Operative ubiquitin-specific protease 22 deubiquitination confers a more invasive phenotype to cholangiocarcinoma.
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ABSTRACT:
SUBMITTER: Tian Y
PROVIDER: S-EPMC8257691 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe G-protein-coupled formylpeptide receptor (FPR) that mediates chemotaxis of phagocytic leucocytes induced by bacterial and host-derived chemotactic peptides is selectively expressed by highly malignant human gliomas and contributes to tumour growth and angiogenesis. As invasion of surrounding normal tissues is one of the important features of tumour malignancy, we investigated the function of FPR in the invasive behaviour of human glioblastoma cells.MethodsCells (FPR(+) and FPR(-)) were isolated by single-cell cloning from a human glioblastoma cell line U-87MG. The FPR expression was assayed by flow cytometry and reverse transcription PCR. The function of FPR was investigated by chemotaxis and calcium flux induced by FPR agonist fMLF. Tumour cell motility was assayed by a wound-healing model in vitro. The growth and invasive phenotype were observed with subcutaneous implantation of tumour cells in nude mice. Over-expression of FPR in FPR(-) cells was performed by transfection of a plasmid vector-containing human FPR gene.ResultsOne of the glioma clones F9 that expressed high level of FPR showed a more 'motile' phenotype in vitro as compared with a clone G3 without FPR expression. Although F9 and G3 clones both formed subcutaneous tumours in nude mice, only F9 tumours invaded surrounding mouse connective tissues. Over-expression of FPR in G3 clone (G3F) increased the cell motility in vitro and the capacity of the cells to form more rapidly growing and invasive tumours in nude mice. We further found that, in addition to supernatant of necrotic tumour cells, foetal calf serum and human serum used in culture media contained FPR agonist activity and increased the motility of FPR-expressing glioblastoma cells.ConclusionThe expression of FPR is responsible for increased motility of human glioblastoma cells and their formation of highly invasive tumours.
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Project description:Protein ubiquitination plays a critical role in Toll-like receptor (TLR) signaling and innate immunity. Although several E3 ubiquitin ligases have been identified downstream of TLRs, the regulation of protein deubiquitination in TLR-triggered innate immune responses is poorly understood. We identified ubiquitin-specific protease 25 (USP25) as a regulator of TLR signaling. USP25 was recruited to the TLR4 signaling complex, and it associated with the adaptor proteins tumor necrosis factor receptor-associated factor 3 (TRAF3) and TRAF6 after stimulation of TLR4 with its ligand lipopolysaccharide (LPS). USP25 specifically reversed the Lys(48)-linked ubiquitination of TRAF3 that was mediated by the E3 ubiquitin ligase cIAP2 (cellular inhibitor of apoptosis 2). Deficiency in USP25 enhanced the extent of ubiquitination of TRAF3 and accelerated its degradation after TLR4 activation, which potentiated TLR4-induced activation of NF-?B (nuclear factor ?B) and MAPK (mitogen-activated protein kinase) signaling, but inhibited activation of the transcription factor IRF3 (interferon regulatory factor 3). USP25-deficient mice exhibited increased susceptibility to LPS-induced septic shock compared to their wild-type counterparts, which was associated with enhanced production of proinflammatory cytokines and decreased production of interferon-?. Thus, by inhibiting the degradation of TRAF3 during TLR4 activation, USP25 enables a balanced innate immune response.
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Project description:BackgroundIntrahepatic Cholangiocarcinoma (iCCA) is characterized by a strong stromal reaction playing a role in tumor progression. Thymus cell antigen 1 (THY1), also called Cluster of Differentiation 90 (CD90), is a key regulator of cell-cell and cell-matrix interaction. In iCCA, CD90 has been reported to be associated with a poor prognosis. In an iCCA PDX model, we recently found that CD90 was downregulated in mice treated with the Notch γ-secretase inhibitor Crenigacestat. The study aims to investigate the role of CD90 in relation to the NOTCH pathway.MethodsTHY1/CD90 gene and protein expression was evaluated in human iCCA tissues and xenograft models by qRT-PCR, immunohistochemistry, and immunofluorescence. Notch1 inhibition was achieved by siRNA. THY1/CD90 functions were investigated in xenograft models built with HuCCT1 and KKU-M213 cell lines, engineered to overexpress or knockdown THY1, respectively.ResultsCD90 co-localized with EPCAM, showing its epithelial origin. In vitro, NOTCH1 silencing triggered HES1 and THY1 down-regulation. RBPJ, a critical transcriptional regulator of NOTCH signaling, exhibited putative binding sites on the THY1 promoter and bound to the latter, implying CD90 as a downstream NOTCH pathway effector. In vivo, Crenigacestat suppressed iCCA growth and reduced CD90 expression in the PDX model. In the xenograft model, Crenigacestat inhibited tumor growth of HuCCT1 cells transfected to overexpress CD90 and KKU-M213 cells constitutively expressing high levels of CD90, while not affecting the growth of HuCCT1 control cells and KKU-M213 depleted of CD90. In an iCCA cohort, patients with higher expression levels of NOTCH1/HES1/THY1 displayed a significantly shorter survival.ConclusionsiCCA patients with higher NOTCH1/HES1/THY1 expression have the worst prognosis, but they are more likely to benefit from Notch signaling inhibition. These findings represent the scientific rationale for testing NOTCH1 inhibitors in clinical trials, taking the first step toward precision medicine for iCCA.
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