Nölting Svenja, Rentsch Jakob, Freitag Helma, Detjen Katharina, Briest Franziska, Möbs Markus, Weissmann Victoria, Siegmund Britta, Auernhammer Christoph J, Aristizabal Prada Elke Tatjana, Lauseker Michael, Grossman Ashley, Exner Samantha, Fischer Christian, Grötzinger Carsten, Schrader Jörg, Grabowski Patricia
Department of Internal Medicine II, Klinikum der Universität München (KUM), Ludwig-Maximilians-Universität München, München, Bavaria, Germany.
Department of Internal Medicine IV, Klinikum der Universität München (KUM), Ludwig-Maximilians-Universität München, München, Bavaria, Germany.
PLoS One. 2017 Aug 11;12(8):e0182852. doi: 10.1371/journal.pone.0182852. eCollection 2017.
BACKGROUND/AIMS: The therapeutic options for metastatic neuroendocrine tumors (NETs) are limited. As PI3K signaling is often activated in NETs, we have assessed the effects of selective PI3Kp110α inhibition by the novel agent BYL719 on cell viability, colony formation, apoptosis, cell cycle, signaling pathways, differentiation and secretion in pancreatic (BON-1, QGP-1) and pulmonary (H727) NET cell lines.
Cell viability was investigated by WST-1 assay, colony formation by clonogenic assay, apoptosis by caspase3/7 assay, the cell cycle by FACS, cell signaling by Western blot analysis, expression of chromogranin A and somatostatin receptors 1/2/5 by RT-qPCR, and chromogranin A secretion by ELISA.
BYL719 dose-dependently decreased cell viability and colony formation with the highest sensitivity in BON-1, followed by H727, and lowest sensitivity in QGP-1 cells. BYL719 induced apoptosis and G0/G1 cell cycle arrest associated with increased p27 expression. Western blots showed inhibition of PI3K downstream targets to a varying degree in the different cell lines, but IGF1R activation. The most sensitive BON-1 cells displayed a significant, and H727 cells a non-significant, GSK3 inhibition after BYL719 treatment, but these effects do not appear to be mediated through the IGF1R. In contrast, the most resistant QGP-1 cells showed no GSK3 inhibition, but a modest activation, which would partially counteract the other anti-proliferative effects. Accordingly, BYL719 enhanced neuroendocrine differentiation with the strongest effect in BON-1, followed by H727 cells indicated by induction of chromogranin A and somatostatin receptor 1/2 mRNA-synthesis, but not in QGP-1 cells. In BON-1 and QGP-1 cells, the BYL719/everolimus combination was synergistic through simultaneous AKT/mTORC1 inhibition, and significantly increased somatostatin receptor 2 transcription compared to each drug separately.
Our results suggest that the agent BYL719 could be a novel therapeutic approach to the treatment of NETs that may sensitize NET cells to somatostatin analogs, and that if there is resistance to its action this may be overcome by combination with everolimus.
背景/目的：转移性神经内分泌肿瘤（NETs）的治疗选择有限。由于PI3K信号通路在NETs中常被激活，我们评估了新型药物BYL719选择性抑制PI3Kp110α对胰腺（BON-1、QGP-1）和肺（H727）NET细胞系的细胞活力、集落形成、凋亡、细胞周期、信号通路、分化和分泌的影响。
通过WST-1法检测细胞活力，通过克隆形成试验检测集落形成，通过caspase3/7试验检测凋亡，通过流式细胞术检测细胞周期，通过蛋白质印迹分析检测细胞信号，通过RT-qPCR检测嗜铬粒蛋白A和生长抑素受体1/2/5的表达，通过ELISA检测嗜铬粒蛋白A的分泌。
BYL719剂量依赖性地降低细胞活力和集落形成，在BON-1细胞中敏感性最高，其次是H727细胞，在QGP-1细胞中敏感性最低。BYL719诱导凋亡和G0/G1细胞周期停滞，伴有p27表达增加。蛋白质印迹显示在不同细胞系中PI3K下游靶点受到不同程度的抑制，但IGF1R被激活。最敏感的BON-1细胞在BYL719处理后显示出显著的GSK3抑制，H727细胞显示出不显著的GSK3抑制，但这些作用似乎不是通过IGF1R介导的。相反，最耐药的QGP-1细胞未显示GSK3抑制，而是有适度激活，这将部分抵消其他抗增殖作用。因此，BYL719增强神经内分泌分化，在BON-1细胞中作用最强，其次是H727细胞，表现为嗜铬粒蛋白A和生长抑素受体1/2 mRNA合成增加，但在QGP-1细胞中未增强。在BON-1和QGP-1细胞中，BYL719/依维莫司联合用药通过同时抑制AKT/mTORC1具有协同作用，与单独使用每种药物相比，显著增加了生长抑素受体2的转录。
我们的结果表明，药物BYL719可能是治疗NETs的一种新的治疗方法，它可能使NET细胞对生长抑素类似物敏感，如果对其作用产生耐药性，可能通过与依维莫司联合使用来克服。
