METHODS: We investigated the effects of PAX3-FOXO1 on the therapeutic efficacy of anlotinib using the CCK-8 assay, flow cytometry, invasion assay, wound healing assay, western blotting, quantitative polymerase chain reaction(qPCR), and xenograft experiments. RNA-seq and co-immunoprecipitation assays were conducted to determine the specific mechanism by which anlotinib regulates PAX3-FOXO1 expression.
RESULTS: Anlotinib effectively inhibited RMS cell proliferation and promoted apoptosis and G2/M phase arrest while impeding tumor growth in vivo. Downregulation of PAX3-FOXO1 enhances the antitumor effects of anlotinib. Anlotinib upregulates protein kinase NEK2 and increases the degradation of PAX3-FOXO1 via the ubiquitin-proteasome pathway, leading to a reduction in PAX3-FOXO1 protein levels.
CONCLUSIONS: Anlotinib effectively inhibited the malignant progression of RMS and promoted degradation of the fusion protein PAX3-FOXO1. Anlotinib could be a targeted therapeutic approach to treat PAX3-FOXO1 fusion-positive RMS.
方法:我们使用CCK-8试验研究了PAX3-FOXO1对安洛替尼疗效的影响,流式细胞术,侵袭试验,伤口愈合试验,西方印迹,定量聚合酶链反应(qPCR),和异种移植实验。进行RNA-seq和共免疫沉淀测定以确定安洛替尼调节PAX3-FOXO1表达的具体机制。
结果:安洛替尼有效抑制RMS细胞增殖,促进细胞凋亡和G2/M期阻滞,同时阻碍体内肿瘤生长。PAX3-FOXO1的下调增强了安洛替尼的抗肿瘤作用。安洛替尼上调蛋白激酶NEK2,并通过泛素-蛋白酶体途径增加PAX3-FOXO1的降解,导致PAX3-FOXO1蛋白水平降低。
结论:安洛替尼有效抑制RMS的恶性进展并促进融合蛋白PAX3-FOXO1的降解。安洛替尼可能是治疗PAX3-FOXO1融合阳性RMS的靶向治疗方法。