Modeling PCa Bone Metastasis: Dual Role of E-selectin Ligands and Integrins

PCa 骨转移建模：E-选择素配体和整合素的双重作用

• 批准号: 8474863
• 负责人: Steven Russell Barthel
• 金额: $ 6.04万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-24 至 2013-05-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8474863
• 关键词: Adherence; Adhesions; American; Animals; Avidity; Binding; Biological Assay; Bioluminescence; Blocking Antibodies; Blood; Blood Circulation; Blood flow; Bone Marrow; Cell Adhesion Molecules; Cells; Cessation of life; Comprehension; Data; Detection; E-Selectin; Endothelial Cells; Goals; Hematopoietic; Hematopoietic stem cells; Homing; Image; Immigration; Immunoprecipitation; Integrins; Knowledge; Lead; Life; Ligands; Link; Malignant Neoplasms; Malignant neoplasm of prostate; Mediating; Metabolic; Metastatic Neoplasm to the Bone; Metastatic Prostate Cancer; Metastatic to; Methods; Migration Assay; Mission; Modeling; Molecular; Movement; Mus; Neoplasm Metastasis; Organ; PC3 cell line; Patients; Phospho-Specific Antibodies; Physiological; Proteins; Public Health; Publications; Published Comment; Publishing; RNA Interference; Research Design; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Signal Transduction; Solid Neoplasm; Suggestion; Testing; Time; United States National Institutes of Health; Up-Regulation; base; bone; cancer cell; cellular engineering; cytokine; galactoside 3-fucosyltransferase; hemodynamics; imaging modality; in vivo; inhibitor/antagonist; innovation; men; migration; neoplastic cell; novel; public health relevance; small molecule; treatment strategy

DESCRIPTION (provided by applicant): Metastatic prostate cancer (PCa) claimed the lives of 28,660 American men in 2008. Of note, 80- 90% of these patients presented with bone metastasis. Unfortunately, our comprehension of how bone metastasis is initiated is ill-defined. Our data shows that ligands for endothelial (E)-selectin are upregulated and functional on bone-metastatic PCa cells, while other groups have shown that b1 and b3 integrins correspond to PCa cell avidity for bone marrow endothelial cells (BMEC) and to metastatic potential. Since bone-homing hematopoietic stem cells also utilize E-selectin ligands and integrins for adherence and transendothelial migration (TEM) into bone marrow (BM), we and others believe that a similar molecular repertoire is necessary for TEM of PCa cells. Objective/Hypothesis: We hypothesize that circulating PCa cells adhere to BMEC through adhesion to E- selectin and then firmly adhere to and traverse BMEC via b1 and b3 integrin adhesion molecules. Our objective is to determine how PCa cell rolling on BMEC E-selectin causes PCa cell TEM into BM via b1 and b3 integrins. Specific Aims: (1) To investigate the role of E-selectin ligands and b1 and b3 integrins in TEM of PCa cells; (2) To investigate the role of E-selectin ligands and b1 and b3 integrins in the migration of PCa cells into bone in vivo. Study Design: (1) TEM of PCa cells engineered to express E-selectin ligand and expressing b1 and b3 integrin heterodimers, E-selectin ligand+/b1+/b3+ PCa cells, will be analyzed using state-of-the-art migration assays under physiologic blood flow conditions. Control PCa cell lines, blocking antibodies, RNAi, small molecule antagonists and metabolic inhibitors will be employed to control for E-selectin ligand/integrin function. (2) In an experimental bone metastasis model, we will assay the migration of PCa cells engineered to express E-selectin ligand and expressing b1 and b3 integrins using a highly sensitive PCR detection method and bioluminescence imaging of live animals. Control PCa cell lines, blocking antibodies, RNAi, small molecule antagonists and metabolic inhibitors will also be employed to control for E-selectin ligand/integrin function. Signaling pathways regulating E-selectin ligand expression in PCa cells will be assessed by RT-PCR, phospho-specific antibodies and pharmacologic inhibitors; physical interaction between E-selectin ligands and integrins will be determined by immunoprecipitation; role of an E-selectin ligand regulator, a1,3 fucosyltransferase 7, in integrin activation will be assessed by flow cytometric and E-selectin cell binding assays. Cancer relevance: Determining how PCa cells enter bone is vital to public health and to the mission of the NIH, as results from our studies could lead to treatment strategies that would diminish metastasis-related deaths. PUBLIC HEALTH RELEVANCE: These studies will help define a critical, yet under-appreciated and ill-characterized step in the metastasis of a solid tumor cell into a vital organ. Determining how PCa cells enter bone is vital to public health and to the mission of the NIH as results from our studies could lead to treatment strategies that might greatly diminish metastasis- related deaths.

描述（由申请人提供）：转移性前列腺癌（PCA）在2008年夺走了28,660名美国男性的生命。不幸的是，我们对如何启动骨转移的理解是错误的定义。我们的数据表明，内皮（E） - 选择蛋白的配体上调并在骨替代PCA细胞上起作用，而其他组则表明B1和B3整合素对应于骨髓内皮细胞（BMEC）和转移性潜力的PCA细胞的伴侣。由于骨骼造血干细胞还利用E-选择蛋白配体和整联蛋白来依从性和跨内皮迁移（TEM）进入骨髓（BM），因此我们和其他人认为，类似的分子库对于PCA细胞的TEM是必需的。客观/假设：我们假设通过粘附到e-selectin的粘附，然后通过B1和B3整合蛋白粘附分子牢固地粘附BMEC并牢固地遵循BMEC。我们的目标是确定BMEC E-选择蛋白上的PCA细胞滚动如何通过B1和B3整合素导致PCA细胞TEM进入BM。具体目的：（1）研究e-选择蛋白配体以及B1和B3整联蛋白在PCA细胞中的作用； （2）研究E-选择蛋白配体，B1和B3整合素在PCA细胞迁移到体内骨骼中的作用。研究设计：（1）将使用最新的生理血流条件下的最先进的迁移分析，将分析旨在表达E-选择蛋白配体和表达B1和B3整合蛋白异二聚体的PCA细胞的TEM，并表达B3整合素异二聚体，E-选择蛋白配体+/B1+/B3+PCA细胞。将使用对照PCA细胞系，阻断抗体，RNAi，小分子拮抗剂和代谢抑制剂来控制E-选择蛋白配体/整合素功能。 （2）在实验性骨转移模型中，我们将使用高度敏感的PCR检测方法和活动物的生物发光成像来测定用于表达E-选择蛋白配体和表达B1和B3整合蛋白的PCA细胞的迁移。还将使用控制PCA细胞系，阻断抗体，RNAi，小分子拮抗剂和代谢抑制剂来控制E-选择蛋白配体/整合素功能。 RT-PCR，磷酸特异性抗体和药理学抑制剂将评估调节PCA细胞中E-选择蛋白配体表达的信号通路；电子选择配体和整联蛋白之间的物理相互作用将由免疫沉淀确定。 E-选择蛋白配体调节剂A1,3岩藻糖基转移酶7在整联蛋白激活中的作用将通过流式细胞仪和E-选择素细胞结合测定法进行评估。癌症相关性：确定PCA细胞如何进入骨骼对公共卫生和NIH的使命至关重要，因为我们的研究的结果可能导致治疗策略会减少与转移相关的死亡。 公共卫生相关性：这些研究将有助于定义一个关键但被低估的且特征不足的步骤，使实体瘤细胞转移为重要器官。确定PCA细胞如何进入骨骼对公共卫生和NIH的使命至关重要，因为我们的研究结果可能导致治疗策略可能会大大减少与转移相关的死亡。