Role of Slowly Cycling Stem Cells in Cancer

描述(申请人提供)：肠上皮由一群快速循环(Lgr5+)的肠道干细胞(ISCs)维持。然而，据推测，肠道中也必须存在缓慢循环的ISCs，以保护基因组免受有害突变的积累，并允许对组织损伤做出反应。我们已经确定了一组以端粒酶(MTert)表达为标志的缓慢周期的ISCs。MTert+细胞是一种罕见的单个细胞，它沿着隐窝绒毛轴呈类似于长期标记保留细胞的模式分布，并对组织损伤具有抵抗力。血统追踪研究表明，mTert+细胞可以产生所有分化的肠道细胞类型，并长期存在。此外，mTert表达的细胞似乎通过Lgr5依赖和Lgr5非依赖的途径促进肠道谱系的发育。为了研究它们在肠癌中的作用，我们稳定了mTert+细胞中的β-连环蛋白，这导致了它们的戏剧性(1500倍)激活。然而，令人惊讶的是，即使在6个月后，这些细胞中持续的规范Wnt信号并没有引起肠腺瘤的形成。这一结果与在快速循环的Lgr5+ISCs中稳定？-catenin后的效果形成了鲜明对比，后者在4周内会产生广泛的腺瘤。我们的研究支持慢周期和快周期的ISCs对肿瘤启动信号表现出明显不同的反应。我们推测，mTert+细胞中没有腺瘤形成是由于一种“强制静止”机制调节这些缓慢循环的ISCs，而快速循环的Lgr5+ISCs缺乏这种机制。为了更好地确定导致腺瘤形成或抑制的机制，这些研究将研究在稳定的？-catenin存在或不存在的情况下表达mTert和Lgr5的ISCs的基因表达谱。为了确定microRNAs(MiRNAs)在腺瘤形成和抑制中的作用，我们将在mTert和Lgr5表达的ISCs中存在或不存在稳定的？-catenin的情况下，对其表达模式进行整体分析。为了确定miRNAs在稳定表达mTert的ISCs中的β-catenin后是否在抑制腺瘤形成中发挥作用，我们将删除这些细胞中关键的miRNA加工酶DICER，以确定该调节系统的失活是否会导致腺瘤的形成。同样，我们将删除表达Lgr5的ISCs中的DICER，以确定miRNAs是否在腺瘤形成中具有功能作用。这一结果将使人们对肠道干细胞腺瘤发生的机制有新的认识。 公共卫生相关性：我们的研究支持慢循环和快循环的肠道干细胞对肿瘤启动信号表现出明显不同的反应的概念。我们假设，慢循环干细胞中没有腺瘤形成是由于快速循环干细胞缺乏增殖抑制机制。这些实验将分离和鉴定通过靶向稳定慢循环和快循环干细胞中的β-连环蛋白而产生的可能的肠癌干细胞。基因表达和microRNA分析将确定癌症干细胞形成的关键调控因素，并为确定导致癌症发展的关键变化提供基础。对这些细胞的比较分析将为调控癌症干细胞形成的最初步骤提供独特的洞察力。

DESCRIPTION (provided by applicant): The intestinal epithelium is maintained by a population of rapidly cycling (Lgr5+) intestinal stem cells (ISCs). It has been postulated, however, that slowly cycling ISCs must also be present in the intestine to protect the genome from accumulating deleterious mutations and allow for response to tissue injury. We have identified a population of slowly cycling ISCs that is marked by telomerase (mTert) expression. mTert+ cells are rare single cells that distribute in a pattern along the crypt-villus axis similar to long-term label-retaining cells and are resistant to tissue injury. Lineage-tracing studies demonstrate that mTert+ cells give rise to all differentiated intestinal cell types and persist long term. Moreover, mTert-expressing cells appear to contribute to intestinal lineage development through both Lgr5-dependent and Lgr5- independent pathways. In order to investigate their role in intestinal cancer we stabilized ?-catenin within mTert+ cells, which resulted in their dramatic (1,500-fold) activation. Surprisingly, however, continuous canonical Wnt signaling in these cells did not give rise to intestinal adenoma formation, even after 6 months. This result stands in stark contrast to the effect seen following stabilization of ?-catenin within rapidly cycling Lgr5+ ISCs, which generate extensive adenomas within 4 weeks. Our studies support the concept that slowly cycling and rapidly cycling ISCs exhibit distinctly different responses to tumor initiating signals. We hypothesize that the lack of adenoma formation in mTert+ cells is due to a mechanism of "enforced quiescence" regulating these slowly cycling ISCs, which is lacking in rapidly cycling Lgr5+ ISCs. In order to better define the mechanisms leading to adenoma formation or repression, these studies will investigate the gene expression profiles of mTert- and Lgr5-expressing ISCs in the presence or absence of stabilized ?-catenin. To determine the role of microRNAs (miRNAs) in adenoma formation and repression we will perform global analysis of their expression patterns in the presence or absence of stabilized ?-catenin in mTert- and Lgr5-expressing ISCs. To determine whether miRNAs play a functional role in the repression of adenoma formation following stabilization of ?-catenin in mTert-expressing ISCs we will delete Dicer, the critical miRNA processing enzyme, within these cells to establish whether inactivation of this regulatory system results in adenoma formation. Similarly, we will delete Dicer in Lgr5-expressing ISCs to determine whether miRNAs have a functional role in the induction of adenoma formation. The results will generate new insight into the mechanisms underlying adenoma initiation in intestinal stem cells. PUBLIC HEALTH RELEVANCE: Our studies support the concept that slowly cycling and rapidly cycling intestinal stem cells exhibit distinctly different responses to tumor initiating signals. We hypothesize that the lack of adenoma formation in slowly cycling stem cells is due to a proliferation blocking mechanism, which is lacking in rapidly cycling stem cells. These experiments will isolate and characterize putative intestinal cancer stem cells generated by targeted stabilization of ?-catenin in both slowly cycling and rapidly cycling stem cells. Gene expression and microRNA analysis will identify key regulators of cancer stem cell formation and provide a basis to determine the critical changes leading to the development of cancer. Comparative analysis of these cells will provide unique insight into the mechanisms regulating the initial steps of cancer stem cell formation.