Reduced Expression of the Androgen Receptor by Third Generation of Antisense Shows Antitumor Activity in Models of Prostate Cancer

Abstract

The androgen receptor (AR) is a member of a unique class of transcription factors because it contains a ligand-binding domain that, when activated, results in nuclear translocation and the transcriptional activation of genes associated with prostate cancer development. Although androgen deprivation therapies are effective initially for the treatment of prostate cancer, the disease eventually relapses and progresses to castrationresistant prostate cancer (CRPC). Nonetheless, the AR still plays a critical role because late-stage investigational agents that deplete testosterone (abiraterone) or block ligand binding (MDV3100) can still control tumor growth in patients with CRPC. These findings indicate that downmodulation of AR expression may provide a complementary strategy for treating CRPC. In this article, we describe a novel, locked, nucleic acid-based antisense oligonucleotide, designated EZN-4176. When administered as a single agent, EZN-4176 specifically downmodulated AR mRNA and protein, and this was coordinated with inhibition of the growth of both androgen-sensitive and CRPC tumors in vitro as well as in animal models. The effect was specific because no effect on growth was observed with a control antisense oligonucleotide that does not recognize AR mRNA, nor on tumors derived from the PC3, AR-negative, tumor cell line. In addition, EZN-4176 reduced AR luciferase reporter activity in a CRPC model derived from C4-2b cells that were implanted intratibially, indicating that the molecule may control prostate cancer that has metastasized to the bone. These data, together with the continued dependency of CRPC on the AR signaling pathway, justify the ongoing phase I evaluation of EZN-4176 in patients with CRPC.

Androgen receptor signaling in castration-resistant prostate cancer: a lesson in persistence

“…Another means to more specifically target AR gene expression is the use of antisense oligonucleotides (ASOs). EZN-4176, an ASO that targets exon 4, demonstrated an impressive activity in pre-clinical studies (Zhang et al 2011). Unfortunately, EZN-4176 failed phase I trials in CRPC due to poor AR knockdown in the clinical setting and minimal antitumor activity (Bianchini et al 2013).…”

Downregulation of HER3 by a Novel Antisense Oligonucleotide, EZN-3920, Improves the Antitumor Activity of EGFR and HER2 Tyrosine Kinase Inhibitors in Animal Models

“…Capital letters denote LNA monomers, and lowercase letters denote DNA monomers. 5 0 -Cy5.5-labeled EZN-3920 and a scrambled oligonucleotide, designated as EZN-3046 (5 0 -CGCAgattagaaACCt-3 0 ) and abbreviated as EZN-SCR, also were made according to the method described previously (29).…”

“…Western blot analysis was conducted as previously described (29). The antibodies used are as follows: HER3 (sc-285), EGFR (sc-03), Akt (5G3, #2966), p-Akt (ser473, #4051), p-HER3(Tyr1289, #4791), p-p53 (Ser15, #9284), and p53 (1C12, #2524).…”

“…with 40 mg/kg unlabeled or Cy5.5-labeled EZN-3920 on day 0 (first day of dosing) and day 4. The tumors were excised 24 hours later and imaged whole by the Xenogen Lumina as described previously (29). Following imaging, tumors were cryopreserved with 4% paraformaldehyde, sectioned, and imaged with fluorescent microscopy.…”

“…LNA-ASOs have shown very high binding affinity to mRNA, excellent potency for target mRNA downmodulation, improved resistance to nuclease digestion, and excellent stability in plasma and tissues in preclinical studies (24). Furthermore, LNA-ASOs simply prepared in saline are highly effective in vitro and in vivo (25)(26)(27)(28)(29). It is shown here that EZN-3920 suppressed tumor growth both in vitro and in vivo in tumors that co-express HER1 or HER2 or develop resistance to HER1/2 inhibitors.…”