血管生成素类似物3（ANGPTL3）的可变剪接在肾癌TKI靶向药物耐药中的作用及机制研究

One of the most frustrating clinical challenges to the metastatic renal cell carcinoma is the tyrosine kinase inhibitor (TKI) targeted resistance. Moreover, research shows that its internal mechanism may be a multi-level, multi-molecular regulatory process. Recently, by comparing the chip-based high throughput expression profiles of the Sunitinib-sensitive renal cell carcinoma samples with the Sunitinib-resistant ones, we have identified many differentially expressed genes. Angiopoietin Analogue 3 (ANGPTL3) is the very hit that is of our interest, due to its Sunitinib-relevant alternative splicing. Except a known polypeptide-coding transcript, ANGPTL3-001, we newly identified ANGPTL3-003 as a transcript of lncRNA. ANGPTL3-001 is highly expressed in the Sunitinib/sorafenib-sensitve samples while the ANGPTL3-003 prefers to the Sunitinib/sorafenib-resistant samples. Further, manipulating the ANGPTL3-001/ANGPTL3-003 expression level can change the renal cancer cells’ response to Sunitinib/sorafenib treatment. Thus, we propose that the ANGPTL3 alternative-splicing mode is the determining factor for the resistance to TKI targeted treatment. In this proposal, we will further investigate the mechanisms underlying the TKI targeted resistance related ANGPTL3 alternative splicing from the cellular and molecular levels. We will also explore the feasibility for the ANGPTL3 alternative splicing as a diagnostic/therapeutic target using mouse tumor-bearing models and clinical samples. The outcome of this project will provide new theoretical basis to the efficacy prediction and resistance reversal of TKI targeted treatment to the renal cell carcinoma.

转移性肾癌酪氨酸激酶抑制剂（TKI）靶向治疗药物耐受是临床面临的难题，研究表明其内在机制可能是一个多层面、多分子调控的过程。前期我们对术后接受舒尼替尼治疗敏感和耐药肾癌患者的标本进行高通量芯片筛选，以获得其中差异表达基因，并对芯片结果进行分析和验证，发现ANGPTL3（血管生成素类似物3）的可变剪接与肾癌舒尼替尼耐药密切相关。ANGPTL3可变剪接生成两种转录本：ANGPTL3-001（编码蛋白RNA）和ANGPTL3-003（长链非编码RNA），扩大样本在舒尼替尼、索拉非尼两个独立的组织标本队列中分别进行验证，发现两种转录本在敏感组和耐药组之间均差异表达，并且对治疗敏感性具有反向调控作用。本课题拟从分子和细胞水平研究ANGPTL3可变剪接调控肾癌TKI靶向药物耐药的机制；从荷瘤模型和临床样本中探讨ANGPTL3可变剪接体作为诊疗靶标的可行性，为预测疗效、逆转耐药提供新的理论依据。

肾癌是人类常见的恶性肿瘤，近年来发病率逐年上升。目前肾癌靶向治疗的难点在于耐药机制不明，缺乏有效的生物学标志物来预测索拉非尼的疗效，在出现耐药后没有很好的方法逆转耐药。血管生成素类似物（ANGPTL）在肾癌靶向耐药过程中所起到的作用以及机制尚未见报道。本研究拟探讨血管生成素类似物在肾癌索拉非尼耐药过程中所起到的作用，寻找相关的分子标志物以及逆转耐药的联合治疗靶点，为提高肾癌靶向治疗的效果、进一步延长患者的总生存期提供依据。在对索拉非尼耐药的组织和细胞系中ANGPTL3低表达，而在对索拉非尼敏感的组织和细胞系中ANGPTL3高表达，蛋白水平的验证同样证实ANGPTL3表达量和肾癌索拉非尼敏感性相关。ANGPTL3的表达量和预后相关：ANGPTL3高表达组中，服用索拉非尼的肾癌病人预后要好于未服用索拉非尼的肾癌病人；但是ANGPTL3低表达组中服用索拉非尼无效。ANGPTL3的FBG-like结构域和Focal adhesion kinase （FAK）相互结合。过表达FAK可以促进肾癌细胞对索拉非尼的耐药，而过表达ANGPTL3可以逆转此效应。敲低ANGPTL3可以提高肾癌细胞对索拉非尼的耐药性，同时敲低FAK可以抵消此效应，而使用FAK的激酶抑制剂PF-562271则并不能逆转ANGPTL3敲低所导致的耐药性提高。进一步研究发现索拉非尼可以抑制FAK的磷酸化，并且促进FAK的入核，进而导致了p53的降解；而过表达ANGPTL3可以抑制FAK的入核，从而抑制p53的泛素化降解，提高p53的表达量。ANGPTL3可能作为预测肾癌索拉非尼敏感性的生物学标志物，其下游FAK的入核以及p53的泛素化降解也有望成为肾癌靶向治疗的新靶点，有着较好的临床转化前景。