Dysregulated Polyamine Metabolism in H. pylori-associated Gastric Inflammation and Disease Progression

幽门螺杆菌感染的胃和由此产生的慢性胃炎的临床后果，消化性 溃疡疾病和疾病进展为胃癌仍然是退伍军人的主要健康问题。这 病原体感染了世界上一半的人口，胃癌是癌症死亡的第三大原因 国际吧H. pylori在退伍军人中的患病率很高;部署相关的感染是一个问题， 获得与较高致癌风险相关的菌株。三种多胺，腐胺（Put）， 亚精胺（Spd）和精胺（Spm）是具有许多生物学效应的普遍存在的分子。Put是合成的 通过限速酶，鸟氨酸脱羧酶（ODC），并依次转化为Spd和Spm， 其通过精胺氧化酶（SMOX）反向转化为Spd。我们的实验室一直对以下角色感兴趣： 多胺在胃肠道炎症和癌变中的作用，我们有以下令人兴奋的发现与此相关 更新补助：1）感染H.幽门螺杆菌表现出减少的胃炎症，DNA损伤， 趋化因子的表达，与Spd的消耗有关; 2）这些表型在H.幽门- Smox-/-小鼠感染的2D类器官; 3）H.幽门诱导的促癌激活 包括Smox-/-小鼠和沙鼠用SMOX抑制剂治疗后， 4）使用人胃类器官培养物来证明β-连环蛋白在体内活化的有效性。 新的和有效的SMOX抑制剂阻断H.幽门螺杆菌诱导的b-连环蛋白激活，与Spd相关 5）通过脱氧羟腐胺赖氨酸的作用，暗示了蛋白质翻译的独特形式，羟腐胺赖氨酸化 合成酶（DHPS），一个与致癌作用有关的过程，似乎被Smox减弱 缺失; 6）通过全外显子组测序（WES）证实胃炎和胃炎中的体细胞基因组异常， INS-GAS小鼠的异型增生组织，H.幽门诱发癌我们现在将使 使用重要的分子工具，包括有价值的突变小鼠模型，一种关键的SMOX抑制剂， 使用来自小鼠和VA患者的3D和2D胃类器官。我们假设多胺失调 由于SMOX和相关的Spd和羟腐胺酸化的产生， 导致胃病进展为致癌风险的途径。我们的具体目标是：1） 直接决定SMOX和Spd在胃癌发生中的作用。我们将测试：A）SMOX在 癌症易感的INS-GAS小鼠，分析H.幽门螺杆菌诱导的致癌作用，DNA损伤，β-连环蛋白激活， 和GEC功能; B）一种新的、有效的、第二代SMOX C）SMOX/Spd对INS-GAS小鼠和沙鼠中的体细胞生长抑制剂SLH150 - 54的影响; 使用全外显子组测序的基因组异常。2)为了确定上皮DHPS是否介导有害的 SMOX/Spd在胃癌发生中的作用我们将分析：A）SMOX在H。 C57BL/6和FVB/N INS-GAS Smox-/-vs. WT小鼠+/-H. pylori +/-Spd; B） 使用C57 BL/6和FVB/N在胃上皮细胞（GECs）中的羟腐胺赖氨酸化对炎症和癌变的影响 在GEC中特异性缺失Dhps的INS-GAS小鼠; C）DHPS/羟腐胺赖氨酸化对体细胞基因组的影响 不稳定3)利用来自VA患者的组织中的胃类器官报告系统来建立人 致癌作用的生物标志物。H.幽门螺杆菌诱导的DNA损伤，b-连环蛋白激活，以及 A）使用CRISPR/CAS9介导的缺失，SMOX的诱导和作用 和B）使用CRISPR/CAS 9介导的SMOX和SMOX抑制剂的缺失，DHPS的诱导和作用。 DHPS和DHPS抑制剂GC 7。这项研究计划将提供重要的新见解H。幽门- 诱导炎症和疾病进展沿着胃癌发生的途径。的翻译 从有价值的动物模型到强大的人类类器官系统的发现有望促进验证 VA患者癌症风险评估、化学预防和治疗的新策略。

Helicobacter pylori infection of the stomach and the resulting clinical consequences of chronic gastritis, peptic ulcer disease, and disease progression to gastric cancer remains a major health concern for Veterans. This pathogen infects half of the world’s population, and gastric cancer is the third leading cause of cancer deaths worldwide. H. pylori prevalence in Veterans is high; deployment-related infection is a problem, especially acquisition of strains associated with higher risk for carcinogenesis. The three polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm), are ubiquitous molecules with many biological effects. Put is synthesized by the rate-limiting enzyme, ornithine decarboxylase (ODC), and is converted sequentially to Spd and Spm, which is back-converted to Spd by spermine oxidase (SMOX). Our Lab has had a long interest in the role of polyamines in GI inflammation and carcinogenesis, and we have the following exciting findings pertinent to this renewal grant: 1) Smox–/– mice infected with H. pylori exhibit decreased gastric inflammation, DNA damage, and chemokine expression, associated with depletion of Spd; 2) These phenotypes are recapitulated in H. pylori- infected 2D organoids from Smox–/– mice; 3) A new link between the H. pylori-induced pro-carcinogenic activation of b-catenin and SMOX, including findings that Smox–/– mice and gerbils treated