Hedgehog (Hh) signaling plays an important role in prostate development and is also a characteristic feature of prostate cancer (PCa). The ability of Hh pathway activation to promote cell invasiveness, epithelial to mesenchymal transition and metastasis suggest that targeting Hh pathway may provide an important clinical avenue for the treatment of advanced PCa. Loss of PTEN function is considered a characteristic feature of advanced PCa. To gain insight into the status of Hh signaling in PCa in relation to PTEN status we selected three cell lines based on their PTEN expression. Gli1 mRNA was upregulated 2000 fold in PC3 (PTEN-/- null) cells, 800 fold in DU145 (PTEN+/-) cells when compared with normal prostate epithelial cells suggesting that PTEN loss is associated with increased Hh signaling. We also observed significant increase in the mRNA levels of Hh ligand (Shh), Hh receptors (PTCH2 and SMO) and effectors of Hh signaling (Gli1 and Gli2) in the prostate tissues of PTEN knockout mice. A 400 fold increase in the mRNA levels of Gli1 and 15 fold increase in the levels of Gli2 mRNA levels was also observed. We also observed significant decrease in the mRNA levels of the negative regulators of Hh signaling, HHIP and SuFu in the prostate tissues of PTEN knockout mice with similar inhibition in the transcript levels of GAS1. We next investigated the effect on Hh signaling in PCa cells treated with itraconazole and cyclopamine alone and in combination. PCa 22Rν1 and PC-3 cells transfected with Gli-dependent luciferase reporter construct were treated with itraconazole (1 and 2 μM), cyclopamine (5 and 10 nM) and combination of itraconazole (1 μM) and cyclopamine (5 nM) for 40 hours. Gli-dependent luciferase reporter activity was inhibited by itraconazole and cyclopamine treatments; however the inhibition was significantly greater in combination. Treatment of PCa cells with the combination of itraconazole and cyclopamine led to increased cell growth inhibition that was greater than the sum of the individual agents alone suggesting synergism. We next analyzed the effect of the combination on the growth of CWR22Rν1 tumors under in vivo conditions using athymic nude mice. In control animals, targeted tumor volume of 1200 mm3 was reached at 6 weeks post cell inoculation and significantly later time in animals treated with itraconazole alone (9 weeks) and cyclopamine alone (8 weeks). Notably, tumor volumes stayed under 165 mm3 in mice that received both itraconazole and cyclopamine. In the prostate specific Pten knockout mice combination of itraconazole and cyclopamine produced 57% inhibition of tumor growth compared to 30% in cyclopamine alone and 45% in itraconazole alone treated mice. These data show that a combination of itraconazole and cyclopamine is considerably more efficient in inhibiting tumor growth highlighting the synergistic efficacy of this combination for the treatment of PCa.
Citation Format: Vaqar M. Adhami, Imtiaz A. Siddiqui, Mohammad Imran Khan, Islam Rady, Leanna Sako, Hasan Mukhtar. Hedgehog pathway inhibitors itraconazole and cyclopamine produce synergistic suppression of Pten deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5808.