内质网蛋白RCN1通过调节选择性剪接促进肿瘤多药耐药的机制研究

Identification of novel therapeutic targets via uncovering the drug resistance mechanism is an effective strategy to overcome drug resistance. Recently, analysis of data from high-throughput deep sequencing reveals that alternative splicing (AS) occurs in almost all genes. Therefore, abnormal regulation of alternative splicing becomes a new mechanism driving drug resistance and a potential target as well. Reticulocalbin-1（RCN1）is selected from proteomics data of drug resistance cells in our early stage of study. It is found that its overexpression facilitates DNA repair and multidrug resistance. Besides, RCN1 affects alternative splicing, RNA processing and other signals obviously based on IP/MS group analysis. And it is confirmed that RCN1 can be located at nucleus and accelerate splicing reaction, regulate expression and nuclear import of splicing-related factors, such as hnRNPA1, DHX9, and affect expression of DNA repair proteins including BRCA1, RAD51, which shows novelty of RCN1 induced drug resistance. Based on foundations of early study and cutting edge research, our project is supposed to discuss: 1) Key splicing molecule in RCN1 regulation and its complex, and determination of splicing regulation mechanism. 2) The network of RCN1 regulating downstream key molecules. It’s mainly focused on splicing mode and products of repairing-related protein pre-mRNA, clarifying its mechanism in drug resistance. 3) With conditional knockout mice and clinical specimens, it is supposed to illuminate biological function of RCN1 and its clinical significance of inducing drug resistance, in order to provide new proof and thinking for reversing drug resistance.

探索耐药新机制、发现新靶标是克服肿瘤耐药的有效策略。高通量深度测序数据揭示95％以上的基因都发生选择性剪接（AS）、产生剪接变异体，因此AS的异常调节是驱动耐药的新机制。我们通过耐药细胞蛋白质组学筛选出内质网蛋白Reticulocalbin1（RCN1），其高表达促进DNA损伤修复、对多种药物产生耐受；通过IP/MS组学分析发现RCN1显著影响AS等信号，并证实RCN1可定位于细胞核，促进剪接反应的发生，调节剪接因子如hnRNP-A1等表达及入核，进而影响修复蛋白BRCA1的表达，表明RCN1通过调节AS而介导耐药。本项目拟探讨：RCN1调控的关键剪接因子及形成的复合物如何影响剪接反应，明确其调节AS的机制；RCN1调控的下游关键分子网路，重点分析修复相关蛋白转录本的成熟方式及剪接产物，明确其介导耐药的机制；利用特异性条件敲除鼠和临床样本，明确RCN1的生物学功能及介导耐药的临床意义。

多药耐药是肿瘤治疗失败的主因之一，基于耐药机制的研究是发现逆转耐药方案及药靶的有效策略。本项目根据前列腺癌多药耐药细胞的差异蛋白质组学、RNA-seq以及蛋白互作组学数据，分析内质网钙离子结合蛋白RCN1介导肿瘤耐药的作用机制，发现RCN1主要通过外显子跳跃的方式调节可变剪接，促进如TOP2A、RAD51等DNA损伤修复相关基因的成熟剪接而介导耐药。下调RCN1后显著诱导DNA损伤、促进肿瘤细胞、成纤维细胞呈现衰老样表型，衰老相关的炎性分泌显著增加，并在肺上皮特异敲除小鼠的肺组织得到验证。利用常用药物/抑制剂、天然产物等筛选能调节RCN1、逆转耐药的小分子化合物，发现大多数药物/抑制剂等都不同程度增加RCN1表达，而HDACi可通过抑制转录因子NF-κB(p65)和TFAP4下调RCN1的表达，下调RCN1或联合HDACi可显著增加耐药小鼠对DNA损伤药物的敏感性。根据RCN1敲除鼠的表型及组织细胞分布，发现其通过炎性机制加剧帕金森的运动障碍。另外，对筛选的其他具有逆转耐药活性的化合物进行机制及药效毒性研究，发现地钱素、RDN等潜力化合物，并通过化学合成标签衍生物/质谱筛选及结构、功能分析，确定RDN可直接结合于靶点VPS18的末端RING结构域，并明确了新靶点VPS18促肿瘤及耐药的功能。由于RCN1的表达与内质网蛋白AGR2具有相关性，研究发现AGR2可作为抗血管新生靶向药的用药标志物，并根据泛素化AGR2通过自噬受体NBR1介导的溶酶体途径降解的机制，采用蛋白酶体抑制剂联用血管靶向药可逆转其介导的耐药。同时AGR2基因敲除/转基因鼠表现出显著的代谢异常、影响降脂药的药效，提出分泌AGR2高表达时采用联合用药方案来提高降脂药的药效。综上，本项目在内质网蛋白RCN1和AGR2介导耐药的机制、逆转耐药的药物选择、联合用药方案以及用药标志物方面取得良好的成果，发表1区文章4篇，2区文章2篇，在投文章2篇；获得省科技进步二等奖2项（分别为第1位和第2位），申请专利2项。