An investigation into the potential use of the histone deacetylase inhibitor, valproic acid for the prevention of gastric cancer in high-risk families

Abstract

Hereditary diffuse gastric cancer (HDGC) is a cancer syndrome caused by germline mutation of the CDH1 gene that encodes the cell adhesion tumor suppressor protein E-cadherin. Mutation carriers have a lifetime risk higher than 70% of developing diffuse gastric cancer, along with an elevated risk of lobular breast cancer. Mutation carriers develop multi focal stage T1a signet ring cell carcinoma as early as 16-years. Development of these foci follow after somatic inactivation of the second CDH1 allele by mechanisms that include DNA promoter hypermethylation, followed by an indolent stage that can last years before further tumor progression. Valproic acid (VPA) and Decitabine (DAC) were to be used as an epigenetic therapy, they each have been identified in the literature for their anticancer properties. In this study a combination of VPA and DAC were used to test chemopreventive capabilities on three cell lines, non-malignant breast MCF10a cells, positive control non-small lung carcinoma NCI-H460 cells and a gastric adenocarcinoma AGS cells. Gene expression of CDH1 was analysed by real-time qPCR and western blotting. VPA was further characterised in MCF10a cells by analysing transcriptome sequence (RNA-Seq) and DNA methylation status changes (Illumina 450k Methyl-array). Both VPA and DAC increased E-cadherin expression in each of the cell lines, however E-cadherin up regulation was transient after VPA treatment withdrawal in MCF10a cells. No synergistic effects between VPA and DAC were detected in MCF10a cells, but a synergistic effect was observed in NCI-H460 cells. VPA treatment in MCF10a cells resulted in gene expression changes associated with cell proliferation and cell cycle. VPA had no measurable effect on DNA methylation levels in MCF10a cells. To further study VPA gene expression changes eleven migraine and epilepsy patients treated with VPA from the Dunedin hospital had buccal and whole blood samples taken before and after VPA treatment. No identifiable trends were detected by real-time qPCR either in the target gene, CDH1 or potential VPA surrogate genes identified from transcriptome data, TGM2 and ADAM23. Overall the characterised effects of VPA in MCF10a cells, were, down-regulation of genes associated with cell proliferation and cell cycle. Because of the transient increase in CDH1 expression in MCF10a cells and the inability to detect a change in expression in neurological patients after VPA treatment, VPA will require further evidence to support its use as a chemopreventive agent in the HDGC syndrome.