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

描述（由申请人提供）：2008年，转移性前列腺癌（PCa）夺去了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开始的理解是不明确的。我们的数据表明，内皮（E）-选择素的配体上调和功能的骨转移性前列腺癌细胞，而其他小组已经表明，b1和b3整合素对应于骨髓内皮细胞（BMEC）的前列腺癌细胞亲合力和转移潜力。由于骨归巢造血干细胞也利用E-选择素配体和整合素粘附和跨内皮迁移（TEM）到骨髓（BM），我们和其他人认为，一个类似的分子库是必要的PCa细胞的TEM。目的/假设：我们假设循环PCa细胞通过粘附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细胞如何滚动BMEC E-选择素导致PCa细胞TEM进入BM通过b1和b3整合素。具体目标：（1）探讨E-选择素配体和整合素b1、b3在PCa细胞TEM中的作用;（2）探讨E-选择素配体和整合素b1、b3在PCa细胞体内向骨迁移中的作用。研究设计：（1）在生理血流条件下，使用最先进的迁移测定法分析经工程改造以表达E-选择素配体并表达b1和b3整联蛋白异源二聚体的PCa细胞（E-选择素配体+/b1+/b3+ PCa细胞）的TEM。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/整联蛋白功能。(2)在实验性骨转移模型中，我们将使用高灵敏度PCR检测方法和活体动物的生物发光成像来测定经工程化以表达E-选择素配体并表达b1和b3整联蛋白的PCa细胞的迁移。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/整联蛋白功能。将通过RT-PCR、磷酸化特异性抗体和药理学抑制剂评估调节PCa细胞中E-选择素配体表达的信号通路;将通过免疫沉淀法确定E-选择素配体和整联蛋白之间的物理相互作用;将通过流式细胞术和E-选择素细胞结合试验评估E-选择素配体调节剂α 1，3岩藻糖基转移酶7在整联蛋白活化中的作用。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，因为我们的研究结果可能会导致减少转移相关死亡的治疗策略。 公共卫生相关性：这些研究将有助于确定实体瘤细胞转移到重要器官中的关键，但未被充分认识和表征的步骤。确定前列腺癌细胞如何进入骨骼对公共卫生和国家卫生研究院的使命至关重要，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.