Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute - Donostia University Hospital -, University of the Basque Country (UPV/EHU), San Sebastian, Spain.
Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute - Donostia University Hospital -, University of the Basque Country (UPV/EHU), San Sebastian, Spain; Department of Gastroenterology & Hepatology, Radboud University Nijmegen Medical Center, The Netherlands.
J Hepatol. 2022 Jul;77(1):177-190. doi: 10.1016/j.jhep.2022.02.007. Epub 2022 Feb 23.
BACKGROUND & AIMS: Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors associated with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Herein, we investigate the role of NEDDylation in CCA development and progression.
Levels and functions of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated in vitro, in vivo and/or in patients with CCA. The development of preneoplastic lesions in Nae1 mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry.
The NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in the cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation in vitro. NEDDylation depletion (pevonedistat or Nae1 mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogene-driven models of CCA in vivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells in vitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, impaired NEDDylation in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration.
Aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell survival and proliferation. Moreover, NEDDylation impacts the CCA-stroma crosstalk. Inhibition of NEDDylation with pevonedistat may represent a potential therapeutic strategy for patients with CCA.
Little is known about the role of post-translational modifications of proteins in cholangiocarcinoma development and progression. Herein, we show that protein NEDDylation is upregulated and hyperactivated in cholangiocarcinoma, promoting tumor growth. Pharmacological inhibition of NEDDylation halts cholangiocarcinogenesis and could be an effective therapeutic strategy to tackle these tumors.
胆管癌（CCA）是一组与预后不良相关的恶性肿瘤，其包含多种不同的肿瘤。翻译后修饰（PTMs）的改变，包括 NEDDylation，会导致异常的蛋白质动力学、细胞紊乱和疾病。在此，我们研究了 NEDDylation 在 CCA 发展和进展中的作用。
在体外、体内和/或 CCA 患者中评估 NEDDylation 的水平和功能，以及对 pevonedistat（NEDDylation 抑制剂）或针对 NAE1 的 CRISPR/Cas9 的反应。使用致癌基因驱动的 CCA 模型研究 Nae1 小鼠的癌前病变发展。使用 CCA 衍生的癌症相关成纤维细胞（CAFs）评估 NEDDylation 在 CCA 细胞中的作用对肿瘤-基质串扰的影响。通过质谱进行蛋白质组学分析。
在人 CCA 细胞和肿瘤中发现 NEDDylation 机制过度表达和过度激活。在 CCA 细胞中，经 NEDD8-免疫沉淀和进一步蛋白质组学分析后发现，上调的大多数 NEDDylated 蛋白参与细胞周期、增殖或存活。NEDDylation 的遗传（CRISPR/Cas9-NAE1）和药理学（pevonedistat）抑制减少了 CCA 细胞的增殖，并抑制了体外集落形成。体内，NEDDylation 耗竭（pevonedistat 或 Nae1 小鼠）阻止了 CCA 的皮下、原位和致癌基因驱动模型中的肿瘤发生。此外，pevonedistat 增强了体外 CCA 细胞中化疗诱导的细胞死亡。从机制上讲，受损的 NEDDylation 会导致 cullin RING 连接酶和 NEDD8 底物的积累，从而诱导 DNA 损伤和细胞周期停滞。此外，CCA 细胞中 NEDDylation 的受损会降低参与成纤维细胞激活、血管生成和致癌途径的蛋白的分泌，最终阻碍 CAF 的增殖和迁移。
异常的蛋白质 NEDDylation 通过促进细胞存活和增殖促进胆管癌的发生。此外，NEDDylation 影响 CCA-基质串扰。用 pevonedistat 抑制 NEDDylation 可能是治疗 CCA 患者的一种潜在治疗策略。
人们对蛋白质翻译后修饰在胆管癌发生和发展中的作用知之甚少。在此，我们表明蛋白质 NEDDylation 在胆管癌中上调并过度激活，促进肿瘤生长。NEDDylation 的药理学抑制可阻止胆管癌发生，可能是治疗这些肿瘤的有效治疗策略。
