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.
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.
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