Diluvio Giulia, Del Gaudio Francesca, Giuli Maria Valeria, Franciosa Giulia, Giuliani Eugenia, Palermo Rocco, Besharat Zein Mersini, Pignataro Maria Gemma, Vacca Alessandra, d'Amati Giulia, Maroder Marella, Talora Claudio, Capalbo Carlo, Bellavia Diana, Checquolo Saula
Department of Molecular Medicine, Sapienza University, Rome, Italy.
Department of Cell and Molecular Biology, Karolinska Institutet, 17177, Stockolm, Sweden.
Oncogenesis. 2018 May 25;7(5):42. doi: 10.1038/s41389-018-0051-9.
Notch dysregulation has been implicated in numerous tumors, including triple-negative breast cancer (TNBC), which is the breast cancer subtype with the worst clinical outcome. However, the importance of individual receptors in TNBC and their specific mechanism of action remain to be elucidated, even if recent findings suggested a specific role of activated-Notch3 in a subset of TNBCs. Epidermal growth factor receptor (EGFR) is overexpressed in TNBCs but the use of anti-EGFR agents (including tyrosine kinase inhibitors, TKIs) has not been approved for the treatment of these patients, as clinical trials have shown disappointing results. Resistance to EGFR blockers is commonly reported. Here we show that Notch3-specific inhibition increases TNBC sensitivity to the TKI-gefitinib in TNBC-resistant cells. Mechanistically, we demonstrate that Notch3 is able to regulate the activated EGFR membrane localization into lipid rafts microdomains, as Notch3 inhibition, such as rafts depletion, induces the EGFR internalization and its intracellular arrest, without involving receptor degradation. Interestingly, these events are associated with the EGFR tyrosine dephosphorylation at Y1173 residue (but not at Y1068) by the protein tyrosine phosphatase H1 (PTPH1), thus suggesting its possible involvement in the observed Notch3-dependent TNBC sensitivity response to gefitinib. Consistent with this notion, a nuclear localization defect of phospho-EGFR is observed after combined blockade of EGFR and Notch3, which results in a decreased TNBC cell survival. Notably, we observed a significant correlation between EGFR and NOTCH3 expression levels by in silico gene expression and immunohistochemical analysis of human TNBC primary samples. Our findings strongly suggest that combined therapies of TKI-gefitinib with Notch3-specific suppression may be exploited as a drug combination advantage in TNBC treatment.
Notch失调与多种肿瘤有关，包括三阴性乳腺癌（TNBC），这是临床预后最差的乳腺癌亚型。然而，即使最近的研究结果表明活化的Notch3在一部分TNBC中具有特定作用，但单个受体在TNBC中的重要性及其具体作用机制仍有待阐明。表皮生长因子受体（EGFR）在TNBC中过表达，但抗EGFR药物（包括酪氨酸激酶抑制剂，TKIs）尚未被批准用于治疗这些患者，因为临床试验结果令人失望。对EGFR阻滞剂的耐药性普遍存在。在这里，我们表明Notch3特异性抑制可增加TNBC耐药细胞对TKI-吉非替尼的敏感性。从机制上讲，我们证明Notch3能够将活化的EGFR膜定位调节到脂筏微结构域中，因为Notch3抑制（如脂筏耗竭）会诱导EGFR内化及其细胞内滞留，而不涉及受体降解。有趣的是，这些事件与蛋白酪氨酸磷酸酶H1（PTPH1）在Y1173残基（而非Y1068）处对EGFR酪氨酸去磷酸化有关，因此表明其可能参与了观察到的Notch3依赖性TNBC对吉非替尼的敏感性反应。与此观点一致，在联合阻断EGFR和Notch3后，观察到磷酸化EGFR的核定位缺陷，这导致TNBC细胞存活率降低。值得注意的是，通过对人类TNBC原发性样本的计算机基因表达和免疫组织化学分析，我们观察到EGFR和NOTCH3表达水平之间存在显著相关性。我们的研究结果强烈表明，TKI-吉非替尼与Notch3特异性抑制的联合疗法可能被用作TNBC治疗中的药物联合优势。
