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Tissue Factor splicing and pancreatic tumor progression: pilot studies

组织因子剪接和胰腺肿瘤进展：初步研究

基本信息

批准号：
8508892
负责人：
VLADIMIR BOGDANOV
金额：
$ 19.26万
依托单位：
UNIVERSITY OF CINCINNATI
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-11 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8508892
关键词：
Adenocarcinoma Cell Animals Apoptosis Cancer Etiology Cell Adhesion Molecules Cell Line Cells Cessation of life Coagulants Correlative Study Coupled Data Disease Progression Doxycycline Duct (organ) structure Endothelial Cells Endothelium Evaluation Gene Structure Genes Growth Hemostatic function Human Image In Situ In Vitro Incidence Infiltration Integral Membrane Protein Integrins Introns Label Laboratories Length Lesion Ligation Light Malignant Neoplasms Malignant neoplasm of pancreas Mediating Membrane Methodology Modeling Mus Nature Neoplasm Metastasis Pancreas Pancreatic Ductal Adenocarcinoma Phosphatidylserines Pilot Projects Primary Neoplasm Process Proteins Publishing RNA Splicing Research Resected SCID Mice Serine Protease Signal Pathway Signal Transduction Solid Specimen Subfamily lentivirinae Techniques Testing Thromboplastin Time Tissues Tumor Biology Tumor-Derived Up-Regulation Variant Vascularization Vesicle Western World angiogenesis autocrine base cancer cell density fluorophore in vivo indexing innovation mRNA Precursor macrophage monocyte neoplastic cell new technology novel strategies overexpression pancreatic cancer cells pancreatic neoplasm pancreatic tumorigenesis response small hairpin RNA tumor tumor growth tumor progression

项目摘要

DESCRIPTION (provided by applicant): We will ascertain whether (over)expression of alternatively spliced Tissue Factor (asTF) promotes pancreatic tumorigenesis, and whether a decrease in asTF expression suppresses pancreatic tumorigenesis. High TF activity has been long observed in pancreatic ductal adenocarcinoma (PDAC). Until recently, the functional significance of high TF expression in PDAC and other forms of solid cancer was thought to be inseparable from the proteolytic function of full-length TF, an integral membrane protein that serves as a co-factor for the serine protease VIIa. We found that asTF - the secreted alternatively spliced TF variant - non-proteolytically activates cancer-associated signaling pathways via ligation of integrin ?6?1, the major contributor to primary tumor growth and metastatic spread in PDAC. ?6?1 expression on PDAC cells correlates with the rate of metastases; thus, asTF-?6?1interactions may enhance metastatic spread in an angiogenesis-independent, cell-autonomous fashion. Our newest data indicate that asTF protein is abundant in PDAC lesions, but not normal ducts, and promotes monocyte recruitment via upregulation of cell adhesion molecules on endothelial cells. Compounds selectively targeting asTF may have a major impact on cancer progression while having minimal impact on hemostasis. We will perform in vitro studies, as well as the orthotopic model in athymic nude/SCID mice with WT/low levels on endogenous TF and human PDAC cell lines expressing asTF. To evaluate the effects of elevated asTF expression, we will generate subclones of PDAC cells (over)expressing asTF in response to doxycycline. To evaluate the effects of lowering asTF levels, we will transduce PDAC cells with a lentivirus expressing a highly effective and specific anti-asTF shRNA. In vivo imaging of cancer cells and tumor vessels will be carried out at regular intervals; the animals wil be sacrificed, tissue specimens collected, and the degree of vascularization / microvessel density / monocyte infiltration evaluated along with the proliferation and apoptosis indices. The central objectives of the analysis: 1. Following doxycycline administration, i) do we observe enhanced cancer progression and/or decreased survival? ii) Is the primary tumor growth augmented? iii) Is there an increased incidence of metastases? 2. Does a decrease in asTF levels inhibit tumor growth and/or spread? To explore the possibility that the levels of tumor asTF correlate with disease progression, clinicopathological studies utilizing resected PDAC tissue will be performed. Our findings will comprise a qualitative gain in the understanding of asTF's contribution to the pathobiology of pancreatic cancer. To generate the asTF-(over)expressing PDAC cells, we will employ an innovative mini-gene developed in our laboratory. In vivo studies will be carried out using cutting-edge real-time imaging methodology - labeled SapC(H2)-DOPS vesicles. SapC-conjugated vesicles present advantage over other methodologies as they selectively target tumor vasculature and phosphatidylserine-enriched tumor cells, and allow rapid in vivo evaluation while being fully compatible with conventional immunohistochemical techniques.

描述（由申请人提供）：我们将确定选择性剪接的组织因子（asTF）的（过）表达是否促进胰腺肿瘤发生，以及asTF表达的降低是否抑制胰腺肿瘤发生。长期以来，在胰腺导管腺癌（PDAC）中观察到高TF活性。直到最近，高TF表达在PDAC和其他形式的实体癌中的功能意义被认为与全长TF的蛋白水解功能不可分割，全长TF是一种整合的膜蛋白，其充当丝氨酸蛋白酶VIIa的辅因子。我们发现，asTF -分泌的选择性剪接的TF变体-非蛋白水解激活癌症相关的信号通路，通过连接整合素？六个？1，PDAC中原发性肿瘤生长和转移性扩散的主要贡献者。?六个？PDAC细胞上的TF-1表达与转移率相关，因此，asTF-？六个？1相互作用可能以不依赖血管生成、细胞自主的方式增强转移扩散。我们的最新数据表明，asTF蛋白是丰富的PDAC病变，但不是正常的管道，并促进单核细胞募集通过上调内皮细胞上的细胞粘附分子。选择性靶向asTF的化合物可能对癌症进展有重大影响，而对止血的影响最小。我们将对内源性TF和表达asTF的人PDAC细胞系进行体外研究，以及WT/低水平无胸腺裸/SCID小鼠的原位模型。为了评估升高的asTF表达的影响，我们将产生响应于多西环素而（过）表达asTF的PDAC细胞的亚克隆。为了评估降低asTF水平的效果，我们将用表达高效特异性抗asTF shRNA的慢病毒感染PDAC细胞。将定期进行癌细胞和肿瘤血管的体内成像;处死动物，收集组织样本，并沿着增殖和凋亡指数评价血管化/微血管密度/单核细胞浸润的程度。分析的主要目的：1。多西环素给药后，i）我们是否观察到癌症进展加快和/或生存期降低？（二）原发性肿瘤生长是否增加？iii）转移的发生率是否增加？2. asTF水平的降低是否抑制肿瘤生长和/或扩散？为了探索肿瘤asTF水平与疾病进展相关的可能性，将利用切除的PDAC组织进行临床病理学研究。我们的研究结果将包括一个定性的增益的理解asTF的贡献胰腺癌的病理生物学。为了产生asTF-（过）表达PDAC细胞，我们将采用我们实验室开发的创新的迷你基因。将使用尖端的实时成像方法-标记的SapC（H2）-DOPS囊泡进行体内研究。SapC缀合的囊泡呈现出优于其他方法的优势，因为它们选择性地靶向肿瘤脉管系统和富含磷脂酰丝氨酸的肿瘤细胞，并且允许快速体内评价，同时与常规免疫组织化学技术完全兼容。