Role of Sox2 in stomach development, regeneration and cancer

描述（由申请人提供）：胃上皮包括两个主要的隔室，具有不同的周转率和细胞组成，称为胃窦或幽门和体或主体。虽然经典的诱变实验表明存在不断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，Lgr5+干细胞在胃窦中具有长期的多系分化能力，并且可以在肿瘤抑制基因Apc缺失时作为腺瘤的细胞类型起源。我们最近在小鼠的上腔和体中发现了罕见的Sox2+细胞。遗传谱系追踪表明，Sox2+细胞可以像胃窦Lgr5+细胞一样，在长达22个月的时间里在胃中产生所有成熟细胞类型，从而成为真正的干细胞。这些观察结果提出了以下与胃转换和胃癌生物学相关的关键问题：(1)Sox2+胃干细胞在发育过程中何时形成；(2) Sox2+干细胞和Lgr5+干细胞是否属于同一谱系；(3) Sox2+干细胞在胃窦和体中的分子和细胞特征；(4) Sox2+干细胞是对称分裂还是不对称分裂，分裂速率是多少？(5) Sox2+干细胞是否对组织损伤和炎症有反应？(6) Sox2+细胞是否比分化后的胃细胞更容易发生肿瘤；(7)胃部发育、组织稳态和癌症是否需要Sox2 ？我们已经在小鼠身上开发了几种新的转基因工具，以解决三个主要目标和多个次要目标背景下的这些基本问题。具体来说，我们已经生成了Sox2- gfp报告小鼠以及Sox2- creer谱系追踪小鼠，以在不同发育阶段的分子和细胞水平上表征Sox2+细胞。目的1：表征Sox2+细胞的超微结构和转录组及其自我更新和分化动力学，建立体外培养体系，评估Sox2+细胞与Lgr5+细胞的谱系关系。在Aim 2中，我们将把Sox2的一个新的条件等位基因与不同的Cre驱动基因杂交，以评估在产前和产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，而对其潜在的遗传和细胞起源知之甚少，我们建议在Aim 3中测试Sox2+干细胞对恶性转化的易感性。在这里，我们将评估在Apc肿瘤抑制基因缺失后，Sox2+干细胞是否易于转化为腺瘤/腺癌，以及肿瘤形成是否需要Sox2蛋白本身。最后，我们将通过删除E-Cadherin基因来验证胃细胞的分化状态影响其转化能力的假设，该基因在50%的人类弥漫性胃癌病例中，在Sox2+干细胞，转运扩增细胞和分化的主细胞中发生突变。

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.