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信号通路参与了肠道早期肿瘤的发展。将Wnt信号转换为靶基因表达变化的转录因子是淋巴细胞增强因子/T细胞因子（LEF/TCF）家族的成员。LEF/ tcf有全长激活型和截断型显性阴性型，也有选择性剪接型。LEF/TCF的活性可通过信号通路和特定LEF/TCF同工异构体的作用进行修饰，这些信号通路通过靶基因的共调节进行串扰。在这方面，已经发现了两种重要的途径。首先，Notch通路与Wnt信号协同维持干细胞生态位，并在肠道内积极循环祖细胞。这种合作在结肠癌中维持癌起始细胞（干细胞或祖细胞）的增殖可能是重要的。其次，涉及钙钙调蛋白激酶II （CAMKII）的激酶级联改变了LEF/TCF的定位和活性。我们的数据显示，TCF-1的定位在人类结肠癌中发生了巨大的变化。提出以下假设：首先，Notch和CAMKII在结肠癌中是活性信号的假设将在原发性人类结肠癌和新衍生的结肠癌起始细胞培养（CCICs） （Aim1）中进行测试。其次，Notch信号改变Wnt靶点表达的假设将通过阻断结肠癌和CCIC培养中的Notch信号来验证。额外的重点将放在分析LEF1及其与Notch信号成分（Aim2）的活动上。第三，CAMKII导致显性TCF-1阴性异构体的核输出以增强Wnt信号的假设将在结肠癌细胞和生化分析中得到验证。dnTCF-1核排斥的生物学后果将通过多西环素诱导shRNA敲低（Aim3）来评估。这些项目的总体目标是确定Notch串扰和CAMKII信号是否增强Wnt信号以促进肿瘤发生，或调节维持癌症启动干细胞的途径。公共卫生相关性：该项目旨在确定对正常肠功能（Wnt）至关重要的信号转导通路如何被改变以促进癌症。在肠道中，Wnt信号控制着干细胞、细胞增殖和成熟细胞功能。其他途径与wnt合作，确保肠道维持适当的这些生长模式。Wnt信号在癌症中异常强烈，这些相同的协同途径可能有助于推动细胞进入致瘤状态。该项目研究了两个这样的合作途径（Notch和Calmodulin Kinase II）如何在结肠癌和新建立的结肠癌干细胞系中修改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.