Abstract 5179: Manipulating the ribosomal protein RPL24 by depletion, truncation, or acetylation alters ribosome formation and inhibits cancer cell growth.
Authors: Wilson-Edell Kathleen A. , Scott Gary K. , Gabriel Bianca S. , Yevtushenko Mariya , Held Jason M. , Hanson Ingrid M. , Benz Christopher C. 
Published: August 2014

Abstract
Control of protein synthesis is critical for cell growth and oncogenic transformation. Inhibiting the PI3K/AKT/mTOR pathway upstream of ribosomes is one approach currently in clinical trials to treat various cancers, including breast cancer. However, many cancers are resistant to such therapies and may respond to translation inhibition via alternate mechanisms. Our previous studies identify a link between histone deacetylase inhibition (HDACi) and ribosomes, in that pan-HDAC inhibitors like Trichostatin A (TSA) induce the decay of HER2 and other oncogeneic transcripts via a polyribosome-dependent mechanism. In recent studies using HER2-overexpressing SKBR3 breast cancer cells, we also found that TSA inhibits phosphorylation of the ribosomal protein S6 and the translation initiation factor 4eBP1. Thus, we investigated the effect of TSA on polysome dynamics and post-translational modifications of ribosomal proteins to better understand how HDACi inhibits cancer cell proliferation and to identify novel cancer therapeutic targets.
We found that TSA reduces the amount of 80S ribosomes relative to free 40S and 60S subunits, implicating impaired ribosome assembly. Eukaryotic translation initiation factor 6 (eIF6) promotes the maturation of nucleolar 60S but must be released from cytoplasmic 60S in order for the 40S and 60S to join and form functional 80S ribosomes. Therefore, we asked if TSA impedes eIF6 release from the 60S and found that eIF6 remains 60S-associated in TSA relative to control treated SKBR3 cells. To determine how TSA affects ribosomal proteins, we performed immunoblotting and 2D-electrophoresis/mass spectrometry screening of ribosomal proteins and identified the large ribosomal subunit protein, RPL24, as acetylated after TSA treatment. Additional interest in this acetylation candidate was driven by two recent reports. One provides structural evidence that the lysine-containing C-terminus of RPL24 resides in proximity to eIF6 on the 60S and also contacts the 40S subunit. The other reports that mice haploinsufficient for RPL24 exhibit greater survival of AKT-driven tumorigenesis. Our analyses produced three additional observations: i) depletion of RPL24 in SKBR3 cells via shRNA, similar to TSA treatment, impairs 80S formation and increases the amount of eIF6 associated with ribosomal 60S; ii) a mutant form of RPL24 with truncated C terminus increases 60S retention of eIF6; and iii) depletion of RPL24, as well as TSA treatment, inhibits SKBR3 proliferation. In summary, altering polysome dynamics by impairing 80S formation and prolonging 60S retention of eIF6, by TSA and/or altered RPL24 function, inhibits cancer cell growth and proliferation. Furthermore, RPL24 acetylation may serve as a biomarker for therapeutic response to pan-HDAC inhibitors and as a drug target for the design of novel cancer therapeutics.
Citation Format: Kathleen A. Wilson-Edell, Gary K. Scott, Bianca S. Gabriel, Mariya Yevtushenko, Jason M. Held, Ingrid M. Hanson, Christopher C. Benz. Manipulating the ribosomal protein RPL24 by depletion, truncation, or acetylation alters ribosome formation and inhibits cancer cell growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5179. doi:10.1158/1538-7445.AM2013-5179