Angiomotin通过Hippo-YAP信号通路调控肾癌干细胞样特性的机制研究

Despite offering significant clinical benefits in renal cell carcinoma (RCC), the effectiveness of current targeted therapies eventually declines with the development of resistance to treatment, become refractory and relapse in the majority of patients. It has gradually gained acceptance that survival and accumulation of cancer stem cells (CSCs) following chemo or radiotherapy was tightly associated with the recurrence of increasingly invasive and malignant tumors, including RCC. We found previously that highly expression of Angiomotin (Amot) in RCC is tightly related to the increased number of CSCs population, enhanced nuclear translocation of YAP/TAZ, and higher invasion/migration ability in vitro. Since it has been proved that Hippo-YAP signaling pathway plays important roles in maintaining the stemness of stem cells, we proposed in this study that the expression of Amot is involved in regulating of CSCs characteristics in RCC via Hippo-YAP signal pathway. To prove our hypothesis, we will isolate CSCs from RCC and culture CSCs. Moreover, we will investigate the association between Amot expression and CSCs stemness by using knock-in or knock-down strategy. More importantly, we will also clarify how Hippo-YAP signaling is involved in and regulated by Amot in the process by using in vitro and in vivo models. We believe that the anticipated results will provide new insight into the mechanisms of RCC pathology, and provide a theoretical basis for creating effective clinical treatment strategy or novel target for RCC.

目前肾癌临床治疗所面临的主要挑战是对放化疗不敏感、易复发和转移。有证据显示，肾癌干细胞与肾癌复发、转移及化学药物抵抗密切相关。我们在前期研究中发现，当上调Angiomotin(Amot)表达后，肾癌细胞的侵袭转移能力增强，并伴随有明显的肾癌干细胞亚群细胞增多，以及核定位YAP/TAZ表达升高。鉴于已有证据表明Hippo-YAP通路在干细胞的自我更新与维持干细胞特性方面发挥着重要作用。据此，我们提出假说：Amot可通过Hippo-YAP信号通路调控肾癌干细胞样特性，进而影响肾癌的恶性生物学行为。在本研究中，我们将分选和鉴定肾癌干细胞，比较干预Amot表达对肾癌干细胞样亚群的影响，并利用体内、外模型来研究Amot与Hippo-YAP信号通路如何通过调控肾癌干细胞样的演进来影响肾癌的发展。预期研究结果不仅能进一步丰富肾癌病理机制方面的理论，而且有望为肾癌的治疗提供新的靶点和理论依据。

目前肾癌临床治疗所面临的主要挑战是对放化疗不敏感、易复发和转移。有证据显示，肾癌干细胞与肾癌复发、转移及化学药物抵抗密切相关。课题组在前期研究中发现，Angiomotin(Amot)可作为肾癌特异性蛋白标志物预示疾病的不良预后，其高表达与肾癌的侵袭转移能力和肿瘤干细胞样特性呈现正相关，并伴随着核定位YAP/TAZ的明显升高。因此我们提出假说：Amot可通过Hippo-YAP信号通路调控肾癌干细胞样特性，进而影响肾癌的恶性生物学行为。基于肾癌干细胞亚群的成功分选及稳定干预Amot-p130肾癌干样细胞株的构建，我们对比观察了各干样细胞亚群的生物学特性，发现Amot-p130能够在体内外促进肾癌干细胞样特性的获得；利用RNA-seq和iTRAQ等技术，筛选出Hippo-YAP信号通路为Amot-p130的重要下游靶点，进一步通过免疫荧光、核浆分离、荧光素酶报告基因实验、免疫共沉淀等技术，证实Amot-p130能够通过与YAP相互作用，促进YAP入核，进而激活YAP-TEAD介导的下游转录活性；耐药相关实验证实Amot-p130在肾癌干细胞亚群中能够降低对化疗药物5-氟尿嘧啶的敏感性，并且调节耐药蛋白的表达水平。通过验证Amot在肾癌中可破坏YAP/TAZ与Hippo激酶蛋白的抑制关联，维持肾癌干细胞特性，我们的研究结果丰富了肾癌干细胞理论，为肾癌的治疗提供了新的靶点和理论依据。 TCGA数据库分析结果提示Amot在其他肿瘤中可以发挥不同作用，在乳腺癌中对Amot功能进行初探发现，Amot-p130亚型可通过抑制Wnt/β-catenin信号通路的激活，抑制乳腺癌干细胞样特性的获得，起到抑制乳腺癌发生发展的作用，提示我们将进一步探索Amot及其亚型在其他恶性肿瘤中发挥的不同作用。