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

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 much-needed 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.

Figure 1

In vitro characterization of HDACi, LBH589 and vorinostat, in HCT116 and HT29 colon cancer cells. HCT116 and HT29 colon cancer cells were exposed to increasing concentrations of either (A) LBH589 or (B) vorinostat alone for 72 h and subsequent growth inhibition was measured by MTS assay (Promega). Values are presented as percent control, calculated from the growth inhibition induced by a given concentration of drug compared to the untreated control. Values are averages of 3 independent experiments ± SEM. The IC_{50(72 h)} values were calculated from the sigmoidal dose-response curves in Prism 5.0 (GraphPad). (C-D) Western blot analysis of acetyl-H3 and acetyl-H4 in (C) HCT116 and (D) HT29 cells treated with 2 μM vorinostat (Vor) or 50 nM LBH589 for 0.5, 1, 2 and 4 h. β-actin was used to control for loading.

Figure 2

Cell cycle and apoptotic anlaysis of HDACi-treated colon cancer cells. Flow cytometric analysis of (A) HCT116 and (B) HT29 cells treated with 2 μM vorinostat (Vor) or 50 nM LBH589. Histogram bars represent mean ± SEM. (C-D) Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage as a measure of the induction of apoptosis in HCT116 and HT29 cells treated with 1 and 2 μM vorinostat or 25 and 50 nM LBH589 for 12 and 24 h. β-actin was used to control for loading.

Figure 3

Hierarchical cluster analysis of HDACi-treated HCT116 and HT29 colon cancer cells. Cells were treated with 2 μM vorinostat or 50 nM LBH589 for 24 h and gene expression was analyzed using the Illumina Human-6 V2 BeadChip array. Hierarchical cluster heat map and tree was generated from HDACi-induced changes in gene expression (1-way ANOVA, p < 0.05).

Figure 4

Venn analysis of differentially expressed genes in vorinostat and LBH589-treated HCT116 and HT29 colon cancer cells. HCT116 and HT29 cells were treated with either 2 μM vorinostat or 50 nM LBH589 for 24 h and gene expression analyzed on the Illumina Human-6 V2 BeadChip array. Genes with an FDR-adjusted p-value of < 0.05 were considered differentially expressed and subjected to Venn analysis. Venn analysis was first performed by analyzing cell-line-specific alterations in each individual cell line; (A) HCT116 cells treated with vorinostat or LBH589. (B) HT29 cells treated with vorinostat or LBH589. Subsequent Venn analysis demonstrates the drug-specific alterations induced by (C) vorinostat (Vor) and (D) LBH589 in both cell lines. Numbers within each circle represent the total number of genes modulated in that experimental condition, the numbers immediately below each Venn diagram indicate the total number of modulated genes by both experimental conditions in that Venn diagram.

Figure 5

Top 12 canonical pathways that were significantly modulated by HDACi as identified by Ingenuity^® Pathway Analysis (IPA). HCT116 colon cancer cells treated for 24 h with (A) 2 μM vorinostat (Vor) or (B) 50 nM LBH589 (LBH); HT29 colon cancer cells treated for 24 h with (C) 2 μM vorinostat (Vor) or (D) 50 nM LBH589 (LBH). 2289 of the 3043 differentially expressed genes (DEGs) in the HCT116 and 1679 of the 2232 DEGs in the HT29 cancer cell lines mapped to defined genetic networks in IPA. Fisher's exact test was used to calculate a p-value determining the probability that the association between the genes in the dataset and the canonical pathway is explained by chance alone. A ratio of the number of genes from the dataset that map to the pathway divided by the total number of molecules in a given pathway that meet the cut criteria, divided by the total number of molecules that make up that pathway is displayed.

Figure 6

qPCR validation of house-keeping and cell-line specific HDACi-induced gene expression changes. HCT116 and HT29 cells were treated with 2 μM vorinostat or 50 nM LBH589 for 6, 12 and 24 h. Total RNA was extracted and qPCR analysis was performed as described in the 'materials and methods' using the primer sets given in Table 4. Histogram bars represent the mean ± SD for two independent RNA isolations analyzed in triplicate. (A) Verification of unaffected 18s and GAPDH expression with HDACi treatment. GAPDH was normalized to 18s and 18s was normalized to GAPDH. qPCR validation of the induction of (B) THBS-1, (C) AVEN (D) AURKB (E) HIST1H1C. All genes were normalized to GAPDH, * denotes a p-value < 0.05 for both HDACi treatment groups when compared to respective time-matched control.

Figure 7

qPCR time-dependent validation of core HDACi-induced gene expression changes in HCT116 and HT29 cells. HCT116 and HT29 cells were treated with 2 μM vorinostat (Vor) or 50 nM LBH589 for 6, 12 and 24 h. Total RNA was extracted, reverse transcribed and qPCR analysis was performed as described in the 'materials and methods' using the primer sets given in Table 4. Histogram bars represent the mean ± SD for two independent RNA isolations analyzed in triplicate. All genes were normalized to GAPDH, * denotes p-value < 0.05 for both HDACi treatment groups when compared to respective time-matched control.

Figure 8

qPCR time-dependent validation of core HDACi-repressed gene expression changes in HCT116 and HT29 cells. HCT116 and HT29 cells were treated with 2 μM vorinostat (Vor) or 50 nM LBH589 for 6, 12 and 24 h. Total RNA was extracted, reverse transcribed and qPCR analysis was performed as described in the 'materials and methods' using the primer sets given in Table 4. Histogram bars represent the mean ± SD for two independent RNA isolations analyzed in triplicate. All genes were normalized to GAPDH, * denotes p-value < 0.05 for both HDACi treatment groups when compared to respective time-matched control.