HSP90 acetylation following histone deacetylase inhibition: A role for the histone acetyltransferases p300 and PCAF

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Histone deacetylase inhibitors (HDACI) are a promising new class of chemotherapeutic agent that result in hyperacetylation of histones and other cellular proteins. HDACI have multifactorial cellular effects but the precise mechanisms underlying antitumour activity have not been defined. HSP90 is a widely expressed molecular chaperone essential for the appropriate folding, stability and function of a range of cellular proteins. As many HSP90 client proteins are involved in signal transduction pathways commonly deregulated in cancer, HSP90 inhibitors have emerged as attractive anti-cancer agents. HDACI cause increased acetylation of HSP90 resulting in inhibition of chaperone activity. HDAC6 has been identified as an HSP90 deacetylase but the HAT responsible for acetylation of HSP90 has not been identified. In this study, western blot and immunoprecipitation techniques were used to analyse the effects of HDACI on HSP90. HCT116 human colorectal tumour cells were exposed to the HDACI SAHA (Vorinostat) or the HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17AAG). SAHA inhibited the growth of HCT116 cells with a GI50 (growth inhibitory concentration) of 1.5±0.7μM and induced marked hyperacetylation of histones H3 and H4 as early as 1h following exposure (0.1-5XGI50). The molecular signature of HSP90 inhibition (increased HSP70 expression and depletion of ERBB2, CDK4 and CRAF) was observed using 17AAG (GI50 32±8 nM) treatment by 8-24h (5xGI50). SAHA (5XGI50) also elicited changes in the expression of HSP70 and client proteins consistent with the molecular signature of HSP90 inhibition, but to a lesser extent than 17AAG. HSP90 was immunoprecipitated from SAHA-treated cells (5XGI50) and probed with acetyl lysine antibody (Upstate). Increased acetylation of HSP90 (150-170% above control) was observed in a time-dependent manner over 24h. CDK4, CRAF and ERBB2 were lost from the immunoprecipitated HSP90 complex by 4-6hrs. Four additional acetylated protein bands co-immunoprecipitated with HSP90, the identification of which is underway. To identify the HAT(s) responsible for acetylation of HSP90 in HDACI treated cells, HSP90 was incubated with [3H]-acetyl coenzyme A (0.5μCi) and the HAT enzymes p300 and PCAF at 30C and the incorporation of radiolabel determined in a filter assay. Two selective peptide coenzyme A conjugate inhibitors of p300 and PCAF HAT activity (Lau et al., 2000, Mol Cell, 5, 589) were used as negative controls. HSP90 showed concentration-dependent increased acetylation in the presence of both PCAF and p300. Time-dependent increases in HSP90 acetylation were blocked by the HAT inhibitors. These findings suggest that HSP90 may be a cellular substrate of p300 and PCAF. Interestingly, HSP90 co-immunoprecipitated with both enzymes. Targeted inhibition by RNAi is presently being used to determine if these HATs plays a cellular role in the acetylation of HSP90.