Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, NHC Key Laboratory of Living Donor Liver Transplantation (Nanjing Medical University), Jiangsu Provincial Medical Innovation Center, Jiangsu Provincial Medical Key Laboratory, Nanjing, Jiangsu Province, China.
Department of Anesthesiology, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, Jiangsu Province, China.
J Hepatol. 2024 Jan;80(1):82-98. doi: 10.1016/j.jhep.2023.10.006. Epub 2023 Oct 12.
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is among the most prevalent and lethal cancers worldwide. The tumor microenvironment (TME) contributes to the poor response of patients with HCC to current therapies, while tumor vascular endothelial cells (ECs) are fundamental TME components that significantly contribute to tumor progression. However, the specific functions and mechanisms of tumor vascular ECs in HCC remain unclear. METHODS: We screened and validated diacylglycerol kinase gamma (DGKG) hyper-expression specifically in HCC tumor vascular ECs. Single-cell RNA-sequencing, cytometry by time-of-flight, and in vitro and in vivo studies were performed to investigate the functions of endothelial DGKG. Multiplexed immunohistochemistry staining and flow cytometry were used to evaluate changes in the TME. RESULTS: Functionally, endothelial DGKG promotes tumor angiogenesis and immunosuppressive regulatory T-cell differentiation in HCC. Of significance, we found that HIF-1α activates DGKG transcription by directly binding to its promoter region under hypoxia. Upregulated DGKG promotes HCC progression by recruiting ubiquitin specific peptidase 16 to facilitate ZEB2 deubiquitination, which increases TGF-β1 secretion, thus inducing tumor angiogenesis and regulatory T-cell differentiation. Importantly, targeting endothelial DGKG potentiated the efficiency of dual blockade of PD-1 and VEGFR-2. CONCLUSION: Hypoxia-induced EC-specific DGKG hyper-expression promotes tumor angiogenesis and immune evasion via the ZEB2/TGF-β1 axis, suggesting EC-specific DGKG as a potential therapeutic target for HCC. IMPACT AND IMPLICATIONS: Here, we reported that hypoxia-induced endothelial cell-specific DGKG hyper-expression promotes angiogenesis and immune evasion in HCC by recruiting USP16 for K48-linked deubiquitination and inducing the subsequent stabilization of ZEB2, leading to increased TGF-β1 secretion. Most importantly, endothelial DGKG inhibition greatly improved the efficacy of the dual combination of anti-VEGFR2 and anti-PD-1 treatment in a mouse HCC model, significantly inhibiting the malignant progression of HCC and improving survival. This preclinical study supports the targeting of endothelial DGKG as a potential strategy for precision HCC treatment.
背景与目的：肝细胞癌（HCC）是全球最常见和最致命的癌症之一。肿瘤微环境（TME）导致 HCC 患者对当前治疗方法的反应不佳，而肿瘤血管内皮细胞（EC）是 TME 的重要组成部分，对肿瘤进展有重要贡献。然而，肿瘤血管 EC 在 HCC 中的具体功能和机制仍不清楚。
方法：我们筛选并验证了二酰基甘油激酶γ（DGKG）在 HCC 肿瘤血管 EC 中特异性高表达。进行单细胞 RNA 测序、飞行时间细胞术以及体外和体内研究，以研究内皮细胞 DGKG 的功能。采用多重免疫组化染色和流式细胞术评估 TME 的变化。
结果：功能上，内皮细胞 DGKG 促进 HCC 中的肿瘤血管生成和免疫抑制性调节性 T 细胞分化。值得注意的是，我们发现 HIF-1α 在缺氧下通过直接结合其启动子区域激活 DGKG 转录。上调的 DGKG 通过招募泛素特异性肽酶 16 促进 ZEB2 去泛素化，从而增加 TGF-β1 的分泌，进而诱导肿瘤血管生成和调节性 T 细胞分化，从而促进 HCC 的进展。重要的是，靶向内皮细胞 DGKG 增强了 PD-1 和 VEGFR-2 双重阻断的效率。
结论：缺氧诱导的 EC 特异性 DGKG 高表达通过 ZEB2/TGF-β1 轴促进肿瘤血管生成和免疫逃逸，提示 EC 特异性 DGKG 可能成为 HCC 的潜在治疗靶点。
影响和意义：本研究报道，缺氧诱导的内皮细胞特异性 DGKG 高表达通过招募 USP16 进行 K48 连接的去泛素化，并诱导随后 ZEB2 的稳定，从而增加 TGF-β1 的分泌，从而促进 HCC 中的血管生成和免疫逃逸。最重要的是，内皮细胞 DGKG 抑制极大地提高了抗 VEGFR2 和抗 PD-1 双重联合治疗在小鼠 HCC 模型中的疗效，显著抑制 HCC 的恶性进展并提高生存率。这项临床前研究支持将内皮细胞 DGKG 作为 HCC 精准治疗的潜在策略。
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