Histone Deacetylase Inhibitors Induce Ribosomal Protein Acetylation And Modulate Breast Cancer Cell Viability

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Dysregulation of protein synthesis is crucial for tumorigenesis. The PI3K/AKT/mTOR pathway regulates ribosomes and mRNA translation. Inhibition of this pathway is an important approach under clinical evaluation for the treatment of malignancies including breast cancer. However, many are resistant to PI3K/AKT/mTOR inhibitors, necessitating alternative inhibitors of oncoprotein synthesis. Our studies in breast cancer cells implicate histone deacetylase inhibition (HDACi) in the impairment of ribosome function and oncoprotein synthesis. In particular, pan-HDAC inhibitors like Trichostatin A (TSA) induce decay of specific oncogenic transcripts (e.g. HER2) within 6 h of treatment via a polyribosome-dependent mechanism. Furthermore, we found that TSA induces the acetylation of the large ribosomal subunit protein L24. TSA also decreased 80S ribosome formation and selectively reduced the expression of the cap-dependently translated oncoproteins NBS1, cyclin D1, and survivin without inhibition of β-tubulin and GAPDH. To elucidate the link between ribosomes and HDACi, we used tandem mass spectrometry to interrogate global acetylation changes within ribosomal proteins following treatment of HER2-positive SKBR3 breast cancer cells by either TSA or the HDAC6-selective inhibitor tubacin. Control and treated SKBR3 ribosomal proteins were immunoprecipitated by an anti-acetyl-lysine antibody and subsequently digested by trypsin. The resulting acetyl-lysine peptides were analyzed by LC-MS/MS (AB SCIEX TripleTOF 5600) and identified from Mascot and ProteinPilot databases. Peptides were quantitated by Skyline MS1 Filtering. We identified three ribosomal proteins (RPL24, RPL37A, RPL7A) with acetylation induced at least two-fold by 2 h of TSA treatment and 12 others whose acetylation was induced two-fold or greater by 6 h of TSA treatment (RPL19, RPL22, RPL27, RPL28, RPL3, RPL35A, RPL3L, RPL6, RPS13, RPS17L, RPS23, RPS27A). The profile of ribosomal proteins acetylated by treatment with the HDAC6 selective inhibitor tubacin was significantly different. To further evaluate the cancer-association of these ribosomal proteins with pan-HDACi-induced acetylation, we analyzed a publically available microarray dataset including mRNA from breast cancers and normal mammary tissue from the same patient. Of the ribosomal protein genes with HDACi-inducible acetylation, we found 6 whose mRNA expression levels were significantly higher in breast cancers relative to matched normal mammary tissue: RPL19 (p=0.001), RPL24 (p=0.001), RPL3 (p=0.001), RPL7a (p=0.002), RPS23 (p=0.004), and RPL22 (p=0.05). These findings point toward a novel role of HDACi in modulating the acetylation of ribosomal proteins, some of which are upregulated in breast cancer. This acetylation could result in altered ribosome function, decreased oncoprotein translation, and ultimately reduced breast cancer cell growth and survival. Citation Format: Kathleen Ann Wilson-Edell, Amanuel Kehasse, Christina Yau, Gary K. Scott, Jason M. Held, Bianca S. Gabriel, Bradford W. Gibson, Christopher C. Benz. Histone deacetylase inhibitors induce ribosomal protein acetylation and modulate breast cancer cell viability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4224. doi:10.1158/1538-7445.AM2014-4224