Inhibition of Sp1 activity by a decoy PNA–DNA chimera prevents urokinase receptor expression and migration of breast cancer cells

Abstract

Sp1 regulates the activation of many genes involved in tumor growth, apoptosis, and angiogenesis. We have previously shown the involvement of Sp1 in the up-regulation of urokinase receptor (uPAR) expression, a key molecule in tumor invasion and metastasis. Here, we investigated whether a marked down-regulation of Sp1 activity may inhibit uPAR expression and migration ability of MDA-MB-231 breast cancer cells. To this end, we tested the decoy ability of a novel peptide nucleic acid (PNA)–DNA chimera which carries a central DNA strand, containing Sp1-binding sequence, covalently linked to two PNA fragments at both ends (PNA–DNA–PNA, PDP). The chimera was synthesized, annealed with complementary DNA (PDP–DNA), and then tested for its ability to bind Sp1 both in vitro and in living MDA-MB-231 breast cancer cells in the presence of urokinase (uPA). This PDP–DNA decoy molecule efficiently competes for the binding to endogenous Sp1 in nuclear extracts, and upon transfection with liposomal vectors, causes a marked decrease of available Sp1 in both untreated and uPA-treated MDA-MB-231 cells. Accordingly, both uPA-dependent enhancement of uPAR expression and cell migration were strongly reduced in transfected cells. Interestingly, a detectable inhibitory effect is also observed in breast cancer cells exposed to PDP–DNA in the absence of transfection reagents. Finally, the inhibitory effect of PDP–DNA appeared to be stronger than that observed with oligonucleotides carrying Sp1 consensus sequence. Our findings show that this novel PNA–DNA chimera, containing Sp1 consensus sequence, effectively inhibits Sp1 activity, uPAR expression, and motility of breast cancer cells indicating its potential therapeutic use to prevent tumor dissemination.