Determining the role of lipid droplets and their therapeutic potential in glioblastoma

摘要 在过去的二十年里，作为最致命的脑瘤，胶质母细胞瘤的预后一直没有改变。 令人沮丧，确诊后中位生存期只有12-16个月。我们最近证明了GBM细胞 通过显著上调脂肪酸和胆固醇的从头合成来获得大量的脂肪酸和胆固醇 和摄取以促进肿瘤的快速生长。然而，过量的脂肪酸和胆固醇可以改变膜的动力学和 功能，导致细胞损伤。GBM细胞如何避免这种脂毒性以维持不同时期的适当血脂水平 对于细胞室，尤其是线粒体中的细胞室，人们知之甚少。在过去5年的资助中， 我们在了解GBM如何控制FA动态平衡方面取得了很大进展。我们证明了GBM 细胞上调二酰甘油酰基转移酶1(DGAT1)，使它们能够储存丰富的脂肪酸作为 含有三酰甘油的脂滴(LDS)可防止过量的FA蓄积而导致毒性。在这 更新提案，我们将解决两个悬而未决的关键问题：1)胆固醇是如何稳态的 在GBM细胞中被调控吗？2)是否可以通过破坏脂质来开发有效的治疗方法 动态平衡？我们最近发现胆固醇酯(CES)，它形成LDS来存储多余的细胞 胆固醇，主要存在于GBM组织中，阻止CE的合成会导致戏剧性的线粒体 基底膜细胞碎裂。此外，我们的初步数据显示，胆固醇是从CE- 含有低密度脂多糖(CE-LDS)的细胞膜，在抑制自噬的同时阻止这种转移。这些数据 提示CE-LDS通过自噬维持适当的细胞胆固醇水平。我们的初步数据进一步 研究表明，硬脂酰辅酶A去饱和酶1(SCD1)，已被证明可以防止内质网(ER) 应激和铁性下垂，在DGAT1抑制后上调。最后，初步数据显示， DGAT1抑制后，多种抗氧化剂基因的表达显著增加。这些结果 强烈提示GBM细胞可以激活防御机制，以减轻由 FA储存中断，可能导致肿瘤对DGAT1抑制产生抵抗力。因此，我们假设 CE-LDs是控制胆固醇稳态和线粒体功能的关键储存库， 将胆固醇储存或重新分配的中断与线粒体胆固醇的干扰结合在一起 通过抑制SCD1或阻断抗氧化途径来进口或中断FA储存是有效的 针对GBM的策略。在目标1中，我们将研究抑制胆固醇储存或 CE-LDS对胆固醇稳态和线粒体功能的再分配以及这种阻断是否 可以协同干扰胆固醇进入线粒体，有效地杀死GBM中的肿瘤细胞 异种移植模型。在目标2中，我们将研究抑制scd1或抗氧化途径是否能强烈地 与DGAT1抑制剂协同作用可有效抑制GBM的体内外生长。成功完成 这项研究将为靶向GBM的潜在新策略提供强有力的临床前数据。

ABSTRACT Over the past two decades, the prognosis for glioblastoma (GBM), the most lethal brain tumor, has remained dismal, with a median survival of only 12-16 months from diagnosis. We recently demonstrated that GBM cells acquire large amounts of fatty acids (FAs) and cholesterol by dramatically upregulating their de novo synthesis and uptake for rapid tumor growth. However, excess FAs and cholesterol can alter membrane dynamics and function, leading to cellular damage. How GBM cells avoid this lipotoxicity to sustain proper lipid levels in different cellular compartments, particularly in the mitochondria, is poorly understood. During the past 5 years of funding, we have made great progress in understanding how GBM controls FA homeostasis. We demonstrated that GBM cells upregulate diacylglycerol acyltransferase 1 (DGAT1), allowing them to store abundant FAs as triacylglycerol-containing lipid droplets (LDs) to prevent excess FA accumulation to induce toxicity. In this renewal proposal, we will address two unanswered critical questions: 1) how is cholesterol homeostasis regulated in GBM cells? and 2) can effective therapeutic approaches be developed for GBM by disrupting lipid homeostasis? We recently found that cholesteryl esters (CEs), which form LDs to store excess cellular cholesterol, are largely present in GBM tissues, and blocking CE synthesis results in dramatic mitochondrial fragmentation in GBM cells. Moreover, our preliminary data showed that cholesterol is transferred from CE- containing LDs (CE-LDs) to the plasma membrane, while inhibition of autophagy blocks this transfer. These data suggest that CE-LDs maintain proper cellular cholesterol levels via autophagy. Our preliminary data further showed that stearoyl-CoA desaturase 1 (SCD1), which has been shown to prevent endoplasmic reticulum (ER) stress and ferroptosis, is upregulated upon DGAT1 inhibition. Finally, preliminary data showed that the expression of multiple antioxidant genes is significantly elevated in response to DGAT1 inhibition. These results strongly suggest that GBM cells can activate defense mechanisms to alleviate the lipotoxicity triggered by disruption of FA storage, possibly leading to tumor resistance to DGAT1 inhibition. Thus, we hypothesize that CE-LDs serve as critical reservoirs for controlling cholesterol homeostasis and mitochondrial function, and that combining disruption of storage or redistribution of cholesterol with interference with mitochondrial cholesterol import, or disruption of FA storage with either inhibition of SCD1 or blockade of antioxidant pathways are effective strategies for targeting GBM. In Aim 1, we will examine the impact of inhibiting cholesterol storage or redistribution from CE-LDs on cholesterol homeostasis and mitochondrial function, and whether such blockade can synergize with interfering in cholesterol import into mitochondria to efficiently kill tumor cells in GBM xenograft models. In Aim 2, we will examine whether inhibiting SCD1 or antioxidant pathways can strongly synergize with DGAT1 inhibition to effectively inhibit GBM growth in vitro and in vivo. Successful completion of this study will provide strong pre-clinical data on potential novel strategies to target GBM.