Abstract C144: A locked nucleic acid antisense oligonucleotode against androgen receptor, down-modulates target mRNA and causes antitumor effects in xenograft models of prostate cancer
Authors: Sapra Puja , Zhang Yixian , Castaneda Stephen , Kim Steven , Kraft Patricia , Bandaru Raj , Greenberger Lee M. , Horak Ivan D. 
Published: June 2012

Abstract
Background: Androgen-deprivation therapies remain the first line treatment for prostate cancer. Despite an excellent initial response, most of these patients will succumb to the castration resistant form of the disease. Interestingly, even in the presence of castration levels of circulating androgens, these tumors are still dependent on a functional androgen receptor (AR). Therefore, inhibition of AR expression, rather than systemic androgen deprivation, may provide a novel strategy for treatment of advanced prostate cancer. We have developed a novel locked nucleic acid (LNA)-based antisense oligonucleotide (ASO), EZN-4176, that silence AR and is associated with tumor growth inhibition.
Methods: Target mRNA and protein knockdown, growth inhibition, and apoptosis induction effects of EZN-4176 were evaluated by qRT-PCR, western blot analysis, ELISA, MTS assay, and caspase3/7 activity assay, respectively, in AR-positive cancer cell lines (LNCaP, 22RV1) or AR-negative cell line (PC3) in the presence or absence of lipofection (transfection reagent). In vivo, therapeutic efficacy of EZN-4176 was evaluated in AR-positive CWR22 (androgen dependent) and 22Rv1 (castration refractory) tumor models.
Results: EZN-4176 resulted in potent down-modulation of AR mRNA or protein (IC50 &lt;5 nM) when transfected in LNCaP or 22Rv1 cells. Consequently, cell growth was inhibited (IC50 &lt; 10 nM), and caspase 3/7 activity was significantly increased. Additionally, the PSA mRNA and protein levels were significantly decreased after EZN-4176 treatment (IC50 &lt; 5 nM). The biological effects of EZN-4176 were specific as the scrambled LNA-ASO did not down regulate target mRNA, nor were inhibitory effects on growth observed with EZN-4176 in an AR-negative cell line (PC3). In non-transfected cells, EZN-4176 but not the scrambled LNA-ASO down-regulated AR and inhibited the growth of DHT-induced LNCaP cells. In vivo, dose-dependent reduction of AR mRNA (up to 80%) in the liver was observed following intravenous administration of the EZN-4176. Additionally, EZN-4176 treatment dose-dependently inhibited AR (up to 40%), PSA (up to 85%) and TMPRSS2 (up to 50%), mRNA in, 22Rv1 or CWR22 tumor xenografts. In CWR22 tumor xenografts, EZN-4176 treatment also resulted in significant tumor growth inhibition (TGI) (∼55%) (similar to high-dose Casodex® [bicalutamide] treatment). TGI and mRNA knockdown effects in xenografts were specific as scrambled LNA-ASO resulted in no to insignificant response and EZN-4176 had no TGI in PC3 xenograft model. Cy5.5-labeled EZN-4176 was shown to localize in subcutaneous CWR22 or LnCaP tumor xenografts using xenogen imaging system.
Conclusions: Our data suggest that EZN-4176 is a potent LNA-based ASO of AR that causes target down-modulation and TGI effects in vivo and hence may provide a novel strategy to treat advanced prostate cancer.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C144.