Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Second Department of Internal Medicine, Third Faculty of Medicine, Charles University, Prague, Czech Republic.
Gastroenterology. 2013 Feb;144(2):381-391. doi: 10.1053/j.gastro.2012.10.048. Epub 2012 Nov 7.
BACKGROUND & AIMS: The Wnt signaling pathway is required for maintenance of the intestinal epithelia; blocking this pathway reduces the proliferative capacity of the intestinal stem cells. However, aberrant Wnt signaling leads to intestinal cancer. We investigated the roles of the Wnt pathway in homeostasis of the intestinal epithelium and during malignant transformation in human cells and mice.
We performed chromatin immunoprecipitation (ChIP) with DNA microarray analysis (ChIP-on-chip) to identify genes regulated by Wnt signaling in human colorectal cancer cells Colo320, DLD1, LS174T, and SW480. Formation of intestinal tumor was induced in C57BL/6J mice using azoxymethane and dextran sulfate. Intestinal tissues from these mice, as well as Apc(+/Min) and Apc(CKO/CKO)/Lgr5-EGFP-IRES-CreERT2 mice, were analyzed by immunohistochemistry and in situ hybridization.
We identified promoter regions of 960 genes that interacted with the Wnt pathway nuclear effector T-cell factor 4 in 4 different human colorectal cancer-derived cell lines; 18 of these promoters were present in all chromatin precipitates. Wnt signaling up-regulated a member of the tumor necrosis factor receptor superfamily called TROY. Levels of TROY messenger RNA were increased in human cells with deficiencies in the adenomatous polyposis coli (APC) gene and in cells stimulated with the Wnt3a ligand. Expression of Troy was significantly up-regulated in neoplastic tissues from mice during intestinal tumorigenesis. Lineage tracing experiments revealed that Troy is produced specifically by fast-cycling intestinal stem cells. TROY associated with a unique marker of these cells, leucine-rich repeat-containing G-protein coupled receptor (LGR) 5. In organoids established from the intestinal crypts, Troy suppressed signaling mediated by R-spondin, a Wnt agonist.
TROY is up-regulated in human colorectal cancer cell lines and in intestinal tumors in mice. It functions as a negative modulator of the Wnt pathway in LGR5-positive stem cells.
Wnt 信号通路对于维持肠道上皮细胞至关重要；阻断该通路会降低肠道干细胞的增殖能力。然而，异常的 Wnt 信号会导致肠道癌症。我们研究了 Wnt 通路在人类细胞和小鼠中维持肠道上皮细胞稳态和恶性转化过程中的作用。
我们使用染色质免疫沉淀（ChIP）和 DNA 微阵列分析（ChIP-on-chip）来鉴定在人结直肠癌细胞 Colo320、DLD1、LS174T 和 SW480 中受 Wnt 信号调节的基因。使用氧化偶氮甲烷和葡聚糖硫酸钠在 C57BL/6J 小鼠中诱导肠肿瘤形成。通过免疫组织化学和原位杂交分析来自这些小鼠以及 Apc(+/Min) 和 Apc(CKO/CKO)/Lgr5-EGFP-IRES-CreERT2 小鼠的肠道组织。
我们鉴定了在 4 种不同的人结直肠癌细胞系中与 Wnt 通路核效应因子 T 细胞因子 4 相互作用的 960 个基因的启动子区域；在所有染色质沉淀中都存在其中 18 个启动子。Wnt 信号上调了肿瘤坏死因子受体超家族的一个成员，称为 TROY。在 APC 基因缺陷的人类细胞和受 Wnt3a 配体刺激的细胞中，TROY 的信使 RNA 水平增加。在肠道肿瘤发生期间，小鼠的肿瘤组织中 Troy 的表达显著上调。谱系追踪实验表明，Troy 是由快速循环的肠道干细胞特异性产生的。TROY 与这些细胞的独特标记物富含亮氨酸重复的 G 蛋白偶联受体 (LGR) 5 相关。在从肠隐窝建立的类器官中，Troy 抑制了 Wnt 激动剂 R-spondin 介导的信号。
TROY 在人结直肠癌细胞系和小鼠的肠道肿瘤中上调。它作为 LGR5 阳性干细胞中 Wnt 通路的负调节剂发挥作用。
