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隔室，具有不同的周转率和细胞组成，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成体干细胞，不断补充这些细胞类型，其身份和定位仍然难以捉摸。值得注意的是，最近的一份报告鉴定了胃窦中的Lgr 5+干细胞，其能够长期进行多谱系分化，并且可以在肿瘤抑制基因Apc缺失后作为腺瘤的起源细胞类型。我们最近在小鼠胃窦和体部发现了罕见的Sox 2+细胞。遗传谱系追踪表明，Sox 2+细胞可以像胃窦Lgr 5+细胞一样，在长达22个月的时间内在胃中产生所有成熟细胞类型，从而有资格成为真正的干细胞。 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）Sox 2+胃干细胞在发育过程中形成;（2）Sox 2+干细胞和Lgr 5+干细胞是同一谱系的一部分吗？（3）胃窦和胃体中Sox 2+干细胞的分子和细胞特征是什么？（4）Sox 2+干细胞是对称分裂还是不对称分裂，以及以何种速率分裂;（5）Sox 2+干细胞是否对组织损伤和炎症有反应;（6）Sox 2+细胞是否比分化的胃细胞更容易发生肿瘤;（7）Sox 2本身是否是胃发育、组织稳态和癌症所必需的？我们已经在小鼠中开发了几种新型转基因工具，以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。 具体而言，我们已经产生了Sox 2-GFP报告小鼠以及Sox 2-CreER谱系示踪小鼠，以在不同发育阶段在分子和细胞水平上表征Sox 2+细胞。目的1：研究Sox 2+细胞的超微结构、转录组、自我更新和分化动力学，建立体外培养体系，探讨Sox 2+细胞与Lgr 5+细胞的谱系关系。在目标2中，我们将Sox 2的一种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各个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Sox 2的需求。鉴于胃癌是全球癌症相关死亡的第二大常见原因，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Sox 2+干细胞对恶性转化的易感性。在这里，我们将评估Sox 2+干细胞是否适合于在Apc肿瘤抑制因子缺失后转化为腺瘤/腺癌，以及Sox 2蛋白本身是否是肿瘤形成所需的。最后，我们将通过删除在50%的人弥漫性胃癌病例中突变的E-Cadherin基因，在Sox 2+干细胞，transit-amplifying细胞和分化的主细胞中，检验胃细胞的分化状态影响其转化的可能性的假设。 公共卫生相关性：胃癌是世界范围内第二大常见的癌症相关死亡原因，在美国弥漫性胃癌中尤其如此。阐明胃发育和体内平衡的基本生物学对于理解胃癌如何发展以及识别治疗的细胞和分子靶点至关重要。因此，通过研究正常和恶性胃中的Sox 2和Sox 2+干细胞，我们将获得新的基本见解，可用于再生医学以及胃癌预防和治疗。

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.