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

描述(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。值得注意的是，这些患者中有80%-90%出现骨转移。不幸的是，我们对骨转移是如何启动的理解是模糊的。我们的数据显示，内皮(E)-选择素的配体在骨转移的PCa细胞上上调并发挥作用，而其他组的研究表明，b1和b3整合素对应于PCa细胞对骨髓内皮细胞(BMEC)的亲和力和转移潜能。由于骨归巢的造血干细胞也利用E-选择素配体和整合素进行黏附和跨内皮细胞向骨髓(BM)的迁移，我们和其他人认为类似的分子谱系对于PCa细胞的TEM是必要的。目的/假设：我们假设循环中的PCa细胞通过与E-选择素的黏附与BMEC黏附，然后通过b1和b3整合素黏附分子与BMEC牢固黏附和穿透。我们的目的是确定PCa细胞如何通过b1和b3整合素在BMEC E-选择素上滚动导致PCa细胞通过b1和b3整合素进入BM。具体目的：(1)探讨E-选择素配体和b1、b3整合素在前列腺癌细胞透射电子显微镜中的作用；(2)探讨E-选择素配体和b1、b3整合素在前列腺癌细胞体内向骨迁移中的作用。研究设计：(1)构建表达E-选择素配体并表达b1和b3整合素异二聚体的PCa细胞，即E-选择素配体+/b1+/b3+细胞，在生理性血流条件下用最先进的迁移分析方法进行分析。控制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，我们将使用高灵敏的PCR检测方法和活体动物的生物发光成像来检测表达E-选择素配体和表达b1和b3整合素的PCa细胞的迁移。控制PCa细胞系、阻断抗体、RNAi、小分子拮抗剂和代谢抑制剂也将被用于控制E-选择素配体/整合素的功能。调节E-选择素配体表达的信号通路将通过RT-PCR、磷酸化特异性抗体和药物抑制剂来评估；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-选择素配体调节因子-1，3岩藻糖基转移酶7在整合素激活中的作用将通过流式细胞仪和E-选择素细胞结合分析来评估。癌症相关性：确定PCA细胞如何进入骨骼对公共健康和NIH的使命至关重要，因为我们的研究结果可能会导致减少与转移相关的死亡的治疗策略。 公共卫生相关性：这些研究将有助于确定实体肿瘤细胞转移到重要器官的关键步骤，但尚未得到充分认识和描述。确定PCA细胞如何进入骨骼对公共健康和NIH的使命至关重要，因为我们的研究结果可能会导致治疗策略，可能会大大减少与转移相关的死亡。

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.