with a SMOX inhibitor have loss of b-catenin activation in vivo; 4) Use of human gastric organoid cultures to demonstrate the effectiveness of a novel and potent SMOX inhibitor in blocking H. pylori-induced b-catenin activation, associated with Spd depletion; 5) Implication of a unique form of protein translation, hypusination, via the action of deoxyhypusine synthase (DHPS), a process which has been linked to carcinogenesis and appears to be attenuated by Smox deletion; 6) Demonstration of somatic genomic abnormalities by whole exome sequencing (WES) in gastritis and dysplasia tissues of INS-GAS mice, a model of H. pylori-induced carcinogenesis. We will now make effective use of important molecular tools, including valuable mutant mouse models, a key SMOX inhibitor, and advanced use of 3D and 2D gastric organoids from mice and VA patients. We hypothesize that dysregulated polyamine metabolism, due to SMOX and associated generation of Spd and hypusination, provides a molecular pathway leading to risk for gastric disease progression to carcinogenesis. Our specific aims are: 1) To directly determine the role of SMOX and Spd in gastric carcinogenesis. We will test: A) The effect of SMOX in cancer-prone INS-GAS mice, analyzing H. pylori-induced carcinogenesis, DNA damage, b-catenin activation, and GEC function in FVB/N INS-GAS Smox–/– mice +/- Spd; B) A novel, potent, second-generation SMOX inhibitor, SLH150-54, in INS-GAS mice and gerbils; C) The effect of SMOX/Spd on the formation of somatic genomic abnormalities using whole exome sequencing. 2) To determine if epithelial DHPS mediates deleterious effects of SMOX/Spd in gastric carcinogenesis. We will analyze: A) The role of SMOX in hypusination during H. pylori infection in C57BL/6 and FVB/N INS-GAS Smox–/– vs. WT mice +/- H. pylori +/- Spd; B) The effect of hypusination in gastric epithelial cells (GECs) on inflammation and carcinogenesis, using C57BL/6 and FVB/N INS-GAS mice with specific deletion of Dhps in GECs; C) the effect of DHPS/hypusination on somatic genomic instability. 3) To utilize gastric organoid reporter systems in tissues from VA patients to establish human biomarkers for carcinogenesis. H. pylori-induced DNA damage, b-catenin activation, and pathways identified in Aims 1 and 2 will be studied, related to: A) Induction and role of SMOX, using CRISPR/CAS9-mediated deletion of SMOX and SMOX inhibitors; and B) Induction and role of DHPS, using CRISPR/CAS9-mediated deletion of DHPS and the DHPS inhibitor, GC7. This research program will provide crucial new insights into H. pylori- induced inflammation and disease progression along the pathway to gastric carcinogenesis. The translation of findings from valuable animal models into robust human organoid systems is expected to facilitate the validation of new strategies for cancer risk assessment, chemoprevention, and treatment for VA patients.