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

结直肠癌是美国癌症死亡的第三大原因。我们的长期目标是 研究的目的是开发新的干预措施，以防止结直肠癌（CRC）。CRC侵袭性，a CRC进展过程中的关键不利步骤，需要某些基因突变（例如APC， KRAS和Trp 53突变），这是驱动CRC的关键事件。然而，CRC进展还需要 增加异常β-连环蛋白（B-连环蛋白）活化超过由APC/B- 连环蛋白突变亚油酸（LA），最常消耗的ω-6多不饱和脂肪酸， 人，增加化学（AOM）和APC突变诱导的小鼠CRC肿瘤发生。 尽管如此，关于饮食LA对CRC的影响，人类研究尚未得出结论。测定 LA在CRC中的作用是重要的，因为美国人的饮食富含LA，而主要的 LA的代谢酶15-脂氧合酶-1（ALOX 15）在人CRC中丢失。最近，我们发现1） 高饮食水平的LA通过增加含LA的磷脂酰肌醇3-磷酸（PI 3 P）促进CRC (PI3P_LA），其增加LRP 5膜再循环和随后的B-连环蛋白活化; 2）ALOX 15- PI3P_LA向PI3P_13-HODE的诱导转化抑制; LRP 5膜再循环，B-连环蛋白 活化、CRC干性和LA促进CRC，尤其是与CRC相关的大肿瘤的形成 侵袭性; 3）ALOX 15功能丧失（LOF）促进由氧化偶氮甲烷引起的大CRC肿瘤形成， 12/15 LOX-KO-12 LOX（ALOX 15-LOF）小鼠。ALOX 15表达缺失是否促进CRC侵袭 仍然未知。我们的初步数据表明，ALOX 15-LOF小鼠增加了CRC侵袭性，并靶向了 APC突变为Lgr 5+结直肠干细胞诱导小鼠结直肠癌，转基因阻断 ALOX 15表达。因此，我们假设ALOX 15功能丧失通过以下途径促进CRC侵袭性： 增加PI3P_LA水平，其增强LRP 5膜再循环，从而增强Wnt/B-连环蛋白 信号和随后的干性。目标1将确定ALOX 15功能增益和ALOX 15 LOF对LRP 5、B-连环蛋白激活、CRC干性和侵袭性的影响，使用CRC小鼠模型，其中CRC APC、KRASG 12 D和Trp 53 R172 H突变的组合或Trp 53 R172 H突变的组合促进侵袭性。 AOM突变诱导B-连环蛋白和KRAS突变。目标2将确定ALOX 15 LOF的作用 对PI 3 P-LA、LRP 5、B-连环蛋白活化、干性和侵袭性的影响，并检查 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ALOX 15， 体外和体内侵袭性研究。预计拟议的研究将提供重要的机制 了解结肠ALOX 15表达作为宿主因子是否影响CRC侵袭风险，尤其是 高膳食LA摄入量。获得的知识可以告知患有结直肠ALOX 15 LOF的受试者， LA摄入和刺激干预措施的发展，以靶向ALOX 15重新表达，以预防侵袭性CRC。

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.