多环芳烃诱导BRCC3/PRKDC复合体去泛素化修饰c-Myc在宣威肺癌增殖中的作用及机制研究

The incidence and mortality of Xuanwei lung cancer are the highest in the world. Polycyclic aromatic hydrocarbons (PAH) from indoor air are the primary cause of Xuanwei lung cancer, but the target of PAH and the molecular mechanisms by which PAH inducing Xuanwei lung cancer have not been elucidated. To solve this difficult problem, we used mRNA microarray and obtained the Xuanwei-specific gene BRCC3. The preliminary study found that, as a deubiquitinating enzyme, BRCC3 was highly expressed in Xuanwei lung cancer and suppressed its expression in lung cancer cells inhibited the expression of c-Myc to decrease the cell proliferation, hinting that BRCC3 may decrease the ubiquitination of c-Myc to promote cell proliferation in lung cancer of Xuanwei area. Interestingly, the BRCC3 is switched to an active conformation by contacts with other proteins. Then, we performed a Co-IP combined with mass spectrometry and found that BRCC3 was contacted with PRKDC to regulate the ubiquitination level of c-Myc. To further analyze the influence of PAH to BRCC3/PRKDC, we treated the lung caner cells with Benzo(a)pyrene (BaP) and found that BaP treatment increased the BRCC3 and PRKDC expression, decreased the P53 level. Further bioinformatics analysis predicted that the human BRCC3/PRKDC promoter region contain the P53-binding sites, and the P53 promoter region contains the AhR/XRE-binding sites. Taken together, we hypothesize the new mechanism by which PAH inducing Xuanwei lung cancer, PAH mediated AhR to inhibit P53 which increases BRCC3/PRKDC levels to decrease the ubiquitination level of c-Myc, promoting the cell proliferation of lung cancer cells. This project intends to obtain reliable evidences for BaP-mediated BRCC3/PRKDC promote cell proliferation of lung cancer, and to provide scientific evidence for the prevention and treatment of Xuanwei lung cancer.

云南宣威肺癌全球高发。多环芳烃污染是导致宣威肺癌高发的主要原因，然而其作用靶标及发病机制远未阐明。我们利用基因芯片，从宣威肺癌组织中筛选出差异基因BRCC3。初步研究发现，去泛素化酶BRCC3在宣威肺癌高表达，与PRKDC互作可能去泛素化修饰c-Myc，促进肺癌细胞增殖。进一步研究显示，苯并芘（BaP）可上调BRCC3、PRKDC，抑制P53。生物信息学分析提示：BRCC3/PRKDC启动子区含有P53结合基序，而P53启动子区含有多环芳烃受体（AhR）结合域，提示AhR可能直接靶向P53，P53直接靶向BRCC3/PRKDC。由此，我们提出多环芳烃致宣威肺癌增殖可能的新机制：多环芳烃通过AhR抑制P53，致BRCC3/PRKDC去泛素化修饰c-Myc，促进肺癌细胞增殖。本项目旨在获得BRCC3/PRKDC参与BaP诱导肺癌增殖的可靠证据，为多环芳烃诱发宣威肺癌的发病机制提供科学依据。

肺癌是全球第一高发肿瘤，在我国，宣威肺癌发病率及死亡率居高不下，使其成为我国乃至全世界的肺癌高发研究现场。病因学研究证实：室内空气多环芳烃（PAH）污染是导致宣威肺癌高发的主要病因。此外，宣威肺癌可能具有特殊的内在驱动基因，环境致癌因素一旦激活这类基因，其将在体内长期持续作用，诱发肿瘤。我们采用mRNA芯片从宣威肺癌和非宣威肺癌组织标本中筛选出差异表达基因BRCC3，进一步采用免疫组化检测发现，BRCC3在宣威肺癌的表达较非宣威肺癌表达更高，与T分期和临床分期显著相关，提示BRCC3可能是宣威肺癌特异性驱动基因。功能学研究显示：BRCC3可促进A549细胞和宣威肺癌细胞系XLA-07细胞的细胞周期进展、细胞增殖能力和集落形成能力。机制研究发现，敲减BRCC3后，A549和XLA-07细胞中c-Myc、Cyclin D1、CDK4表达降低，提示BRCC3可能通过c-Myc介导Cyclin D1/CDK4诱导肺癌细胞增殖。值得注意的是，BRCC3作为去泛素化酶，能够促进泛素链从底物解聚，挽救蛋白的降解命运；而c-Myc在肿瘤中主要通过泛素-蛋白酶体途径降解。由此，抑制BRCC3可能干预c-Myc的泛素化降解途径。利用Co-IP结合质谱分析发现，BRCC3与PRKDC存在相互作用关系，BRCC3与PRKDC相互作用形成复合体，去泛素化调控c-Myc，进而调控肺癌细胞增殖。进一步研究显示，苯并芘（BaP）使A549和XLA-07细胞中BRCC3、PRKDC、c-Myc和CyclinD1表达增加，P53表达降低，细胞增殖加快。生物信息学和ChIP分析发现：芳香烃受体（AhR）可直接靶向P53启动子，抑制P53转录，进而靶向促进BRCC3/PRKDC。由此，我们提出PAH促进肺癌细胞增殖可能存在的机制：PAH进入细胞后，在胞质与AhR结合，靶向抑制P53的转录，进而促进BRCC3/PRKDC复合体表达，使其去泛素化修饰c-Myc，促进肺癌细胞增殖。该研究从多环芳烃类环境致癌物促进肿瘤细胞增殖这一研究视野，在更深层次上探讨多环芳烃类致癌物通过AhR通路诱导BRCC3/PRKDC介导c-Myc泛素化途径异常，进而促进宣威肺癌增殖的作用机制，为环境致癌物诱导肿瘤发生发展及宣威肺癌高发的分子机制研究提供更充分的实验依据。