Osteosarcoma (OS) is the most common primary bone sarcoma, chemoresistance becomes an obstacle to its treatment. Metabolic reprogramming is a hallmark of malignancy, targeting the metabolic pathways might provide a reasonable therapeutic strategy for OS. Here we demonstrated that Ailanthone (AIL), a major component of the Chinese medicine Ailanthus altissima, significantly suppressed OS cell growth in vitro and in vivo. Furthermore, AIL dose-dependently inhibited cell migration and invasion, induced cell cycle arrest and apoptosis in OS cells. Combined transcriptomics, proteomics and metabolomics analyses revealed that AIL induced widespread changes in metabolic programs in OS cells, while the serine biosynthetic pathway (SSP) was the most significantly altered pathway. qRT-PCR and Western blot assay confirmed that the transcript and protein levels of the SSP genes (PHGDH, PSAT1 and PSPH) were downregulated dose-dependently by AIL. In addition, we found out that many downstream pathways of the SSP including the one-carbon pool by folate, purine metabolism, pyrimidine metabolism, DNA replication and sphingolipid metabolism were downregulated after AIL treatment. In the revere test, PHGDH overexpression but not exogenous serine supplementation clearly attenuated the effects of AIL on OS cells. Taken together, AIL exerts antitumor effects on OS through mediating metabolic reprogramming, at least in part, by suppressing the SSP. Our findings suggest that AIL could emerge as a potential therapeutic strategy in OS.

Objective: To investigate the expression of alpha-smooth muscle actin (ACTA2) in osteosarcoma tissues and its relationship with prognosis. Methods: Prognostic analysis of lung metastasis-related genes in osteosarcoma using the TCGA database. Single-cell sequencing detected the expression of ACTA2 in 11 osteosarcoma tissues. Paraffin-embedded tissues of 74 osteosarcoma patients treated at the Sixth People's Hospital of Shanghai Jiao Tong University from 2014 to 2019 were collected, and tissue microarrays were prepared. ACTA2 expression was detected and scored by immunohistochemistry. According to the median value of the ACTA2 histochemical score, 74 patients were divided into two groups, the high-expression group and low-expression group, and the relationship of expression with clinicopathological characteristics and prognosis was analyzed by Cox regression. Results: Through analysis of ACTA2 expression by single-cell sequencing of osteosarcoma samples together with an immuno-microarray, we found moderate ACTA2 expression. Upon analyzing the prognostic impact of ACTA2, CCL2, TGFBI, VEGFA, PDGFB, PDGFC, COL1A1, COL14A1, CXCL12, CXCL14, CSPP1, LUM, DES, MYL9, and SFRP2 on osteosarcoma patients using the TCGA database, we found that patients with high ACTA2 expression had a significantly better prognosis than those with low ACTA2 expression. Patients with high expression of ACTA2 in osteosarcoma lung metastases showed longer progression-free survival and overall survival than those with low expression. Conclusion: High expression of ACTA2 in patients with osteosarcoma lung metastasis suggests a better prognosis.

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PurposeTopoisomerase 1 (TOP1) plays a crucial role in various cell cycle processes and its dysregulation can lead to the development of multiple tumours. However, conventional TOP1 inhibitors such as topotecan and irinotecan have poor clinical efficacy in osteosarcoma (OS) patients. This is partly due to the activation of the ataxia telangiectasia and Rad3‐related/checkpoint kinase 1 (ATR/CHEK1) DNA damage repair pathway, which repairs TOP1 poison‐induced DNA lesions, compromises the cytotoxicity of TOP1 inhibitors and contributes to drug resistance. Therefore, there is a need to develop more effective TOP1 inhibitors for OS.Experimental designIn this study, we evaluated the antitumor effects of isovalerylspiramycin I (ISP‐I), a novel macrolide antibiotic, using various assays including CCK‐8 proliferation assays, wound healing migration assays, Transwell invasion assays, apoptosis, cell cycle, DNA replication and damage analyses on OS cells. We also performed a surface plasmon resonance‐high‐performance liquid chromatography‐mass spectrometry assay to identify ISP‐I's direct target protein in OS. Molecular docking analysis, thermoshift assays, enzyme activity assays and reverse tests were used to confirm ISP‐1′s target. Finally, we tested the efficacy of ISP‐I in vivo using a tumour xenograft model.ResultsOur results showed that ISP‐I significantly suppressed the growth of OS cells both in vitro and in vivo. Furthermore, ISP‐I dose‐dependently inhibited cell migration and invasion, and induced apoptosis and cell cycle arrest in OS cells. Mechanistically, ISP‐I directly bound to TOP1 and inhibited DNA replication. Additionally, ISP‐I significantly downregulated the ATR/CHEK1 pathway, which led to the suppression of DNA damage repair, ultimately augmenting DNA damage and triggering cell death.ConclusionsIn conclusion, our study suggests that ISP‐I could be a novel TOP1 inhibitor that does not activate the ATR/CHEK1 DNA damage repair pathway. This characteristic allows ISP‐I to synergistically inhibit OS cell proliferation, migration and invasion. ISP‐I may represent a promising candidate for the treatment of OS.