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Inhibition of VEGF Signaling Pathway and Metastasis

抑制 VEGF 信号通路和转移

基本信息

批准号：
7903568
负责人：
YI LU
金额：
$ 5.52万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-08-01 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7903568
关键词：
Adenoviruses Affect Air Sacs American Binding Binding Sites Biochemical Biological Assay Breast Cancer Cell Breast Cancer Treatment CDKN2A gene Cancer Cell Growth Cancer Etiology Cancer Patient Cause of Death Cell Extracts Cell Proliferation Cells Cessation of life Chloramphenicol O-Acetyltransferase Co-Immunoprecipitations Complementary DNA Complex DNA Development Disease Dorsal Down-Regulation Ectopic Expression Endothelial Cells Environment Enzyme-Linked Immunosorbent Assay Evaluation Failure Fatty acid glycerol esters Gene Expression Genes Genetic Transcription Growth Heterodimerization Human Immunoprecipitation Implant In Vitro Injection of therapeutic agent Intravenous Ligands Luciferases Lung Mammary Neoplasms Mammary gland Measures Mediating Messenger RNA Modality Modeling Molecular Mus Mutagenesis Neoplasm Metastasis Nude Mice Organ Patients Phosphorylation Phosphotransferases Plasmids Play Primary Neoplasm Protein Tyrosine Kinase Proteins Radiosurgery Recombinants Reporter Genes Research Personnel Reverse Transcriptase Polymerase Chain Reaction Role Second Primary Cancers Secondary to Signal Pathway Signal Transduction Site Tail Testing Tetanus Helper Peptide Tetracyclines Therapeutic Transcription Coactivator Transcriptional Activation Translations Tubular formation Tumor Angiogenesis Tumor Suppressor Genes Umbilical vein VEGFA gene Vascular Endothelial Growth Factor Receptor Vascular Endothelial Growth Factor Receptor-1 Vascular Endothelial Growth Factors Veins Western Blotting Woman Work analog angiogenesis cancer diagnosis chemotherapy effective therapy expression vector gel mobility shift assay gene therapy hypoxia inducible factor 1 in vivo in vivo Model intraperitoneal malignant breast neoplasm matrigel migration neoplastic cell neovascularization prevent programs promoter protein expression receptor receptor expression research study response tumor growth

项目摘要

Metastasis is a major cause of death of breast cancer (BCa) patients. Angiogenesis is a necessary step for breast cancer growth and progression. Signaling via vascular endothelial growth factor (VEGF) plays a vital role in tumor angiogenesis. Our working hypothesis is that p16 downregulates VEGF gene expression at the transcriptional level, thus inhibiting angiogenesis, which contributes to p16-mediated suppression in BCa growth and metastasis. AIM 1: To elucidate the mechanism by which p16 downregulates the VEGF signaling pathway. Aim 1.1. To confirm that p16 downrequlates VEGF at both mRNA and protein levels in BCa cells. Aim 1.2. To determine whether p16 downregulates VEGF gene expression at the transcriptional level. Aim 1.3. To determine the molecular mechanism by which p16 downregulatesVEGF expression at the transcriptional level. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator for the VEGF gene promoter. HIF-1 is composed of an inducible subunit, HIF-1a and a constitutively expressed subunit, HIF-1R. Heterodimerization of HIF-1a and HIF-1fi is required to form the transcriptionally active HIF-1. We hypothesize that p16 downregulates VEGF gene expression at the level of transcription. Binding of p16 to HIF-1a prevents HIF-1 transcriptional activity via decreased HIF-1a interaction with HIF-1II. We will use the following three subaims to test this hypothesis. 1.3.1. To determine whether p16 directly binds to HIF-1a and alters HIF-1a cellular localization. 1.3.2. To determine whether p16 blocks (i) the formation of the HIF-1 a and HIF-1li complex and (ii)HIF-1 binding activity to HRE (HIF-1 binding site) DMAregion. 1.3.3. To determine whether p16 specifically inhibits HIF-1a-induced VEGF transcription. Aim. 1.4. To determine whether p16 affects VEGF receptor expression and phosphorvlation. AIM 2: To determine p16's effects in suppression of breast tumor growth, angiogenesis and metastasis. Aim. 2.1. To determine whether p16 inhibits BCa cell growth in vitro. Aim. 2.2. To determine whether p16 inhibits BCa-induced angiogenesis. The effects of p16 on angiogenesis will be evaluated in both in vitro and in vivo angiogenic assays. Aim. 2.3. To determine whether p16 suppresses breast tumor metastasis in an in vivo model. BCa cells stably transfected with Tet-on regulated p16 expression vector will be administrated into mice by mammary fat pad injection (spontaneous metastasis model) and by tail vein injection (experimental metastasis model). The effects of inducible p16 expression on suppression of primary tumor growth and secondary tumor formation (lung metastases) will be evaluated in both models. The long-term objectives are (I) to clarify the mechanism of how p16 downregulates VEGF gene expression; and (II) to determine whether inhibition of tumor angiogenesis via downregulation of VEGF by p16 is an effective way to suppress BCa growth and metastasis.

