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

 描述（由申请人提供）：亚油酸（LA）在人体中的消耗量很高。LA增加了氧化偶氮甲烷（AOM）诱导的啮齿动物结直肠肿瘤发生，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LA氧化代谢产生13-S-HODE（一种天然的PPAR-γ活化配体）的限速酶。PPAR-gamma抑制异常Wnt/B-连环蛋白信号传导，这是启动和驱动CRC肿瘤发生的关键事件。15-LOX-1在人类结直肠息肉和CRC中下调，但15-LOX-1对CRC风险的饮食LA调节的影响尚不清楚。在小鼠体内，我们发现转基因15-LOX-1表达抑制了CRC肿瘤的发生，并且在所有实验诱导的肿瘤中15-LOX-1表达下调。此外，在15-LOX-1-Gut小鼠中，用高LA饮食浓度的AOM诱导的CRC的增加被抑制;并且15-LOX-1抑制B-连环蛋白活化。因此，我们假设结肠上皮细胞中的15-LOX-1缺失对于过量LA通过增强Wnt/B-连环蛋白信号传导促进CRC肿瘤发生至关重要。我们将通过3个具体目的来检验这一假设：目的1和2：确定结肠上皮细胞中15-LOX-1表达（目的1）和表达缺失（目的2）对饮食LA促进CRC肿瘤发生和异常Wnt/B-连环蛋白信号传导的影响。为了实现目标1，我们将饲养15-LOX-1-Gut和Apc 580 mu小鼠，以产生Apc 580 mu-15-LOX-1-Gut小鼠，并检查喂食高或低LA含量饮食的小鼠中15-LOX-1表达对CRC的影响（肿瘤发生率和多样性），隐窝增殖区长度（Ki-67 IHC）、活化B-连环蛋白水平和Wnt/B-连环蛋白靶基因（c-Myc、细胞周期蛋白D1和Axin 2）mRNA水平。对于目标2，我们将替换12/15-LOX敲除小鼠的肠上皮细胞中的12-S-LOX以产生具有功能性15-LOX-1损失的小鼠，并检查喂食高或低LA含量饮食的小鼠对AOM诱导的CRC、隐窝增殖区长度和13- HODE水平的影响。我们还将评估15-LOX-1在人结肠癌性和正常类器官（从患者的结肠隐窝分离）中的功能获得（目的1）和下调（目的2）并与各种浓度的LA一起培养对13-HODE产生、细胞增殖、细胞分化和Wnt/B-连环蛋白信号传导的影响。目标3：确定结肠上皮细胞中15-LOX-1表达对CRC肿瘤发生的时间和空间效应与饮食LA摄入量的关系。我们将产生具有条件性15-LOX-1表达的小鼠，用AOM治疗它们，并给它们喂食高或低LA含量的饮食。15-LOX-1表达将在CRC肿瘤发生的开始或进展期间被诱导，并且我们将检查对目标1中测量的结果的影响。我们期望这些研究提供重要的新信息，指导开发新的CRC预防干预措施，并将15-LOX-1表达低的受试者确定为高LA摄入增加了CRC肿瘤发生的风险。

 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.