Molecular targeting of PPAR-delta in colon cancer

描述（由申请人提供）：我们的长期目标是帮助开发新的分子靶向结肠癌治疗方法。血管生成是结肠肿瘤发生的关键。过氧化物酶体增殖物激活受体- δ (PPAR-d)在人结肠癌中表达上调。关于种系PPAR-d基因敲除（KO）对APCMin小鼠肠道肿瘤发生的影响以及PPAR-d激动剂对APCMin小鼠肿瘤细胞系和肠腺瘤血管内皮生长因子（VEGF）表达的影响，已发表的数据不一致。癌细胞中PPAR-d上调对血管生成和肿瘤发生的影响仍有待确定。我们的初步数据显示，靶向肠道PPAR-d KO深刻抑制偶氮甲烷诱导的小鼠结肠肿瘤发生和VEGF表达；PPAR-d在PPAR-d缺失的HCT-116 （PPAR-d- ko）细胞中的重新表达恢复了它们形成肝转移的能力，并增强了VEGF和白细胞介素-8 （IL-8）的表达；脂质体PPAR-d siRNA抑制小鼠HCT-116的肿瘤发生和PPAR-d、VEGF和IL-8的表达。我们假设PPAR-d在结肠癌细胞中的过表达可上调VEGF和IL-8的表达，从而促进肿瘤血管生成和肿瘤发生。目的1：通过检测PPAR-d- ko细胞中PPAR-d再表达对裸鼠血管生成标志物（如CD31免疫组化、fitc凝集素测定）、肿瘤发生（肝和肺转移形成）、VEGF和IL-8表达（定量RT-PCR mRNA、ELISA蛋白）的影响，确定PPAR-d在结肠癌细胞中的特异性表达是否促进肿瘤发生和血管生成，并上调VEGF和IL-8的表达。目的2是通过检测PPAR-d siRNA脂质体对血管生成、肿瘤发生（裸鼠HCT-116和HT-29细胞形成肝和肺转移）以及PPAR-d、VEGF和IL-8表达的影响，确定PPAR-d通过体内向结肠癌细胞全身递送PPAR-d siRNA敲低PPAR-d是否足以抑制肿瘤发生和血管生成，下调VEGF和IL-8表达。目的3：通过裸鼠实验检测PPAR-d- ko细胞中VEGF过表达对血管生成和肿瘤发生的影响，确定VEGF在PPAR-d促进血管生成和肿瘤发生中的作用；评估VEGF脂质体sirna敲低对PPAR-d- ko细胞中PPAR-d再表达促进血管生成和肿瘤发生的影响；比较PPAR-d和VEGF下调（通过脂质体siRNA）对HCT-116和HT-29细胞血管生成和肝肺转移形成的影响。目的4是确定IL-8在PPAR-d促进血管生成和肿瘤发生中的作用，就像在特定的目的3中对VEGF所做的那样。在Aims 3和4中，我们还将研究PPAR-d是否与VEGF和IL-8启动子结合以增强其转录，在野生型PPAR-d表达、PPAR-d过表达或PPAR-d- ko的HCT- 116细胞中使用VEGF和IL-8启动子荧光素酶缺失构建体、EMSA和ChIP/real-time PCR。证实这一假设可能会导致开发ppar -d靶向治疗来抑制肿瘤发生。

DESCRIPTION (provided by applicant): Our long-term goal is to help develop new molecularly targeted colon cancer treatments. Angiogenesis is critical for colonic tumorigenesis. The peroxisome proliferator-activated receptor-delta (PPAR-d) is upregulated in human colon cancer. Published data are discordant on the effects of germ line PPAR-d genetic knock out (KO) on intestinal tumorigenesis in APCMin mice and the effects of PPAR-d agonist on vascular endothelial growth factor (VEGF) expression in cancer cell lines and intestinal adenomas of APCMin mice. The impact of PPAR-d upregulation in cancer cells on angiogenesis and tumorigenesis remains to be determined. Our preliminary data show that targeted intestinal PPAR-d KO profoundly inhibited azoxymethane-induced murine colonic tumorigenesis and VEGF expression; PPAR-d re-expression in PPAR-d null HCT-116 (PPAR-d-KO) cells restored their ability to form liver metastases and enhanced VEGF and interleukin-8 (IL-8) expression; and liposomal PPAR-d siRNA inhibited tumorigenesis and PPAR-d, VEGF, and IL-8 expression in HCT-116 in mice. We hypothesize that PPAR-d overexpression in colon cancer cells upregulates VEGF and IL-8 expression to promote tumor angiogenesis and tumorigenesis. Aim 1 is to determine if PPAR-d specific expression in colon cancer cells promotes tumorigenesis and angiogenesis and upregulates VEGF and IL-8 expression, by examining the effects of PPAR-d re-expression in PPAR-d-KO cells on angiogenesis markers (e.g. CD31 immunohistochemistry; FITC-lectin assay), tumorigenesis (liver and lung metastasis formation), and VEGF and IL-8 expression (mRNA by quantitative RT-PCR, protein by ELISA) in nude mice. Aim 2 is to determine whether PPAR-d knockdown via systemic delivery of liposomal PPAR-d siRNA to colon cancer cells in vivo is sufficient to inhibit tumorigenesis and angiogenesis and downregulate VEGF and IL-8 expression, by examining the effects of liposomal PPAR-d siRNA on angiogenesis, tumorigenesis (liver and lung metastasis formation by HCT-116 and HT-29 cells in nude mice), and PPAR-d, VEGF, and IL-8 expression. Aim 3 is to determine VEGF role in PPAR-d promotion of angiogenesis and tumorigenesis, by examining in nude mice the effects of VEGF overexpression in PPAR-d-KO cells on angiogenesis and tumorigenesis; assessing the effects of VEGF liposomal-siRNA knockdown on angiogenesis and tumorigenesis promoted by PPAR-d re-expression in PPAR-d-KO cells; and comparing the effects of PPAR-d and VEGF downregulation (via liposomal siRNA) on angiogenesis and liver and lung metastasis formation by HCT-116 and HT-29 cells. Aim 4 is to determine IL-8 role in PPAR-d promotion of angiogenesis and tumorigenesis, as done for VEGF in specific Aim 3. In Aims 3 and 4, we will also investigate if PPAR-d binds to the VEGF and IL-8 promoters to enhance their transcription, using VEGF and IL-8 promoter-luciferase deletion construct assays, EMSA, and ChIP/real-time PCR in HCT- 116 cells with wild-type PPAR-d expression, PPAR-d overexpression, or PPAR-d-KO. Confirmation of the tested hypothesis could lead to developing PPAR-d-targeted therapy to inhibit tumorigenesis.