UNDERSTANDING TRAIL SENSITIVITY IN HUMAN GLIOMA

描述(申请人提供)：这项建议的长期目标是通过更好地了解TRAIL抵抗的决定因素来改进人脑胶质瘤的治疗。TRAIL是一种有吸引力的治疗分子，因为它可以诱导肿瘤细胞的凋亡，但不能诱导正常细胞的凋亡。然而，即使在TRAIL敏感的多形性胶质母细胞瘤(GBM)细胞系中，TRAIL耐药也很常见。尽管TRAIL耐药可以由许多因素控制，但我们发现，caspase-8抑制剂Flips的过度表达在原发GBM常见的高水平TRAIL耐药中发挥了关键作用。Flips的过度表达与Flips mRNA水平的升高无关，但与Flips mRNA与翻译多聚核糖体的关联增加有关。Flips mRNA的翻译又受到RAS途径的两个臂的影响，这两个臂都在GBM中激活：该途径的RAS臂阻止Flips mRNA与多聚体的结合，下调Flips蛋白，并使GBM细胞对TRAIL敏感，而该途径的Ras-Akt臂加强Flips mRNA的多体结合，上调Flips蛋白，保护GBM细胞免受TRAIL的影响。然而，RAS途径的Ral和Raf臂似乎也抑制了翻转RNA的水平，这表明对翻转的转录控制也可能是重要的。我们不知道Ral Akt和Raf通路是如何控制Flips mRNA水平和翻译的，Flips mRNA是如何差异地靶向翻译调控的，如果Flips mRNA是一个更大的靶向翻译控制的凋亡相关mRNAs的一部分，GBM细胞系中注意到的调节是否也发生在原发胶质瘤中，或者如何最好地操纵这些途径以获得治疗效益。然而，根据我们的初步研究，我们假设RAS相关的FLOPS的翻译和转录调控控制着GBM中TRAIL的敏感性。这一假设将在以下具体目标中得到检验。1：确定Ral/Akt、翻译调节因子、Flips水平与GBM细胞TRAIL敏感性之间的关系。2：鉴定与RAS依赖的Flips翻译调控相关的Flips mRNA序列。3：确定Flips是否是一个更大的翻译调控分子组的一部分，这些分子参与了TRAIL诱导的细胞凋亡的控制。4：明确Raf/Ral、Flips RNA下调与GBM中TRAIL敏感性之间的关系。5：确定人原发低级别胶质瘤(LGG)、间变性星形细胞瘤(AA)和基底膜(GBM)短期培养的TRAIL敏感性，并确定RAS通路和/或翻转蛋白的水平/活性是否与TRAIL敏感性相关。6：确定翻译控制的调节是否能使原代人GBM对TRAIL敏感。这项工作与公众健康相关，因为它将提供识别TRAIL敏感脑瘤的方法，并使TRAIL耐药肿瘤对TRAIL诱导的杀伤敏感。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to improve the therapy of human glioma by better understanding the determinants of resistance to TRAIL. TRAIL is an attractive therapeutic molecule because it induces apoptosis in tumor cells, but not in normal cells. Even in TRAIL sensitive glioblastoma multiforme (GBM) cell lines, however, TRAIL resistance is common. Although TRAIL resistance can be controlled by many factors, we have found that over-expression of FLIPs, a caspase-8 inhibitor that blocks activation of the extrinsic apoptotic cascade, plays a key role in the high level TRAIL resistance common in primary GBM. FLIPs over- expression is not associated with increased FLIPs mRNA levels, but with increased association of FLIPs mRNA with translating polyribosomes. FLIPs mRNA translation is in turn influenced by two arms of the Ras pathway, both activated in GBM: the Ras-Ral arm of the pathway blocks association of FLIPs mRNA with polysomes, downregulates FLIPs protein, and sensitizes GBM cells to TRAIL, while the Ras-Akt arm of the pathway enhances FLIPs mRNA polysomal association, upregulates FLIPs protein, and protects GBM cells from TRAIL. The Ral and Raf arms of the Ras pathway, however, also appear to suppress FLIPs RNA levels, suggesting that transcriptional control of FLIPs may also be important. We do not know how the Ral Akt, and Raf pathways control FLIPs mRNA levels and translation, how the FLIPs mRNA is differentially targeted for translation regulation, if the FLIPs mRNA is part of a larger group of apoptosis-related mRNAs targeted for translational control, whether the regulation noted in GBM cell lines also occurs in primary gliomas, or how to best manipulate the pathways for therapeutic benefit. Based on our preliminary studies, however, we hypothesize that Ras-related translational and transcription regulation of FLIPs controls TRAIL sensitivity in GBM. This hypothesis will be tested in the following specific aims. 1: To define the connections between Ral/Akt, regulators of translation, FLIPs levels, and TRAIL sensitivity in GBM cells. 2: To identify FLIPs mRNA sequences responsible for Ras-dependent FLIPs translational regulation. 3: To determine if FLIPs is part of a larger group of translationally regulated molecules that contribute to the control TRAIL-induced apoptosis. 4: To define the connections between Raf/Ral, downregulation of FLIPs RNA, and TRAIL sensitivity in GBM. 5: To define TRAIL sensitivity in a panel of short term cultures of primary human low grade gliomas (LGG), anaplastic astrocytoma (AA), and GBM, and to determine if levels/activity of Ras pathways and/or FLIPs correlate with TRAIL sensitivity. 6: To determine if modulation of translation control can sensitize primary human GBM to TRAIL. This work has relevance to public health in that it will provide the ways to identify TRAIL-sensitive brain tumors and also to make TRAIL-resistant tumors sensitive to TRAIL-induced killing.