Abstract 4698: Inhibition of histone deacetylase (HDAC) 3 induces hyperacetylation and inhibition of nuclear heat shock protein (hsp) 90 leading to depletion of ATR and CHK1 with sensitization to DNA damage in breast and cervical cancer cells

Abstract

Abstract We have recently reported that in addition to checkpoint kinase 1 (CHK1), ataxia-telangiectasia mutated and Rad3- related (ATR) is chaperoned by hsp90. Treatment of breast and cervical cancer cells with hsp90 inhibitor induces proteasomal degradation and depletion of ATR and CHK1. This results in impairment of the DNA damage response (DDR) and causes sensitization to α-irradiation and replication stress due to hydroxyurea (Mol Cancer Ther 10:1194, 3011). In the present studies, we determined that treatment with pan-histone deacetylase (HDAC) inhibitor panobinostat (20 to 50 nM) or vorinostat (0.5 to 2 μM) induced polyubiquitylation and depletion of ATR and CHK1. Co-treatment with bortezomib, a proteasome inhibitor, restored PS-mediated depletion of ATR and CHK1. Notably, treatment with panobinostat induced hyperacetylation of both nuclear and cytoplasmic hsp90. This inhibited the chaperone association of nuclear hsp90 with ATR and CHK1, thereby promoting their degradation. PS treatment induced γ-H2AX levels, which were inhibited by co-treatment with N-acetylcysteine, suggesting that PS-induced ROS is involved in DNA damage. Moreover, PS treatment further increased α-irradiation induced DNA damage and its repair, characterized by the increase in α-irradiation-induced comet tail moment and the lack of DNA repair-mediated attenuation of the comet tail moment. To determine which class I HDAC is involved in deacetylation of nuclear hsp90, we individually knocked down (KD) HDAC 1, 2 and 3, by utilizing specific shRNA, and determined the effect on nuclear hsp90 hyperacetylation and the levels of ATR and CHK1. Our findings show that HDAC3 binds to hsp90 and KD of HDAC3, but not of HDAC1 or 2, caused hyperacetylation of nuclear hsp90 and depletion of ATR and CHK1. This was also observed in HDAC3 null mouse embryonic fibroblasts (MEFs). Ectopic overexpression of HDAC3 inhibited nuclear hsp90 acetylation in transformed cells. These findings demonstrate that HDAC3 is the nuclear hsp90 lysine deacetylase. They also demonstrate that genetic KD of HDAC3, or its inhibition by pan-HDAC inhibitors, induces hyperacetylation of hsp90, mediates loss of hsp90 chaperone association and depletion of ATR and CHK1, abrogates α-irradiation-induced DDR, and results in sensitization of transformed cells to DNA damage due to α-irradiation or replication stress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4698. doi:1538-7445.AM2012-4698