Molecular targeting of PPAR-delta in colon cancer

结肠癌中 PPAR-δ 的分子靶向

基本信息

批准号：
8091356
负责人：
Imad Shureiqi
金额：
$ 31.8万
依托单位：
UNIVERSITY OF TX MD ANDERSON CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8091356
关键词：
Agonist Angiogenic Factor Azoxymethane Binding Biological Assay Bladder Blood Vessels Breast Cancer Etiology Cancer cell line Cells Cessation of life Clinical Colon Carcinoma Colonic Adenoma Colonic Neoplasms Colonic Polyps Data Development Dinoprostone Disease Down-Regulation Dyslipidemias Electrophoretic Mobility Shift Assay Ensure Enzyme-Linked Immunosorbent Assay Epithelial Cells Event Extracellular Fluid Fluorescein Future Genetic Genetic Transcription Germ Lines Goals Growth Growth Factor Overexpression HCT116 Cells HT29 Cells Human Image Immunohistochemistry In Vitro Incidence Interleukin-8 Intervention Intestines Isothiocyanates Knock-out Label Lead Lectin Ligands Liposomes Liver Longitudinal Studies Luciferases Lung Magnetic Resonance Imaging Malignant Neoplasms Malignant neoplasm of pancreas Metastatic Neoplasm to the Liver Modeling Molecular Molecular Target Morbidity - disease rate Mus Neoplasm Metastasis Nude Mice Null Lymphocytes PECAM1 gene PPAR delta Perfusion Pharmaceutical Preparations Pharmacologic Substance Polymerase Chain Reaction Pre-Clinical Model Process Production Protein Secretion Proteins Publishing Relative (related person)Reporting Research Resistance Role Safety Signal Pathway Small Interfering RNA Staging Stromal Cells System Testing Theoretical model Therapeutic Intervention Therapy Evaluation Time Tumor Angiogenesis United States Up-Regulation VEGFA gene Vascular Endothelial Growth Factors Xenograft procedure adenoma angiogenesis base cancer cell cancer therapy chemotherapy chromatin immunoprecipitation clinical efficacy colon cancer cell line density improved in vivo inhibitor/antagonist knock-down liver xenograft mRNA Expression mortality mouse model neoplastic cell novel strategies overexpression pre-clinical promoter public health relevance success therapeutic target treatment strategy tumor tumor growth tumorigenesis

项目摘要

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. PUBLIC HEALTH RELEVANCE: Increased production of the protein peroxisome proliferator-activated receptor delta (PPAR-d) is associated with colon cancer development; however, the molecular mechanisms involved need to be clarified. The proposed project aims to improve understanding of the molecular mechanisms by which PPAR-d contributes to aspects of colon cancer development, especially the ability of tumors to make new blood vessels. This improved understanding is expected to help determine whether PPAR-d would be a suitable molecular target for development of new drugs to treat 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。血管生成对于结肠肿瘤的发生至关重要。过氧化物酶体增殖物激活受体 δ (PPAR-d) 在人类结肠癌中表达上调。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 激动剂对癌细胞系和肠道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APCMin 小鼠。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。我们的初步数据表明，靶向肠道 PPAR-d KO 深度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 缺失的 HCT-116 (PPAR-d-KO) 细胞中 PPAR-d 重新表达恢复了其形成肝转移的能力，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 中的肿瘤发生以及 PPAR-d、VEGF 和 IL-8 表达。我们假设结肠癌细胞中 PPAR-d 过表达上调 VEGF 和 IL-8 表达，从而促进肿瘤血管生成和肿瘤发生。目的 1 是通过检查 PPAR-d-KO 细胞中 PPAR-d 重新表达对血管生成标记物的影响，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 表达。例如 CD31 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 VEGF 和 IL-8 表达（通过定量 RT-PCR 检测 mRNA）蛋白质通过 ELISA）在裸鼠中进行。目标 2 是通过检查脂质体 PPAR-d 的作用，确定通过体内系统性递送脂质体 PPAR-d siRNA 至结肠癌细胞来敲低 PPAR-d 是否足以抑制肿瘤发生和血管生成并下调 VEGF 和 IL-8 表达。 siRNA 对血管生成、肿瘤发生（裸鼠中 HCT-116 和 HT-29 细胞形成的肝和肺转移）以及 PPAR-d、VEGF 和IL-8 表达。目标3是通过在裸鼠中检查PPAR-d-KO细胞中VEGF过表达对血管生成和肿瘤发生的影响来确定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 重新表达促进的血管生成和肿瘤发生的影响；并比较 PPAR-d 和 VEGF 下调（通过脂质体 siRNA）对 HCT-116 和 HT-29 细胞血管生成以及肝和肺转移形成的影响。目标 4 是确定 IL-8 在 PPAR-d 促进血管生成和肿瘤发生中的作用，就像特定目标 3 中对 VEGF 所做的那样。在目标 3 和 4 中，我们还将研究 PPAR-d 是否与 VEGF 和 IL-8 结合在野生型 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 来增强启动子的转录PPAR-d 表达、PPAR-d 过表达或 PPAR-d-KO。验证假设可能会导致开发 PPAR-d 靶向疗法来抑制肿瘤发生。公共卫生相关性：蛋白质过氧化物酶体增殖物激活受体 δ (PPAR-d) 的产生增加与结肠癌的发展相关；然而，所涉及的分子机制需要澄清。该项目旨在加深对 PPAR-d 促进结肠癌发展的分子机制的理解，特别是肿瘤产生新血管的能力。这种加深的了解预计将有助于确定 PPAR-d 是否是开发治疗结肠癌新药的合适分子靶点。