Role of Slowly Cycling Stem Cells in Cancer

描述（由申请方提供）：肠上皮由快速循环（Lgr 5+）肠干细胞（ISC）群体维持。然而，据推测，缓慢循环的ISC也必须存在于肠中以保护基因组免受累积的有害突变并允许对组织损伤的反应。我们已经确定了一个人口的缓慢循环的ISCs是由端粒酶（mTert）表达的标志。mTert+细胞是罕见的单细胞，其以类似于长期标记保留细胞的模式沿着隐窝-绒毛轴分布，并且对组织损伤具有抗性。谱系追踪研究表明，mTert+细胞产生所有分化的肠细胞类型，并长期存在。此外，表达mTert的细胞似乎通过Lgr 5依赖性和Lgr 5非依赖性途径促进肠谱系发育。为了研究它们在肠癌中的作用，我们稳定了？mTert+细胞内的连环蛋白，这导致其显著（1，500倍）活化。然而，令人惊讶的是，即使在6个月后，这些细胞中连续的经典Wnt信号传导也没有引起肠腺瘤形成。这一结果与稳定后的效果形成鲜明对比？-在快速循环的Lgr 5 + ISCs内的连环蛋白，其在4周内产生广泛的腺瘤。我们的研究支持这样的概念，即缓慢循环和快速循环的ISCs对肿瘤起始信号表现出明显不同的反应。我们假设mTert+细胞中腺瘤形成的缺乏是由于调节这些缓慢循环的ISC的“强制静止”机制，其在快速循环的Lgr 5 + ISC中缺乏。为了更好地确定导致腺瘤形成或抑制的机制，这些研究将研究在存在或不存在稳定的？连环蛋白。为了确定microRNA（miRNAs）在腺瘤形成和抑制中的作用，我们将在稳定的？在表达mTert和Lgr 5的ISC中的连环蛋白。为了确定miRNAs是否在稳定？-在表达mTert的ISC中的连环蛋白，我们将删除这些细胞内的关键miRNA加工酶Dicer，以确定该调节系统的失活是否导致腺瘤形成。类似地，我们将在表达Lgr 5的ISC中删除Dicer，以确定miRNA是否在诱导腺瘤形成中具有功能性作用。这些结果将对肠干细胞中腺瘤发生的机制产生新的认识。 公共卫生相关性：我们的研究支持了慢循环和快循环肠干细胞对肿瘤起始信号表现出明显不同的反应的概念。我们推测，在缓慢循环的干细胞中缺乏腺瘤形成是由于增殖阻断机制，而快速循环的干细胞中缺乏这种机制。这些实验将分离和表征通过靶向稳定？缓慢循环和快速循环干细胞中的连环蛋白。基因表达和microRNA分析将确定癌症干细胞形成的关键调控因子，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节癌症干细胞形成的初始步骤的机制提供独特的见解。

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.