Cholangiocarcinoma (CCA) is a hepatobiliary carcinoma with uncontrolled cell proliferation, poor prognosis, and high mortality. The ovarian tumor structural domain (OTU) containing protein 6B (OTUD6B) belongs to the OTU deubiquitin family and is vital in tumor development. However, its expression and biological function in CCA remain unknown. The expression of OTUD6B in CCA was analyzed using TIMER2.0, UALCAN, and GEO databases. MTT, clonal formation assay, immunofluorescence staining, immunohistochemistry staining, and flow cytometry examined the regulation of OTUD6B on cell proliferation, cycle, and apoptosis. The effects of OTUD6B on tumor volume and weight were assessed using the xenograft tumor model. The activities of PTK2 and STAT3 were detected by western blot and CO-IP. The biological database identified that OTUD6B was upregulated in CCA. In CCA cells, OTUD6B knockdown reduced CCA cell proliferation and promoted apoptosis. Cell cycle analysis indicated that the cycle stopped at the G0/G1 phase after OTU6B downregulation. Furthermore, OTUD6B knockdown resulted in a decrease in tumor volume and weight in xenograft tumor models. Mechanistically, OTUD6B is involved in the deubiquitination of PTK2. PTK2 further affected the phosphorylation of STAT3 thereby regulating the CCA process. Our study demonstrates that OTUD6B knockdown participates in the ubiquitination of PTK2 and phosphorylation of STAT3 to alleviate the process of CCA. These results suggest that OTUD6B may be a potential new strategy for CCA treatment.
Yang Dian,Liu Yichao,Hong Yueshun,Miao Enming,Wang Peng,Sun Yuming,Zhou Lina,Liu Shuyan,Zhang Yingqiu,Qin Hongqiang,Ye Mingliang,Liu Han
Stress granules (SGs) are membrane-less organelles formed through liquid-liquid phase separation of proteins and RNAs, serving as temporary repositories for biomacromolecules to protect cells under stress conditions. Impaired SG disassembly is closely implicated in neurodegenerative diseases and aging, yet the mechanisms regulating SG dynamics are incompletely investigated. The constituents of heterogenous SGs are complicated and broadly categorized as core and shell components. In contrary to the relatively stable core components, our understanding of the diverse SG shell is deficient. By combining interactomic and proximity proteomic approaches, we reveal that the deubiquitinating enzyme OTUD6B is associated with SG-related functions. Immunofluorescence assays showed that OTUD6B localized to SGs, as well as regulated their early assembly and clearance, partially dependent on its enzymatic activity. Further proximity proteomics and interactomics results uncover the ATPase VCP/p97, a key SG disassembly factor, as an OTUD6B-associated protein. OTUD6B and VCP association is governed through their disordered regions normally participated in biomolecular condensation. VCP knockdown or pharmacological inhibition phenocopied OTUD6B silencing by leading to defects in SG dynamics. Mechanistically, SG coalescence of VCP incurred by OTUD6B in a partially enzymatic activity-dependent manner functions to accelerate not only the early assembly, but also SG clearance following stress removal. Therefore, our findings establish OTUD6B as a critical modulator of SG dynamics, linking its function to stress responses and potential disease mechanisms.
危险品化学品经营许可证（带存储）