Investigating the role of CRAT as a driver of triple negative breast cancer chemoresistance

项目摘要/摘要 三阴性乳腺癌(TNBC)是一种侵袭性乳腺癌亚型，其靶向性有限 治疗方法是可用的。因此，对TNBC患者的护理治疗标准是新辅助。 化疗中约50%的患者治疗后仍有肿瘤残留，预后较差。 近来发现线粒体氧化磷酸化(OXPHOS)表达上调 和化疗耐药的TNBC的治疗脆弱性，然而，这一发现背后的机制并不是 明白了。三羧酸循环(TCA)产生OXPHOS所需的还原当量，是 需要乙酰辅酶A(AcCoA)分子。乙酰辅酶A可以从长链脂肪的分解中衍生出来 脂肪酸氧化过程中的酸度(粮农组织)。因为脂肪酸新陈代谢增强与 提高TNBC的存活率，化疗耐药的TNBC可能从脂肪酸中获得AcCoA，从而促进 TCA和OXPHOS。肉毒碱乙酰转移酶基因(Crat)产生一种酶(Crat)，催化 乙酰基在线粒体内辅酶A和肉碱之间的可逆转移。据认为， CRAT缓冲了游离AcCoA池，以最大限度地满足TCA循环的能量需求，并防止丙酮酸 通过过量的AcCoA抑制脱氢酶。在耐药的TNBC中，更高的转录和/或 CRAT的翻译可能会最大限度地增加OXPHOS所需的TCA衍生的还原当量，有助于 化疗耐药。我们已经确定脂肪酸代谢途径和crat显著富含 化疗(多西紫杉醇联合卡铂，标准的TNBC)耐药与化疗- 敏感的TNBC PDX和患者活检组织(NCT02547987)在RNA和蛋白质水平上。在预赛中 分析手术后与手术前TNBC患者来源的异种移植(PDX)肿瘤，我也发现 卡铂处理的PDXs与载药PDXs胞浆脂滴(LDs)的透射电子比较 显微镜(透射电子显微镜)，提示脂肪酸代谢增强。因此，我假设提升了 CRAT在化疗耐药的TNBC中的表达可增强代谢可塑性，缓冲脂质 AcCoA的衍生积累最大限度地增加了TCA循环通量和OXPHOS，有助于抗药性。我 我将通过确定AcCoA是否优先来自增加的脂肪酸来解决这一假说 化疗耐药的TNBC中的氧化。我将调查克雷特消融术是否会损害化疗敏感性和存活率。 使用体外和体内模型。我还将决定哪个亚型的crat是必要的和充分的。 以促进化疗耐药。综上所述，这些研究将提高我们对增长的机械理解 OXPHOS在残留的TNBC中的作用，为针对CrAT功能的治疗改善预后提供了理论依据 化疗耐药患者。

Project Summary/Abstract Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype in which limited targeted therapies are available. Therefore, the standard of care treatment for TNBC patients is neoadjuvant chemotherapy where ~50% of patients have residual tumor burden and poor prognosis after treatment. Recently, it has been demonstrated that mitochondrial oxidative phosphorylation (oxphos) is both upregulated and a therapeutic vulnerability in chemoresistant TNBC, however, the mechanism behind this finding is not understood. The tricarboxylic acid cycle (TCA), which produces reducing equivalents necessary for oxphos, is requires the molecule acetyl-CoA (AcCoA). AcCoA can be derived from the breakdown of long chain fatty acids during fatty acid oxidation (FAO). Because heightened fatty acid metabolism has been associated with improved survival in TNBC, it is possible that chemoresistant TNBC derives AcCoA from fatty acids, fueling the TCA and oxphos. The gene carnitine acetyl transferase (CRAT) produces an enzyme (CrAT) that catalyzes the reversible transfer of an acetyl group between CoA and carnitine within the mitochondria. It is thought that CrAT buffers the pool of free AcCoA to maximize the energetic needs of the TCA cycle and to prevent pyruvate dehydrogenase inhibition via excess AcCoA. Within chemoresistant TNBC, greater transcription and/or translation of CRAT may maximize TCA-derived reducing equivalents needed for oxphos, aiding in chemoresistance. We have identified the fatty acid metabolism pathway and CRAT as significantly enriched in chemotherapy (docetaxel combined with carboplatin, standard NACT for TNBC)-resistant versus chemo- sensitive TNBC PDXs and patient biopsies (NCT02547987) at the RNA and protein levels. In a preliminary analysis of post-versus pre-NACT TNBC patient derived xenograft (PDX) tumors, I also found increased cytosolic lipid droplets (LDs) in carboplatin treated PDXs compared to vehicle by transmission electron microscopy (TEM), suggestive of enhanced fatty acid metabolism. Therefore, I hypothesize that elevated expression of CRAT in chemoresistant TNBC provides enhanced metabolic plasticity, buffering lipid derived accumulation of AcCoA to maximize TCA cycle flux and oxphos, aiding in chemoresistance. I will address this hypothesis by determining if AcCoA is preferentially derived from increased fatty acid oxidation in chemoresistant TNBC. I will investigate if CRAT ablation impairs chemosensitivity and survival using both in vitro and in vivo models. I will also determine which isoform of CRAT is necessary and sufficient to drive chemoresistance. Together, these studies will improve our mechanistic understanding of increased oxphos in residual TNBC and provide rationale for therapies targeting CrAT function to improve prognosis for chemoresistant patients.