Inhibition of VEGF Signaling Pathway 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 乳腺癌的生长和发展。通过血管内皮生长因子(VEGF)的信号转导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下调血管内皮生长因子基因的表达。 转录水平，从而抑制血管生成，这有助于p16介导的抑制在 BCA的生长和转移。目的1：阐明p16下调血管内皮细胞生长的机制。 血管内皮生长因子信号通路。目标1.1。证实p16在mRNA和蛋白水平下调血管内皮生长因子 BCA细胞中的水平。目标1.2。确定p16是否下调血管内皮细胞生长因子基因的表达 转录水平。目标1.3。确定p16下调血管内皮生长因子的分子机制 在转录水平上的表达。缺氧诱导因子-1(HIF-1)是一种转录激活因子 血管内皮生长因子基因启动子。HIF-1由诱导性亚基HIF-1a和结构型HIF-1a组成 表达亚基HIF-1R。需要HIF-1a和HIF-1FI的异二聚化才能形成 转录活性HIF-1。我们假设p16在一定程度上下调了血管内皮生长因子基因的表达。 抄写。P16与HIF-1a结合通过降低HIF-1a抑制HIF-1转录活性 与HIF-1II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。要确定 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-1a和HIF-1li复合体的形成和(Ii)HIF-1与HRE(HIF-1)的结合活性 结合部位)。1.3.3。确定p16是否特异性抑制HIF-1a诱导的血管内皮细胞生长因子 抄写。瞄准。1.4.确定p16是否影响血管内皮生长因子受体的表达和磷酸化。 目的2：探讨p16‘S基因在抑制乳腺肿瘤生长、血管生成和肿瘤生长中的作用。 转移。瞄准。2.1.目的：探讨p16基因在体外对BCA细胞生长的抑制作用。瞄准。2.2.要确定 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试验。瞄准。2.3.确定p16是否抑制乳腺肿瘤 体内模型中的转移。Tet-on调控的p16表达载体稳定转染BCA细胞 将通过乳房脂肪垫注射(自发转移模型)和尾部注射给药 静脉注射(实验性转移模型)。可诱导的p16基因表达对肿瘤细胞增殖抑制的影响 原发肿瘤生长和继发肿瘤形成(肺转移)将在这两个模型中进行评估。 其长期目标是：(1)阐明p16下调血管内皮生长因子基因的机制。 以及(Ii)确定是否通过下调血管内皮生长因子抑制肿瘤血管生成 P16是抑制BCA生长和转移的有效途径。

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.