Role of Sox2 in stomach development, regeneration and cancer

描述(申请人提供)：胃上皮由两个主要隔室组成，具有不同的周转率和细胞成分，称为胃窦或幽门和胃体或主体。虽然经典的诱变实验表明存在持续补充这些细胞类型的成体干细胞，但它们的身份和定位仍然难以捉摸。值得注意的是，最近的一份报告发现了胃窦部的Lgr5+干细胞，它们能够长期多向分化，在肿瘤抑制基因APC缺失后，可以作为腺瘤的起源细胞类型。我们最近在小鼠的胃窦和体部发现了罕见的Sox2+细胞。遗传谱系追踪表明，Sox2+细胞可以像胃窦Lgr5+细胞一样，在胃中培养出所有类型的成熟细胞长达22个月，从而符合真正的干细胞的条件。这些观察提出了与胃周转和胃癌生物学有关的下列关键问题：(1)SOX2+胃干细胞在发育过程中是形成的；(2)SOX2+干细胞和LGr5+干细胞是同一谱系的一部分；(3)Sox2+干细胞在胃窦和胃体中的分子和细胞特征是什么；(4)Sox2+干细胞是对称还是不对称分裂的，分裂的速度是多少；(5)Sox2+干细胞对组织损伤和炎症反应敏感；(6)Sox2+干细胞比胃细胞更容易分化成肿瘤；以及(7)Sox2本身是否对胃发育、组织动态平衡和癌症是必需的？我们已经开发了几种新的小鼠转基因工具，在三个主要目标和多个子目标的背景下解决这些基本问题中的每一个。具体地说，我们已经培育了Sox2-GFP报告小鼠以及Sox2-Creer谱系追踪小鼠，以在分子和细胞水平上表征不同发育阶段的Sox2+细胞。目的1研究Sox2+细胞的超微结构和转录组特征及其自我更新和分化动力学，建立Sox2+细胞体外培养体系，评价Sox2+细胞与Lgr5+细胞的亲缘关系。在目标2中，我们将Sox2的一个新的条件等位基因交叉到不同的CRE驱动因素中，以评估在出生前和出生后发育的不同阶段以及在几种遗传和化学毒性模型造成的胃细胞损伤的背景下对Sox2的需求。鉴于胃癌是全球癌症相关死亡的第二大常见原因，人们对其潜在的遗传和细胞起源知之甚少，我们建议在目标3中测试Sox2+干细胞对恶性转化的敏感性。在这里，我们将评估Sox2+干细胞在APC肿瘤抑制基因缺失后是否能够转化为腺瘤/腺癌，以及Sox2蛋白本身是否是肿瘤形成所必需的。最后，我们将通过在Sox2+干细胞、转运放大细胞和分化的主细胞中删除E-钙粘附素基因来检验胃细胞的分化状态影响其转化能力的假设。在50%的人弥漫性胃癌病例中，E-钙粘附素基因发生突变。 公共卫生相关性：胃癌是全球第二大最常见的癌症相关死亡原因，在美国弥漫性胃癌的增加尤为明显。阐明胃发育和动态平衡的基本生物学对于了解胃癌的发展和确定治疗的细胞和分子靶点是必不可少的。因此，通过研究正常和恶性胃组织中的Sox2和Sox2+干细胞，我们将获得可用于再生医学以及胃癌预防和治疗的新的基本见解。

DESCRIPTION (provided by applicant): The stomach epithelium comprises two main compartments with distinct turnover rates and cell compositions termed antrum or pylorus and corpus or main body. While classical mutagenesis experiments suggested the existence of adult stem cells that continuously replenish these cell types, their identity and localization remains elusive. Notably, a recent report identified Lgr5+ stem cells in the antrum, which are capable of multilineage differentiation over long-term and can serve as the cell type of origin for adenomas upon deletion of the tumor suppressor gene Apc. We have recently identified rare Sox2+ cells in the antrum and corpus of mice. Genetic lineage tracing demonstrates that Sox2+ cells can, like antral Lgr5+ cells, give rise to all mature cell types in the stomach for up to 22 months, thus qualifying as bona fide stem cells. These observations raise the following key questions relevant to the biology of stomach turnover and stomach cancer: (1) When in development are Sox2+ stomach stem cells formed; (2) Are Sox2+ stem cells and Lgr5+ stem cells part of the same lineage; (3) What are the molecular and cellular features of Sox2+ stem cells in antrum and corpus; (4) Do Sox2+ stem cells divide symmetrically or asymmetrically and at which rate; (5) Are Sox2+ stem cells responsive to tissue injury and inflammation; (6) Are Sox2+ cells more amenable to tumorigenesis than differentiated stomach cells; and (7) Is Sox2 itself required for stomach development, tissue homeostasis and cancer? We have developed several novel transgenic tools in mice to address each of these fundamental questions in the context of three major aims and multiple subaims. Specifically, we have generated Sox2-GFP reporter mice as well as Sox2-CreER lineage tracing mice to characterize Sox2+ cells at the molecular and cellular levels at different stages of development. Aim 1 entails characterization of the ultrastructure and transcriptome of Sox2+ cells as well as their self-renewal and differentiation kinetics, establishment of an in vitro culture system and evaluation of the lineage relationship between Sox2+ cells and Lgr5+ cells. In Aim 2, we will cross a novel conditional allele for Sox2 to different Cre drivers to assess the requirement for Sox2 at various stages of pre-and postnatal development and in the context of stomach cell injury inflicted by several genetic and chemotoxic models. Given that stomach cancer is the second-most common cause of cancer-related deaths worldwide with relatively little known about its underlying genetic and cellular origins, we propose to test in Aim 3 the susceptibility of Sox2+ stem cells to malignant transformation. Here, we will evaluate whether Sox2+ stem cells are amenable to transformation into adenomas/adenocarcinomas upon deletion of the Apc tumor suppressor and whether Sox2 protein itself is required for tumor formation. Lastly, we will test the hypothesis that the differentiation state of stomach cells influences their amenability to transformation by deleting the E-Cadherin gene, which is mutated in 50% of human diffuse gastric cancer cases, in Sox2+ stem cells, transit-amplifying cells and differentiated chief cells. PUBLIC HEALTH RELEVANCE: Stomach cancer is the second-most common cancer-related cause of death worldwide with an increase seen specifically in diffuse gastric cancer in the United States. Elucidating the fundamental biology of stomach development and homeostasis is imperative for understanding how stomach cancer develops and for identifying cellular and molecular targets for treatment. Thus, by studying Sox2 and Sox2+ stem cells in the normal and malignant stomach, we will gain new basic insights that can be exploited for regenerative medicine as well as for stomach cancer prevention and treatment.