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

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

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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.

目的1。研究TRKB激活化学耐药性BDNF激活的生物学后果。 A. AKT途径以前使糖原合成酶3Beta（GSK3）灭活，我们鉴定出PI-3激酶途径是介导BDNF/TRKB诱导的对依托泊苷，vincristine和adrimycin的抗性。去年，我们确定AKT在调节NB细胞的化学耐药性方面起着关键作用，并确定了AKT的抑制剂，可以增强化学疗法的疗效。现在，我们已经确定AKT抑制了GSK3B活性，而GSK3的失活通过改变固有的凋亡途径来减轻化学疗法的影响。此外，我们发现，如果我们可以增强GSK3的活性，那么神经母细胞瘤肿瘤细胞对化学疗法更敏感。许多药理学抗抑制剂（例如LICL）抑制GSK3活性，我们发现这些活性会使肿瘤细胞对化学疗法的耐药性较低。这增加了某些类型的抗抑制剂可能影响化疗的功效的可能性。 B.通过MAPK途径激活TRKB的BDNF激活可降低促凋亡的BH-3蛋白BIM，这有助于紫杉醇耐药性。肿瘤细胞在新的微环境中生存或生存的肿瘤细胞能力取决于外部压力（化学疗法，营养和O2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，根据所使用的化学治疗药物，BDNF/TRKB激活通过不同的信号通路减弱了细胞毒性剂的影响。我们使用了候选基因方法来研究参与化学抗性的AKT下游的靶标。激活的AKT磷酸化并抑制几种促凋亡蛋白，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，我们没有检测到不良或caspase 9的磷酸化或总蛋白水平的显着变化。在我们的AKT研究中，AKT活性的遗传和药物抑制剂将我们的注意力集中在FKHRL1上。 FKHRL1通过调节p27与caspase 8的相互作用以及与凋亡的BH-3蛋白BIM的相互作用在凋亡过程中起作用。由于研究中的NB细胞系未表达caspase 8，因此我们专注于BIM。 BIM是许多其他系统中AKT的目标。我们发现BDNF/TRKB激活会导致BIM水平的降低，而BIM水平是由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。但是，将BIM或MAPK途径抑制的沉默使NB细胞敏感到紫杉醇。 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，在该轻链中与健康细胞中与微管相关的动力蛋白运动复合物隔离。某些凋亡刺激（例如紫杉醇）稳定微管，并破坏LC8与动力蛋白运动复合物之间的相互作用，从而使BIM与LC8一起释放BCL-2并中和其抗凋亡活性。在我们的研究中，BIM siRNA减少BIM，降低了紫杉醇诱导的凋亡。越来越明显的是，不同的药物或有毒的损伤使用不同的凋亡信号通路，就像生存信号通路使用不同的途径来减轻死亡信号一样。在临床上，只有8/33 NB患者使用“前期II期窗口”方法对紫杉醇的客观反应限制了其与当前多机构方案相结合时的潜在效用。我们的研究表明，当适当的信号转导抑制剂可用时，可能需要在组合研究中重新评估以前认为具有边际活性的药物。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A。在一个协作项目中，我们发现TRKB的BDNF激活可以刺激大脑中趋化因子的表达。这具有巨大的意义，因为趋化因子已经与细胞运动有关，这是肿瘤细胞转移所必需的。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。