CXCR7/CXCR4异二聚体(CXCR4/CXCR7 heterodimer)通过信号转导偏移促进组蛋白去甲基化：结直肠癌发病新机制研究及防治策略探索

We previously found that CXCR/CXCR4 heterodimerization was associated with increased histone demethylase JMJD2A in human colorectal rectal cancer tissues. In order to investigate the mechanism of colorectal tumorigenesis, we generated mice models with high expression of CXCR4/CXCR7 heterodimer. CXCR4/CXCR7 heterodimer was determined a major stimulator of the pathogenesis of colitis and colorectal tumorigenesis. we hypothesize that CXCR7 as an atypical G protein-coupled receptor (GPCR) might lead to CXCR4/CXCR7 heterodimer to bind with β-arrestin through mechanism of signal bias and then induce histone demethylation. We aim to investigate the mechanism of signal bias for CXCR4/CXCR7 heterodimer in the pathogenesis of colitis and colorectal tumorigenesis. Fluorescence resonance energy transfer (FRET) assay will be used to analyze signal bias of CXCR4/CXCR7 heterodimer in inducing nuclear β-arrestin. Ch-IP kits will be used to determine histone demethylase activity. Chromatin immunoprecipitation (Ch-IP) assay with antibodies to demethylase will be performed to determine the transcription of downstream genes. ChIP, PCR and Western blot assays will be used to confirm the levels of the transcriptions and promoters bound to the histones in colorectal cancer tissues. We try to design CXCR4/CXCR7 heterodimer inhibitors for investigating new methods of CRC therapies through inhibition of CXCR7/CXCR4 heterodimer-induced histone demethylation.

在调研结肠癌细胞形成CXCR7/CXCR4异二聚体（heterodimer）与组蛋白去甲基化酶JMJD2A表达升高密切相关基础上，建立villin-CXCR4-CXCR7小鼠，发现CXCR4/CXCR7 heterodimer促进炎癌转化。假设CXCR7通过信号偏移促使CXCR4/CXCR7 heterodimer直接与β-arrestin1结合并促进入核，核内β-arrestin1促使JMJD2A催化组蛋白H3K9me3和H3K36me3去甲基化。拟以荧光共振能量转移（FRET）技术分析CXCR4/CXCR7 heterodimer与β-arrestin1直接结合，分析信号转导偏移；以ChIP、PCR及Western blot分析关联基因转录；以甲基化芯片分析参入应答信号偏移所致组蛋白去甲基化位点；设计靶向CXCR4/CXCR7 heterodimer抑制剂，探索结直肠癌治疗新策略。

激活趋化因子受体CXCR4和CXCR7（ACKR3）能够促进免疫炎症相关细胞迁移，参与慢性炎症发生及其炎癌转化病理学过程。本项目揭示了CXCR/CXCR4异二聚体的形成机制及促进结肠癌发生新机制。临床癌组织标本研究显示，与癌旁组织比较，人结肠癌标本CXCR4和CXCR7表达水平明显增加，激活的CXCR4和CXCR7共定位于癌细胞，形成异二聚体，而CXCR7/CXCR4异二聚体与组蛋白去甲基酶JMJD2A表达水平成正相关性。转基因动物实验显示，与Villin-CXCR7和Villin-CXCR4小鼠模型比较，形成异二聚体的Villin-CXCR7-CXCR4小鼠极易通过促进炎癌转化进程而形成结肠癌。进一步分析表明，CXCR7/CXCR4异源二聚体能够通过募集和诱导β-arrestin 1入核方式而提高核内JMJD2A表达水平，进而导致组蛋白H3K9me3和H3K36me3去甲基化，而这些由于CXCR7/CXCR4异二聚体激活所致的改变导致炎症因子和癌基因转录增加，促进肿瘤发生发展。本项目临床意义在于，抑制CXCR7/CXCR4异源二聚体形成或者阻止其诱导组蛋白去甲基化可能发展成为结直肠癌防治新策略。另外，我们还深入研究了CRCR7激活诱导结直肠癌发生新机制，通过临床标本和动物模型发现CRCR7激活通过活化NOLC1与Fibrillarin互作而诱导核糖体rRNA转录，促进结直肠癌恶性发展。机制研究发现，激活ACKR3诱导β-arretin-1 (β-arr1)核转位，导致β-arr1与核仁蛋白1 (NOLC1)互作，促进磷酸化形式NOLC1上调，而磷酸化NOLC1进一步激活Fibrillarin，导致组蛋白H2A甲基化，促进核糖体rRNA转录。