Osteosarcoma (OS) is a malignant bone neoplasm, mostly occurring in pediatric patients. OS is characterized by a highly aggressive and metastatically active tumor. Chemotherapy followed by surgical excision is the treatment of choice but is often associated with both chemoresistance and relapse. Hence, it is important to develop further understanding of OS pathogenesis and identify potential therapeutic targets. Both the signal transducer and activator of transcription 3 (STAT3) and mammalian target of rapamycin (mTOR) have been implicated in OS pathogenesis. Crosstalk between mTOR and STAT3 signaling has been shown to regulate hypoxia‐induced angiogenesis in other diseases. In this study, we determined using OS cell lines if there is a crosstalk between these two pathways and how that impacts sensitivity to treatment with Rapamycin. OS cell lines exhibited differential sensitivity to mTOR inhibitor Rapamycin. Evaluation of phosphorylated STAT3 showed that in Rapamycin‐sensitive 143B cells, the inhibitor decreased phosphorylation of STAT3 at Y705, but not at S727 whereas, in Rapamycin‐resistant U2OS cells, the inhibitor decreased S727 phosphorylation but not Y705. However, knockdown of STAT3 in U2OS cells made them sensitive to Rapamycin. Immunofluorescence (IF) analysis showed that mTOR is constitutively activated in the 143B cells but is suppressed in the U2OS cells, indicating that this might be their reason for being resistant to Rapamycin. Both cell lines were sensitive to treatment with the STAT3 inhibitor Napabucasin (NP). Treatment with NP inhibited STAT3 activation at Y705 and additionally inhibited mTOR activation, indicating crosstalk between STAT3 and mTOR signaling pathways. Rapamycin could effectively prevent lung metastasis in an orthotropic OS mice model using 143B cells. However, Rapamycin could not inhibit lung metastasis in mice injected with U2OS cells. The STAT3 inhibitor NP attenuated lung metastasis with the U2OS cells. Our results thus established yet undefined crosstalk of STAT3 and mTOR signaling pathways in OS and highlight the possibility of using mTOR inhibitors for treatment in patients with OS.

中文翻译：

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STAT3 和 mTOR 信号之间的串扰在驱动骨肉瘤细胞系化疗敏感性中的作用

骨肉瘤（OS）是一种恶性骨肿瘤，多发生于儿科患者。OS 的特征是高度侵袭性和转移性活跃的肿瘤。化疗后手术切除是首选的治疗方法，但通常与化疗耐药和复发有关。因此，进一步了解 OS 发病机制并确定潜在的治疗靶点非常重要。信号转导和转录激活因子 3 (STAT3) 和哺乳动物雷帕霉素靶标 (mTOR) 都与 OS 发病机制有关。mTOR 和 STAT3 信号之间的串扰已被证明可以调节其他疾病中缺氧诱导的血管生成。在这项研究中，我们使用 OS 细胞系确定这两种途径之间是否存在串扰，以及这如何影响对雷帕霉素治疗的敏感性。OS 细胞系对 mTOR 抑制剂雷帕霉素表现出不同的敏感性。对磷酸化 STAT3 的评估表明，在雷帕霉素敏感的 143B 细胞中，抑制剂降低了 Y705 处 STAT3 的磷酸化，但在 S727 处不降低，而在雷帕霉素抗性 U2OS 细胞中，抑制剂降低了 S727 磷酸化但不降低 Y705。然而，U2OS 细胞中 STAT3 的敲低使它们对雷帕霉素敏感。免疫荧光 (IF) 分析显示 mTOR 在 143B 细胞中被组成型激活，但在 U2OS 细胞中被抑制，表明这可能是它们对雷帕霉素产生抗性的原因。两种细胞系都对 STAT3 抑制剂 Napabucasin (NP) 处理敏感。用 NP 处理抑制了 Y705 的 STAT3 激活，并另外抑制了 mTOR 激活，表明 STAT3 和 mTOR 信号通路之间的串扰。在使用 143B 细胞的正交各向异性 OS 小鼠模型中，雷帕霉素可有效预防肺转移。然而，雷帕霉素不能抑制注射 U2OS 细胞的小鼠的肺转移。STAT3 抑制剂 NP 减弱了 U2OS 细胞的肺转移。因此，我们的结果确定了 STAT3 和 mTOR 信号通路在 OS 中尚未确定的串扰，并强调了使用 mTOR 抑制剂治疗 OS 患者的可能性。