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：
7733226
负责人：
Carol J Thiele
金额：
$ 50.8万
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/7733226
关键词：
1-Phosphatidylinositol 3-Kinase Accounting Adriamycin PFS Affect Apoptosis Apoptotic Attention Attenuated BAD gene BCL2L11 gene BIM Bcl-2-binding protein Bad protein Binding Biological Models Biology Brain Brain-Derived Neurotrophic Factor Candidate Disease Gene Cell Death Cell Line Cell Survival Cells Cessation of life Clinical Complex Cytotoxic agent Disease Dose Drug resistance Drug usage Dynein ATPase Etoposide/Vincristine FOXO1A gene Genetic Glycogen (Starch) Synthase Investigation Ligands Location MAP Kinase Gene MYCN gene Mediating Microtubules Modality Modeling Motor Mutation Neoplasm Metastasis Neuroblastoma Neurons Neurotrophic Tyrosine Kinase Receptor Type 2 Nutrient Outcome Paclitaxel Pathway interactions Patients Pharmaceutical Preparations Phase Phosphorylation Physiological Play Process Prognostic Marker Proteins Proto-Oncogene Proteins c-akt Resistance Retinoids Risk Role Signal Pathway Signal Transduction Signal Transduction Inhibitor Signal Transduction Pathway Small Interfering RNA Stimulus Stress System TP53 gene Testing Thinking Treatment Protocols Tumor Biology Tumor Cell Biology Xenograft procedure angiogenesis base caspase-8 caspase-9 cell motility chemokine chemotherapeutic agent chemotherapy cisplatin/etoposide protocol cytotoxicity dynein light chain forkhead protein in vivo in vivo Model inhibitor/antagonist metastatic process mouse model neoplastic cell neurotrophic factor novel outcome forecast p27 Cell Cycle Protein p27 Enzyme Inhibitor pro-apoptotic protein response tumor

项目摘要

Aim 1. To investigate biologic consequences of BDNF activation of TrkB on chemoresistance. A. AKT pathway inactivates Glycogen synthetase 3beta (GSK3) Previously, we identified the PI-3 kinase pathway as mediating BDNF/TrkB-induced resistance to etoposide, vincristine and adriamycin. Last year we identified that AKT played a key role in regulating chemoresistance in NB cells and identified inhibitors of the AKT that could enhance the efficacy of chemotherapy. Now we have identified that AKT suppresses GSK3B activity and the inactivation of GSK3 attenuates the effects of chemotherapy by altering the intrinsic apoptotic pathway. Moreover we find that if we can enhance the activity of GSK3, the neuroblastoma tumor cells are more sensitive to chemotherapy. Many pharmacologic anti-deppresants such as LiCl inhibit GSK3 activity and we found that these can make the tumor cells less resistant to chemotherapy. This raises the possibility that some types of anti-depressants may affect the efficacy of chemotherapy. B. BDNF activation of TrkB via MAPK pathway decreases pro-apoptotic BH-3 protein BIM, which contributes to paclitaxel resistance. The ability of a tumor cell to survive an apoptoic insult or survive in a new microenvironment depends on both the external stress (chemotherapy, nutrient and O2 levels), the microenvironment (e.g. metastasize) and the cells' internal signaling systems. Our study on Bim indicates that, depending on the chemotherapeutic drugs utilized, BDNF/TrkB activation attenuates the effects of cytotoxic agents by distinct signaling pathways. We used a candidate gene approach to investigate targets downstream of AKT involved in chemoresistance. Activated Akt phosphorylates and inhibits several pro-apoptotic proteins, such as Bad, caspase-9 and Forkhead transcription factors (FKHRL1, FKHR and AFX), leading to cell survival. We did not detect significant changes in phosphorylation or total protein levels for BAD or caspase 9 after BDNF activation of TrkB. In our AKT study, the genetic and pharmacologic inhibiton of AKT activity focused our attention on FKHRL1. FKHRL1 functions in apoptotic processes by regulating p27 interaction with caspase 8 and with the pro-apoptotic BH-3 protein BIM. As the NB cell lines under study did not express caspase 8, we focused on BIM. BIM is a target of AKT in many other systems. We found that BDNF/TrkB activation leads to a decrease in BIM levels that is mediated by activation of MAPK and not AKT. SiRNA mediated BIM knockdown had no effect on cisplatin/etoposide induced cytotoxicity. However, silencing of BIM or inhibition of the MAPK pathway sensitized NB cells to paclitaxel. Bim is thought to induce cell death by binding to LC8 cytoplasmic dynein light chain where it is sequestered with the microtubule-associated dynein motor complex in healthy cells. Certain apoptotic stimuli such as paclitaxel stabilize microtubules and disrupt the interaction between LC8 and the dynein motor complex, freeing Bim to translocate together with LC8 to Bcl-2 and neutralize its anti-apoptotic activity. In our study, reduction of Bim by Bim siRNA, decreases paclitaxel induced apoptosis. It is becoming increasingly apparent that different drugs or toxic insults utilize distinct apoptotic signaling paths just as survival signaling pathways use distinct pathways to attenuate death signals. Clinically, only 8/33 NB patients using an "up-front phase II window" approach had objective responses with paclitaxel which limited its potential utility when combined with current multi-agent regimens. Our study would indicate that drugs previously thought to have marginal activity may need to be re-evaluated in combination studies when appropriate signal transduction inhibitors become available. Specific Aim 2. To investigate biologic consequences of BDNF activation of TrkB on metastasis and angiogenesis A. In a collaborative project we have found that BDNF activation of TrkB can stimulate expression of chemokines in the brain. This has enormous implications as chemokines have been implicated in cell movement which is required for metastasis of tumor cells. This finding contributes to our understanding of how BDNF activation of TrkB stimulates tumor cell movement.

