STUB1 介导的 UHRF1 的 K63 连接泛素化通过维持 PLA2G2A 的 DNA 高甲基化促进胆管癌的进展。

STUB1-mediated K63-linked ubiquitination of UHRF1 promotes the progression of cholangiocarcinoma by maintaining DNA hypermethylation of PLA2G2A.

机构信息

Department of Biliary-Pancreatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, Hubei, 430074, China.

Department of General Surgery, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, China.

出版信息

J Exp Clin Cancer Res. 2024 Sep 13;43(1):260. doi: 10.1186/s13046-024-03186-6.

DOI:10.1186/s13046-024-03186-6

PMID:39267107

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11395162/

Abstract

BACKGROUND

Cholangiocarcinoma (CCA) is a highly malignant tumor characterized by a lack of effective targeted therapeutic strategies. The protein UHRF1 plays a pivotal role in the preservation of DNA methylation and works synergistically with DNMT1. Posttranscriptional modifications (PTMs), such as ubiquitination, play indispensable roles in facilitating this process. Nevertheless, the specific PTMs that regulate UHRF1 in CCA remain unidentified.

METHODS

We confirmed the interaction between STUB1 and UHRF1 through mass spectrometry analysis. Furthermore, we investigated the underlying mechanisms of the STUB1-UHRF1/DNMT1 axis via co-IP experiments, denaturing IP ubiquitination experiments, nuclear‒cytoplasmic separation and immunofluorescence experiments. The downstream PLA2G2A gene, regulated by the STUB1-UHRF1/DNMT1 axis, was identified via RNA-seq. The negative regulatory mechanism of PLA2G2A was explored via bisulfite sequencing PCR (BSP) experiments to assess changes in promoter methylation. The roles of PLA2G2A and STUB1 in the proliferation, invasion, and migration of CCA cells were assessed using the CCK-8 assay, colony formation assay, Transwell assay, wound healing assay and xenograft mouse model. We evaluated the effects of STUB1/UHRF1 on cholangiocarcinoma by utilizing a primary CCA mouse model.

RESULTS

This study revealed that STUB1 interacts with UHRF1, resulting in an increase in the K63-linked ubiquitination of UHRF1. Consequently, this facilitates the nuclear translocation of UHRF1 and enhances its binding affinity with DNMT1. The STUB1-UHRF1/DNMT1 axis led to increased DNA methylation of the PLA2G2A promoter, subsequently repressing its expression. Increased STUB1 expression in CCA was inversely correlated with tumor progression and overall survival. Conversely, PLA2G2A functions as a tumor suppressor in CCA by inhibiting cell proliferation, invasion and migration.

CONCLUSIONS

These findings suggest that the STUB1-mediated ubiquitination of UHRF1 plays a pivotal role in tumor progression by epigenetically silencing PLA2G2A, underscoring the potential of STUB1 as both a prognostic biomarker and therapeutic target for CCA.

摘要

背景

胆管癌（CCA）是一种恶性程度很高的肿瘤，其特征是缺乏有效的靶向治疗策略。UHRF1 蛋白在 DNA 甲基化的维持中起着关键作用，并与 DNMT1 协同作用。转录后修饰（PTMs），如泛素化，在促进这一过程中起着不可或缺的作用。然而，CCA 中调节 UHRF1 的特定 PTMs 仍未确定。

方法

我们通过质谱分析证实了 STUB1 和 UHRF1 之间的相互作用。此外，我们通过 co-IP 实验、变性 IP 泛素化实验、核质分离和免疫荧光实验研究了 STUB1-UHRF1/DNMT1 轴的潜在机制。通过 RNA-seq 鉴定了受 STUB1-UHRF1/DNMT1 轴调控的下游 PLA2G2A 基因。通过亚硫酸氢盐测序 PCR（BSP）实验探索 PLA2G2A 的负调控机制，以评估启动子甲基化的变化。通过 CCK-8 测定、集落形成测定、Transwell 测定、划痕愈合测定和异种移植小鼠模型评估 PLA2G2A 和 STUB1 在 CCA 细胞增殖、侵袭和迁移中的作用。我们利用原发性 CCA 小鼠模型评估了 STUB1/UHRF1 对胆管癌的影响。

结果

本研究表明，STUB1 与 UHRF1 相互作用，导致 UHRF1 的 K63 连接泛素化增加。因此，这促进了 UHRF1 的核转位，并增强了其与 DNMT1 的结合亲和力。STUB1-UHRF1/DNMT1 轴导致 PLA2G2A 启动子的 DNA 甲基化增加，进而抑制其表达。CCA 中 STUB1 表达的增加与肿瘤进展和总生存期呈负相关。相反，PLA2G2A 在 CCA 中作为肿瘤抑制因子发挥作用，抑制细胞增殖、侵袭和迁移。