OVOL2, an Inhibitor of WNT Signaling, Reduces Invasive Activities of Human and Mouse Cancer Cells and Is Down-regulated in Human Colorectal Tumors
OVOL2, an Inhibitor of WNT Signaling, Reduces Invasive Activities of Human and Mouse Cancer Cells and Is Down-regulated in Human Colorectal Tumors
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OVOL2 是一种 WNT 信号传导抑制剂，可减少人类和小鼠癌细胞的侵袭活性，并在人类结直肠肿瘤中下调
DOI：
10.1053/j.gastro.2015.11.041
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发表时间：
2016-03-01
期刊：
影响因子：
29.4
通讯作者：
Li, Bo-An
中科院分区：
文献类型：
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作者：
Ye, Guo-Dong;Sun, Guang-Bin;Li, Bo-An
背景与目的：WNT信号通路的激活促进了多种肿瘤细胞的侵袭活动，但目前尚不清楚WNT信号通路是否调节结直肠癌细胞的侵袭过程，以及参与的机制是什么。我们研究了WNT信号通路调控的锌指转录因子Ovo家族成员Ovol2在小鼠和人类肠道肿瘤中的表达和功能。方法：收集厦门大学中国医院2009-2012年间手术切除的结直肠癌标本，分析Ovol2蛋白和信使核糖核酸在结直肠癌细胞系和组织阵列中的表达，并收集临床资料。将表达Ovol2的慢病毒感染CRC细胞系(SW620)，进行迁移和侵袭实验，并注射到裸鼠体内以观察肿瘤的生长和转移情况。采用串联亲和纯化的方法从结肠癌细胞中分离纯化Ovol2复合物，并对其进行了液相色谱、串联质谱分析和免疫沉淀实验。用荧光素酶报告试剂盒检测基因启动子活性。我们分析了Ovol2(Ovol2(Flox/+)转基因小鼠)和APC(Min/+)小鼠的肠道特异性干扰小鼠；对这些小鼠进行了杂交和分析。结果：结肠癌组织中Ovol2信使RNA的表达水平明显低于腺瘤，且随肿瘤分级的增加而降低。免疫组织化学分析显示275例结直肠癌组织中Ovol2表达水平与肿瘤分期呈负相关。Ovol2在SW620细胞中的过表达减少了细胞的迁移和侵袭，减少了上皮细胞向间充质细胞转化的标志物，并抑制了其作为裸鼠移植瘤的转移；Ovol2基因的敲除导致LS174T细胞从上皮表型向间充质表型转化。Ovol2结合T细胞因子(TCF)4和β-连环蛋白，促进组蛋白脱乙酰基酶1重新聚集到TCF4-β-连环蛋白复合体中；这抑制了受WNT调控的上皮向间充质转化相关基因的表达，如Slug。Ovol2是LS174T和SW480细胞中WNT信号的下游靶点。在晚期结直肠癌患者标本和SW620细胞中，Ovol2启动子高甲基化；高甲基化导致Ovol2下调，并无法抑制WNT信号。阻断APC(min/+)小鼠的Ovol2可增加肠道组织中WNT的活性，并促进侵袭性肠道肿瘤的形成。结论：Ovol2是一种结直肠肿瘤抑制物，通过促进组蛋白脱乙酰酶1向TCF4-β-连环蛋白复合体的募集来阻断WNT信号。增加Ovol2水平的策略可能会被开发出来，以减少结直肠癌的进展和转移。
BACKGROUND & AIMS: Activation of WNT signaling promotes the invasive activities of several types of cancer cells, but it is not clear if it regulates the same processes in colorectal cancer (CRC) cells, or what mechanisms are involved. We studied the expression and function of OVOL2, a member of the Ovo family of conserved zinc-finger transcription factors regulated by the WNT signaling pathway, in intestinal tumors of mice and human beings. METHODS: We analyzed the expression of OVOL2 protein and messenger RNA in CRC cell lines and tissue arrays, as well as CRC samples from patients who underwent surgery at Xiamen University in China from 2009 to 2012; clinical information also was collected. CRC cell lines (SW620) were infected with lentivirus expressing OVOL2, analyzed in migration and invasion assays, and injected into nude mice to assess tumor growth and metastasis. Tandem affinity purification was used to purify the OVOL2-containing complex from CRC cells; the complex was analyzed by liquid chromatography, tandem mass spectrometry, and immunoprecipitation experiments. Gene promoter activities were measured in luciferase reporter assays. We analyzed mice with an intestine-specific disruption of Ovol2 (Ovol2(flox/+) transgenic mice), as well as Apc(min/+) mice; these mice were crossed and analyzed. RESULTS: Analysis of data from patients indicated that the levels of OVOL2 messenger RNA were significantly lower in colon carcinomas than adenomas, and decreased significantly as carcinomas progressed from grades 2 to 4. Immunohistochemical analysis of a tissue array of 275 CRC samples showed a negative association between tumor stage and OVOL2 level. Overexpression of OVOL2 in SW620 cells decreased their migration and invasion, reduced markers of the epithelial-to-mesenchymal transition, and suppressed their metastasis as xenograft tumors in nude mice; knockdown of OVOL2 caused LS174T cells to transition from epithelial to mesenchymal phenotypes. OVOL2 bound T-cell factor (TCF) 4 and beta-catenin, facilitating recruitment of histone deacetylase 1 to the TCF4-beta-catenin complex; this inhibited expression of epithelial-to-mesenchymal transition-related genes regulated by WNT, such as SLUG, in CRC cell lines. OVOL2 was a downstream target of WNT signaling in LS174T and SW480 cells. The OVOL2 promoter was hypermethylated in late-stage CRC specimens from patients and in SW620 cells; hypermethylation resulted in OVOL2 down-regulation and an inability to inhibit WNT signaling. Disruption of Ovol2 in Apc(min/+) mice increased WNT activity in intestinal tissues and the formation of invasive intestinal tumors. CONCLUSIONS: OVOL2 is a colorectal tumor suppressor that blocks WNT signaling by facilitating the recruitment of histone deacetylase 1 to the TCF4-beta-catenin complex. Strategies to increase levels of OVOL2 might be developed to reduce colorectal tumor progression and metastasis.