Pathophysiologic Consequences of Neurotrophin Activation of Trk in Neuroblastoma

神经营养蛋白激活神经母细胞瘤中 Trk 的病理生理学后果

基本信息

批准号：
7965671
负责人：
Carol J Thiele
金额：
$ 69.9万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7965671
关键词：
AKT inhibition Accounting Adult Affect Apoptotic Attenuated Biological Models Biology Brain-Derived Neurotrophic Factor Cell Line Cell Survival Cells Clinical Cytotoxic agent Disease Dose Drug resistance Drug usage Etoposide Evaluation Genetic Hypoxia In Vitro Investigation Ligands Location MYCN gene Malignant Neoplasms Mediating Modality Modeling Mus Mutate Mutation Neoplasm Metastasis Neuroblastoma Neurons Neurotrophic Tyrosine Kinase Receptor Type 2 Nutrient Outcome Pathway interactions Patients Perifosine Phase I Clinical Trials Phase I/II Trial Physiological Process Production Prognostic Marker Proto-Oncogene Proteins c-akt Reporter Resistance Response Elements Retinoids Role Signal Pathway Signal Transduction Signal Transduction Pathway System Systemic Therapy TP53 gene Testing Tetanus Helper Peptide Tetracyclines Toxic effect Tumor Biology Tumor Cell Biology Vascular Endothelial Growth Factors Xenograft Model Xenograft procedure angiogenesis base cell growth chemotherapeutic agent chemotherapy cytotoxic deprivation high risk in vitro Model in vitro testing in vivo in vivo Model inhibitor/antagonist metastatic process migration mouse model neoplastic cell neurotrophic factor novel outcome forecast pre-clinical promoter response tumor

项目摘要

Aim 1. To investigate biologic consequences of BDNF activation of TrkB on chemoresistance. A. Inhibition of AKT pathway in neuroblastoma inhibits tumor cell growth in vitro and in vivo. Our previous studies have identified activation of the PI3Kinase/Akt/GSK3 pathway mediates resistance to chemotherapy in neuroblastoma cells. Moreover our genetic and pharmacologic studies indicated that activation of AKT alone attenuated the effects of chemotherapy in neuroblastoma cells. Since activated AKT is more highly expressed in tumors of Neuroblastoma patients with a poor prognosis, we screened a number of inhibitors of the AKT that could enhance the efficacy of chemotherapy in our pre-clinical in vitro models. We focused on the AKT inhibitor, Perifosine, because it is Phase I/II trials in adult cancers, with a toxicity profile that is controllable with systemic therapy. Perifosine was tested in a number of our pre-clinical in vitro models as well as our in vivo murine heterotypic and orthotopic xenograft models. Perifosine inhibits activation of AKT and more impressively inhibits tumor cell growth in all 4 cell line models tested in vitro and in vivo. In the AS neuroblastoma model system there was complete tumor regression. In the least sensitive model, the addition of chemotherapy with Perifosine caused dramatic tumor regressions. These studies provide proof of principle that targeting Akt alone will inhibit neuroblastoma cell growth and will synergize with cytotoxics such as etoposide. B. Evaluation of Trk inhibitor. Elevated levels of TrkB are associated with poor prognosis in patients with Neuroblastoma. Functionally we have found that activation of TrkB stimulates neuroblastoma migration and enhances survival under nutrient deprivation and when exposed to chemotherapy in in vitro models. We have developed an in vivo model in which the level of TrkB expression in tumor cells is regulated by tetracycline. We have found that mice whose tumors have increased levels of TrkB are less sensitive to low dose chemotherapy than mice whose tumors have lower levels of TrkB. Current studies are aimed at evaluating whether inhibition of the TrkB/PI-3Kinase/Akt pathway re-sensitizes tumor cells to chemotherapy. A trk inhibitor AZ6819 for which a Phase I study in planned in adults has been evaluated to assess whether inhibition of TrkB in neuroblastoma tumor cells alters cell growth or modulates effects of chemotherapy. Specific Aim 2. To investigate biologic consequences of BDNF activation of TrkB on metastasis and angiogenesis Our previous studies have identified that activation of TrkB stimulated increases in HIF-1&#945; and production of VEGF in Neuroblastoma. Moreover we have found that increases in TrkB stimulate increased invasiveness. In order to model this in vivo additional neuroblastoma Tet-regulated TrkB expression models have been generated so that the cell lines contain the spectrum of genetic alterations found in poor prognosis neuroblastoma tumors. Moreover, we have developed in vitro models that contain more physiologically relevant levels of O2 and evaluated how hypoxia affects the sensitivity of neuroblastoma cells to a number of biologic response modifiers. Hypoxia induces expression of TrkB and we have developed a TrkB promoter reporter system that has mutated the hypoxia response elements to be able to assess the impact of hypoxia on the expression of TrkB.

