Abstract 1626: Polyribosome-associated proteins targeted by HDACi promote the selective degradation of oncogenic transcripts

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL We previously demonstrated that histone deacetylase inhibitors (HDACi), including several HDAC6-selective inhibitors, rapidly induce the accelerated decay of ERBB2 mRNA as well as ∼ 300 other oncogenic transcripts possessing U-rich sequences in their 3’UTR. While the mechanism underlying this oncogene transcript decay switch remains unknown, more recent evidence indicates that HDACi induced ERBB2 mRNA decay occurs on actively translating polyribosomes; and current studies are attempting to define the polyribosome proteins mediating this switch as they might represent novel therapeutic target candidates. A screen for proteins rapidly modified, recruited to, or released from polyribosomes following HDACi exposure (trichostatin-A, TSA) was undertaken in the ERBB2-positive human breast cancer cell line, SKBR3. Monosome and polyribosome enriched cytosols from control or TSA treated cells were either sucrose gradient fractionated for Northern and Western blot analysis of fractions, or the proteins were resolved by 2-dimensional (2-D) gel electrophoresis using three different isoelectric focusing gradients (ranging from pH 12 to pH 4). Western blots confirmed that monosome and polyribosome fractions contained the 40S ribosome scaffold protein, RACK1. Northern blots confirmed that HDACi (TSA × 6h) induced decay of ERBB2 mRNA, which could be completely prevented by co-treatment with the translational inhibitor cycloheximide, was confined to the polyribosome fractions. Comparison of the protein stained 2-D gels from control and treated samples indicated that 2-4 h of TSA exposure changes the isoelectric migration of >80 proteins, suggesting that HDACi induces post-translational modifications (PTMs) in various polyribosome proteins. Altered 2-D protein spots were excised, trypsin digested, and identified by nano-liquid chromatography (LC)-tandem mass spectrometry (MS/MS) using a 4000 QTRAP (Applied Biosystems, with Mascot v. 2.2 search database); in addition to RACK1, other TSA modified proteins include YB-1 (Y-box binding protein), DBPA (DNA-binding protein A), PRKRA (interferon-inducible double stranded RNA-dependent protein kinase activator A), NPM (nucleophosmin), and HNRPC (heterogeneous nuclear ribonucleoproteins C1/C2). Western blots looking for differential lysine acetylation (Ac-K) revealed that TSA induces Ac-K protein isoforms in the sucrose gradient polyribosome fractions containing decaying ERBB2 mRNA; induced Ac-K isoforms were also observed on the 2-D gels. MS/MS analysis of these TSA induced Ac-K isoforms is expected to reveal novel polyribosome HDAC substrates and candidate effectors of oncogene transcript decay. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1626. doi:10.1158/1538-7445.AM2011-1626