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

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. © 2018 Elsevier B.V

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 therapies1. After surgery, patients with estrogen receptor (ERα)-positive breast cancer are treated with adjuvant endocrine therapy2, including selective estrogen receptor modulators (SERMs) and/or aromatase inhibitors (AIs)3. However, more than 20% of patients relapse within 10 years and eventually progress to incurable metastatic disease4. 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

Author Correction: Differential epigenetic reprogramming in response to specific endocrine therapies promotes cholesterol biosynthesis and cellular invasion (Nature Communications, (2015), 6, 1, (10044), 10.1038/ncomms10044)

This Article omits a declaration from the Competing Interests statement, which should have included the following: ‘One of the authors, Y.L., is an editor on the staff of Nature Communications, but was not in any way involved in the journal review process. The other authors declare no competing interests.’ Also, the Article contains an error in the name of the author Alba Rodriguez-Meira, which is incorrectly given as Alba Meira. These errors have not been corrected in either the PDF or HTML versions of the Article. © 2019, The Author(s)