马兜铃酸致尿路上皮癌多中心发病的作用和机制研究

Multifocality is one of the most important clinical features of urothelial cell carcinoma, and clarifying the mechanism is of great significance for the disease prevention and treatment, so that it has always been a hot research topic in this field. In our previous study, through exome sequencing, we deciphered the clonal relationship and mutational signature of multifocal urothelial cell carcinoma from several patients. The results indicated that the pathogenesis of multifocal urothelial cell carcinoma in China is unique. Both the "monoclonal origin" pattern and "field effect" pattern existed, and the detection rate of aristolochic acid mutational signature in multifocal urothelial cell carcinoma lesions is extremely high, which is obviously different from the findings of other countries. And in aristolochic acid associated urothelial cell carcinoma patients, a large number of gene mutations could also be detected in the normal urothelium. Therefore, the scientific hypothesis of this project is as follows: aristolochic acid is one of the risk factors of multifocal urothelial cell carcinoma in China. Aristolochic acid can induce the onset of multifocal urothelial cell carcinoma via “field effect” pattern, and it can also induce the onset of multifocal urothelial cell carcinoma by facilitating the seeding and intraepithelial migration of cancer cells ("monoclonal origin"). In this study we intend to further depict the mutational signature of multifocal urothelial cell carcinoma in our country, and to conduct in vitro and in vivo experiments elucidating the role and mechanism of aristolochic acid in development of multifocal urothelial cell carcinoma.

多中心发病是尿路上皮癌（UCC）的重要特点之一，阐明其发生机制对疾病防治工作具有重要指导意义，一直都是该领域的研究热点。本课题组前期纳入多中心UCC患者对其癌灶及正常尿路上皮组织进行外显子测序分析，结果提示我国多中心UCC的发病机制具有独特性：“单克隆起源”模式与“区域病变起源”模式并存，病灶中马兜铃酸（AA）特征性突变印记检出率极高，且在AA相关UCC患者形态正常的尿路上皮中也可检出大量基因突变。故本课题的科学假说为：AA是我国多中心UCC的发病危险因素之一，AA可通过诱导尿路上皮发生“区域病变”致UCC多中心发病，亦可通过增强细胞迁移、增殖能力，降低细胞间黏附进而通过腔内种植或上皮内迁移机制（“单克隆起源”）促进UCC多中心发病。本研究拟进一步描绘我国多中心UCC独特的基因突变印记特征，并联合体内外实验以期初步阐明AA在我国多中心UCC发病中所发挥的作用及作用机制。

多中心发病尿路上皮癌的重要特点之一，给患者和医生造成了严重的困扰，阐明其发生机制对疾病防治工作具有重要指导意义，一直以来都是该领域的研究热点。但其发病机制较为复杂，既往主要存在两种假说，即“区域病变学说”和“单克隆起源学说”。本课题组前期纳入数例多中心尿路上皮癌患者，对其癌灶及正常尿路上皮组织进行外显子测序分析，初步结果提示我国多中心尿路上皮癌的发病机制具有独特性：“单克隆起源”模式与“区域病变起源”模式并存；且病灶中马兜铃酸特征性印记检出率极高，在其中一例马兜铃酸相关尿路上皮癌患者形态正常的尿路上皮中也可检出大量基因突变。基于此，本课题拟进一步描绘我国多中心尿路上皮癌的基因突变印记特征，寻找可能的致病因素，并初步阐明马兜铃酸在我国多中心尿路上皮癌发生过程中所发挥的作用及其作用机制。.本项目研究过程中，我们对120例多中心尿路上皮癌患者的多中心癌灶及正常尿路上皮组织进行全外显子测序分析，对其中88例尿路上皮癌患者的癌灶进行了转录组学分析，证实了我国尿路上皮癌多中心发病机制中，“单克隆起源”机制和“区域病变”起源机制共存；突变特征检出以APOBEC及马兜铃酸相关突变特征为主，约1/3的患者可检出马兜铃酸突变特征；呈现马兜铃酸突变特征的患者中，尿路上皮更易形成“区域病变”，正常尿路上皮中可见大量突变检出，且呈现更为显著的克隆扩增现象；呈现马兜铃酸突变特征的患者癌灶中FGFR3的表达水平较高，DNA错配修复通路存在缺陷的比例较高，且更易呈现免疫耗竭状态。进一步我们进行了细胞水平的验证，结果提示马兜铃酸可诱导尿路上皮细胞高表达FGFR3，同时抑制DNA错配修复通路基因及免疫活性基因的表达。对于该结论的进一步证实可为今后精准诊疗方案的制定提供依据。