Lombardo Ylenia, Faronato Monica, Filipovic Aleksandra, Vircillo Valentina, Magnani Luca, Coombes R Charles
Breast Cancer Res. 2014 Jun 11;16(3):R62. doi: 10.1186/bcr3675.
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.
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.
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.
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.
抗雌激素疗法耐药是雌激素受体α（ERα）阳性乳腺癌疾病复发和死亡的主要原因。他莫昔芬或雌激素撤除会增加乳腺癌细胞对Notch信号通路的依赖性。在此，我们研究了Nicastrin和Notch信号通路在内分泌耐药乳腺癌细胞中的作用。
我们使用了两种内分泌治疗耐药（ETR）乳腺癌模型：他莫昔芬耐药（TamR）和长期雌激素剥夺（LTED）的MCF7细胞。我们通过Transwell实验评估这些细胞的迁移和侵袭能力。通过实时PCR和蛋白质印迹分析评估上皮-间质转化（EMT）调节因子以及Notch受体和靶点的表达。此外，我们测试了体外抗Nicastrin单克隆抗体（mAb）和γ-分泌酶抑制剂（GSI）作为潜在的EMT逆转治疗剂。最后，我们构建了稳定过表达Nicastrin的MCF7细胞，并评估了它们的EMT特征和对他莫昔芬的反应。
我们发现ETR细胞获得了上皮-间质转化（EMT）表型，并表现出Nicastrin和Notch靶点水平升高。有趣的是，我们检测到Notch4水平较高，但Notch1和Notch2水平较低，这表明在获得耐药性后，信号传导转向通过不同的Notch受体。抗Nicastrin单克隆抗体和GSI PF03084014可有效阻断Nicastrin/Notch4轴，并部分抑制EMT过程。结果，细胞迁移和侵袭减弱，干细胞样群体显著减少。Nicastrin和Notch4的基因沉默导致了类似的效果。最后，Nicastrin的稳定过表达足以通过Notch4激活使MCF7对他莫昔芬无反应。
ETR细胞表达高水平的Nicastrin和Notch4，其激活最终驱动侵袭行为。抗Nicastrin mAb和GSI PF03084014减弱EMT分子的表达，降低细胞侵袭性。Nicastrin过表达本身会诱导与EMT表型获得相关的他莫昔芬耐药。我们的研究结果表明，靶向Nicastrin和/或Notch4作为内分泌耐药乳腺癌的有效治疗策略值得进一步临床评估。
