AMDHD1 acts as a tumor suppressor and contributes to activation of TGF-β signaling pathway in cholangiocarcinoma

Abstract 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.