GNAI1 and GNAI3 Reduce Colitis-Associated Tumorigenesis in Mice by Blocking IL6 Signaling and Down-regulating Expression of GNAI2
GNAI1 and GNAI3 Reduce Colitis-Associated Tumorigenesis in Mice by Blocking IL6 Signaling and Down-regulating Expression of GNAI2
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GNAI1 和 GNAI3 通过阻断 IL6 信号传导和下调 GNAI2 的表达来减少小鼠结肠炎相关的肿瘤发生
DOI：
10.1053/j.gastro.2019.02.040
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发表时间：
2019-06-01
期刊：
影响因子：
29.4
通讯作者：
Chu，Wen-Ming
中科院分区：
文献类型：
--
作者：
Li，Zhi-Wei;Sun，Beicheng;Chu，Wen-Ming
背景与目标白细胞介素 6 (IL6) 和肿瘤坏死因子有助于结肠炎相关癌症 (CAC) 的发展。我们研究了这些信号通路以及 G 蛋白亚基 α i1 (GNAI1)、GNAI2 和 GNAI3 在小鼠和人类 CAC 发展中的参与。方法B6;129 野生型（对照）或 Gnai1、Gnai2 和/或 Gnai3 破坏或 CD11c+ 或上皮细胞中 Gnai2 条件性破坏的小鼠 细胞给予葡聚糖硫酸钠（DSS）诱导结肠炎，然后给予氧化偶氮甲烷（AOM）诱导癌变；一些小鼠被注射了抗 IL6 抗体。从小鼠身上收集粪便，并通过聚合酶链反应分析微生物组的组成。通过流式细胞术分析从脾脏和结肠组织中分离的树突状细胞（DC）和骨髓源性抑制细胞（MDSC）。我们对结肠肿瘤组织、MDSC 和小鼠胚胎成纤维细胞进行了免疫沉淀和免疫印迹分析，以研究 GNAI1、GNAI2 和 GNAI3 的表达水平以及 GNAI1 和 GNAI3 与 IL6 信号通路中蛋白质的相互作用。我们通过 PrimeFlow 分析了结肠固有层中 CD11c+ 细胞 Gnai2 信使 RNA 的表达，通过流式细胞术分析了 DC 中 IL6 的表达，并通过酶联免疫吸附测定分析了血清和结肠组织中细胞因子的分泌。我们从 83 名在中国接受手术的结直肠癌患者和 35 名在美国接受手术的 CAC 患者获得了结肠肿瘤和匹配的非肿瘤组织。通过组织学、免疫印迹、免疫组织化学和/或 RNA 测序分析对小鼠和人类结肠组织进行分析。结果 GNAI1 和 GNAI3 (GNAI1;3) 双敲除 (DKO) 小鼠在给予 DSS 后出现更严重的结肠炎，并且在给予 AOM 加 DSS 后比对照小鼠出现更严重的结肠肿瘤。 DKO 小鼠肿瘤增加与粪便微生物组的变化无关，但与核因子 (NF) κB 和信号转导器和转录激活子 (STAT) 3 的激活有关； GNAI2、一氧化氮合酶 2 和 IL6 水平升高； CD4+DC 和 MDSC 数量增加； CD8+DC 数量减少。 DKO 小鼠中 IL6 主要由 CD4+/CD11b+ 产生，但 CD8+ DC 不产生。给 DKO 小鼠注射 IL6 阻断抗体可减少 MDSC 的扩增以及 CAC 诱导后形成的肿瘤数量。 MDSC 或小鼠胚胎成纤维细胞与 IL6 一起孵育可诱导 JAK2-TRAF6-TAK1-CHUK/IKKB 信号通路激活 NF-κB 或 JAK2 激活 STAT3。这种激活导致 GNAI2、IL6 信号转导器（IL6ST，也称为 GP130）和一氧化氮合酶 2 的表达，以及 MDSC 的扩增；由于细胞和小鼠中缺乏 GNAI1;3，这些蛋白的表达水平和 MDSC 的扩增进一步增加。 DKO 小鼠 CD11c+ 细胞中 Gnai2 的条件性破坏可阻止 NF-κB 和 STAT3 的激活以及 DC 和 MDSC 数量的变化。 与正常组织相比，CAC 患者的结肠肿瘤组织中 GNAI1 和 GNAI3 水平降低，GNAI2 水平升高。对公共人类结直肠肿瘤 DNA 微阵列数据库 (GSE39582) 的进一步分析表明，低 Gani1 和 Gnai3 信使 RNA 表达和高 Gnai2 信使 RNA 表达与无复发生存率降低显着相关。结论 GNAI1;3 抑制 DSS 加 AOM 诱导的小鼠结肠肿瘤发展，而 GNAI2 的表达在 CD11c+ 细胞和 IL6 ...
