Genetic Down-regulation of Phosphoinositide 3-Kinase by Bikunin Correlates with Suppression of Invasion and Metastasis in Human Ovarian Cancer HRA Cells

Abstract

Using a cDNA microarray analysis, we previously found that exposure of a highly invasive ovarian cancer cell line HRA with bikunin, a Kunitz-type protease inhibitor, or bikunin gene overexpression markedly reduced phosphoinositide kinase (PI3K) p85 gene expression, demonstrating that PI3K may be a candidate bikunin target gene. To clarify how reduced levels of PI3K may confer repressed invasiveness, we transfected HRA cells with PI3K p85 antisense-oligodeoxynucleotide (AS-ODN) and compared the properties of the transfected cells with those of parental cells and sense (S)-ODN cells. We have also demonstrated previously that transforming growth factor-β1 (TGF-β1) stimulates urokinase-type plasminogen activator (uPA-dependent invasion and metastasis of HRA cells. Here, we show that 1) TGF-β1 induced a rapid increase of the PI3K activity that was accompanied by increased expression (5-fold) of the uPA mRNA; 2) pharmacological inhibition of PI3K or AS-PI3K ODN transfection inhibited TGF-β1-stimulated Akt phosphorylation; 3) both PI3K pharmacological inhibitors and forced expression of AS-PI3K ODN reduced TGF-β1-stimulated uPA mRNA and protein expression by ∼70% compared with controls; 4) concentrations of PI3K inhibitors, sufficient to inhibit uPA up-regulation, inhibited TGF-β1-dependent HRA cell invasion; 5) the AS-PI3K ODN cells had a decreased ability to invade the extracellular matrix layer as compared with controls; and 6) when the AS-PI3K ODN cells were injected intraperitoneally into nude mice, the mice developed smaller intraperitoneal tumors and showed longer survival. We conclude that PI3K plays an essential role in promoting uPA-mediated invasive phenotype in HRA cells. Our data identify a novel role for PI3K as a bikunin target gene on uPA up-regulation and invasion.