Abstract C229: Targeting STAT3 in prostate cancer: Identification of STAT3 as a direct target of the fungal metabolite galiellalactone.

Abstract

Abstract Introduction: The transcription factor Signal Transducer and Activator 3 (STAT3) is known to affect tumor growth in several malignancies. STAT3 is frequently found to be activated by phosphorylation (p-STAT3) in castration-resistant prostate cancer and constitutes a promising therapeutic target. The fungal metabolite galiellalactone (GL), a potent STAT3-signaling inhibitor, inhibits the growth, both in vitro and in vivo, of prostate cancer cells expressing activated STAT3 and inhibits growth and induces apoptosis of prostate cancer stem cell-like cells expressing p-STAT3. GL inhibits STAT3 signaling without blocking phosphorylation by a mechanism that we here aim to elucidate. Materials and Methods: A biotinylated analogue of galiellalactone (GL-biot) was synthesized for identification of GL target proteins. The p-STAT3 expressing human prostate cancer cell line DU145 was incubated with GL-biot, followed by streptavidin beads to isolate proteins bound to GL-biot. Bound proteins were identified using Western blot analysis. Competition assays were performed by pre-incubating DU145 cell lysates with GL prior to GL-biot treatment. Localization of GL-biot and co-localization of GL-biot with STAT3 was assessed using confocal microscopy. Electrophoretic mobility shift assay (EMSA) was performed on GL treated DU145 cell lysates for analysis of STAT3 binding to DNA. Results: The inhibitory activity of GL-biot on proliferation and inhibition of STAT3-signaling was similar to that of GL. By adding streptavidin beads to GL-biot-treated DU145 cell lysates, STAT3 was isolated and identified as a target protein. Pretreatment with GL prior to addition of GL-biot prevented the binding of GL-biot to STAT3 in a dose dependent manner. GL-biot was observed to interact also with STAT5 but not STAT1. Confocal microscopy revealed GL-biot in both the cytoplasm and nucleus of DU145 cells treated with GL-biot, appearing to co-localize with STAT3 in the nucleus. DU145 cell lysates subjected to EMSA with STAT3 probes showed a dose dependent decrease in STAT3-DNA binding when treated with GL. Conclusions: Our results show that GL binds directly to STAT3 and inhibits DNA binding thus preventing the transcriptional activity of STAT3. This further validates GL as a promising STAT3-inhibitor for the treatment of castration-resistant prostate cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C229. Citation Format: Nicholas Don-Doncow, Zilma Escobar, Martin Johansson, Eduardo Muñoz, Olov Sterner, Anders Bjartell, Rebecka Hellsten. Targeting STAT3 in prostate cancer: Identification of STAT3 as a direct target of the fungal metabolite galiellalactone. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C229.