PO.CL01.03 · 临床研究
BAP1缺失通过TLCD1介导的膜磷脂重塑赋予胆管癌铁死亡抵抗
BAP1 loss confers ferroptosis resistance to cholangiocarcinoma via TLCD1-mediated membrane phospholipid remodeling
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：胆管癌（CCA）是一种起源于肝胆管上皮细胞的致命癌症。表观遗传调控因子——泛素C端水解酶去泛素化酶BRCA1相关蛋白1（BAP1）的功能缺失突变见于26-32%的人类CCA，并与更差的预后和对细胞毒性治疗的抵抗相关。本研究的目标是阐明BAP1缺失如何通过代谢重编程促进细胞死亡逃逸。
材料与方法：使用shRNA/siRNA或CRISPR/Cas9构建了同基因型的野生型和BAP1缺陷型人源及鼠源CCA细胞系，用于通过铁死亡诱导剂处理来评估铁死亡敏感性。在体外，通过磷脂过氧化（C-11 BODIPY）、线粒体磷脂过氧化（MitoPeDPP、MitoCLox）、铁过载（MitoFerro green）和细胞死亡（CellTiter-Glo）评估铁死亡。通过LC-MS/MS对同基因型细胞进行磷脂组学和蛋白质组学分析。采用同基因型肝脏原位CCA鼠模型进行体内临床前研究。
结果：我们的发现表明，BAP1缺失赋予CCA铁死亡抵抗。BAP1缺失保护CCA免受线粒体铁过载、ROS蓄积和磷脂过氧化。值得注意的是，BAP1缺失可防止线粒体磷脂过氧化和心磷脂氧化。无偏倚的磷脂组学分析揭示，BAP1缺失重塑了膜磷脂组成，富集了阻断铁死亡的单不饱和磷脂（MUFA-PLs），而减少了诱导铁死亡的多不饱和磷脂（PUFA-PLs）。我们发现BAP1缺失上调TLCD1，这是一种磷脂酰乙醇胺酰基转移酶，可将MUFA掺入膜磷脂，从而稳定膜以抵抗过氧化。在BAP1缺陷型CCA细胞中敲低TLCD1可恢复铁死亡敏感性。值得注意的是，与WT肿瘤相比，TLCD1在人类BAP1突变型CCA中显著上调。我们在内质网、高尔基体以及线粒体外膜处鉴定到TLCD1。在同系肝脏原位CCA鼠模型中，携带GPX4 siRNA的工程化EpCAM适配体包被乳糖体（lactosome）选择性靶向CCA细胞，诱导铁死亡并强效抑制肿瘤生长而无全身毒性。最后，与WT对照相比，BAP1突变型患者来源异种移植（PDX）和类器官对铁死亡诱导剂具有抵抗性。
结论：我们的发现揭示，BAP1缺失重编程线粒体磷脂代谢，从而赋予CCA铁死亡抵抗，并将TLCD1确定为BAP1突变型CCA中一个可靶向的代谢弱点。
查看英文原文 English abstract
INTRODUCTION: Cholangiocarcinoma (CCA) is a deadly cancer of the hepatic bile duct epithelial cells. Loss-of-function mutations of epigenetic regulator ubiquitin C-terminal hydrolase deubiquitinase BRCA1-associated protein 1 (BAP1) occur in 26-32% of human CCA and are associated with worse prognosis and resistance to cytotoxic therapy. The goal of this study is to identify how BAP1 loss promotes cell death evasion through metabolic rewiring.
MATERIALS & METHODS: Isogenic wild-type and BAP1-deficient human and murine CCA cell lines were generated with shRNA/siRNA or CRISPR/Cas9 and were used to assess ferroptosis sensitivity by treatment with ferroptosis inducers. In vitro, ferroptosis was assessed by phospholipid peroxidation (C-11BODIPY), mitochondrial phospholipid peroxidation (MitoPeDPP, MitoCLox), iron oeverload (MitoFerro green) and cell death (CellTiter-Glo). Phospholipidomics and proteomics were performed in isogenic cells by LC-MS/MS. Isogenic liver orthotopic CCA murine models were used for in vivo pre-clinical studies.
RESULTS: Our findings indicate that BAP1 loss confers CCA ferroptosis resistance. BAP1 loss protects CCA against mitochondrial iron overload, ROS accumulation, and phospholipid peroxidation. Strikingly, BAP1 loss protects from mitochondrial phospholipid peroxidation and cardiolipin oxidation. Unbiased phospholipidomic profiling revealed that BAP1 loss remodels membrane phospholipid composition, enriching for ferroptosis-blocking monounsaturated phospholipids (MUFA-PLs) at the expense of ferroptosis-inducing polyunsaturated (PUFA-PLs). We found that BAP1 loss upregulates TLCD1, a phosphatidylethanolamine acyltransferase that incorporates MUFAs into membrane phospholipids, thereby stabilizing membranes against peroxidation. TLCD1 knockdown in BAP1-deficient CCA cells, restored ferroptosis sensitivity Notably, TLCD1 is significantly upregulated in human BAP1-mutant CCA as compared to WT tumors. We identified TLCD1 in the endoplasmic reticulum, Golgi and by mitochondrial outer membrane. In syngeneic liver orthotopic CCA murine models, engineered EpCAM-aptamer-coated lactosomes carrying GPX4 siRNA selectively targeted CCA cells, inducing ferroptosis and robustly suppressing tumor growth without systemic toxicity. Lastly, BAP1-mutant patient-derived xenografts (PDXs) and organoids were resistant to ferroptosis inducers, as compared to WT controls.
CONCLUSIONS: Our findings reveal that BAP1 loss rewires mitochondrial phospholipid metabolism thus conferring CCA resistance to ferroptosis, identifying TLCD1 as a targetable metabolic vulnerability in BAP1-mutant CCA.
利益披露 Disclosure
Y. Zhao, None.