15-LOX-1 Modulation of Colon Cancer Promotion by Linoleic Acid

 描述(申请人提供)：亚油酸(LA)在人类中的消耗量很高。LA增加了偶氮甲烷(AOM)诱导的啮齿动物结直肠癌的发生，但LA对人类结直肠癌(CRC)的影响尚不清楚。15-脂氧合酶-1(15-LOX-1)是乳酸氧化代谢生成PPAR-γ的天然激活配体13-S-HODE的限速酶。PPAR-γ抑制Wnt/B-catenin信号的异常，Wnt/B-catenin是启动和驱动CRC肿瘤发生的关键事件。15-LOX-1在人类大肠息肉和癌组织中表达下调，但15-LOX-1对饮食LA调节结直肠癌风险的影响尚不清楚。在肠道靶向人15-LOX-1(15-LOX-1-Gut)的小鼠中，我们发现转基因15-LOX-1表达抑制了结直肠癌的发生，并且15-LOX-1在所有实验诱导的肿瘤中表达下调。此外，高LA饮食浓度的AOM诱导的结直肠癌在15-LOX-1-肠道小鼠中被抑制；15-LOX-1抑制B-连环蛋白的激活。因此，我们假设，结肠上皮细胞中15-LOX-1的丢失对于过量LA通过增强Wnt/B-catenin信号促进结直肠癌的发生至关重要。我们将通过三个特定的目标来验证这一假设：目标1和目标2：确定结肠上皮细胞中15-LOX-1的表达(目标1)和表达缺失(目标2)对饮食LA促进结直肠癌发生和Wnt/B-catenin信号异常的影响。第一，我们将培育15-LOX-1-Gut和Apc580-Mu小鼠，产生Apc580-15-LOX-1-Gut小鼠，并检测15-LOX-1在饲喂高或低LA饲料的小鼠中的表达对肿瘤发生率和多发性(CRC)、隐窝增殖区长度(Ki-67 IHC)、活化的B-catenin蛋白水平和Wnt/B-catenin靶基因(c-Myc、Cyclin D1和Axin2)mRNA水平的影响。对于目的2，我们将替换12/15-LOX基因敲除小鼠肠上皮细胞中的12-S-LOX，以产生功能性15-LOX-1缺失的小鼠，并观察高或低LA含量饮食对AOM诱导的结直肠癌小鼠、隐窝增殖区长度和13-HODE水平的影响。我们还将评估15-LOX-1功能增强(目标1)和下调(目标2)在人结肠癌和正常器官(从患者的结肠隐窝分离)中的作用，并与不同浓度的LA培养对13-HODE的产生、细胞增殖、细胞分化和Wnt/B-catenin信号转导的影响。目的：研究15-LOX-1在结肠上皮细胞中的表达与膳食LA摄入量对结直肠癌发生的时间和空间效应。我们将产生条件性15-LOX-1表达的小鼠，用AOM治疗它们，并给它们喂高或低LA含量的饮食。15-LOX-1的表达将在结直肠癌发生的起始或进展过程中被诱导，我们将检验对AIM 1中测量的结果的影响。我们期望这些研究将提供重要的新信息，指导用于预防结直肠癌的新干预措施的开发，并确定15-LOX-1低表达的受试者具有高LA摄入量增加结直肠癌发生的风险。

 DESCRIPTION (provided by applicant): Linoleic acid (LA) consumption is high in humans. LA increases azoxymethane (AOM)-induced colorectal tumorigenesis in rodents, but the impact of LA on colorectal cancer (CRC) in humans is unknown. 15- Lipoxygenase-1 (15-LOX-1) is the rate-limiting enzyme for the oxidative metabolism of LA to generate 13-S- HODE, a natural activating ligand of PPAR-gamma. PPAR-gamma suppresses aberrant Wnt/B-catenin signaling, a critical event initiating and driving CRC tumorigenesis. 15-LOX-1 is downregulated in human colorectal polyps and CRCs, but the influence of 15-LOX-1 on dietary LA modulation of CRC risk is unknown. In mice with intestinally targeted human 15-LOX-1 (15-LOX-1-Gut), we found that transgenic 15-LOX-1 expression suppressed CRC tumorigenesis, and 15-LOX-1 expression was downregulated in all experimentally-induced tumors. Furthermore, the increase in AOM-induced CRC with high LA dietary concentrations was repressed in 15-LOX-1-Gut mice; and 15-LOX-1 inhibited B-catenin activation. We therefore hypothesize that 15-LOX-1 loss in colonic epithelial cells is critical for excess LA to promote CRC tumorigenesis via augmenting Wnt/B-catenin signaling. We will test this hypothesis via 3 specific aims: Aims 1 and 2: Determine the effects of 15-LOX-1 expression (aim 1) and loss of expression (aim 2) in colonic epithelial cells on dietary LA promotion of CRC tumorigenesis and aberrant Wnt/B-catenin signaling. For aim 1, we will breed 15-LOX-1-Gut and Apc580mu mice to generate Apc580mu-15-LOX-1-Gut mice and examine the effects of 15-LOX-1 expression in mice fed high- or low-LA-content diets on CRC (tumor incidence and multiplicity), crypt proliferative zone length (Ki-67 IHC), activated B-catenin protein levels, and Wnt/B-catenin target gene (c-Myc, Cyclin D1, and Axin2) mRNA levels. For aim 2, we will replace 12-S-LOX in intestinal epithelial cells of 12/15-LOX knockout mice to generate mice with functional 15-LOX-1 loss and examine the effects in mice fed high- or low-LA-content diets on AOM-induced CRC, crypt proliferative zone length, and 13- HODE levels. We will also evaluate the effect of 15-LOX-1 gain of function (aim 1) and downregulation (aim 2) in human colonic cancerous and normal organoids (isolated from patients' colonic crypts) and cultured with various concentrations of LA on 13-HODE generation, cell proliferation, cell differentiation, and Wnt/B-catenin signaling. Aim 3: Determine the tempora and spatial effects of 15-LOX-1 expression in colonic epithelial cells on CRC tumorigenesis in relation to dietary LA intake. We will generate mice with conditional 15-LOX-1 expression, treat them with AOM, and feed them high- or low-LA-content diets. 15-LOX-1 expression will be induced during initiation or progression of CRC tumorigenesis, and we will examine the effects on the outcomes measured in aim 1. We expect these studies to provide important new information to direct development of novel interventions for CRC prevention and to identify subjects with low 15-LOX-1 expression as having increased risk of CRC tumorigenesis with high LA intake.