目标 1. 研究 TrkB 的 BDNF 激活对化疗耐药性的生物学影响。 A. 抑制神经母细胞瘤中的 AKT 通路可抑制体外和体内肿瘤细胞的生长。我们之前的研究已经确定 PI3Kinase/Akt/GSK3 通路的激活介导神经母细胞瘤细胞对化疗的耐药性。此外，我们的遗传和药理学研究表明，单独激活 AKT 会减弱化疗对神经母细胞瘤细胞的作用。由于活化的 AKT 在预后不良的神经母细胞瘤患者的肿瘤中表达更高，因此我们在临床前体外模型中筛选了多种可以增强化疗疗效的 AKT 抑制剂。我们重点关注 AKT 抑制剂 Perifosine，因为它是针对成人癌症的 I/II 期试验，其毒性特征可通过全身治疗控制。哌立福辛在我们的许多临床前体外模型以及体内小鼠异型和原位异种移植模型中进行了测试。在体外和体内测试的所有 4 个细胞系模型中，哌立福辛抑制 AKT 的激活，更令人印象深刻的是抑制肿瘤细胞的生长。在 AS 神经母细胞瘤模型系统中，肿瘤完全消退。在最不敏感的模型中，添加哌立福辛化疗导致肿瘤显着消退。这些研究提供了原理证明，即单独靶向 Akt 将抑制神经母细胞瘤细胞生长，并与依托泊苷等细胞毒素产生协同作用。 B. Trk 抑制剂的评价。 TrkB 水平升高与神经母细胞瘤患者预后不良相关。从功能上讲，我们发现 TrkB 的激活可刺激神经母细胞瘤迁移，并在营养缺乏和体外模型中接受化疗时提高存活率。我们开发了一种体内模型，其中肿瘤细胞中 TrkB 的表达水平受四环素调节。我们发现肿瘤 TrkB 水平升高的小鼠对低剂量化疗的敏感性低于肿瘤 TrkB 水平较低的小鼠。目前的研究旨在评估抑制 TrkB/PI-3Kinase/Akt 通路是否会使肿瘤细胞对化疗重新敏感。 trk 抑制剂 AZ6819 已被评估，计划在成人中进行 I 期研究，以评估抑制神经母细胞瘤细胞中的 TrkB 是否会改变细胞生长或调节化疗效果。具体目标 2. 研究 TrkB 的 BDNF 激活对转移和血管生成的生物学影响我们之前的研究已经发现，TrkB 的激活会刺激 HIF-1 的增加，从而导致 HIF-1 的增加。以及神经母细胞瘤中 VEGF 的产生。此外，我们发现 TrkB 的增加会刺激侵袭性的增加。为了在体内模拟这种情况，已经生成了额外的神经母细胞瘤 Tet 调节的 TrkB 表达模型，以便细胞系包含在预后不良的神经母细胞瘤肿瘤中发现的遗传改变谱。此外，我们还开发了含有更多生理相关水平 O2 的体外模型，并评估了缺氧如何影响神经母细胞瘤细胞对多种生物反应调节剂的敏感性。缺氧诱导 TrkB 表达，我们开发了 TrkB 启动子报告系统，该系统使缺氧反应元件发生突变，从而能够评估缺氧对 TrkB 表达的影响。