Targeting STAT3 in castration-resistant prostate cancer: Galiellalactone as a direct inhibitor of STAT3 in prostate cancer cells.

e16065 Background: The transcription factor STAT3 is constitutively active in castration-resistant prostate cancer (CRPC) and constitutes a promising therapeutic target. The reactive fungal metabolite galiellalactone (GL), a 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 pSTAT3. We aim in this study to identify the STAT3 signaling inhibiting mechanism of GL. Methods: A biotinylated analogue of galiellalactone (GL-biot) was synthesized to be used for identification of GL target proteins. The STAT3 inhibitory activity of GL-biot was confirmed with STAT3 luciferase reporter gene assay. The pSTAT3 expressing human prostate cancer cell line DU145 was incubated with GL-biot, followed by streptavidin beads to isolate proteins bound to GL-biot. Localization of GL-biot in DU145 cells was assessed using confocal microscopy. EMSA was performed on GL treated DU145 cell lysates for analysis of STAT3 binding to DNA. Mass spectrometry analysis (MS) was performed on recombinant STAT3 pretreated with GL in order to identify modified cysteines. Results: By adding streptavidin beads to GL-biot treated DU145 cell lysates, STAT3 was isolated and identified as a target protein. Using confocal microscopy of DU145 cells treated with GL-biot, STAT3 appeared to co-localize with GL-biot in the nucleus. GL inhibited STAT3 binding to DNA in DU145 cell lysates without affecting phosphorylation status of STAT3. MS analysis of recombinant STAT3 pretreated with GL revealed three modified cysteines (C367, C468 and C542). Conclusions: We here demonstrate with chemical and molecular pharmacological methods that galiellalactone is a cysteine reactive inhibitor that covalently binds to one or more cysteines on STAT3 and that this leads to inhibition of STAT3 binding to DNA and thus blocks STAT3 signaling. This further validates GL as a promising STAT3-inhibitor for the treatment of CRPC.