癌相关成纤维细胞脂肪酸代谢重塑调控肝癌转移复发的机制研究

Cancer-associated Fibroblasts (CAFs) are dominant stromal cells in tumor microenvironment, the interactions between CAF fatty-acid metabolic remodeling and cancer are largely unknown. CAF played as a key role in Hepatocellular Carcinoma (HCC) initiation and progression, CAF deposition involves activation of hepatic stellate cells, which is associated with depletion of intracellular lipid droplets. By high-throughout analyses, we found that β-Hydroxybutyrate (β-OHB) was rich in CAF conditioned-medium, CIDEA was a key protein during this process. Herein, based on the previous work in the field of HCC metabolism and microenvironment and pre-experimental study, we hypothesized that β-OHB which was secreted by CAF inhibited the activity of HDAC, HDAC inhibition contributed to suppression of oxidative stress and induction of immune escape in HCC. To validate this hypothesis, we employed RNA-seq and mass spectrometry-based lipidomic approach to hunt for the key protein. Moreover, through protein array and ubiquitination analyses, we will gain insight into the signal pathway and post-translational modification of CIDEA. Eventually, we adopted mRNA microarray, Ingenuity pathway analyses, epigenetics technology, clinical sample and animal model validation to investigate the mechanism of HDAC-mediated oxidative stress suppression and immune escape in HCC. This study will elucidate how CAF fatty-acid metabolic remodeling will contribute to HCC oxidative stress suppression and immune escape via epigenetic modification, which will shed light on HCC target therapy and metabolic intervention.

癌相关成纤维细胞（CAF）是肿瘤微环境中最主要的间质细胞，但CAF脂代谢重塑与肿瘤的相互关系知之甚少。研究认为，CAF在肝细胞癌（HCC）发生发展中扮演重要角色，脂滴的降解是肝星状细胞激活为CAF的重要机制。课题组在预实验通过测序研究表明：CAF脂肪酸代谢重塑促进癌代谢物β-羟丁酸（β-OHB）胞外分泌，CIDEA是关键分子。基于肝癌代谢和微环境前期研究结果，我们提出假设：CAF通过脂肪酸代谢重塑外分泌的β-OHB抑制组蛋白去乙酰化酶（HDAC）活性，HDAC活性抑制后HCC抗氧化应激增强和CTL失活。为验证这一假说，本研究将采用：1）RNA-seq、脂代谢质谱揭示CAF脂代谢的关键分子；2）蛋白质泛素化研究阐明CIDEA的下游通路；3）基因芯片、表观遗传学技术等验证探索抗氧化应激及免疫逃逸的机制。拟阐明：CAF脂代谢重塑促HCC抗氧化应激和免疫逃逸，为肝癌靶向治疗和代谢干预提供新思路。

肝癌微环境中代谢重塑可表现为两方面：其一，肿瘤微环境中主要成分发生代谢重塑如何影响肝癌的发生发展；其二，肝癌细胞自生代谢重塑如何通过改变微环境以促进自身恶性行为。本课题组前期研究已发现肝星状细胞在活化为肿瘤相关成纤维细胞（Cancer-associated Fibroblasts, CAF）的过程中，细胞内脂滴消失，脂肪酸分解代谢异常激活，但该现象是否参与肝癌的发生发展仍不明确。同时，本课题组前期研究还发现肝癌中负责乳酸外排的单羧酸转运体MCT4（monocarboxylate transporter 4）表达异常升高，其在肝癌免疫逃逸中发挥重要作用。本课题组围绕上述两个关键科学现象，分别从肿瘤细胞和间质细胞代谢重塑两个角度切入，深入阐述肿瘤微环境代谢重编程参与肝癌发生发展的功能和机制。得出以下结论：.(1) 肿瘤相关成纤维细胞脂代谢重塑促进肝癌发生发展的机制：1）CAF脂质分解代谢显著激活。其通过高表达MHGCS2将脂质分解后产生的Ac-CoA合成酮体并向外分泌给肝癌细胞摄取功能，促进其恶性行为。2）CAF中摄取脂质相关的转运体CD36高表达，其可通过两方面作用抑制HCC的恶性表型：其一，CD36可促进CAF分泌IL-10和Lipocalin-2重塑肝癌细胞脂肪酸代谢；其二CD36+CAF可通过上调自身PD-L1水平诱导TCL功能耗竭。.(3) 肝癌细胞MCT4表达参与免疫逃逸的机制：MCT4在肝癌中表达水平明显升高，且其表达与肝癌患者的不良预后相关。抑制MCT4能通过CD8+T细胞依赖的途径抑制肝癌的生长能力。具体机制表现为两方面的综合作用。一方面，抑制MCT4可以明显逆转肝癌酸性微环境，提升CD8+T细胞的抗肿瘤应答活性；另一方面，肝癌MCT4抑制可以通过ROS/NF-kB信号通路明显提高肿瘤细胞趋化因子CXCL9/CXCL10的表达水平，从而增强微环境中趋化招募CD8+T细胞的数量。在利用Hepa1-6细胞构建的C57BL/6小鼠皮下瘤模型中，联用MCT4抑制剂VB124能显著增敏PD-1单抗的治疗效果。.本研究揭示了肝癌的发生、发展与其微环境细胞代谢调控失衡密切相关。在肝癌治疗层面，可通过靶向肿瘤细胞自身代谢重塑或干预间质细胞代谢失调两方面来探究肝癌治疗的潜在治疗策略，也为后续临床转化研究提供了新的思路。