糖酵解限速酶外泌重塑肿瘤微环境促进肝癌进程

Metabolic reprogramming is a hallmark of cancer. With the development of hepatocellular carcinoma (HCC), the aerobic glycolysis is enhanced, while gluconeogenesis is repressed. However, whether and how HCC cells could deliver the metabolic reprogramming signal to stromal cells in tumor microenvironment is largely unknown. Tumor-secreted extracellular vesicles (EVs) are critical mediators of intercellular communication between tumor cells and stromal cells in local and distant microenvironments by delivering signal molecules, including proteins, nucleic acids, and lipids. Therefore, tumor-secreted EVs may induce the differentiation of tumor-associated stromal cells (TASCs, such as tumor associated macrophages and carcinoma associated fibroblasts) and consequently remodel the tumor microenvironment, which further promotes tumor cell proliferation and metastasis, as well as induction of peripheral immune tolerance, ultimately boosts the development of cancer. Our preliminary data indicated that Sumoylation of several glycolytic enzymes in HCC cells promoted their excretion via EVs pathway. After uptaken by stromal cells, the EVs evidently induced the aerobic glycolysis of stromal cells and promoted the differentiation of stromal cells into tumor-associated stromal cells, thereby resulting in the remodeling of tumor microenvironment. In this project, we will focus on the following researches: (1) explore the functions and regulatory mechanisms of Sumoylation in controlling the secretion of glycolytic enzymes via EVs; (2) investigate the functions of tumor-secreted EVs-driven glycolytic enzymes on the metabolic reprogramming of stromal cells, so as to elucidate the mechanisms of how HCC cells deliver metabolic reprogramming signal to stromal cells and co-opt these reactive stromal cells’ transition into the tumor-associated stromal cells; (3) analyze the regulatory functions of the cytokines/chemokines secreted by tumor-associated stromal cells on HCC progression, and elucidate the mechanism of how HCC cells remodel tumor microenvironment to form an unique "tumor-stromal cell-tumor" feedback loop; (4) discover the functions of Nur77 in regulating metabolic reprogramming of tumor-associated stromal cells, and elucidate the regulatory role of Nur77 on glycolytic enzymes’ Sumoylation, EVs secretion and release of inflammatory factors, as well as screening the Nur77 targeting lead compounds for HCC therapy; (5) clarify the clinical significance of glycolytic enzymes’ excretion via EVs in HCC, and determine whether glycolytic enzymes in EVs could function as potential clinical diagnostic markers for HCC. Together, our study will not only demonstrate the communications between HCC cells and tumor-associated stromal cells through EVs, which reprograms glucose metabolism of stromal cells, remodels tumor microenvironment and promotes HCC progression, but also indicate the potential functions of glycolytic enzymes in EVs as biomarkers for HCC diagnosis.

肝癌发生发展伴随着肝癌细胞的代谢重编程，如糖异生的抑制和糖酵解的增强。然而，肝癌细胞是否以及如何将重编程信号传递给肿瘤微环境中的基质细胞还不清楚。胞外囊泡是细胞间通讯的重要方式。肿瘤细胞可通过胞外囊泡将蛋白质、核酸等信号分子传递给基质细胞。预实验我们发现肝癌细胞糖代谢酶SUMO化修饰促进其以胞外囊泡形式传递给基质细胞，促进基质细胞糖酵解并分化为肿瘤相关基质细胞，从而重塑肿瘤微环境。据此，我们提出以下研究计划：①SUMO化修饰调控代谢酶外泌的机制；②代谢酶外泌促进肿瘤基质细胞代谢重编程；③肿瘤基质细胞分泌相关细胞因子促进肝癌恶性进程；④Nur77调控肿瘤微环境糖代谢重编程；⑤确定肝癌代谢酶外泌的临床检测指标。通过研究有望阐明肿瘤细胞代谢异常及代谢酶外泌对基质细胞代谢重编程和肿瘤微环境调控的作用机制，及其对肿瘤发生发展的影响，确定可用于肝癌检测的外泌因子，发现能够调控相关节点的先导化合物。

本项目的研究目标是阐明肿瘤细胞代谢异常及外泌对基质细胞代谢重编程和肿瘤微环境的作用机制，及其产生的生物学效应和对肿瘤发生发展的影响，提出能够用于临床检测肝癌的外泌因子，发现能够调控相关节点的先导化合物。经过5年的努力，我们完成上述提出的所有研究内容；发表研究论文3篇（Nature Metabolism; Molecular Cell; PNAS）；申请专利3件；积极参加线下/线上学术会议（共16次），并在会上报告研究成果；获福建省自然科学一等奖1项（排名第一）。. 研究成果如下：.（1）发现肝癌细胞中SUMO化修饰促进糖酵解代谢酶PKM2靶向质膜以微囊泡（ectosome）形式外泌，进而通过诱导单核细胞分化成为巨噬细胞促进肝癌进展的新机制。临床肝癌病人的血浆微囊泡PKM2水平明显提高，而健康志愿者血浆中几乎检测不到微囊泡PKM2，表明微囊泡PKM2有望成为一个潜在的肝癌早期诊断标志物（Molecular Cell,2020）。.（2）研究肝星状细胞通过己糖激酶HK1外泌加速肝癌进程的机制和信号通路，阐明了一个未见报道的肝纤维化促进肝癌进程的新机制，并筛选到具有潜力的小分子化合物PDNPA，通过阻断HSCs中HK1外泌抑制肝癌进程（Nature Metabolism，2022)。.（3）发现核受体Nur77作为转录因子，在乳腺癌中通过调控脂肪酸吸收和转运基因阻断细胞对外源脂肪酸的摄取，进而抑制乳腺癌细胞增殖的信号转导通路，同时筛选发现Nur77特异性激动剂Csn-B是一种有效的治疗乳腺癌的小分子化合物（PNAS，2020）。.（4）发现Ezrin蛋白对于调控葡萄糖饥饿诱导胞外囊泡外泌至关重要。这些外泌的微囊泡能够进一步重塑肿瘤微环境，促进巨噬细胞向肿瘤相关巨噬细胞分化，形成免疫抑制微环境，使得肝癌细胞逃避免疫攻击（明年上半年投稿）。

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