Abstract

The insulin-like growth factor (IGF) axis consists of two ligands (IGF-I and IGF-II), two receptors (IGF1R and IGF2R), as well as a family of closely related IGF binding proteins (IGFBP 1-6). It is generally considered that altered expression in IGF axis members is associated with the development of several cancers. Elevated expression of IGFBP-2 is directly correlated with both the in vitro aggressive behavior and clinical presentation of metastatic ovarian, breast, adrenal, and central nervous system cancers. However, a role, if any, for IGFBP-2 in metastatic androgen independent prostate cancer (AI PCa) is unknown. Additionally, while alterations in IGFBP-2 expression are associated with progression to AI PCa, published reports regarding the influence of androgen treatment on the most commonly utilized androgen sensitive (AS) PCa cell line, LNCaP are contradictory. Therefore, we undertook an analysis of first, the influence of androgen treatment on IGFBP-2 expression in LNCaP, and second, the role of IGFBP-2 in the metastatic nature of AI PCa. In summary, we report the following in regard to the regulation of IGFBP-2 expression in LNCaP human PCa cells following treatment with the synthetic androgen R1881: (1) R1881 treatment produces an initial increase in steady state levels of IGFBP-2 mRNA, followed by a time dependent decrease in steady state levels of IGFBP-2 mRNA, (2) R1881 treatment results in decreased levels of intracellular IGFBP-2 in Triton-x-100 soluble locales, as well as the increased nuclear localization of IGFBP-2, and (3) through the apparent action of an as yet unidentified serine protease, R1881 treatment results in the limited proteolysis of IGFBP-2 and a subsequent decrease in intact extracellular IGFBP-2. Furthermore, we demonstrate that (4) IGFBP-2 is a substrate for androgen-induced proteolysis in PCa cells with a functional androgen receptor (AR), (5) the ability of IGFBP-2 to undergo androgen-induced proteolysis is attenuated greatly with progression to AI and is inversely correlated with in vivo metastatic potential, and (6) androgen-induced IGFBP-2 proteolysis results in decreased binding to IGFs, and thus has functional consequences. In regard to the role of IGFBP-2 in the facilitation of the aggressive phenotype attributed to metastatic PCa, we show that IGFBP-2 treatment (7) increased LNCaP levels of phosphorylated focal adhesion kinase (FAK) in both a concentration and time dependent manner, (8) significantly increased the invasion of LNCaP cells, and (9) increased the migration of non-metastatic, poorly motile LNCaP cells. In summary, our results indicate that IGFBP-2 expression is repressed in AS LNCaP human PCa cells in an androgen containing environment, resulting in a significant decease in extracellular IGFBP-2 levels. Furthermore, our results indicate that decreases in secreted levels of IGFBP-2 achieved via extracellular proteolysis are lost with progression to AI, and this increase in extracellular IGFBP-2 may facilitate the metastatic behavior of AI PCa cells.