BACKGROUND The treatment of metastatic osteosarcoma (OS) remains a challenge for oncologists, and novel therapeutic strategies are urgently needed. An understanding of the pathways that regulate OS dissemination is required for the design of novel treatment approaches. We recently identified Rho-associated coiled-coil containing protein kinase 2 (ROCK2) as a crucial driver of OS cell migration. In this study, we explored the impact of ROCK2 disruption on the metastatic capabilities of OS cells and analyzed its functional relationship with Yes-associated protein-1 (YAP), the main transcriptional mediator of mechanotransduction signaling. METHODS The effects of ROCK2 depletion on metastasis were studied in NOD Scid gamma (NSG) mice injected with U-2OS cells in which ROCK2 expression had been stably silenced. Functional studies were performed in vitro in human U-2OS cells and in three novel cell lines derived from patient-derived xenografts (PDXs) by using standard methods to evaluate malignancy parameters and signaling transduction. The nuclear immunostaining of YAP and the evaluation of its downstream targets Cysteine Rich Angiogenic Inducer 6, Connective Tissue Growth Factor and Cyclin D1 by quantitative PCR were performed to analyze YAP activity. The effect of the expression and activity of ROCK2 and YAP on tumor progression was analyzed in 175 OS primary tumors. RESULTS The silencing of ROCK2 markedly reduced tumor growth and completely abolished the metastatic ability of U-2OS cells. The depletion of ROCK2, either by pharmacological inhibition or silencing, induced a dose- and time-dependent reduction in the nuclear expression and transcriptional activity of YAP. The nuclear expression of YAP was observed in 80/175 (46%) tumor samples and was significantly correlated with worse patient prognosis and a higher likelihood of metastasis and death. The use of verteporfin, a molecule that specifically inhibits the TEAD-YAP association, remarkably impaired the growth and migration of OS cells in vitro. Moreover to inhibiting YAP activity, our findings indicate that verteporfin also affects the ROCK2 protein and its functions. CONCLUSIONS We describe the functional connection between ROCK2 and YAP in the regulation of OS cell migration and metastasis formation. These data provide support for the use of verteporfin as a possible therapeutic option to prevent OS cell dissemination.

中文翻译：

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ROCK2剥夺可通过调节YAP活性来抑制骨肉瘤细胞中的肿瘤生长和转移潜能。

背景技术转移性骨肉瘤（OS）的治疗仍然是肿瘤学家的挑战，并且迫切需要新的治疗策略。设计新型治疗方法需要了解调节OS传播的途径。我们最近发现，含有Rho相关的含有蛋白激酶2（ROCK2）的卷曲螺旋是OS细胞迁移的关键驱动力。在这项研究中，我们探讨了ROCK2破坏对OS细胞转移能力的影响，并分析了其与Yes-associated protein-1（YAP）的功能关系，Yes-associated protein-1（YAP）是机械转导信号的主要转录介质。方法在注射了稳定表达了ROCK2表达的U-2OS细胞的NOD Scidγ（NSG）小鼠中研究了ROCK2耗竭对转移的影响。通过使用标准方法评估恶性参数和信号转导，在人U-2OS细胞和源自患者的异种移植物（PDXs）的三种新型细胞系中进行了功能研究。通过定量PCR对YAP进行核免疫染色并评估其下游靶标富半胱氨酸血管生成诱导物6，结缔组织生长因子和Cyclin D1，以分析YAP活性。在175例OS原发性肿瘤中分析了ROCK2和YAP的表达和活性对肿瘤进展的影响。结果ROCK2的沉默显着降低了肿瘤的生长，并完全废除了U-2OS细胞的转移能力。通过药理抑制或沉默使ROCK2耗竭，诱导了YAP的核表达和转录活性的剂量和时间依赖性降低。在80/175（46％）肿瘤样品中观察到YAP的核表达，并且与患者预后较差以及转移和死亡的可能性更高有显着相关性。Verteporfin（一种特异性抑制TEAD-YAP缔合的分子）的使用显着损害了OS细胞的体外生长和迁移。除了抑制YAP活性外，我们的发现还表明verteporfin也影响ROCK2蛋白及其功能。结论我们描述了ROCK2和YAP之间在调节OS细胞迁移和转移形成中的功能连接。这些数据为使用维替泊芬作为预防OS细胞扩散的可能治疗选择提供了支持。在80/175（46％）肿瘤样品中观察到YAP的核表达，并且与患者预后较差以及转移和死亡的可能性更高有显着相关性。Verteporfin（一种特异性抑制TEAD-YAP缔合的分子）的使用显着损害了OS细胞的体外生长和迁移。除了抑制YAP活性外，我们的发现还表明verteporfin也影响ROCK2蛋白及其功能。结论我们描述了ROCK2和YAP之间在调节OS细胞迁移和转移形成中的功能连接。这些数据为使用维替泊芬作为预防OS细胞扩散的可能治疗选择提供了支持。在80/175（46％）肿瘤样品中观察到YAP的核表达，并且与患者预后较差以及转移和死亡的可能性更高有显着相关性。Verteporfin（一种特异性抑制TEAD-YAP缔合的分子）的使用显着损害了OS细胞的体外生长和迁移。除了抑制YAP活性外，我们的发现还表明verteporfin也影响ROCK2蛋白及其功能。结论我们描述了ROCK2和YAP之间在调节OS细胞迁移和转移形成中的功能连接。这些数据为使用维替泊芬作为预防OS细胞扩散的可能治疗选择提供了支持。