Abstract
The transcription factor STAT3 is constitutively active in several malignancies including castration-resistant prostate cancer and has been identified as a promising therapeutic target. The fungal metabolite galiellalactone, a STAT3 signaling inhibitor, inhibits the growth, both in vitro and in vivo, of prostate cancer cells expressing active STAT3 and induces apoptosis of prostate cancer stem cell-like cells expressing phosphorylated STAT3 (pSTAT3). However, the molecular mechanism of this STAT3-inhibiting effect by galiellalactone has not been clarified. A biotinylated analogue of galiellalactone (GL-biot) was synthesized to be used for identification of galiellalactone target proteins. By adding streptavidin-Sepharose beads to GL-biot-treated DU145 cell lysates, STAT3 was isolated and identified as a target protein. Confocal microscopy revealed GL-biot in both the cytoplasm and the nucleus of DU145 cells treated with GL-biot, appearing to co-localize with STAT3 in the nucleus. Galiellalactone inhibited STAT3 binding to DNA in DU145 cell lysates without affecting phosphorylation status of STAT3. Mass spectrometry analysis of recombinant STAT3 protein pretreated with galiellalactone revealed three modified cysteines (Cys-367, Cys-468, and Cys-542). Here we demonstrate with chemical and molecular pharmacological methods that galiellalactone is a cysteine reactive inhibitor that covalently binds to one or more cysteines in STAT3 and that this leads to inhibition of STAT3 binding to DNA and thus blocks STAT3 signaling without affecting phosphorylation. This further validates galiellalactone as a promising direct STAT3 inhibitor for treatment of castration-resistant prostate cancer.
Highlights
signal transducer and activator of transcription 3 (STAT3) is constitutively active in castration-resistant prostate cancer and the fungal metabolite galiellalactone inhibits STAT3 signaling
The results indicate that the STAT3 signaling inhibitor galiellalactone binds directly and covalently to STAT3 and that this interaction prevents DNA binding without interfering with upstream activation by phosphorylation
This further validates galiellalactone as a promising STAT3 inhibitor for the treatment of castration-resistant prostate cancer and other malignancies with constitutive activation of STAT3
Summary
STAT3 is constitutively active in castration-resistant prostate cancer and the fungal metabolite galiellalactone inhibits STAT3 signaling. Significance: Galiellalactone is a promising direct STAT3 inhibitor for treatment of castration-resistant prostate cancer. We demonstrate with chemical and molecular pharmacological methods that galiellalactone is a cysteine reactive inhibitor that covalently binds to one or more cysteines in STAT3 and that this leads to inhibition of STAT3 binding to DNA and blocks STAT3 signaling without affecting phosphorylation. This further validates galiellalactone as a promising direct STAT3 inhibitor for treatment of castration-resistant prostate cancer
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