Abstract A249: Potent protein kinase inhibitors block Pim kinase-mediated increase in prostate epithelial cell migration, regulation of p27 protein half-life, and secretion of hepatocyte growth factor

Abstract

Abstract Pim-1 proto-oncogene encodes a serine/threonine protein kinase that regulates apoptosis, cell cycle progression, and transcription. The expression of this protein kinase is elevated in both prostate intraepithelial neoplasia (PIN) and prostatic adenocarcinoma, suggesting an important role for Pim-1 kinase in prostate cancer development and growth. To investigate the role of Pim-1 in controlling tumor growth we have synthesized novel benzylidene-thiazolidine2, 4-dione (J. Med. Chem. (2009) 52:74) inhibitors of this kinase. The most potent members of this chemotype have IC50s of 13 nM for Pim-1. To examine the activity of these agents in prostate cancer we have first set out to define the biochemical activity of Pim-1 in epithelial cells. We have expressed Pim-1 in a mouse prostate epithelial cell (MPECs) line that demonstrates stem cell characteristics. We find that Pim-1 expressing cells produce and secrete markedly increased levels of hepatocyte growth factor/scatter factor (HGF/SF), and this protein kinase stimulates increases in HGF/SF mRNA. Additionally, expression of Pim-1 markedly increases HGF/SF induced migration of MPECs. The contribution of Pim-1 kinase to this biochemical pathway is confirmed in murine embryonic fibroblasts (MEFs) that are deficient for all three Pim protein kinases (TKO) and evidence reduced level of HGF mRNA in TKO versus wild type MEFs. The Pim-1-stimulated migration of MPECs is inhibited by two benzylidene-thiazolidine-2, 4-diones, SMI-4a and 16a, as well as known inhibitors of the HGF receptor, c-Met. HGF treatment of MPECs induced p27 upregulation that could be inhibited by the expression of Pim-1, thus allowing cell cycle progression. Further studies showed that expression of Pim-1 did not alter the mRNA level of p27, but enhanced the cell cycle-dependent degradation and thus decreased the half-life of the p27 protein. Consistent with this finding, p27 ubiquitination assays showed that Pim-1 increases this modification in vivo. We found that the Pim-1-mediated ubiquitination is regulated by complex formation between Pim-1 and Skp2, a protein component of the SCF complex, which is known to regulate the ubiquitination and degradation of p27. Pim-1 does not affect Skp2's E3 ligase activity, but appears to inhibit the degradation of Skp2 through phosphorylation. Incubation of these cells with the Pim protein kinase inhibitor, SMI-4a, decreased Skp2 expression and increased p27 and cyclin E expression. Together our data demonstrates the complex pathway by which Pim-1 protein kinase regulates HGF/SF and p27 levels, thus controlling cell migration, proliferation, and potentially transformation. Potent Pim kinase inhibitors block these two signaling pathways thus inhibiting prostate epithelial cell migration and growth. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A249.