REGULATION OF ATROPHY-INDUCED PROGENITOR CELLS IN THE GASTRIC CORPUS

项目摘要 胃的上皮干细胞的特征很差，这是令人惊讶的，因为它在许多胃肠道疾病中的作用。 疾病高剂量的选择性雌激素受体调节剂（SERM）他莫昔芬（HD-Tam）杀死顶叶 细胞，并导致增殖性、化生性胃反应，其遵循患者中所见的模式 慢性感染幽门螺杆菌，但在一个更同步和可控的模式， 在注射后3天达到峰值。已发表的使用HD-Tam的研究表明，这种反应取决于 CD 44-ERK→ CD 44 → CD 44-STAT 3信号转导轴与峡部干细胞增殖的结合 细胞（iSC）和来自成熟的酶原细胞的贡献，成熟的酶原细胞重新编程以重新进入细胞周期，称为 招募干细胞（rSC）。虽然这些激增的人口是不可分割的，在他们的反应， 我们最近发现，高度特异性的白喉毒素（DT）介导的壁细胞 细胞死亡诱导iSC扩增，但不诱导化生或rSC生成。这两个模型现在允许我们 特异性表征iSC和rSC以确定其相对增殖机制。 我们的目的是：1）表征iSC扩增/恢复的机制并确定CD 44的具体作用 在体内以及在人和鼠胃类器官（“胃类器官”）中的iSC扩增中。我们也将纯 DT后的iSC和HD-Tam后的混合iSC + rSC，并运行RNA-Seq以表征每个群体。 2)使用雷帕霉素和二甲双胍检查管理每个增殖群体的新代谢途径， 这两种都被认为在HD-Tam后降低了增殖。这些药物将在体内进行测试， HD-Tam和DT以及在胃腺中，沿着药物筛选以鉴定参与其表达的其他蛋白质。 信号通路3)我们现在能够确定化生rSC的产生是否会增加 胃肿瘤发生率高于单独增加的iSC增殖。我们将遵循既定的协议， 致癌物MNU在用DT或HD-Tam周期治疗的同时驱动小鼠的肿瘤发生，同时监测 肿瘤发生全过程无创近红外荧光活体成像与新型 在体内追踪胃癌的探针。我们将跟踪特定的突变和蛋白质必要的这一点 癌症进展，并关注我们的大型人类胃癌组织生物库中发现的任何候选者。 我们的DT和HD-Tam模型允许我们首次分离胃中的iSC和rSC增殖， 最后允许每个群体的详细表征。我们的实验旨在利用这个机会， 将我们已知的增殖轴更具体地归因于iSC和/或rSC扩增， 参与控制每个群体中增殖的途径，并进行无偏筛选以确定其他 这些途径的调节剂。最后，我们的模型使我们能够测试实体组织癌症如何发生的新理论 研究成熟的有丝分裂后的细胞（rSC）是否会重新编程和重新进入细胞周期， 生成）相对于单独增加的iSC增殖增加肿瘤发生的风险。

PROJECT SUMMARY The epithelial stem cell of the stomach is poorly characterized, which is surprising given its role in many gastric diseases. High doses of the Selective Estrogen Receptor Modulator (SERM) tamoxifen (HD-Tam) kill parietal cells and causes a proliferative, metaplastic gastric response which follows the pattern seen in patients chronically infected with Helicobacter pylori, but in a more synchronous and controllable model, with the effect peaking at 3 days post injections. Published studies using HD-Tam show that this response is dependent on a ℗-ERK→CD44→℗-STAT3 signaling axis and combines proliferation both from expansion of the isthmal stem cell (iSC) and contribution from mature zymogenic cells which reprogram to re-enter the cell cycle, termed recruited stem cells (rSCs). While these proliferating populations are inseparable in their response to any metaplasia-causing agent, we recently discovered that highly specific diphtheria toxin (DT)-mediated parietal cell death induces iSC expansion but no metaplasia or rSC generation. These two models now allow us to specifically characterize iSCs and rSCs to determine their relative mechanisms of proliferation. Our Aims are: 1) Characterize mechanisms of iSC expansion/recovery and determine the specific role of CD44 in iSC expansion both in vivo and in human and murine gastric organoids (“gastroids”). We will also sort pure iSCs following DT and mixed iSCs+rSCs following HD-Tam and run RNA-Seq to characterize each population. 2) Examine new metabolic pathways governing each proliferative population using rapamycin and metformin, both of which are seen to decrease proliferation following HD-Tam. These drugs will be tested in vivo following HD-Tam and DT as well as in gastroids, along with a drug screen to identify other proteins involved in their signaling pathways. 3) We are now able to determine whether metaplastic rSC generation increases risk of gastric tumorigenesis over increased iSC proliferation alone. We will follow an established protocol using the carcinogen MNU to drive tumorigenesis in mice while treating with cycles of DT or HD-Tam while monitoring the entire course of tumorigenesis non-invasively with Near Infrared Fluorescence live imaging with novel probes found to track gastric cancer in vivo. We will track specific mutations and proteins necessary for this cancer progression and focus on any candidates found with our large biobank of human gastric cancer tissues. Our DT and HD-Tam models allow us to separate iSC and rSC proliferation in the stomach for the first time, finally allowing for detailed characterization of each population. Our experiments aim to use this opportunity to attribute our known proliferative axis more specifically to iSC and/or rSC expansion, define new metabolic pathways involved in governing proliferation in each population, and run unbiased screens to identify additional modulators of these pathways. Finally, our models allow us to test a new theory for how solid tissue cancers arise, investigating whether rounds of reprogramming and cell cycle re-entry of mature, post-mitotic cells (rSC generation) increase risk for tumorigenesis over increased iSC proliferation alone.