressive role in different tumors, including breast cancer. Herein, we demonstrated, for the first time, that the synthetic FXR agonist GW4064, inhibiting leptin signaling, affects the tumor-promoting activities of CAFs in breast malignancy. GW4064 inhibited growth, motility and invasiveness induced by leptin as well as by CAF-conditioned media in different breast cancer cell lines. These effects rely on the ability of activated FXR to increase the expression of the suppressor of the cytokine signaling 3 (SOCS3) leading to inhibition of leptin-activated signaling and downregulation of leptin-target genes. In vivo xenograft studies, using MCF-7 cells alone or co-injected with CAFs, showed that GW4064 administration markedly reduced tumor growth. Interestingly, GW4064-treated tumors exhibited decreased levels of leptin-regulated proteins along with a strong staining intensity for SOCS3. Thus, FXR ligands might represent an emerging potential anti-cancer therapy able to block the tumor supportive role of activated fibroblasts within the breast microenvironment.

Differential epigenetic reprogramming in response to specific endocrine therapies promotes cholesterol biosynthesis and cellular invasion.

Endocrine therapies target the activation of the oestrogen receptor alpha (ERα) via distinct mechanisms, but it is not clear whether breast cancer cells can adapt to treatment using drug-specific mechanisms. Here we demonstrate that resistance emerges via drug-specific epigenetic reprogramming. Resistant cells display a spectrum of phenotypical changes with invasive phenotypes evolving in lines resistant to the aromatase inhibitor (AI). Orthogonal genomics analysis of reprogrammed regulatory regions identifies individual drug-induced epigenetic states involving large topologically associating domains (TADs) and the activation of super-enhancers. AI-resistant cells activate endogenous cholesterol biosynthesis (CB) through stable epigenetic activation in vitro and in vivo. Mechanistically, CB sparks the constitutive activation of oestrogen receptors alpha (ERα) in AI-resistant cells, partly via the biosynthesis of 27-hydroxycholesterol. By targeting CB using statins, ERα binding is reduced and cell invasion is prevented. Epigenomic-led stratification can predict resistance to AI in a subset of ERα-positive patients.