Inhibition of VEGF Signaling Pathway and Metastasis

抑制 VEGF 信号通路和转移

• 批准号: 7904012
• 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
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904012
• 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; 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

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。通过血管内皮生长因子（VEGF）信号传导在肿瘤血管生成中起着至关重要的作用。我们的工作假设是，p16在转录水平下下调了VEGF基因表达，从而抑制了血管生成，这有助于P16介导的BCA生长和转移抑制。目标1：阐明p16下调VEGF信号通路的机制。目标1.1。为了确认p16在BCA细胞中的mRNA和蛋白质水平上都降低了VEGF。目标1.2。确定p16是否在转录水平下下调VEGF基因表达。目标1.3。确定p16在转录水平下下调VEGF表达的分子机制。低氧诱导因子-1（HIF-1）是VEG​​F基因启动子的转录激活剂。 HIF-1由诱导型亚基HIF-1Alpha和组成型表达的亚基HIF-1Beta组成。需要HIF-1Alpha和HIF-1BETA的异二聚化以形成转录活性的HIF-1。我们假设p16在转录水平下下调了VEGF基因表达。 P16与HIF-1Alpha的结合通过降低HIF-1Alpha与HIF-1Beta的相互作用来防止HIF-1转录活性。我们将使用以下三个子iaim来检验这一假设。 1.3.1。确定p16是否直接与HIF-1Alpha结合并改变HIF-1Alpha细胞定位。 1.3.2。确定p16块（i）是否形成了HIF-1Alpha和HIF-1Beta复合物以及（II）HIF-1结合活性与HRE（HIF-1结合位点）DNA区域的形成。 1.3.3。确定p16是否特异性抑制了HIF-1Alpha诱导的VEGF转录。目的。 1.4。确定p16是否影响VEGF受体表达和磷光化。目标2：确定P16在抑制乳腺肿瘤生长，血管生成和转移中的影响。目的。 2.1。确定p16是否在体外抑制了BCA细胞的生长。目的。 2.2。确定p16是否抑制了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是否抑制体内模型中的乳腺肿瘤转移。用Tet-On调节的P16表达载体稳定转染的BCA细胞将通过乳腺脂肪垫注射（自发转移模型）和尾静脉注射（实验转移模型）将其施加到小鼠中。在这两种模型中，将评估诱导p16表达对原发性肿瘤生长和继发性肿瘤形成（肺转移）抑制的影响。长期目标是（i）阐明p16如何下调VEGF基因表达的机制； （ii）确定通过p16对VEGF下调抑制肿瘤血管生成是否是抑制BCA生长和转移的有效方法。