DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

BMC Medical Genomics BioMed Central Research article Open Access DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines Melissa J LaBonte1, Peter M Wilson1, William Fazzone1, Susan Groshen2, Heinz-Josef Lenz3 and Robert D Ladner*1 Address: 1Department of Pathology Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA, 2Department of Biostatistics Core Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA and 3Division of Medical Oncology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Email: Melissa J LaBonte - ; Peter M Wilson - ; William Fazzone - ; Susan Groshen - ; Heinz-Josef Lenz - ; Robert D Ladner* - * Corresponding author Published: 30 November 2009 BMC Medical Genomics 2009, 2:67 doi:10.1186/1755-8794-2-67 Received: 19 June 2009 Accepted: 30 November 2009 This article is available from: http://www.biomedcentral.com/1755-8794/2/67 © 2009 LaBonte et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes. Methods: HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity® Pathway Analysis. Results: Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed. Conclusion: This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing muchneeded information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer. Page 1 of 20 (page number not for citation purposes) BMC Medical Genomics 2009, 2:67 Background Within the cellular microenvironment, regulation of gene expression can occur post-transcriptionally through modification of histones and non-histone proteins by acetylation, phosphorylation, methylation, ubiquitination and sumoylation. Two distinct families of enzymes, histone acetyltransferases (HAT) and histone deacetylases (HDAC), work in concert by performing opposing functions to maintain a tightly regulated pattern of acetylation homeostasis. HDACs are zinc-dependent hydrolases which can be classified into 4 different families (class I, IIa, IIb, and IV) that are involved in the remodeling of chromatin by deacetylation of specific lysine residues on histone tails [1,2]. The action of HDACs occurs through formation of large multi-protein complexes with co-activating, co-repressing, and chromatin-remodeling proteins. It has further been demonstrated that the actions of HDACs and the resultant deacetylation of specific lysine residues is not limited to histones, but occurs on non-histone proteins such as α-tubulin, Hsp90, gluccocorticoid receptors, DNA methyltransferase 1 (DNMT 1) and multiple transcription factors (p53, E2F, GATA1, TFIIE and TFIIF) [3-5]. As such, the role of HDACs in the regulation of cellular processes is more complex than first thought, extending far beyond regulating gene expression and involving active roles in cell-cycle-related processes [6-8]. It is therefore not surprising that dysregulation of HDAC and HAT activity has been identified and reported to contribute to the progression of a number of cancers including leukemia, lymphoma, gastric, prostate, breast and colon [9-13]. Multiple HDAC inhibitors (HDACi) have been developed to date and their administration results in the acetylation of both histone and non-histone proteins, leading to the modulation of between 2 and 10% of expressed genes [14]. The classes of compounds identified as HDACi include: short-chain fatty acids (such as valproic acid), hydroxamic acids (such as TSA, PXD101, LBH589 and vorinostat), cyclic tetrapeptides (such as depsipeptide, FK228) and benzamides (such as MS-275) [15]. Mechanistically, HDACi have been shown to induce G1 and G2/ M cell cycle arrest, promote differentiation, induction of apoptotic signaling cascades, mitotic failure, polyploidy and increased generation of reactive oxygen species [1618]. The hydroxamic acid-based HDACis, vorinostat (SAHA, Merck) [19,20] and LBH589 (panobinostat, Novartis) [21] are pan-inhibitors of class I and II HDACs that have demonstrated potent cytotoxicity in vitro against a variety of solid tumor cell lines. Vorinostat is currently FDA-approved for the treatment of cutaneous T-cell lymphoma (CTCL) and is currently in clinical investigation for mesothelioma, non-small cell lung cancer and colon http://www.biomedcentral.com/1755-8794/2/67 cancer. LBH589 is also under extensive clinical investigation in CTCL and a variety of solid tumors. Colorectal cancer is the third most commonly diagnosed cancer i (...truncated)