Combination of an SRC kinase inhibitor with a novel pharmacological antagonist of the urokinase receptor diminishes in vitro colon cancer invasiveness.

Abstract

The urokinase-type plasminogen activator receptor (u-PAR) contributes to colon cancer invasion and metastases. We have shown previously that u-PAR expression in colon cancer is driven by the Src tyrosine kinase. In the current study, we determined the ability of PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a Src kinase inhibitor, to reduce u-PAR expression and colon cancer invasion. Western blotting, Northern blotting, and u-PAR promoter-reporter assays were performed to determine whether PP2 represses u-PAR expression. In vitro invasion assays were used to determine whether this kinase inhibitor, with or without a novel u-PAR antagonist, diminished cultured colon cancer invasiveness. A constitutively active c-Src increased in vitro invasiveness of SW480 cells, whereas HT-29 cells expressing antisense c-Src showed diminished invasiveness, validating c-Src as a target for low molecular weight compound(s). The Src inhibitor PP2 reduced u-PAR transcription in HT-29 cells over the concentration range that blocked Src kinase activity. PP2 also reduced u-PAR protein amounts in three other colon cancer cell lines with modest to high constitutive Src activity. Treatment of HT-29 cells and 2C8 cells (a SW480 clone expressing a constitutively active Src) with PP2 diminished their in vitro invasiveness. Furthermore, combination of the Src inhibitor with a novel u-PAR peptide antagonist (NI-5.12) proved superior to the individual agents in suppressing invasiveness. A c-Src kinase inhibitor represses u-PAR expression and, alone or in combination with a u-PAR antagonist, diminishes colon cancer invasiveness. Thus, concurrent targeting of c-Src expression and pharmacological blockade of the u-PAR may represent a novel means of controlling colon cancer spread.