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

摘要 在过去的二十年里，胶质母细胞瘤（GBM），最致命的脑肿瘤，的预后一直保持不变。 令人沮丧，从诊断开始的中位生存期只有12 - 16个月。我们最近证明GBM细胞 获得大量的脂肪酸（脂肪酸）和胆固醇通过显着上调他们的从头合成 和摄取以促进肿瘤快速生长。然而，过量的脂肪酸和胆固醇可以改变膜动力学， 功能，导致细胞损伤。GBM细胞如何避免这种脂毒性，以维持适当的脂质水平，在不同的 细胞区室，特别是线粒体中的区室，知之甚少。在过去五年的资助中， 我们在了解GBM如何控制FA稳态方面取得了很大进展。我们证明了GBM 细胞上调二酰基甘油酰基转移酶1（DGAT 1），使它们能够储存丰富的脂肪酸， 含有三酰甘油的脂滴（LD），以防止过量的FA积累，从而诱导毒性。在这 更新建议，我们将解决两个悬而未决的关键问题：1）如何胆固醇稳态 在GBM细胞中调节？和2）是否可以通过破坏脂质来开发有效的治疗GBM的方法 体内平衡？我们最近发现，胆固醇酯（CE），形成LD储存多余的细胞 胆固醇，主要存在于GBM组织中，阻断CE合成导致显著的线粒体损伤。 GBM细胞中的碎片。此外，我们的初步数据表明，胆固醇是从CE- 自噬的抑制可使含有LD的细胞（CE-LD）转移至质膜，而自噬的抑制则阻断这种转移。这些数据 表明CE-LD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 显示硬脂酰辅酶A去饱和酶1（SCD 1），已被证明可以阻止内质网（ER） 应激和铁凋亡，在DGAT 1抑制后上调。最后，初步数据显示， 多种抗氧化剂基因的表达响应DGAT 1抑制而显著升高。这些结果 这强烈表明GBM细胞可以激活防御机制，以减轻由 破坏FA储存，可能导致肿瘤对DGAT 1抑制的抗性。因此，我们假设， CE-LD作为控制胆固醇稳态和线粒体功能的关键储库， 结合胆固醇储存或再分布的破坏与线粒体胆固醇的干扰 通过抑制SCD 1或阻断抗氧化剂途径来输入或破坏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 再分配CE-LD对胆固醇稳态和线粒体功能的影响，以及这种阻断是否 可协同干扰胆固醇输入线粒体，以有效杀死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SCD 1或抗氧化途径是否能强烈地 与DGAT 1抑制协同有效地抑制体外和体内GBM生长。成功完成 该研究将提供关于靶向GBM的潜在新策略的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.