Histone deacetylase turnover and recovery in sulforaphane-treated colon cancer cells: competing actions of 14-3-3 and Pin1 in HDAC3/SMRT corepressor complex dissociation/reassembly

Abstract

BackgroundHistone deacetylase (HDAC) inhibitors are currently undergoing clinical evaluation as anti-cancer agents. Dietary constituents share certain properties of HDAC inhibitor drugs, including the ability to induce global histone acetylation, turn-on epigenetically-silenced genes, and trigger cell cycle arrest, apoptosis, or differentiation in cancer cells. One such example is sulforaphane (SFN), an isothiocyanate derived from the glucosinolate precursor glucoraphanin, which is abundant in broccoli. Here, we examined the time-course and reversibility of SFN-induced HDAC changes in human colon cancer cells.ResultsCells underwent progressive G2/M arrest over the period 6-72 h after SFN treatment, during which time HDAC activity increased in the vehicle-treated controls but not in SFN-treated cells. There was a time-dependent loss of class I and selected class II HDAC proteins, with HDAC3 depletion detected ahead of other HDACs. Mechanism studies revealed no apparent effect of calpain, proteasome, protease or caspase inhibitors, but HDAC3 was rescued by cycloheximide or actinomycin D treatment. Among the protein partners implicated in the HDAC3 turnover mechanism, silencing mediator for retinoid and thyroid hormone receptors (SMRT) was phosphorylated in the nucleus within 6 h of SFN treatment, as was HDAC3 itself. Co-immunoprecipitation assays revealed SFN-induced dissociation of HDAC3/SMRT complexes coinciding with increased binding of HDAC3 to 14-3-3 and peptidyl-prolyl cis/trans isomerase 1 (Pin1). Pin1 knockdown blocked the SFN-induced loss of HDAC3. Finally, SFN treatment for 6 or 24 h followed by SFN removal from the culture media led to complete recovery of HDAC activity and HDAC protein expression, during which time cells were released from G2/M arrest.ConclusionThe current investigation supports a model in which protein kinase CK2 phosphorylates SMRT and HDAC3 in the nucleus, resulting in dissociation of the corepressor complex and enhanced binding of HDAC3 to 14-3-3 or Pin1. In the cytoplasm, release of HDAC3 from 14-3-3 followed by nuclear import is postulated to compete with a Pin1 pathway that directs HDAC3 for degradation. The latter pathway predominates in colon cancer cells exposed continuously to SFN, whereas the former pathway is likely to be favored when SFN has been removed within 24 h, allowing recovery from cell cycle arrest.

Regulated Clearance of Histone Deacetylase 3 Protects Independent Formation of Nuclear Receptor Corepressor Complexes

“…Although histone deacetylase inhibitors are useful in the treatment of these cancers, they are associated with nonspecific effects. It has been reported that treatment with histone deacetylase inhibitors can induce concomitant degradation of HDAC3 and corepressors in cancer cells (48), confirming the inverse correlation between HDAC3 clearance and corepressor expression as observed here. Along with the presently observed HDAC3 reduction upon corepressor depletion in all cancer cell lines tested here, these studies suggest that the pathways driving HDAC3 degradation in tumors are intact.…”

Sulforaphane Inhibits Growth of Human Breast Cancer Cells and Augments the Therapeutic Index of the Chemotherapeutic Drug, Gemcitabine

“…Its chemopreventive effects have assigned to multiple mechanisms such as modulatory action on phase 2 enzymes via Keap1-Nrf2 signaling and antioxidant response element (ARE)-driven gene expression as well as by induction of cell cycle arrest and apoptosis and inhibition of histone deacetylase (HDAC) (Clarke el al., 2011;Rajendran et al, 2011). Conspicuously, SFN has been shown to selectively target the precancerous and cancerous cells (Clarke el al., 2011).…”

Dietary Sulforaphane in Cancer Chemoprevention: The Role of Epigenetic Regulation and HDAC Inhibition

“…Studies involved in the evaluation of sulforaphane as an HDAC inhibitor, and those aimed at providing insights into the mechanisms associated with sulforaphane-regulated inhibition of HDAC activity, have focused on the roles of HDAC3 and HDAC6 as important targets (43, 89,223,224). After the incubation of HCT116 human colon cancer cells with 15 lM sulforaphane, a significant reduction in HDAC1, HDAC2, HDAC3, and HDAC8 was observed compared with vehicle-treated cells at 36 h post administration (223).…”