Osteopontin (OPN) is a secreted, integrin binding and phosphorylated acidic glycoprotein which has an important role in tumor development including adhesion, invasion and metastasis. High OPN expression in the primary tumour is associated with early metastasis and poor outcome, in human beast and other cancers. Forced OPN overexpression in benign cells may induce neoplastic-like cell behaviour including increased attachment and invasion in vitro as well as the ability to metastasise in vivo. Conversely, OPN inhibition by antisense cDNA impedes cell growth and tumour forming capacity. In this study, subtractive hybridization (SSH) is utilized suppressive to evaluate OPN activated gene expression, using the Rama 37 (R37) rat mammary cell line and a subclone rendered invasive and metastatic by stable transfection with an expression vector for OPN. Sequence and expression array analysis of the respective cDNA libraries of over 1600 subtracted cDNA fragments revealed 982 ESTs, 45 novel sequences and 659 known genes. RAN GTPase (RAN) was one of the genes highly expressed in association with OPN. Here we show that transfection of noninvasive R37 cells with an expression vector for RAN induced the invasive phenotype in vitro and metastasis in syngeneic rats characterized by mechanisms that were independent of OPN. Furthermore, transfection of invasive and metastatic R37-0PN cells with RAN specific siRNA inhibited invasion, anchorageindependent growth, adhesion to laminin and metastasis in vivo. This study identifies RAN GTPase as a novel effector of OPN mediated anchorageindependent growth, invasion and metastasis in a mammary epithelial model of cancer progression.