Downregulation of Homologous Recombination DNA Repair Genes by HDAC Inhibition in Prostate Cancer Is Mediated through the E2F1 Transcription Factor

Sushant K Kachhap,Shabana Shabbeer,Michael A Carducci,Justin Deangelis,Nadine Rosmus,Mohammad Hedayati,Madeleine S Q Kortenhorst,Michel D Wissing,Luigi Marchionni,Spencer J Collis,Hans Hammers,Janet Mendonca,Naseruddin Höti,Theodore L Deweese,Johan W R Nortier,Jianqing Lin,Kerstin Borgmann

doi:10.1371/journal.pone.0011208

Abstract

BackgroundHistone deacetylase inhibitors (HDACis) re-express silenced tumor suppressor genes and are currently undergoing clinical trials. Although HDACis have been known to induce gene expression, an equal number of genes are downregulated upon HDAC inhibition. The mechanism behind this downregulation remains unclear. Here we provide evidence that several DNA repair genes are downregulated by HDAC inhibition and provide a mechanism involving the E2F1 transcription factor in the process.Methodology/Principal FindingsApplying Analysis of Functional Annotation (AFA) on microarray data of prostate cancer cells treated with HDACis, we found a number of genes of the DNA damage response and repair pathways are downregulated by HDACis. AFA revealed enrichment of homologous recombination (HR) DNA repair genes of the BRCA1 pathway, as well as genes regulated by the E2F1 transcription factor. Prostate cancer cells demonstrated a decreased DNA repair capacity and an increased sensitization to chemical- and radio-DNA damaging agents upon HDAC inhibition. Recruitment of key HR repair proteins to the site of DNA damage, as well as HR repair capacity was compromised upon HDACi treatment. Based on our AFA data, we hypothesized that the E2F transcription factors may play a role in the downregulation of key repair genes upon HDAC inhibition in prostate cancer cells. ChIP analysis and luciferase assays reveal that the downregulation of key repair genes is mediated through decreased recruitment of the E2F1 transcription factor and not through active repression by repressive E2Fs.Conclusions/SignificanceOur study indicates that several genes in the DNA repair pathway are affected upon HDAC inhibition. Downregulation of the repair genes is on account of a decrease in amount and promoter recruitment of the E2F1 transcription factor. Since HDAC inhibition affects several pathways that could potentially have an impact on DNA repair, compromised DNA repair upon HDAC inhibition could also be attributed to several other pathways besides the ones investigated in this study. However, our study does provide insights into the mechanism that governs downregulation of HR DNA repair genes upon HDAC inhibition, which can lead to rationale usage of HDACis in the clinics.

Highlights

Epigenetic regulation of gene expression is thought to be brought about by both chromatin modulators that modify Nterminal tails of histones and DNA methylating enzymes that methylate CpG clusters in the promoter regions of eukaryotic genomes [1,2,3]
Applying Analysis of Functional Annotation (AFA) on the data set from the above microarray experiment we found that several genes of the homologous recombination (HR) repair and DNA damage response pathway are downregulated upon HDAC inhibition
Protein-protein interaction analysis on the microarray data revealed several genes related to the E2F1 and BRCA1 pathways were downregulated with both Histone deacetylase inhibitors (HDACis) more than 1.3 fold in both prostate cancer (PCa) cells lines (Fig. 1a)

Summary

Introduction

Epigenetic regulation of gene expression is thought to be brought about by both chromatin modulators that modify Nterminal tails of histones and DNA methylating enzymes that methylate CpG clusters in the promoter regions of eukaryotic genomes [1,2,3]. Cancer cells, including prostate cancer (PCa), recruit different HDACs associated with these large multi-protein co-repressor complexes to silence tumor suppressor genes and this serves as one rationale for the use of HDACis to treat cancer [8,9]. The activity of both class I and class II HDACs is inhibited by short chain fatty acids (Phenylbutyrate, Valproic acid (VPA)) and hydroxamic acids (Vorinostat, Trichostatin A), while benzamides. We demonstrate transcriptional downregulation of several DNA damage response genes in PCa cells upon HDAC inhibition, provide functional evidence of the involvement of HR repair pathway in compromised DNA repair, and provide a role of the E2F1 transcription factor in the downregulation of DNA damage response genes

Results

Discussion

Materials and Methods