Edelfosine Sensitizes Androgen Deprived Prostate Tumor Growth by Modulating ATF3 and Inhibiting Androgen Receptor Activity

Purpose/Objectives: Edelfosine is a synthetic alkyl-lysophospholipid and has been shown to possess significant antitumor activity in several human tumor models. Here, we investigated the effects of edelfosine combined with androgen deprivation (AD) in androgen sensitive prostate cancer cells (LNCaP and VCaP).Materials/Methods: LNCaP and VCaP cells were grown for 3 days in respective medium. AD was achieved by culturing in medium containing 10% charcoal-stripped serum. Cell proliferation was examined using a real-time cell monitoring system. Apoptosis was measured by Annexin-V and caspase-3/7 activity assays. Reporter gene assays and transfections were performed for measuring PSA and androgen receptor response element (ARE) luciferase activity. Western analyses, TUNEL and immunohistochemistry were carried out on tumor samples. For in vivo studies, male athymic nude mice with orthotopic LNCaP tumors were androgen deprived by bilateral orchiectomy. These mice were then treated with edelfosine by oral gavage. Tumor volume (TV), and serum PSA levels were obtained weekly after treatments.Results: Treatment of androgen sensitive cells in vitro with AD combined with edelfosine resulted in higher antiproliferative activity and apoptosis, as compared to edelfosine or AD alone. AD combined with Edelfosine resulted in a dose-dependent decrease in pAKT, while not effecting the expression of total AKT. Furthermore, edelfosine treatment inhibited AKT mediated phosphorylation of the androgen receptor (AR-Ser213-210) and was associated with an increase in ATF3 protein expression, a stress response gene and a negative regulator of AR signaling. Co-immunoprecipitation with AR antibody in edelfosine+AD treated LNCaP cell lysates confirmed the interaction between AR and ATF3. ATF3 binds to AR after AD+edelfosine treatment and represses the transcriptional activation of AR that was shown here by PSA promoter studies, where edelfosine or overexpression of ATF3 inhibited PSA promoter activity. Knockdown of ATF3 using a siRNA-ATF3 reversed the inhibition of PSA promoter activity by AD+edelfosine, suggesting that the growth inhibition effect of edelfosine was ATF3 dependent. Further, in vivo LNCaP experiments showed significant reductions in TV (TV=15±6mm3) and PSA (2±1ng/ml) after edelfosine+AD, compared to edelfosine (TV=192±63mm3; PSA=39±12ng/ml) or AD alone (TV=112±52mm3; PSA=30±12ng/ml) alone.Conclusions: Here we report for the first time that AKT and ATF3 are involved in Edelfosine mediated apoptosis. Edelfosine is a promising agent for enhancing the inhibitory effect of AD on androgen sensitive cells for a range of patients, from those without metastasis and intermediate to high risk features to those with metastasis.

SSJ24-05

Edelfosine Sensitizes Androgen Deprived Prostate Tumor Growth by Modulating ATF3 and Inhibiting Androgen Receptor Activity