Cholangiocarcinoma (CCA) is a malignant tumor of the digestive system, characterized by its aggressive behavior and the absence of effective therapeutic biomarkers. Although recent studies have implicated AMDHD1 in tumor formation, its role in CCA development has been insufficiently explored. We utilized multiple bioinformatic datasets alongside 108 clinical samples to examine AMDHD1 expression in CCA. Then, in vitro and in vivo experiments were conducted to assess its impact on tumor growth and metastasis. Furthermore, proteomic analysis and immunoprecipitation mass spectrometry were employed to identify the downstream effectors of AMDHD1. We discovered that AMDHD1 was down-regulated in CCA and this down-regulation was associated with adverse clinicopathological features and prognosis. We also demonstrated that overexpression of AMDHD1 hindered G1/S progression in the cell cycle and promoted apoptosis, thereby inhibiting tumor growth and metastasis. Mechanistically, we found that AMDHD1 operated in a TGF-β-dependent manner and the inhibition of TGF-β signaling abrogated the effect of AMDHD1 overexpression on CCA cells. Specifically, AMDHD1 inhibited the ubiquitination and degradation of the SMAD4 protein through binding to the MH2 domain and synergistically enhanced SMAD2/3 phosphorylation, which activated of TGF-β signaling pathway and resulted in the suppression of CCA cell proliferation and migration. Our study identifies AMDHD1 as a significant prognostic biomarker and a tumor suppressor in CCA. It underscores the pivotal role of the AMDHD1/TGF-β signaling pathway in the development and progression of CCA.

胆管癌（CCA）是一种消化系统恶性肿瘤，其特点是具有侵袭性且缺乏有效的治疗生物标志物。尽管最近的研究表明 AMDHD1 与肿瘤形成有关，但其在 CCA 发展中的作用尚未得到充分探索。我们利用多个生物信息学数据集以及 108 个临床样本来检查 CCA 中的 AMDHD1 表达。然后，进行体外和体内实验来评估其对肿瘤生长和转移的影响。此外，采用蛋白质组分析和免疫沉淀质谱法来鉴定 AMDHD1 的下游效应子。我们发现 AMDHD1 在 CCA 中下调，这种下调与不良的临床病理特征和预后相关。我们还证明AMDHD1的过度表达会阻碍细胞周期的G1/S进程并促进细胞凋亡，从而抑制肿瘤的生长和转移。从机制上讲，我们发现AMDHD1以TGF-β依赖性方式运作，抑制TGF-β信号消除了AMDHD1过表达对CCA细胞的影响。具体而言，AMDHD1通过与MH2结构域结合抑制SMAD4蛋白的泛素化和降解，并协同增强SMAD2/3磷酸化，从而激活TGF-β信号通路，从而抑制CCA细胞增殖和迁移。我们的研究确定 AMDHD1 是 CCA 中重要的预后生物标志物和肿瘤抑制因子。它强调了 AMDHD1/TGF-β 信号通路在 CCA 发生和进展中的关键作用。