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

• 批准号: 8157460
• 负责人: Carol J Thiele
• 金额: $ 68.66万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/8157460
• 关键词: Neuroblastoma; neurotrophic factor

Aim 1. To investigate biologic consequences of BDNF activation of TrkB on chemoresistance. 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. 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-1alpha 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. Since TrkA expression is associated with good prognosis and elevated TrkA levels had been previously identified to suppress VEGF levels we initiated a study to assess whether NGF activation of TrkA altered VEGF expression. Our evidence supported previous study that elevated TrkA in the absence of NGF suppressed VEGF. However we found that NGF activation in TrkA expressing NB cells stimulated VEGF production via activation of HIF-1a. However, in assessing normal dorsal root ganglion TrkA expressing neurons from mice we determined that NGF activation of TrkA induces HIF-1a and increases in VEGF production. Specific Aim 3. Develop in vivo model systems to test targeted agents to the BDNF/TrkB signal transduction pathway 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. The Akt inhibitor Perifosine was evaluated and found to re-sensitize NB cells expressing high levels of TrkB to etoposide. These studies were first performed in "in vitro" assays and similar results were also seen in our murine model of TrkB induced chemoresistance. This indicates that targeting Akt in NB tumors should sensitize them to chemotherapy. This may be an effective strategy for relapsed neuroblastoma tumors.

目的1。研究TRKB激活化学耐药性BDNF激活的生物学后果。抑制神经母细胞瘤中AKT途径的抑制会抑制体外和体内肿瘤细胞的生长。我们以前的研究已经确定了PI3KINASE/AKT/GSK3途径的激活介导神经母细胞瘤细胞中化学疗法的抗性。此外，我们的遗传学和药理学研究表明，仅AKT的激活减弱了化学疗法对神经母细胞瘤细胞的影响。由于活化的AKT在预后较差的神经母细胞瘤患者的肿瘤中高度表达，因此我们筛选了许多AKT的抑制剂，这些抑制剂可以增强化学疗法在临床前体外模型中的功效。我们专注于AKT抑制剂Perifosine，因为它是成人癌症中的I/II期试验，具有全身治疗可控制的毒性特征。在我们的许多临床前体外模型以及我们的体内鼠异型和原位异种移植模型中测试了perifosine。 Perifosine抑制AKT的激活，并更令人印象深刻地抑制所有在体外和体内测试的4个细胞系模型中的肿瘤细胞生长。在AS神经母细胞瘤模型系统中，有完全的肿瘤回归。在最小敏感的模型中，核苷化学疗法的添加引起了巨大的肿瘤回归。这些研究提供了原理证明，仅靶向AKT将抑制神经母细胞瘤细胞的生长，并将与依托泊苷这样的细胞毒素协同作用。 具体目的2。研究TRKB激活BDNF对转移和血管生成的生物学后果，我们先前的研究已经确定，TRKB的激活刺激了HIF-1Alpha的增加和神经母细胞瘤中VEGF的产生。此外，我们发现TRKB的增加刺激了侵入性的提高。为了模拟该体内的其他神经母细胞瘤TER调节的TRKB表达模型，以便细胞系包含不良预后神经母细胞瘤肿瘤中发现的遗传改变的频谱。此外，我们开发了体外模型，其中包含更多生理相关水平的O2水平，并评估了缺氧如何影响神经母细胞瘤细胞对许多生物学反应修饰剂的敏感性。由于TRKA表达与良好的预后有关，并且先前已经鉴定出TRKA水平升高以抑制VEGF水平，因此我们开始了一项研究，以评估TRKA的NGF激活是否改变了VEGF的表达。我们的证据支持先前的研究，该研究在没有NGF抑制VEGF的情况下升高TRKA。但是，我们发现表达NB细胞的TRKA中的NGF激活通过HIF-1A激活刺激VEGF的产生。但是，在评估来自小鼠的正常背根神经节TRKA时，我们确定TRKA的NGF激活会诱导HIF-1A并增加VEGF的产生。 具体目标3。开发体内模型系统，以测试靶向剂至BDNF/TRKB信号转导途径升高TRKB水平的较低水平与神经母细胞瘤患者的预后不良有关。从功能上讲，我们发现TRKB的激活刺激神经母细胞瘤的迁移，并在营养剥夺下以及在体外模型中接受化学疗法时增强生存率。我们已经开发了一个体内模型，其中肿瘤细胞中TRKB表达水平受四环素调节。我们发现，肿瘤水平升高的小鼠对低剂量化学疗法的敏感性较小，而肿瘤的TRKB水平较低。目前的研究旨在评估抑制TRKB/PI-3Kinase/AKT途径是否会重新敏感性肿瘤细胞进行化学疗法。评估了AKT抑制剂perifosine并发现将高水平TRKB的NB细胞重新敏感为依托泊苷。这些研究首先是在“体外”测定中进行的，在我们的TRKB诱导化学耐药性的鼠模型中也观察到了相似的结果。这表明靶向NB肿瘤中的AKT应该使它们对化疗敏感。这可能是复发神经母细胞瘤肿瘤的有效策略。