Lo Yuan-Hung, Noah Taeko K, Chen Min-Shan, Zou Winnie, Borras Ester, Vilar Eduardo, Shroyer Noah F
Integrative Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, Houston, Texas; Department of Medicine and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas.
Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
Gastroenterology. 2017 Jul;153(1):205-218.e8. doi: 10.1053/j.gastro.2017.03.048. Epub 2017 Apr 5.
BACKGROUND & AIMS: The canonical Wnt signaling pathway activates the transcriptional activity of β-catenin. This pathway is often activated in colorectal cancer cells, but strategies to block it in tumors have not been effective. The SAM pointed domain containing ETS transcription factor (SPDEF) suppresses formation of colon tumors by unclear mechanisms. We investigated these mechanisms and the effects of SPDEF on β-catenin activity in mouse models of colorectal cancer (CRC), CRC cell lines, and mouse and human normal and cancer colonoids.
We performed studies of Lgr5; β-catenin; Rosa26; TRE-Spdef mice, which express an oncogenic form of β-catenin in Lgr5-positive ISCs upon administration of tamoxifen and SPDEF upon administration of tetracycline. CRC lines (HCT116 and SW480) were engineered to express inducible tagged SPDEF or vector (control) and subcutaneously injected into immunodeficient NSG mice. We generated SPDEF-inducible human colonoids, including a line derived from normal rectal mucosa (control) and an adenocarcinoma line derived from a patient with germline MUTYH mutation. Full-length and truncated forms of SPDEF were expressed in CRC cells; cells were assayed for β-catenin activity and studied in immunoprecipitation and chromatin immunoprecipitation assays.
Expression of SPDEF was sufficient to inhibit intestinal tumorigenesis by activated β-catenin, block tumor cell proliferation, and restrict growth of established tumors. In tumor cells with activated β -catenin, expression of SPDEF induced a quiescent state, which was reversed when SPDEF expression was stopped. In mouse and human normal and tumor-derived enteroids/colonoids, those that expressed SPDEF for 3 days were significantly smaller. SPDEF inhibited the transcriptional activity of β-catenin via a protein-protein interaction, independent of SPDEF DNA binding capacity. SPDEF disrupted β-catenin binding to TCF1 and TCF3, displacing β-catenin from enhancer regions of genes that regulate the cell cycle but not genes that regulate stem cell activities.
In studies of mice and human CRC, we found that SPDEF induces a quiescent state in CRC cells by disrupting binding of β-catenin to TCF1 and TCF3 and regulation of genes that control the cell cycle. In this model, β-catenin activity determines the proliferation or quiescence of CRC cells based on the absence or presence of SPDEF.
经典Wnt信号通路可激活β-连环蛋白的转录活性。该通路在结直肠癌细胞中常被激活，但在肿瘤中阻断它的策略尚未取得有效成果。含ETS转录因子的SAM结构域（SPDEF）通过不明机制抑制结肠肿瘤的形成。我们在结直肠癌（CRC）小鼠模型、CRC细胞系以及小鼠和人类正常及癌性结肠类器官中研究了这些机制以及SPDEF对β-连环蛋白活性的影响。
我们对Lgr5;β-连环蛋白;Rosa26;TRE-Spdef小鼠进行了研究，该小鼠在给予他莫昔芬后在Lgr5阳性肠干细胞中表达致癌形式的β-连环蛋白，在给予四环素后表达SPDEF。对CRC细胞系（HCT116和SW480）进行改造，使其表达可诱导的带标签SPDEF或载体（对照），并皮下注射到免疫缺陷的NSG小鼠体内。我们构建了SPDEF可诱导的人类结肠类器官，包括一个源自正常直肠黏膜的细胞系（对照）和一个源自种系MUTYH突变患者的腺癌细胞系。在CRC细胞中表达全长和截短形式的SPDEF；检测细胞的β-连环蛋白活性，并进行免疫沉淀和染色质免疫沉淀分析。
SPDEF的表达足以抑制由激活的β-连环蛋白引发的肠道肿瘤发生，阻断肿瘤细胞增殖，并限制已形成肿瘤的生长。在β-连环蛋白激活的肿瘤细胞中，SPDEF的表达诱导了静止状态，当SPDEF表达停止时这种状态会逆转。在小鼠和人类正常及肿瘤来源的类肠/结肠类器官中，那些表达SPDEF 3天的明显更小。SPDEF通过蛋白质-蛋白质相互作用抑制β-连环蛋白的转录活性，与SPDEF的DNA结合能力无关。SPDEF破坏了β-连环蛋白与TCF1和TCF3的结合，将β-连环蛋白从调节细胞周期的基因增强子区域置换出来，但不影响调节干细胞活性的基因。
在对小鼠和人类CRC的研究中，我们发现SPDEF通过破坏β-连环蛋白与TCF1和TCF3的结合以及对控制细胞周期的基因的调控，在CRC细胞中诱导静止状态。在这个模型中，β-连环蛋白的活性根据SPDEF的缺失或存在决定CRC细胞的增殖或静止。
