LEF/TCF Expression in colon cancer

描述(申请人提供)：结肠癌的分子遗传学分析已经证实，Wnt信号通路参与了肠道肿瘤的早期发展。将Wnt信号转化为靶基因表达变化的转录因子是淋巴增强因子/T细胞因子(Lef/Tcf)家族的成员。有LEF/TCFs的全长激活和截短显性负型，以及交替剪接的异构体。LEF/TCFs的活性被通过共同调节靶基因而产生串扰的信号通路以及改变特定LEF/TCF亚型的活动的信号通路所改变。在这方面，已经发现了两条重要的途径。首先，Notch通路与Wnt信号合作，维持干细胞的生态位，并积极地在肠道内循环祖细胞。这种合作可能对结肠癌维持增殖的癌症启动细胞(干细胞或祖细胞)很重要。第二，涉及钙-钙调蛋白激酶II(CaMKII)的激动级联会改变LEF/TCF的定位和活性。我们的数据显示TCF-1在人类结肠癌中的定位发生了戏剧性的变化。提出了以下假设。首先，Notch和CaMKII在结肠癌中是活跃信号的假设将在原发人类结肠癌和新衍生的结肠癌起始细胞培养(CCICs)(Aim1)中得到验证。第二，在结肠癌和CCIC培养中，Notch信号改变Wnt靶标表达的假说将通过阻断Notch信号来检验。此外，还将重点分析LEF1及其与Notch信号组件(AIM2)的活动。第三，CaMKII导致显性负TCF-1亚型核输出以增强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的shRNA敲除(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，或缓和维持启动癌症的干细胞的途径。与公共健康相关：该项目建议定义对正常肠道功能(WNT)至关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的功能。其他途径与WNTS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。这个项目研究了两个这样的协作通路(Notch和钙调蛋白激酶II)如何改变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.