Divergent JNK Phosphorylation of HDAC3 in Triple-Negative Breast Cancer Cells Determines HDAC Inhibitor Binding and Selectivity

Abstract

Summary Histone deacetylase (HDAC) catalytic activity is regulated by formation of co-regulator complexes and post-translational modification. Whether these mechanisms are transformed in cancer and how this affects the binding and selectivity of HDAC inhibitors (HDACi) is unclear. In this study, we developed a method that identified a 3-16-fold increase in HDACi selectivity for HDAC3 in triple negative breast cancer cells (TNBC) in comparison to luminal subtypes that was not predicted by current practice measurements with recombinant proteins. We found this increase was caused by c-Jun N-terminal kinase (JNK) phosphorylation of HDAC3, was independent of HDAC3 complex composition or subcellular localization, and was associated with a 5-fold increase in HDAC3 enzymatic activity. This study points to HDAC3 and the JNK axes as targets in TNBC, highlights how HDAC phosphorylation affects HDACi binding and selectivity, and outlines a method to identify changes in individual HDAC isoforms catalytic activity, applicable to any disease state.

EGFR–c-Src-Mediated HDAC3 Phosphorylation Exacerbates Invasion of Breast Cancer Cells

“…Strikingly, the decreased invasiveness was re-increased by the transient transfection of the shRNA-resistant HDAC3 wt plasmid, but not by rshHDAC3 Y321/331 , strongly confirming that EGFR–c-Src mediated HDAC3 phosphorylation at Y321 and 331 was crucial for the invasion of breast cancer cells. At the same time, our results support the previous reports, highlighting the importance of HDAC3 selective inhibition in the regulation of breast cancer [16,34,52].…”

Revisiting Histone Deacetylases in Human Tumorigenesis: The Paradigm of Urothelial Bladder Cancer

“…Remarkably, the JNK kinase (a typical MAPK family member)-dependent phosphorylation of HDAC3 is associated with a five times upregulation of its enzyme activity and three to 16 times increase in HDAC inhibitor selectivity for HDAC3 in triple-negative breast cancer cells, as compared to luminal subtypes. These findings highlight how HDAC phosphorylation affects HDAC inhibitor binding and selectivity, and underscore the significance of JNK-HDAC3 axis in -breast- cancer clinical management and therapy [66,140,141,142,143,144,145,146].…”

Scaffold dependent histone deacetylase (HDAC) inhibitor induced re-equilibration of the subcellular localization and post-translational modification state of class I HDACs

“…In addition to subcellular localization, we also investigated HDAC3 phosphorylation state in response to HDACi treatment, as this isoform’s subcellular localization was not affected ( S1 Fig ). Using an antibody found to be specific for non-phosphorylated HDAC3 [ 19 ], we analyzed the amount of non-phosphorylated HDAC3 after treatment with 0.2, 10 and 50 μM panobinostat, trichostatin A, SAHA, entinostat or PCI-34051 ( Fig 6A ). We observed 1.8–3.8 and 1.8–2.3-fold increase in non-phosphorylated HDAC3 in response to panobinostat and trichostatin A, respectively ( Fig 6B ).…”

“…Equal protein loading was confirmed with Ponceau S staining (Sigma-Aldrich). After staining, the membranes were blocked with Odyssey blocking buffer (Li-Cor) for 2 hours at RT, and then incubated with primary antibodies in blocking buffer for HDAC1 (Abcam, ab7028, lot GR188529-1, rabbit), HDAC2 (Abcam, ab124974, lot GR97402-7, rabbit), HDAC3 (Abcam, ab7030, lot GR121157, rabbit), non-phosphorylated HDAC3 (Ref [ 19 ]; Millipore, 05–813, lot 2726719, mouse), GAPDH (Abcam, ab128915, lot GR90965-22, rabbit), TATA-binding protein (Abcam, ab818, lot GR131329-14, mouse), H3 (Abcam, ab1791, lot GR242835-1, rabbit) and Acetyl-histone H3 (Millipore, 06–599, lot 2153150, rabbit) overnight at 4°C. The membranes were then washed three times with PBS containing 0.1% Tween-20 (PBST) for 5 minutes at RT.…”