Abstract C146: LNA (locked nucleic acid) oligonucleotide based GLI2 RNA antagonist EZN-4482 effectively inhibited tumor growth and increased survival of animals with liver metastasis
Authors: Zhang Yixian , Sapra Puja , Kim Steven , Zhu Peifang , Qu Zhengxing , Castaneda Stephen , Wang Maoliang , Filpula David , Greenberger Lee M. , Horak Ivan D. 
Published: June 2012

Abstract
Background: The Hedgehog (Hh) pathway has been implicated in the growth of a number of human malignancies and cancer stem cells. In the presence of Hh ligands, PTCH1 inhibition of a membrane protein, Smoothened (SMO), is relieved. SMO activation triggers a cascade of downstream events that ultimately activate the zinc finger transcription factors GLI2 and GLI3 and induce transcription of their target genes such as GLI1, PTCH1, Cyclin D, and BCL2. Emerging evidence suggests that GLI1 and GLI2 represent the main activators of Hh pathway due to amplification, overexpression of Hh ligand, and mutations of components along the Hh pathway. Therefore, specific down regulation of GLIs with RNA antagonists such as siRNA and antisense molecules may offer an effective therapeutic approach for cancer treatment. We reveal here the biological activities of LNA-based GLI2 mRNA antagonist, EZN-4482 and 4496.
Material and Methods: In vitro, the ability of the antagonists to down modulate mRNA and cell growth was evaluated by qRT-PCR and MTS respectively. In vivo, GLI2 mRNA down-modulation in tumors, which were grown on the flank of nude mice, was evaluated after intravenous administration of the antagonists. The effect of antagonists on tumor growth and survival of mice were evaluated in PC3 prostate tumor grown on the flank and in A549 lung carcinoma cells that had metastasized to the liver respectively.
Results: Both antagonists, when transfected into tumor cells, were potent down-modulators of GLI2 mRNA (IC50 ≤ 2 nM), resulting caspase ativation and growth inhibition. Interestingly, we found that without any transfection reagent, 1–10 micromolar concentrations of these antagonists were able to down modulate GLI2 mRNA and inhibit the growth of multiple cell lines while their scrambled control oligonucleotides showed no effect, suggesting that the effect was specific. In mice, EZN-4482 (3 mg/kg given q3d x4) down modulated GLI2 mRNA in PC3 prostate tumors. Moreover, using murine specific probes, we found EZN-4482 also inhibited GLI1/2 and PTCH1 in the same tumors. Moderate tumor growth inhibition was found with EZN-4482 in the PC3 xenograft model. Additionally, we show that EZN-4482 prolonged the survival of animals with liver metastasis derived from A549 cells.
Conclusions: GLI2 antagonists potently and specifically inhibited GLI2 mRNA expression and tumor growth in two tumor models. The possible mechanisms of efficacy include inhibition of mRNA levels of GLI1/2 and PTCH1 in mouse stromal cells. While small molecule inhibitors of SMO are being evaluated clinically, this approach is limited to tumors where pathway activation is upstream of GLIs or tumors that are resistant to such inhibitors. Therefore, a GLI2 RNA antagonist may be an effective therapy to treat a broad spectrum of cancers including ones that fail treatment with SMO inhibitor therapy.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C146.