目标 1. 研究 TrkB 的 BDNF 激活对化疗耐药性的生物学影响。 A. AKT 途径使糖原合成酶 3beta (GSK3) 失活此前，我们确定 PI-3 激酶途径介导 BDNF/TrkB 诱导的依托泊苷、长春新碱和阿霉素耐药性。去年，我们发现 AKT 在调节 NB 细胞的化疗耐药性中发挥着关键作用，并确定了可以增强化疗疗效的 AKT 抑制剂。现在我们已经确定 AKT 抑制 GSK3B 活性，并且 GSK3 失活通过改变内在的细胞凋亡途径来减弱化疗的效果。此外，我们发现如果能够增强GSK3的活性，神经母细胞瘤肿瘤细胞对化疗更加敏感。许多药理学抗抑郁药（例如 LiCl）会抑制 GSK3 活性，我们发现这些药物可以降低肿瘤细胞对化疗的抵抗力。这增加了某些类型的抗抑郁药可能影响化疗疗效的可能性。 B. BDNF 通过 MAPK 途径激活 TrkB 会降低促凋亡 BH-3 蛋白 BIM，从而导致紫杉醇耐药。肿瘤细胞在细胞凋亡损伤中存活或在新的微环境中存活的能力取决于外部应激（化疗、营养和氧气水平）、微环境（例如转移）和细胞的内部信号系统。我们对 Bim 的研究表明，根据所使用的化疗药物，BDNF/TrkB 激活可通过不同的信号通路减弱细胞毒性药物的作用。我们使用候选基因方法来研究 AKT 下游参与化疗耐药的靶标。激活的 Akt 磷酸化并抑制多种促凋亡蛋白，例如 Bad、caspase-9 和 Forkhead 转录因子（FKHRL1、FKHR 和 AFX），从而导致细胞存活。在 BDNF 激活 TrkB 后，我们没有检测到 BAD 或 caspase 9 磷酸化或总蛋白水平的显着变化。在我们的 AKT 研究中，AKT 活性的遗传和药理学抑制将我们的注意力集中在 FKHRL1 上。 FKHRL1 通过调节 p27 与 caspase 8 和促凋亡 BH-3 蛋白 BIM 的相互作用在细胞凋亡过程中发挥作用。由于所研究的 NB 细胞系不表达 caspase 8，因此我们将重点放在 BIM 上。 BIM 是许多其他系统中 AKT 的目标。我们发现 BDNF/TrkB 激活导致 BIM 水平降低，这是由 MAPK 而不是 AKT 激活介导的。 siRNA 介导的 BIM 敲低对顺铂/依托泊苷诱导的细胞毒性没有影响。然而，沉默 BIM 或抑制 MAPK 通路会使 NB 细胞对紫杉醇敏感。 Bim 被认为通过与 LC8 细胞质动力蛋白轻链结合来诱导细胞死亡，轻链与健康细胞中微管相关的动力蛋白运动复合体隔离。某些细胞凋亡刺激物（例如紫杉醇）可稳定微管并破坏 LC8 与动力蛋白运动复合体之间的相互作用，使 Bim 与 LC8 一起易位为 Bcl-2，并中和其抗细胞凋亡活性。在我们的研究中，通过 Bim siRNA 减少 Bim，可减少紫杉醇诱导的细胞凋亡。越来越明显的是，不同的药物或毒性损伤利用不同的细胞凋亡信号传导途径，就像生存信号传导途径使用不同的途径来减弱死亡信号一样。临床上，只有 8/33 的使用“前期 II 期窗口”方法的 NB 患者对紫杉醇产生了客观反应，这限制了其与当前多药治疗方案联合使用时的潜在效用。我们的研究表明，当合适的信号转导抑制剂可用时，以前被认为具有边际活性的药物可能需要在联合研究中重新评估。具体目标 2. 研究 BDNF 激活 TrkB 对转移和血管生成的生物学影响 A. 在一个合作项目中，我们发现 BDNF 激活 TrkB 可以刺激大脑中趋化因子的表达。这具有巨大的意义，因为趋化因子与肿瘤细胞转移所需的细胞运动有关。这一发现有助于我们理解 TrkB 的 BDNF 激活如何刺激肿瘤细胞运动。