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年夺去了28660名美国男性的生命。值得注意的是，80% - 90%的患者出现骨转移。不幸的是，我们对骨转移如何开始的理解是不明确的。我们的数据显示，内皮(E)选择素的配体在骨转移性PCa细胞上被上调并发挥作用，而其他研究小组已经表明b1和b3整合素与骨髓内皮细胞（BMEC）的PCa细胞亲和力和转移潜力相对应。由于骨归巢造血干细胞也利用e-选择素配体和整合素进行粘附和跨内皮迁移（TEM）到骨髓（BM），我们和其他人认为类似的分子库对于PCa细胞的TEM是必要的。目的/假设：我们假设循环PCa细胞通过E-选择素粘附BMEC，然后通过b1和b3整合素粘附分子牢固粘附并穿过BMEC。我们的目标是确定PCa细胞在BMEC e -选择素上滚动是如何通过b1和b3整合素导致PCa细胞TEM转化为BM的。(1)研究e -选择素配体和b1、b3整合素在PCa细胞TEM中的作用；(2)探讨e -选择素配体和b1、b3整合素在体内PCa细胞向骨迁移中的作用。研究设计：(1)对表达e-选择素配体和b1、b3整合素异源二聚体（e-选择素配体+/b1+/b3+ PCa细胞）的PCa细胞进行TEM分析，采用最先进的生理血流条件下迁移实验。对照PCa细胞系，阻断抗体、RNAi、小分子拮抗剂和代谢抑制剂将用于控制e -选择素配体/整合素的功能。(2)在实验性骨转移模型中，我们将采用高灵敏度PCR检测方法和活体生物发光成像技术，检测表达e -选择素配体和表达b1和b3整合素的PCa细胞的迁移情况。对照PCa细胞系，阻断抗体、RNAi、小分子拮抗剂和代谢抑制剂也将用于控制e -选择素配体/整合素的功能。通过RT-PCR、磷酸化特异性抗体和药物抑制剂评估PCa细胞中调节e -选择素配体表达的信号通路；e -选择素配体和整合素之间的物理相互作用将通过免疫沉淀来确定；e -选择素配体调节剂a1,3聚焦转移酶7在整合素激活中的作用将通过流式细胞术和e -选择素细胞结合试验进行评估。癌症相关性：确定前列腺癌细胞如何进入骨骼对公众健康和NIH的使命至关重要，因为我们的研究结果可能导致减少转移相关死亡的治疗策略。