Screening for therapeutic targets of vorinostat by SILAC-based proteomic analysis in human breast cancer cells

Abstract

Histone deacetylases (HDACs) play critical roles in silencing tumor suppressor genes. HDAC inhibitors reactivate tumor suppressor genes and inhibit tumor cell growth in vitro and in vivo, and several HDAC inhibitors are currently being evaluated in clinical trials for cancer therapy. A comprehensive analysis of proteins regulated by HDAC inhibitors would enhance our ability to define and characterize their essential therapeutic targets. Here, we employed stable isotope labeling with amino acids in cell culture-based quantitative proteomics to identify acetylated proteins in human breast cancer cells. Treatment with the clinically relevant HDAC inhibitor, suberoylanilide hydroxamic acid (vorinostat), induces lysine acetylation of 61 proteins in MDA-MB-231 human breast cancer cells. Suberoylanilide hydroxamic acid not only induces lysine acetylation in chromatin-associated proteins, but also acetylates previously unrecognized nonhistone proteins, including transcriptional factors and regulators, chaperones, cell structure proteins, and glycolytic enzymes in a time-dependent manner. Knowledge of the full repertoire of acetylated proteins will provide a foundation for further defining the functions of HDACs in cancer cells.

Fig. 1. Effect of SAHA on lysine acetylation

(A) Western blot with anti-DNMT1 antibodies of whole cell lysates from 10 µM SAHA treated MDA-MB-231 (top) and SILAC-labeled MDA-MB-231 cells (bottom) demonstrates no differences in DNMT1 inhibition. (B) Acetylated proteins were detected by using anti-acetyllysine antibodies in SILAC-labeled MDA-MB-231cells. β-actin was used as a loading control. Three experiments showed similar results. (C) Schematic summary of SILAC-based experimental design.

Fig. 1. Effect of SAHA on lysine acetylation

(A) Western blot with anti-DNMT1 antibodies of whole cell lysates from 10 µM SAHA treated MDA-MB-231 (top) and SILAC-labeled MDA-MB-231 cells (bottom) demonstrates no differences in DNMT1 inhibition. (B) Acetylated proteins were detected by using anti-acetyllysine antibodies in SILAC-labeled MDA-MB-231cells. β-actin was used as a loading control. Three experiments showed similar results. (C) Schematic summary of SILAC-based experimental design.

Fig. 2. Immunoprecipitation of acetylated proteins

Cell lysates from control and SILAC labeled MDA-MB-231 cells treated with SAHA for 24 or 48 h were combined, and lysine-acetylation containing proteins were immunoprecipitated with anti-acetyllysine antibodies followed by colloidal Coomassie staining (left panel) or immunoblotting (right panel).

Fig. 3

Representative MS/MS spectra of peptides derived from enolase-1, Hsp90, EF-1α and 14-3-3γ. Sequences identified with * are the SILAC labeled residues.

Fig. 4. Confirmation of acetylated proteins by immunoprecipitation and Western blots

Immunoprecipitation was performed with antibodies for candidate lysine acetylated proteins using lysates of MDA-MB-231 cells in the presence or absence of SAHA treatment. Western blotting was done with antibody against the specific protein or an anti-lysine acetylation antibody. Three experiments showed similar results.