转移是乳腺癌（BCa）患者死亡的主要原因。血管生成是一个必要的步骤，乳腺癌的生长和进展。通过血管内皮生长因子（VEGF）的信号转导在肿瘤的发生发展中起着至关重要的作用。在肿瘤血管生成中的作用我们的工作假设是，p16下调VEGF基因表达，转录水平，从而抑制血管生成，这有助于p16介导的抑制， BCa生长和转移。目的1：阐明p16下调细胞凋亡的机制。 VEGF信号通路。目标1.1。为了证实p16在mRNA和蛋白水平下调VEGF， BCa细胞水平。目标1.2。为了确定p16是否下调VEGF基因表达，转录水平。目标1.3。为了确定p16下调VEGF的分子机制，在转录水平表达。缺氧诱导因子-1（HIF-1）是一种转录激活因子， VEGF基因启动子。HIF-1由诱导型亚基HIF-1a和组成型亚基HIF-1a组成。表达的亚基HIF-1 R。HIF-1a和HIF-1fi的异源二聚化是形成HIF-1a和HIF-1fi的必需条件。转录活性HIF-1。我们假设p16下调VEGF基因的表达水平，转录。p16与HIF-1a的结合通过降低HIF-1a抑制HIF-1的转录活性与HIF-1 Ⅱ的相互作用我们将使用以下三个子目标来检验这一假设。1.3.1.以确定 p16是否直接与HIF-1a结合并改变HIF-1a的细胞定位。1.3.2.以确定是否 p16阻断（i）HIF-1 a和HIF-1 li复合物的形成和（ii）HIF-1与HRE（HIF-1）的结合活性结合位点）DMA区。1.3.3.确定p16是否特异性抑制HIF-1a诱导的VEGF 转录。瞄准1.4.确定p16是否影响VEGF受体表达和磷酸化。目的2：探讨p16基因在乳腺癌生长抑制、血管生成及肿瘤生长抑制中的作用。转移瞄准2.1.确定p16是否在体外抑制BCa细胞生长。瞄准2.2.以确定 p16是否抑制BCa诱导的血管生成。p16对血管生成的影响将在在体外和体内血管生成测定中。瞄准2.3.确定p16是否抑制乳腺肿瘤在体内模型中的转移。Tet-on调控的p16表达载体稳定转染的BCa细胞将通过乳房脂肪垫注射（自发转移模型）和尾部给药静脉注射（实验转移模型）。诱导型p16基因表达在抑制肿瘤细胞增殖中的作用在两种模型中评价原发性肿瘤生长和继发性肿瘤形成（肺转移）。本研究的远期目标是：（1）阐明p16下调VEGF基因表达的机制表达;和（II）确定是否通过VEGF的下调抑制肿瘤血管生成， p16是抑制BCa生长和转移的有效途径。