Abstract 600: Targeting the hedgehog pathway by LNA (locked nucleic acid) oligonucleotide-based GLI2 RNA antagonists, EZN-4482 and EZN-4496, in vitro and in vivo
Authors: Qu Zhengxing , Zhang Yixian , Shi Victoria , Zhu Peifang , Kim Steven , Castaneda Stephen , Wang Maoliang , Filpula David , Greenberger Lee M. , Horak Ivan D. 
Published: February 2011

Background: Aberrent activation of hedgehog (Hh) signaling has been implicated in the initiation and growth of a number of human malignancies, including those derived from skin, lung, liver, prostate and breast. The GLI family proteins are zinc finger transcription factors and the downstream effectors of Hh pathway, controling expression of the target genes such as GLI1, Ptch1, Cyclin D, and BCL2. Since amplification, overexpression of Hh ligand, and mutations of components along the Hh pathway are found in cancer and cancer stem cells, specific down regulation of GLIs with antisense molecules may offer a novel and effective therapeutic approach for cancer treatment. We report here the biological activities of LNA-based GLI2 mRNA antagonist, EZN-4482 and EZN-4496. Methods: In vitro, the ability of the antagonists to down modulate their direct target, GLI2 mRNA, and to inhibit cell growth was evaluated by qRT-PCR and MTS, respectively, in multiple cell lines of various origins. To identify and investigate the molecular events associated with targeted downmodulation of GLI2, expression of Hh pathway components and downstream effectors were analyzed by gene expression profiling and immnomoblotting analysis. In vivo, GLI2 mRNA down-modulation in PC3 xenograft tumor 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 are specific and potent down-modulators of GLI2 mRNA (by transfection, IC50 &lt; 2 nM). Down-modulation of GLI2 mRNA with or without transfection was shown to result in reduction of GLI2 protein in a human breast cancer cell line, HCC-1937, and human prostate cancer cell lines, 15PC3 and C4-2b. In mice bearing PC3 xenograft tumors, systemic administration of EZN-4482 resulted in down-modulation of GLI2, GLI1, and PTCH1 in both PC3 and host stromal cells. Additionally, we show that EZN-4482 prolonged the survival of animals with A549 cell derived liver metastasis derived from A549 cells. Conclusions: EZN-4482 and EZN-4496 are potent and specific GLI2 antagonists in vitro and in vivo. The possible mechanisms of efficacy in vivo may include inhibition of mRNA levels of GLI1/2 and PTCH1 in mouse stromal cells. Therefore, targeted down-modulation of GLI2 using the LNA-based mRNA antagonists may be an effective therapy to treat a broad spectrum of cancers including ones that fail treatment with SMO inhibitor therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 600.