Abstract：
BACKGROUND: Rhabdomyosarcoma (RMS) is one of the most common soft tissue sarcomas in children and adolescents, in which PAX3-FOXO1 fusion gene positive patients have very poor prognosis. PAX3-FOXO1 has been identified as an independent prognostic predictor in RMS, with no currently available targeted therapeutic intervention. The novel tyrosine kinase inhibitor anlotinib exhibits a wide range of anticancer effects in multiple types of cancers; however, there have been no relevant studies regarding its application in RMS.
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.
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.
摘要：
背景：横纹肌肉瘤（RMS）是儿童和青少年中最常见的软组织肉瘤之一，其中PAX3-FOXO1融合基因阳性患者预后极差。PAX3-FOXO1已被确定为RMS的独立预后预测因子，目前没有可用的有针对性的治疗干预。新型酪氨酸激酶抑制剂安洛替尼在多种类型的癌症中表现出广泛的抗癌作用；然而，关于其在RMS中的应用尚无相关研究。
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。
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。
