Wnt Signaling in IBD-Related Colon Cancer

描述（由申请人提供）： Wnt配体通过与细胞表面卷曲受体结合诱导信号级联反应。这导致细胞β-连环蛋白的增加，并最终诱导生长促进基因的转录。APC基因突变模拟的这一过程与散发性结肠癌发生过程密切相关。APC和β-连环蛋白的突变在炎症性肠病（IBD）相关肿瘤中似乎很少见。然而，已经描述了Wnt途径的其他组分的异常表达，包括卷曲受体、SARP 1和凌乱（dvl）。在这个提议中，Wnt信号转导途径的上游元件、卷曲受体和卷曲抑制分子的表达将在来自IBD患者的正常、发育异常和恶性结肠组织中定义。首先，我们将通过免疫组化检测IBD相关结肠癌组织中Fz受体的表达，并将其与邻近粘膜组织和散发性结肠癌进行比较。然后，将利用对10种Fz受体亚型中的每一种以及对特定卷曲抑制蛋白（FIP）具有特异性的反义RNA探针进行原位杂交来确定它们在这些组织中的表达。接下来，将前瞻性地从接受监测结肠镜检查的IBD患者中获得正常和异型增生粘膜的结肠活检。将评价这些组织的卷曲受体亚型和FIP的表达，以及Wnt信号传导的激活，通过β-连环蛋白的核化和下游途径组分LEF 1的表达和活性增加来指示。最后，特定Fz受体亚型在Wnt途径信号的起始和传播中的作用将利用新型的体外共培养表达系统来定义。将在该系统中评价在IBD组织中表现出差异表达的那些Fz受体亚型。单个Fz受体亚型的表达构建体将用于在结肠癌细胞系中进行更深入的分析。FIP对Wnt信号传导的精确作用将在体外定义，注意不同的特异性Fz受体亚型的潜在不同反应。

DESCRIPTION (provided by applicant): Wnt ligands induce a signaling cascade by binding to cell surface frizzled receptors. This leads to increases in cellular beta-catenin and, ultimately, the induction of transcription of growth promoting genes. This process, mimicked by mutations in the APC gene, is integrally associated with the process of sporadic colon carcinogenesis. Mutations in APC and beta-catenin appear to be rare in inflammatory bowel disease (IBD)-related tumors. However, abnormal expression of other components of the Wnt pathway, including frizzled receptors, SARP1 and disheveled (dvl) have been described. In this proposal, the expression of upstream elements of the Wnt signal transduction pathway, frizzled receptors and frizzled inhibitory molecules will be defined in normal, dysplastic and malignant colonic tissues from patients with IBD. First, we will examine the expression of Fz receptors by immunohistochemistry in IBD-associated colon cancer tissues and compare this expression to adjacent mucosal tissues and sporadic colon cancers. In situ hybridization with anti-sense RNA probes specific for each of the ten Fz receptor subtypes as well as for specific frizzled-inhibitory proteins (FIPs) will then be utilized to define their expression in these tissues. Next, colon biopsies of normal and dysplastic mucosa will be obtained prospectively from patients with IBD undergoing surveillance colonoscopy. These tissues will be evaluated for expression of frizzled receptor subtypes and FIPs and also for activation of Wnt signaling, indicated by nuclearization of beta-catenin and increased expression and activity of a downstream pathway component, LEF1. Finally, the role of specific Fz receptor subtypes in the initiation and propagation of Wnt pathway signals will be defined utilizing a novel, in vitro, co-culture expression system. Those Fz receptor subtypes which exhibit differential expression in IBD tissues will be evaluated in this system. Expression constructs for individual Fz receptor subtypes will be utilized for more in-depth analysis in colon cancer cell lines. The precise effects of FIPs on Wnt signaling will be defined in vitro, with attention to the potentially disparate responses of different, specific, Fz receptor subtypes.