ALOX15 regulation of colon cancer invasiveness via PI3P-linoleic acid metabolism

ALOX15 通过 PI3P-亚油酸代谢调节结肠癌侵袭性

• 批准号: 10545078
• 负责人: Imad Shureiqi
• 金额: $ 63.89万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10545078
• 关键词: ALOX15 gene; APC mutation; Acids; Adenocarcinoma; Adverse event; Affect; American diet; Animals; Arachidonate 15-Lipoxygenase; Automobile Driving; Azoxymethane; Benign; Biological Assay; Biological Markers; Breeding; CD44 gene; CDX2 gene; Cancer Etiology; Cell membrane; Cellular Membrane; Cessation of life; Chemicals; Chemopreventive Agent; Colon; Colon Carcinoma; Colonic Polyps; Colorectal; Colorectal Cancer; Complex; Consumption; Corn Oil; Cyclin D1; DNA Sequence Alteration; Data; Development; Diet; Disease; Doxycycline; Endosomes; Enzymes; Event; Genes; Goals; Human; Immunodeficient Mouse; In Vitro; Incidence; Induced Mutation; Intake; Integration Host Factors; Intervention; Invaded; KRAS2 gene; KRASG12D; Knowledge; LDL-Receptor Related Protein 1; LGR5 gene; Lentivirus; Linoleic Acids; Lipoxygenase 1; Liquid Chromatography; LoxP-flanked allele; Mass Spectrum Analysis; Metabolism; Mucous Membrane; Mus; Mutation; Omega-6 Fatty Acids; Organoids; Phospholipids; Polyunsaturated Fatty Acids; Prevention strategy; Recycling; Regulation; Reporting; Research; Resected; Resolution; Risk; Rodent; Role; Sampling; Signal Transduction; System; Testing; Tissues; Transfection; Transgenic Organisms; United States; adenoma; aldehyde dehydrogenase 1; beta catenin; c-myc Genes; cancer invasiveness; carcinogenesis; colon cancer risk; colon carcinogenesis; dietary; dietary excess; drinking water; gain of function; in vivo; insight; lipoprotein receptor related protein 5; loss of function; mouse model; novel; novel chemoprevention; organoid transplantation; permissiveness; peroxidation; phosphatidylinositol 3-phosphate; prevent; preventive intervention; receptor; stem cell self renewal; stem cells; stemness; therapy development; tumor; tumorigenesis; villin

Colorectal cancer is the third leading cause of cancer deaths in the United States. The long-term goal of our research is to develop novel interventions to prevent colorectal carcinogenesis (CRC). CRC invasiveness, a critical adverse step during CRC progression, requires a combination of certain genetic mutations (e.g. APC, KRAS and Trp53 mutations), which are the key events to drive CRC. However, CRC progression also requires additional factors which increase aberrant Beta-catenin (B-catenin) activation above levels induced by APC/B- catenin mutations. Linoleic acid (LA), the most commonly consumed omega-6 polyunsaturated fatty acids in humans, increases both chemically (AOM)– and APC mutation– induced CRC tumorigenesis in mice. Nonetheless, human studies have been inconclusive regarding the impact of dietary LA on CRC. Determination of LA's role in CRC is important because American diets are enriched with LA while expression of the main metabolizing enzyme for LA,15-lipoxygenase-1 (ALOX15), is lost in human CRC. Recently, we found that 1) high dietary levels of LA promoted CRC by increasing phosphatidylinositol 3-phosphate (PI3P) containing LA (PI3P_LA), which increases LRP5 membranous recycling and subsequently B-catenin activation; 2) ALOX15- induced conversion of PI3P_LA to PI3P_13-HODE suppresses; LRP5 membranous recycling, B-catenin activation, CRC stemness and LA promotion of CRC, especially formation of large tumors, associated with CRC invasiveness; 3) ALOX15 loss of function (LOF) promotes large CRC tumor formation by azoxymethane in 12/15LOX-KO-12LOX (ALOX15-LOF) mice. Whether loss of ALOX15 expression promotes CRC invasiveness remains unknown. Our preliminary data show that ALOX15-LOF mice increased CRC invasiveness and targeted APC mutation into Lgr5+ colorectal stem cells induced CRC in the mice, which was blocked by transgenic ALOX15 expression. We therefore hypothesize that ALOX15 loss of function promotes CRC invasiveness by increasing PI3P_LA levels, which enhances LRP5 membranous recycling, thus potentiating Wnt/B-catenin signaling and subsequently stemness. Aim 1 will determine the effects of ALOX15 gain of function and ALOX15 LOF on LRP5, B-catenin activation, CRC stemness and invasiveness using CRC mouse models in which CRC invasiveness is promoted by either a combination of APC, KRASG12D and Trp53R172H mutations or Trp53R172H mutation with AOM induced B-catenin and KRAS mutations. Aim 2 will determine the effects of ALOX15 LOF on PI3P-LA, LRP5, B-catenin activation, stemness and invasiveness in human CRCs and examine the effects of ALOX15 re-expression via lentivirus Tet-on inducible system in human CRC-derived organoids on invasiveness in-vitro and in-vivo studies. The proposed studies are expected to provide important mechanistic insights into whether colonic ALOX15 expression as a host factor affects CRC invasiveness risk especially with high dietary LA intake. This gained knowledge could inform subjects with colorectal ALOX15 LOF to avoid high LA intake and spur development of interventions to target ALOX15 for re-expression to prevent invasive CRC.

