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。这一过程与 APC 基因突变相似，与散发性结肠癌的发生过程密切相关。 APC 和 β-连环蛋白突变在炎症性肠病 (IBD) 相关肿瘤中似乎很少见。然而，Wnt 通路其他成分的异常表达，包括 frizzled 受体、SARP1 和 disheveled (dvl) 也已被描述。在该提案中，将定义 IBD 患者的正常、发育不良和恶性结肠组织中 Wnt 信号转导途径上游元件、卷曲受体和卷曲抑制分子的表达。首先，我们将通过免疫组织化学检查 IBD 相关结肠癌组织中 Fz 受体的表达，并将该表达与邻近粘膜组织和散发性结肠癌进行比较。然后利用对 10 种 Fz 受体亚型以及特定卷曲抑制蛋白 (FIP) 具有特异性的反义 RNA 探针进行原位杂交，以确定它们在这些组织中的表达。接下来，将从接受监测结肠镜检查的 IBD 患者中前瞻性地获取正常和发育异常粘膜的结肠活检。将评估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，这通过 β-连环蛋白的核化和下游途径成分 LEF1 的表达和活性增加来表明。最后，将利用新型体外共培养表达系统来定义特定 Fz 受体亚型在 Wnt 通路信号启动和传播中的作用。那些在 IBD 组织中表现出差异表达的 Fz 受体亚型将在该系统中进行评估。各个 Fz 受体亚型的表达构建体将用于对结肠癌细胞系进行更深入的分析。 FIP 对 Wnt 信号传导的精确影响将在体外确定，同时关注不同的特定 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.