UNDERSTANDING TRAIL SENSITIVITY IN HUMAN GLIOMA

描述（由申请人提供）：本提案的长期目标是通过更好地了解TRAIL耐药的决定因素来改善人类胶质瘤的治疗。TRAIL是一种有吸引力的治疗分子，因为它在肿瘤细胞中诱导凋亡，但在正常细胞中不诱导凋亡。然而，即使在TRAIL敏感的多形性胶质母细胞瘤（GBM）细胞系中，TRAIL抗性也是常见的。尽管TRAIL耐药可以由许多因素控制，但我们发现FLIPs（一种阻断外源性凋亡级联激活的半胱天冬酶-8抑制剂）的过表达在原发性GBM中常见的高水平TRAIL耐药中起关键作用。FLIPs过表达与FLIPs mRNA水平增加无关，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中被激活：该途径的Ras-Ral臂阻断FLIPs mRNA与多聚体的缔合，下调FLIPs蛋白，并使GBM细胞对TRAIL敏感，而该途径的Ras-Akt臂增强FLIPs mRNA多聚体缔合，上调FLIPs蛋白，并保护GBM细胞免受TRAIL的影响。然而，Ras途径的Ral和Raf臂似乎也抑制FLIPs RNA水平，这表明FLIPs的转录控制也可能是重要的。我们不知道Ral Akt和Raf通路如何控制FLIPs mRNA水平和翻译，FLIPs mRNA如何差异靶向翻译调节，FLIPs mRNA是否是靶向翻译控制的更大组阿尔茨海默病相关mRNA的一部分，GBM细胞系中的调节是否也发生在原发性胶质瘤中，或者如何最好地操纵通路以获得治疗益处。然而，根据我们的初步研究，我们假设Ras相关的FLIPs的翻译和转录调控控制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GBM细胞中Ral/Akt、翻译调节因子、FLIPs水平和TRAIL敏感性之间的联系。2：鉴定负责Ras依赖性FLIPs翻译调控的FLIPs mRNA序列。3：确定FLIPs是否是有助于控制TRAIL诱导的细胞凋亡的更大组的细胞凋亡调节分子的一部分。4：确定Raf/Ral、FLIPs RNA下调和GBM中TRAIL敏感性之间的联系。第五节：在一组原发性人低级别胶质瘤（LGG）、间变性星形细胞瘤（AA）和GBM的短期培养物中确定TRAIL敏感性，并确定Ras通路和/或FLIP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可以使原代人GBM对TRAIL敏感。这项工作与公共卫生有关，因为它将提供鉴定TRAIL敏感性脑肿瘤的方法，并使TRAIL抗性肿瘤对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.