Gene expression signature analysis identifies vorinostat as a candidate therapy for gastric cancer

Abstract

Background: Gastric cancer continues to be one of the deadliest cancers in the world and therefore identification of new drugs targeting this type of cancer is thus of significant importance. The purpose of this study was to identify and validate a therapeutic agent which might improve the outcomes for gastric cancer patients in the future.

Methodology/principal findings: Using microarray technology, we generated a gene expression profile of human gastric cancer-specific genes from human gastric cancer tissue samples. We used this profile in the Broad Institute's Connectivity Map analysis to identify candidate therapeutic compounds for gastric cancer. We found the histone deacetylase inhibitor vorinostat as the lead compound and thus a potential therapeutic drug for gastric cancer. Vorinostat induced both apoptosis and autophagy in gastric cancer cell lines. Pharmacological and genetic inhibition of autophagy however, increased the therapeutic efficacy of vorinostat, indicating that a combination of vorinostat with autophagy inhibitors may therapeutically be more beneficial. Moreover, gene expression analysis of gastric cancer identified a collection of genes (ITGB5, TYMS, MYB, APOC1, CBX5, PLA2G2A, and KIF20A) whose expression was elevated in gastric tumor tissue and downregulated more than 2-fold by vorinostat treatment in gastric cancer cell lines. In contrast, SCGB2A1, TCN1, CFD, APLP1, and NQO1 manifested a reversed pattern.

Conclusions/significance: We showed that analysis of gene expression signature may represent an emerging approach to discover therapeutic agents for gastric cancer, such as vorinostat. The observation of altered gene expression after vorinostat treatment may provide the clue to identify the molecular mechanism of vorinostat and those patients likely to benefit from vorinostat treatment.

Figure 1. In vitro therapeutic efficacy of vorinostat in gastric cancer cell lines.

(A) MTT assays were performed after incubation of AGS, KATO-III, and NCI-N87 with the indicated concentrations of vorinostat for 72 hrs. (B) Change of cell cycle by vorinostat was assessed by fluorescence-activated cell sorting (FACS) analysis of PI stained cells treated with 5 µM vorinostat for 24 hrs. (C) Viability test using PI exclusive solution. AGS and KATO-III cells were treated with 5 µM vorinostat for 72 hrs and assessed by FACS. In representative plot, dead cells were manifested as dots with low forward scatter and high side scatter. (D) Western blot analysis of active caspase-3 from AGS and KATO-III gastric cancer cells without or with 5 µM vorinostat treatment. Cell lysates were analyzed at the indicated time points. Actin was used as a loading control. *, P<0.05. In the bar graph, data represent mean+SD (standard deviation).

Figure 2. Hierarchical Clustering Gene expression analysis of gastric cancer cell lines after vorinostat treatment.

(A) Unsupervised hierarchical clustering of gene expression data from AGS and KATO III before and after 5 µM vorinostat treatment for 48 hours. Genes with an expression level that has at least 2-fold difference relative to median value across cell lines in at least 2 arrays were selected for hierarchical clustering analysis (3,646 gene features). (B) Supervised hierarchical clustering with autophagy related genes (149 probes) of AGS and KATO III after vorinostat treatment.

Figure 3. Autophagy induction by vorinostat treatment in gastric cancer cells.

(A) AGS and KATO-III were treated with 5 µM vorinostat for the indicated time points. Protein levels of LC3 and p62 were analyzed by immunoblot analysis. Actin was used as a loading control. p62 levels were measured by densitometric analysis of the western blots and compared to actin levels. p62 levels of untreated AGS and KATOIII cells were considered as 1. (B) AGS cells were treated for 12 h with 5 µM vorinostat with or without 50 nM bafilomycin A1 (BafA1). Cell lysates were analyzed by immunoblot analysis for LC3 and actin.

Figure 4. Electron microscopy analysis of autophagy in AGS cells.

After 12 h treatment DMSO (A, A') or with 5 µM vorinostat (B, B') (left panels, low magnification, scale bar: 2 µm), EM analysis was performed. High magnification images of boxed areas with Av depicting autophagic vacuoles (left panels, scale bar: 500 nm; N: nucleus, M: mitochondria).

Figure 5. Effect of vorinostat-induced autophagy inhibition.

(A) AGS and KATO-III cells were treated with 5 µM vorinostat and different concentrations of chloroquine (CQ). Cell viability was assessed after 12 hours using crystal violet staining and control (Ctr) was set as 100%. (B) KATO-III cells were transfected with siRNA against Beclin 1 (siBeclin1) or with non-targeting Risc Free siRNA or treated with Dharmafect I alone (mock). siRNA efficiency was confirmed by western blot analysis of Beclin 1 and Risc Free as a control. α-tubulin was used to show equal loading of proteins. Viability was measured after 12 hours vorinostat treatment using crystal violet staining. Viability of untreated control (Ctr) cells was set as 100%. *, P<0.05.

Figure 6. Change of gene expression signature after vorinostat treatment.

Venn Diagram of genes selected by univariate test (two-sample t-test). Genes were selected for p<0.001 between compared groups. The red circle (gene list A) represents gastric cancer specific genes from Yonsei data. The blue circle (gene list B) represents gastric cancer specific genes from Stanford data. The yellow circle (gene list C) represents vorinostat specific gene signature from both AGS and KATO-III cell lines (P<0.001, 2 fold change).