Project description:SaOS2 osteosarcoma cells were cultured with or without atorvastatin (10 µM) for 6, 15 or 24h (2 biological replcates). RNA were isolated and hybridized to RNG microarrays. Background Osteosarcoma is the most common primary tumor of bone. The rapid development of metastatic lesions and resistance to chemotherapy remain major mechanisms responsible for the failure of treatments and poor survival rate for patients. Methods We previously showed that the HMGCoA reductase inhibitors statins exhibit anti-tumoral effects on osteosarcoma cells. Here, using microarray analysis, we identify cyr61/CCN1 as a new target of statins. Modulations of expression of cyr61 were performed in human and murine osteosarcoma cell lines to investigate in vitro cell viability, migratory potential and invasiveness. Cyr61 expression was evaluated in 231 tissue cores from osteosarcoma patients using tissue microarray. Tumor behavior and metastases occurence were analysed by IM injection of modified osteosarcoma cells to BALB/c mice. Results Transcriptome comparisons revealed that statins down-regulate cyr61 expression in SaOS2 cells. Cyr61 silencing in human and murine osteosarcoma cell lines enhanced cell death, but reduced cell migration and cell invasion compared to parental cells whereas cyr61 overexpression had opposite effects. Tissue microarray analysis demonstrated that cyr61 protein expression is higher in human osteosarcoma compared to normal bone tissue and is further increased in metastatic tissues. In vivo, cyr61 overexpression in osteosarcoma cells enhanced lung metastases development whereas cyr61 silencing strongly reduced metastases in mice. Conclusion The results reveal that cyr61 expression increases with tumor grade in human osteosarcoma and demonstrate that cyr61 silencing inhibits in vitro osteosarcoma cell invasion and migration as well as in vivo lung metastases in mice. These data provide a novel molecular target for therapeutic intervention in metastatic osteosarcoma. Dye balance-experiment comparing atorvastatin versus untreated cells at 6, 15 and 24 hours using 2 biological replicates.

Project description:Background: Osteosarcoma, a primary malignant bone tumor predominantly affecting children and adolescents, exhibits a dismal prognosis for patients with metastatic or recurrent disease, characterized by low five-year survival rates. This poor outcome is largely attributed to the incomplete understanding of the molecular mechanisms governing osteosarcoma aggressiveness and metastasis, coupled with a paucity of effective early diagnostic biomarkers and targeted therapeutic strategies. Consequently, elucidating the critical molecular events driving osteosarcoma pathogenesis and identifying novel biomarkers are of paramount importance for improving patient outcomes. Prior research has demonstrated aberrant expression of Zinc Finger DHHC-Type Palmitoyltransferase 9 (ZDHHC9) in various malignancies, but its specific function and clinical significance in osteosarcoma remain poorly defined. Methods: This study employed a comprehensive approach integrating bioinformatic analyses and experimental validation to investigate the functional role and clinical relevance of ZDHHC9 in osteosarcoma. Bioinformatic analyses (TIMER, GEPIA, TCGA databases) were utilized to examine ZDHHC9 expression across a pan-cancer landscape and its association with survival in osteosarcoma patients. Immunohistochemistry (IHC) and Western blotting were employed to analyze ZDHHC9 expression in clinical osteosarcoma specimens and cell lines. In vitro assays (CCK-8, colony formation, wound healing, Transwell, flow cytometry, and Western blotting) were performed to evaluate the impact of ZDHHC9 on osteosarcoma cell proliferation, migration, and invasion. Proteomic sequencing, molecular docking, and co-immunoprecipitation (Co-IP) experiments were conducted to explore the interaction between ZDHHC9 and KRAS. Western blotting was used to analyze the regulation of Raf1, ERK1/2, and p-ERK1/2 expression by ZDHHC9 and KRAS. Finally, a xenograft model was employed to assess the effect of ZDHHC9 on in vivo tumor growth. Results: Bioinformatic analyses revealed that elevated ZDHHC9 expression correlates with poor prognosis in osteosarcoma. IHC and Western blotting confirmed significantly increased ZDHHC9 expression in osteosarcoma tissues compared to adjacent non-tumorous tissues. Upregulation of ZDHHC9 was significantly associated with elevated Ki67 levels and advanced Enneking stage, suggesting a link to tumor aggressiveness. In vitro studies demonstrated that downregulation of ZDHHC9 suppressed osteosarcoma cell proliferation, migration, and invasion, while promoting apoptosis. Conversely, upregulation of ZDHHC9 elicited opposite effects. Proteomic sequencing revealed that ZDHHC9 knockdown significantly downregulated the expression of proteins associated with the RAS/MAPK signaling pathway. Functional experiments showed that overexpression of KRAS partially abrogated the inhibitory effects of ZDHHC9 knockdown on cell proliferation, migration, and invasion. Molecular docking and Co-IP experiments confirmed a specific interaction between ZDHHC9 and KRAS, leading to activation of the RAS/MAPK signaling pathway. Conclusion: ZDHHC9 promotes osteosarcoma cell proliferation, migration, and invasion by enhancing KRAS-mediated activity of the RAS/MAPK signaling pathway, which promotes cell cycle progression while inhibiting apoptosis.

Project description:SaOS2 osteosarcoma cells were cultured with or without atorvastatin (10 µM) for 6, 15 or 24h (2 biological replcates). RNA were isolated and hybridized to RNG microarrays. Background Osteosarcoma is the most common primary tumor of bone. The rapid development of metastatic lesions and resistance to chemotherapy remain major mechanisms responsible for the failure of treatments and poor survival rate for patients. Methods We previously showed that the HMGCoA reductase inhibitors statins exhibit anti-tumoral effects on osteosarcoma cells. Here, using microarray analysis, we identify cyr61/CCN1 as a new target of statins. Modulations of expression of cyr61 were performed in human and murine osteosarcoma cell lines to investigate in vitro cell viability, migratory potential and invasiveness. Cyr61 expression was evaluated in 231 tissue cores from osteosarcoma patients using tissue microarray. Tumor behavior and metastases occurence were analysed by IM injection of modified osteosarcoma cells to BALB/c mice. Results Transcriptome comparisons revealed that statins down-regulate cyr61 expression in SaOS2 cells. Cyr61 silencing in human and murine osteosarcoma cell lines enhanced cell death, but reduced cell migration and cell invasion compared to parental cells whereas cyr61 overexpression had opposite effects. Tissue microarray analysis demonstrated that cyr61 protein expression is higher in human osteosarcoma compared to normal bone tissue and is further increased in metastatic tissues. In vivo, cyr61 overexpression in osteosarcoma cells enhanced lung metastases development whereas cyr61 silencing strongly reduced metastases in mice. Conclusion The results reveal that cyr61 expression increases with tumor grade in human osteosarcoma and demonstrate that cyr61 silencing inhibits in vitro osteosarcoma cell invasion and migration as well as in vivo lung metastases in mice. These data provide a novel molecular target for therapeutic intervention in metastatic osteosarcoma.

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LDHA/Hedgehog signaling pathway axis regulates lung metastasis in osteosarcoma

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