A deguelin analogue, SH48 inhibits STAT3 signaling in breast cancer cells

Abstract

Constitutively activated signal transducer and activator of transcription 3 (STAT3) plays an important role in oncogenesis and tumor development. Depending on the cell type and stimulus, STAT3 mediates different anti-proliferative events effects, such as apoptosis, autophagy and senescence. STAT3 is activated through tyrosine phosphorylation, which leads to its dimerization and translocation to the nucleus. In the present study, a new STAT3 inhibitor, SH48 discovered by virtual screening, was found to inhibit phosphorylation, nuclear translocation and transcriptional activity of STAT3 in human mammary epithelial MCF-10A cells transfected with ras oncogene (MCF10A-ras). SH48 has an α,β-unsaturated carbonyl group in its structure, and is speculated to interact with a thiol residue of STAT3, thereby inactivating this transcription factor. The thiol-reducing agents, dithiothreitol or N-acetylcysteine abolished the SH48-induced suppression of STAT3 activation. In contrast to SH48, non-electrophilic analogues of SH48 failed to inhibit STAT3 activation. By utilizing a biotinylated SH48, we were able to demonstrate the interaction between SH48 and STAT3. SH48 binds to STAT3 directly via Michael addition, and thereby inhibits STAT3 activation. SH48 may also make hydrogen bonding with STAT3, but this interaction is not likely to inhibit STAT3 phosphorylation. SH48 induces cell death in MCF10A-ras cells, but not in normal MCF10A cells. SH48-induced cell death was not associated with apoptosis, but it upregulated the expression of LC3Ⅱ and p62 involved in autophagy. SH48 does induce autophagic cell death (type II cell death) as determined by FACS analysis of Lysotracker fluorescence in MCF10A-ras cells. Autophagy induced by SH48 resulted in suppression of tumor growth, which appears to be dependent on its ability to inhibit STAT3 activation. In conclusion, SH48 induces autophagy by directly targeting STAT3.