Inhibition of VEGF Signaling Pathway and Metastasis

抑制 VEGF 信号通路和转移

基本信息

批准号：
7460670
负责人：
YI LU
金额：
$ 18.85万
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/7460670
关键词：
ARNT gene 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 HIF1A gene 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 Purpose 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 Tetracycline Tetracyclines Therapeutic Today 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 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

DESCRIPTION (provided by applicant): 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 downregulates VEGF 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-1alpha and a constitutively expressed subunit, HIF-1beta. Heterodimerization of HIF-1alpha and HIF-1beta 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-1alpha prevents HIF-1 transcriptional activity via decreased HIF-1alpha interaction with HIF-1beta. We will use the following three subaims to test this hypothesis. 1.3.1. To determine whether p16 directly binds to HIF-1alpha and alters HIF-1alpha cellular localization. 1.3.2. To determine whether p16 blocks (i) the formation of the HIF-1alpha and HIF-1beta complex and (ii) HIF-1 binding activity to HRE (HIF-1 binding site) DNA region. 1.3.3. To determine whether p16 specifically inhibits HIF-1alpha-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.

描述（由申请人提供）：转移是乳腺癌（BCa）患者死亡的主要原因。血管生成是乳腺癌生长和进展的必要步骤。血管内皮生长因子 (VEGF) 信号传导在肿瘤血管生成中发挥着至关重要的作用。我们的工作假设是，p16 在转录水平下调 VEGF 基因表达，从而抑制血管生成，从而有助于 p16 介导的 BCa 生长和转移抑制。目的1：阐明p16下调VEGF信号通路的机制。目标 1.1。证实 p16 在 BCa 细胞中的 mRNA 和蛋白质水平上下调 VEGF。目标 1.2。确定 p16 是否在转录水平下调 VEGF 基因表达。目标 1.3。确定 p16 在转录水平下调 VEGF 表达的分子机制。缺氧诱导因子-1 (HIF-1) 是 VEGF 基因启动子的转录激活剂。 HIF-1 由诱导型亚基 HIF-1α 和组成型表达亚基 HIF-1β 组成。 HIF-1α 和 HIF-1β 的异二聚化是形成具有转录活性的 HIF-1 所必需的。我们假设 p16 在转录水平下调 VEGF 基因表达。 p16 与 HIF-1alpha 的结合通过减少 HIF-1alpha 与 HIF-1beta 的相互作用来阻止 HIF-1 转录活性。我们将使用以下三个子目标来检验这个假设。 1.3.1.确定 p16 是否直接结合 HIF-1α 并改变 HIF-1α 细胞定位。 1.3.2.确定 p16 是否阻断 (i) HIF-1alpha 和 HIF-1beta 复合物的形成以及 (ii) HIF-1 与 HRE（HIF-1 结合位点）DNA 区域的结合活性。 1.3.3.确定 p16 是否特异性抑制 HIF-1α 诱导的 VEGF 转录。目的。 1.4.确定 p16 是否影响 VEGF 受体表达和磷酸化。目标 2：确定 p16 在抑制乳腺肿瘤生长、血管生成和转移方面的作用。目的。 2.1.确定 p16 是否在体外抑制 BCa 细胞生长。目的。 2.2.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体外和体内血管生成测定中进行评估。目的。 2.3.确定 p16 是否在体内模型中抑制乳腺肿瘤转移。将稳定转染Tet-on调节的p16表达载体的BCa细胞通过乳腺脂肪垫注射（自发转移模型）和尾静脉注射（实验性转移模型）给予小鼠。将在两种模型中评估诱导型 p16 表达对抑制原发性肿瘤生长和继发性肿瘤形成（肺转移）的影响。长期目标是（一）阐明p16下调VEGF基因表达的机制； (II)确定p16通过下调VEGF来抑制肿瘤血管生成是否是抑制BCa生长和转移的有效方法。