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ROCK2 mediates osteosarcoma progression and TRAIL resistance by modulating O-GlcNAc transferase degradation.

ABSTRACT: Osteosarcoma is a common bone tumor, with a poor prognosis. New combinatorial therapies that sensitize anticancer drug-resistant osteosarcoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are, therefore, required. The GTPase RhoA effector, Rho-associated coiled-coil forming protein kinase 2 (ROCK2), is well known for its roles in various types of cancer; however, its involvement osteosarcoma has not yet been scrutinized. In this study, we analyzed ROCK2 expression, clinicopathological features, and prognosis in osteosarcoma patients. Apoptosis, colony formation, and cell proliferation were analyzed using flow cytometry, colony formation assays, and CCK8 assays, respectively. Proteomics analysis was used to evaluate osteosarcoma progression. We found that adjacent tissues had lower ROCK2 expression levels than osteosarcoma tissues and the level of expression was related to osteosarcoma tumor size and prognosis. Osteosarcoma prognosis was associated with ROCK2 expression level, which served as an independent marker in multivariate analysis. ROCK2 silencing inhibited proliferation in vivo and in vitro and triggered apoptotic osteosarcoma cell death. ROCK2 inhibited the TRAIL-mediated apoptotic pathway in osteosarcoma cells and promoted activation. Mechanistically, ROCK2 affected osteosarcoma progression and TRAIL resistance by modifying O-GlcNAcylation through O-GlcNAc transferase degradation. Taken together, our results demonstrated a unique mechanism whereby ROCK2 influences osteosarcoma progression and TRAIL resistance, hence improving osteosarcoma management.

SUBMITTER: Deng X

PROVIDER: S-EPMC7136927 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AimsHypoxia induces vascular inflammation by a mechanism not fully understood. Emerging evidence implicates O-GlcNAc transferase (OGT) in inflammation. This study explored the role of OGT in hypoxia-induced vascular endothelial inflammatory response.Methods and resultsHypoxia was either induced (1% O2 chamber) or mimicked by exposure to hypoxia-mimetic agents in cultured endothelial cells. Hypoxia increased hypoxia-inducible factor (HIF-1α) and inflammatory response (gene and protein expression of interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and E-selectin) but, surprisingly, reduced OGT protein (not mRNA) levels. Hypoxia-mimetic CoCl2 failed to reduce OGT when proteasome inhibitors were present, suggesting proteasome involvement. Indeed, CoCl2 enhanced 26S proteasome functionality evidenced by diminished reporter (Ub(G76V)-GFP) proteins in proteasome reporter cells, likely due to increased chymotrypsin-like activities. Mechanistically, β-TrCP1 mediated OGT degradation, since siRNA ablation of this E3 ubiquitin ligase stabilized OGT. Administration of the oxidative stress inhibitors reversed both proteasome activation and OGT degradation. Furthermore, up-regulation of OGT by stabilization, overexpression, or activation mitigated CoCl2-elicited inflammatory response. These observations were recapitulated in a mouse (C57BL/6J) model mimicking hypoxia, in which lung tissues presented higher levels of HIF-1α, proteasome activity, and inflammatory response, but lower levels of OGT (n = 5/group, hypoxia vs. normoxia, P < 0.05). However, administration of an activator of OGT (glucosamine: 1 mg/g/day, vehicle: saline, ip, 5 days) abolished the up-regulation of proteasome activity and inflammatory response (n = 5/group, the treated vs. untreated hypoxia groups, P < 0.05).Conclusions26S proteasome-mediated OGT reduction contributed to hypoxia-induced vascular endothelial inflammatory response. Modulation of OGT may represent a new approach to treat diseases characterized by hypoxic inflammation.

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ROCK2 mediates osteosarcoma progression and TRAIL resistance by modulating O-GlcNAc transferase degradation.

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