Molecular targeting of PPAR-delta in colon cancer

结肠癌中 PPAR-δ 的分子靶向

• 批准号: 7987654
• 负责人: Imad Shureiqi
• 金额: $ 32.79万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 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/7987654
• 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; Fluoresceins; 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; 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; Reverse Transcriptase Polymerase Chain Reaction; 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

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(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。血管生成在结肠肿瘤的发生中起关键作用。PPAR-d在人类结肠癌中表达上调。关于胚系PPAR-d基因敲除(KO)对APCM小鼠肠道肿瘤发生的影响，以及PPAR-d激动剂对APCM小鼠癌细胞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形成的影响仍有待确定。我们的初步数据显示，靶向肠道PPAR-d KO可显著抑制偶氮甲烷诱导的小鼠结肠肿瘤形成和血管内皮生长因子的表达；PPAR-d缺失的HCT-116(PPAR-d-KO)细胞恢复了其形成肝转移的能力，并增强了血管内皮生长因子和白细胞介素8(IL-8)的表达；脂质体PPAR-d siRNA抑制了小鼠的肿瘤形成，并抑制了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上调了VEGF和IL-8的表达，从而促进了肿瘤的血管生成和肿瘤的发生。目的1通过检测PPAR-d-d在PPAR-d-KO细胞中的重新表达对裸鼠体内血管生成标记物(如CD31免疫组织化学、FITC凝集素分析)、肿瘤形成(肝和肺转移形成)以及血管内皮生长因子和白介素8表达(定量RT-PCR检测和蛋白检测)的影响，以确定PPAR-d在结肠癌细胞中的特异性表达是否促进肿瘤的发生和血管生成，并上调血管内皮生长因子和白介素8的表达。目的2通过检测脂质体PPAR-d siRNA对血管生成、肿瘤形成(HCT-116和HT-29细胞在裸鼠体内形成肝和肺转移)以及PPAR-d、VEGF和IL-8表达的影响，确定通过系统地将PPAR-d siRNA导入结肠癌细胞体内是否足以抑制肿瘤的形成和血管生成，并下调血管内皮生长因子和IL-8的表达。目的3通过在裸鼠体内检测PPAR-d-KO细胞中高表达的血管内皮生长因子对血管生成和肿瘤形成的影响；评价血管内皮生长因子脂质体-siRNA敲除对PPAR-d-KO细胞中PPAR-d重新表达促进血管生成和肿瘤形成的影响；比较PPAR-d和血管内皮生长因子下调(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，以确定血管生成和肿瘤发生的机制。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，就像在特定目标3中所做的那样。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实验、EMSA和CHIP/Real-time PCR来研究PPAR-d是否与VEGF和IL-8启动子结合以增强其转录。验证这一假设可能会导致开发PPAR-d靶向疗法来抑制肿瘤的形成。 公共卫生相关性：过氧化物酶体增殖物激活受体增量蛋白(PPAR-d)的增加与结肠癌的发生有关；然而，涉及的分子机制需要澄清。这项拟议的项目旨在提高对PPAR-d在结肠癌发展方面的分子机制的理解，特别是肿瘤生成新血管的能力。这一更好的理解有望帮助确定PPAR-d是否会成为开发治疗结肠癌新药的合适分子靶点。