结直肠癌是美国癌症死亡的第三大原因。我们的长期目标 研究的目的是开发新的干预措施来预防结直肠癌（CRC）。 CRC 侵袭性， CRC进展过程中的关键不利步骤，需要某些基因突变的组合（例如APC、 KRAS 和 Trp53 突变），这是驱动 CRC 的关键事件。然而，CRC 进展还需要 导致异常 β-连环蛋白 (B-连环蛋白) 激活高于 APC/B- 诱导水平的其他因素 连环蛋白突变。亚油酸 (LA)，最常食用的 omega-6 多不饱和脂肪酸 人类，增加了化学（AOM）和 APC 突变诱导的小鼠 CRC 肿瘤发生。 尽管如此，人类研究对于膳食 LA 对 CRC 的影响尚无定论。决心 了解 LA 在 CRC 中的作用非常重要，因为美国饮食中富含 LA，而主要表达 LA 代谢酶 15-脂氧合酶-1 (ALOX15) 在人类结直肠癌中缺失。最近，我们发现1） 高膳食水平的 LA 通过增加含有 LA 的磷脂酰肌醇 3-磷酸 (PI3P) 来促进结直肠癌 (PI3P_LA)，增加 LRP5 膜回收和随后的 B-连环蛋白激活； 2) ALOX15- PI3P_LA 向 PI3P_13-HODE 的诱导转化受到抑制； LRP5 膜回收，B-连环蛋白 CRC 的激活、CRC 干性和 LA 促进 CRC，特别是与 CRC 相关的大肿瘤的形成 侵入性； 3) ALOX15 功能丧失 (LOF) 通过氧化偶氮甲烷促进大结直肠癌肿瘤形成 12/15LOX-KO-12LOX (ALOX15-LOF) 小鼠。 ALOX15 表达缺失是否会促进 CRC 侵袭性 仍然未知。我们的初步数据表明 ALOX15-LOF 小鼠增加了 CRC 侵袭性并有针对性 APC 突变为 Lgr5+ 结直肠干细胞可诱导小鼠结直肠癌，但转基因技术可阻断该现象 ALOX15 表达。因此，我们假设 ALOX15 功能丧失通过以下方式促进 CRC 侵袭性： 增加 PI3P_LA 水平，从而增强 LRP5 膜回收，从而增强 Wnt/B-catenin 信号传递和随后的干性。目标 1 将确定 ALOX15 功能增益和 ALOX15 的影响 使用 CRC 小鼠模型对 LRP5、B-连环蛋白激活、CRC 干性和侵袭性进行 LOF，其中 CRC APC、KRASG12D 和 Trp53R172H 突变的组合或 Trp53R172H 会促进侵袭性 AOM 突变诱导 B-连环蛋白和 KRAS 突变。目标 2 将确定 ALOX15 LOF 的影响 对人类 CRC 中 PI3P-LA、LRP5、B-catenin 激活、干性和侵袭性的影响并检查其影响 通过慢病毒 Tet-on 诱导系统在人 CRC 衍生的类器官中重新表达 ALOX15 侵入性体外和体内研究。拟议的研究预计将提供重要的机制 深入了解结肠 ALOX15 表达作为宿主因素是否会影响 CRC 侵袭风险，尤其是 高膳食 LA 摄入量。所获得的知识可以告知患有结直肠 ALOX15 LOF 的受试者以避免高风险 LA 摄入并刺激针对 ALOX15 重新表达的干预措施的开发，以预防侵袭性 CRC。