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

项目总结/摘要 三阴性乳腺癌（TNBC）是一种侵袭性乳腺癌亚型，其中有限的靶向治疗可能导致乳腺癌的复发。 治疗是可行的。因此，TNBC患者的标准护理治疗是新辅助治疗。 化疗，其中约50%的患者在治疗后具有残留肿瘤负荷和不良预后。 最近的研究表明，线粒体氧化磷酸化（oxphos） 然而，这一发现背后的机制并不是 明白三羧酸循环（TCA），产生氧化膦所需的还原当量， 需要乙酰辅酶A（AcCoA）分子。乙酰辅酶A可来源于长链脂肪酸的分解， 脂肪酸氧化过程中的酸（FAO）。因为高脂肪酸代谢与 提高TNBC的存活率，可能是化疗耐药的TNBC从脂肪酸中获得AcCoA， 三氯乙酸和氧化磷。肉毒碱乙酰转移酶（CRAT）基因产生一种酶（CrAT），催化 乙酰基在线粒体内CoA和肉毒碱之间的可逆转移。人们认为 CrAT缓冲游离AcCoA池，以最大化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。在化学抗性TNBC中，更高的转录和/或表达水平可能与TNBC中的转录和/或表达水平有关。 CRAT的翻译可以使oxphos所需的TCA衍生的还原当量最大化， 化学抗性我们已经确定了脂肪酸代谢途径和CRAT作为显着丰富的， 化疗（多西他赛联合卡铂，TNBC的标准NACT）耐药与化疗耐药 敏感的TNBC PDX和患者活检（NCT 02547987）在RNA和蛋白质水平。在初步 对NACT后与NACT前TNBC患者来源的异种移植物（PDX）肿瘤的分析，我还发现， 通过透射电子显微镜观察卡铂处理的PDX中的细胞溶质脂滴（LD）与溶媒相比 显微镜（TEM）显示脂肪酸代谢增强。因此，我推测， CRAT在化学抗性TNBC中的表达提供了增强的代谢可塑性，缓冲脂质， 衍生AcCoA的积累，以最大限度地提高TCA循环通量和oxphos，从而有助于产生化疗耐药性。我 将通过确定AcCoA是否优先来源于脂肪酸增加来解决这一假设 化学抗性TNBC中的氧化。我将研究CRAT消融是否会损害化疗敏感性和生存率 使用体外和体内模型。我还将确定哪种CRAT亚型是必要和充分的 to drive驱动chemoresistance耐药性.总之，这些研究将提高我们对增加的 并为靶向CrAT功能的治疗提供了基本原理，以改善TNBC患者的预后。 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.