LEF/TCF Expression in colon cancer

描述（由申请人提供）：结肠癌的分子遗传学分析已经确定，Wnt信号通路参与肠道中的早期肿瘤发展。将Wnt信号转换为靶基因表达变化的转录因子是类淋巴细胞增强因子/T细胞因子（LEF/TCF）家族的成员。LEF/TCF有全长激活和截短显性负性形式以及选择性剪接同种型。LEF/TCF的活性通过靶基因的共调节而相互作用的信号传导途径以及通过改变特定LEF/TCF同种型的作用的途径来改变。在这方面，已经发现两种途径是重要的。首先，Notch通路与Wnt信号传导协作以维持干细胞生态位并在肠中活跃地循环祖细胞。这种合作可能在结肠癌中对维持增殖的癌症起始细胞（干细胞或祖细胞）很重要。其次，涉及钙-钙调蛋白激酶II（CAMKII）的激酶级联改变LEF/TCF定位和活性。我们的数据显示，TCF-1的定位在人类结肠癌中发生了显着改变。提出了以下假设。首先，将在原发性人结肠癌和新衍生的结肠癌起始细胞培养物（CCIC）中测试Notch和CAMKII是结肠癌中的活性信号的假设（Aim 1）。第二，Notch信号修饰Wnt靶表达的假设将通过阻断结肠癌和CCIC培养物中的Notch信号来测试。额外的重点将放在分析LEF1及其活动与Notch信号组件（Aim2）。第三，将在结肠癌细胞和生物化学测定中测试CAMK II引起显性阴性TCF-1同种型的核输出以增强Wnt信号传导的假设。将通过多西环素诱导的shRNA敲低（Aim3）评估dnTCF-1核排斥的生物学后果。这些项目的总体目标是确定Notch串扰和CAMKII信号传导是否增强Wnt信号以促进肿瘤发生，或调节维持癌症起始干细胞的途径。公共卫生相关性：该项目旨在确定对正常肠道功能（Wnt）重要的信号转导途径如何改变以促进癌症。在肠道中，Wnt信号控制干细胞、细胞增殖和成熟细胞功能。其他途径与Wnt合作，以确保肠道保持适当的这些生长模式。wnt信号在癌症中异常强烈，这些相同的协作途径可能有助于将细胞推向致瘤状态。该项目研究了两个这样的合作途径（Notch和钙调蛋白激酶II）如何修改结肠癌和新建立的结肠癌干细胞系中的Wnt信号。

DESCRIPTION (provided by applicant): Molecular genetic analysis of colon cancers has established that the Wnt signaling pathway is involved in early tumor development in the intestine. The transcription factors that commute Wnt signals into changes in target gene expression are members of the Lymphoid Enhancer Factor/T Cell Factor (LEF/TCF) family. There are full-length activating and truncated dominant negative forms of LEF/TCFs as well as alternatively spliced isoforms. The activities of LEF/TCFs are modified by signaling pathways that cross-talk through co-regulation of target genes, and by pathways that modify the actions of specific LEF/TCF isoforms. Two pathways have been discovered to be important in this regard. First, the Notch pathway collaborates with Wnt signaling to maintain a stem cell niche and actively cycling progenitor cells in the intestine. This collaboration may be important in colon cancer to maintain proliferating cancer-initiating cells (stem or progenitor cells). Second, a kinase cascade that involves calcium-calmodulin Kinase II (CAMKII) modifies LEF/TCF localization and activity. Our data shows that TCF-1 localization is dramatically altered in human colon cancer. The following hypotheses are proposed. First, the hypothesis that Notch and CAMKII are active signals in colon cancer will be tested in primary human colon cancer and newly derived colon cancer initiating cell cultures (CCICs) (Aim1). Second, the hypothesis that Notch signals modify Wnt target expression will be tested by blocking Notch signaling in colon cancer and CCIC cultures. Additional emphasis will be placed on an analysis of LEF1 and its activities with Notch signaling components (Aim2). Third, the hypothesis that CAMKII causes nuclear export of a dominant negative TCF-1 isoform to enhance Wnt signaling will be tested in colon cancer cells and in biochemical assays. The biological consequences of dnTCF-1 nuclear exclusion will be assessed by doxycycline-induced shRNA knockdown (Aim3). The overall goal of these projects are to determine whether Notch cross-talk and CAMKII signaling enhance Wnt signals to promote tumorigenesis, or moderate the pathway to maintain cancer-initiating stem cells. PUBLIC HEALTH RELEVANCE: This project proposes to define how a signal transduction pathway that is important for normal intestine function (Wnt), is altered to promote cancer. In the intestine, Wnt signals control stem cells, cell proliferation and mature cell function. Other pathways cooperate with Wnts to ensure that the intestine maintains proper these growth patterns. Wnt signals are abnormally strong in cancer, and these same collaborating pathways may be helping push cells into a tumorigenic state. This project examines how two such collaborating pathways (Notch and Calmodulin Kinase II) modify Wnt signals in colon cancer and in newly established colon cancer stem cell lines.