Background & AimsInterleukin 6 (IL6) and tumor necrosis factor contribute to the development of colitis-associated cancer (CAC). We investigated these signaling pathways and the involvement of G protein subunit alpha i1 (GNAI1), GNAI2, and GNAI3 in the development of CAC in mice and humans.MethodsB6;129 wild-type (control) or mice with disruption ofGnai1,Gnai2, and/orGnai3or conditional disruption ofGnai2in CD11c+or epithelial cells were given dextran sulfate sodium (DSS) to induce colitis followed by azoxymethane (AOM) to induce carcinogenesis; some mice were given an antibody against IL6. Feces were collected from mice, and the compositions of microbiomes were analyzed by polymerase chain reactions. Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) isolated from spleen and colon tissues were analyzed by flow cytometry. We performed immunoprecipitation and immunoblot analyses of colon tumor tissues, MDSCs, and mouse embryonic fibroblasts to study the expression levels of GNAI1, GNAI2, and GNAI3 and the interactions of GNAI1 and GNAI3 with proteins in the IL6 signaling pathway. We analyzed the expression ofGnai2messenger RNA by CD11c+cells in the colonic lamina propria by PrimeFlow, expression of IL6 in DCs by flow cytometry, and secretion of cytokines in sera and colon tissues by enzyme-linked immunosorbent assay. We obtained colon tumor and matched nontumor tissues from 83 patients with colorectal cancer having surgery in China and 35 patients with CAC in the United States. Mouse and human colon tissues were analyzed by histology, immunoblot, immunohistochemistry, and/or RNA-sequencing analyses.ResultsGNAI1 and GNAI3 (GNAI1;3) double-knockout (DKO) mice developed more severe colitis after administration of DSS and significantly more colonic tumors than control mice after administration of AOM plus DSS. Development of increased tumors in DKO mice was not associated with changes in fecal microbiomes but was associated with activation of nuclear factor (NF) κB and signal transducer and activator of transcription (STAT) 3; increased levels of GNAI2, nitric oxide synthase 2, and IL6; increased numbers of CD4+DCs and MDSCs; and decreased numbers of CD8+DCs. IL6 was mainly produced by CD4+/CD11b+, but not CD8+, DCs in DKO mice. Injection of DKO mice with a blocking antibody against IL6 reduced the expansion of MDSCs and the number of tumors that developed after CAC induction. Incubation of MDSCs or mouse embryonic fibroblasts with IL6 induced activation of either NF-κB by a JAK2-TRAF6-TAK1-CHUK/IKKB signaling pathway or STAT3 by JAK2. This activation resulted in expression of GNAI2, IL6 signal transducer (IL6ST, also called GP130) and nitric oxide synthase 2, and expansion of MDSCs; the expression levels of these proteins and expansion of MDSCs were further increased by the absence of GNAI1;3 in cells and mice. Conditional disruption ofGnai2in CD11c+cells of DKO mice prevented activation of NF-κB and STAT3 and changes in numbers of DCs and MDSCs. Colon tumor tissues from patients with CAC had reduced levels of GNAI1 and GNAI3 and increased levels of GNAI2 compared with normal tissues. Further analysis of a public human colorectal tumor DNA microarray database (GSE39582) showed that lowGani1andGnai3messenger RNA expression and highGnai2messenger RNA expression were significantly associated with decreased relapse-free survival.ConclusionsGNAI1;3 suppresses DSS-plus-AOM–induced colon tumor development in mice, whereas expression of GNAI2 in CD11c+cells and IL6 in …