ROLES OF NRP-1 IN BREAST CANCER DEVELOPMENT

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。血管生成开关是肿瘤进展的先决条件，也可能是获得侵袭性的先决条件。血管平滑肌细胞的动员是新血管形成的关键事件。研究表明，PDGF由包括乳腺在内的多种肿瘤细胞分泌，是血管平滑肌细胞运动的重要调节因子。最近，我们发现NRP-1在血管SMCs中的表达存在差异。然而，NRP-1在调节血管SMCs运动中的作用尚未完全确定。我们预计NRP-1可能有助于通过肿瘤细胞衍生的pdgf介导的血管SMCs的动员。为了验证这一假设，我们确定了PDGF是否与NRP-1物理相互作用以增强血管SMCs的迁移。western blot检测不同肿瘤细胞源性培养基中PDGF的分泌水平。为了确定PDGF的刺激作用，在Boyden室中，细胞在有或没有PDGF的基础培养基中生长，通过计数Giemsa染色的细胞来确定迁移。RT-PCR和western blot检测NRP-1在含PDGF或不含PDGF的血管SMCs中的表达。通过shRNA沉默NRP-1，证实了NRP-1在pdgf诱导迁移中的重要性。采用免疫沉淀/免疫印迹法检测PDGF与NRP-1的相互作用。研究表明，乳腺肿瘤细胞分泌的b型PDGF增强了血管SMCs的迁移和NRP-1的表达。shRNA沉默NRP-1可阻断pdgf诱导的SMCs迁移。b型PDGF与SMCs的NRP-1相互作用，发挥迁移反应。NRP-1是血管SMCs迁移过程中PDGF信号通路的重要组成部分。接下来，我们将确定与此事件相关的下游信号分子。特别是，我们将确定PDGF受体在PDGF- nrp -1介导的血管SMCs迁移中的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Angiogenic switch is a prerequisite for the tumor progression and presumably for the acquisition of an invasive property. Mobilization of vascular smooth muscle cells (SMCs) is the key event for new blood vessel formation. Studies have demonstrated that PDGF, secreted by various tumor cells including breast, is an important regulator of vascular smooth muscle cells motility. Recently, we have shown that NRP-1 expressed differentially in Vascular SMCs. However, the role of NRP-1 in regulation of vascular SMCs motility has not yet been fully established. We anticipate that NRP-1 may contribute in the mobilization of vascular SMCs that could be mediated through tumor cell-derived-PDGF. To test the hypothesis, we determined if PDGF physically interacts with NRP-1 to potentiate the migration of vascular SMCs. PDGF secretion level was determined by western blot analysis in different tumor cell-derived media. To determine the stimulatory effect of PDGF, cells were grown in basal media with or without PDGF in the Boyden chamber and migration was determined by counting the Giemsa stained cells. NRP-1 expression in vascular SMCs with or without PDGF was established by RT-PCR and western blot analysis. The importance of NRP-1 in PDGF-induced migration was established by silencing of NRP-1 using shRNA. Interaction of PDGF and NRP-1 was determined by immunoprecipitation/immunobloting. The studies indicate that breast tumor cells-secreted B-forms of PDGF augments the migration and NRP-1 expression in vascular SMCs. Silencing of NRP-1 by shRNA blocks the PDGF-induced migration of SMCs. B-forms of PDGF physically interacts with NRP-1 of SMCs to exert the migratory response. NRP-1 is a major component in PDGF signaling pathway in vascular SMCs migration. Next, we will determine the down-stream signaling molecules associated with this event. In particular, we will determine the role of PDGF receptors in PDGF-NRP-1 mediated vascular SMCs migration.