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）在人结肠癌中上调。关于生殖系PPAR-d基因敲除（KO）对APCMin小鼠肠肿瘤发生的影响以及PPAR-d激动剂对APCMin小鼠癌细胞系和肠腺瘤中血管内皮生长因子（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PPAR-d KO显著抑制氧化偶氮甲烷诱导的小鼠结肠肿瘤发生和VEGF表达; PPAR-d无效HCT-116中PPAR-d的再表达（PPAR-d-KO）细胞恢复其形成肝转移的能力，并增强VEGF和白细胞介素-8（IL-8）表达;脂质体PPAR-d siRNA可抑制HCT-116小鼠肿瘤的发生及PPAR-d、VEGF和IL-8的表达。我们推测，PPAR-d在结肠癌细胞中的过表达上调VEGF和IL-8的表达，以促进肿瘤血管生成和肿瘤发生。目的1是通过检测PPAR-d-KO细胞中PPAR-d再表达对血管生成标志物的影响，确定结肠癌细胞中PPAR-d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（例如，CD 31免疫组织化学; FITC-凝集素测定），肿瘤发生（肝和肺转移形成）以及裸小鼠中VEGF和IL-8的表达（定量RT-PCR检测mRNA，ELISA检测蛋白质）。目的2是通过检查脂质体PPAR-d siRNA对血管生成、肿瘤发生、肿瘤转移和肿瘤转移的影响，确定通过脂质体PPAR-d siRNA全身递送至结肠癌细胞体内的PPAR-d敲低是否足以抑制肿瘤发生和血管生成并下调VEGF和IL-8表达。（HCT-116和HT-29细胞在裸鼠中的肝和肺转移形成）和PPAR-d、VEGF和IL-8表达。目的3通过在裸鼠中检测VEGF在PPAR-d-KO细胞中过表达对血管生成和肿瘤发生的影响，评估VEGF脂质体-siRNA敲低对PPAR-d-KO细胞中PPAR-d再表达促进的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。比较PPAR-d和VEGF下调（通过脂质体siRNA）对HCT-116和HT-29细胞的血管生成以及肝和肺转移形成的影响。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，如在特定目的3中对VEGF所做的那样。在目标3和4中，我们还将在野生型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测定、EMSA和ChIP/实时PCR研究PPAR-d是否与VEGF和IL-8启动子结合以增强其转录。验证的假设可能导致开发PPAR-d靶向治疗以抑制肿瘤发生。

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.