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

Histone 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.Applying 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.Our 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

Repair protein levels in VPA treated PCa cells followed by irradiation.

<p>a) PCA cell lines treated with 1.5 mM VPA for 48 h, irradiated with different doses of radiation, probed for various repair proteins by western blotting. Unirradiated (0Gy cells) were also included. b) Total and nuclear extract of VPA (1.5 mM for 48 h) treated DU-145 cells with and without irradiation probed for BRCA1 protein. Unirradiated (0Gy cells) were also included. c) Top panel shows TOPO IIα protein level in nuclear extract from DU-145 cells upon VPA treatment. Bottom panel is an agarose gel showing TOPO IIα activity in the same extracts, lane 4 is a negative control.</p

VPA treated prostate cancer cells show decreased repair capacity and γ-H2AX clearance.

<p>a) Neutral comet assay performed on prostate cancer cells treated with 1.5 mM VPA for 48 h and irradiated with 6 Gy γ- radiation followed by a 4 h repair interval. Unirradiated cells (0Gy) with and without VPA treatment did not show any significant difference in comet tail moments. The graph depicts average tail moment of 50 cells error bar indicates SD value of three experiments. Comparisons have been performed using the student's t-test. b) Immunofluorescence showing H2AX Ser¹³⁹ staining in DU-145 cells treated with 1.5 mM VPA for 48 h and irradiated with 4Gy radiation followed by 4 h repair time. The graph shows quantitation of H2AX foci in control (blue column) and treated (red column) DU-145 and LNCaP cells. Error bar represents standard deviation of three independent experiments.</p

Analysis of Functional Annotation (AFA) in HDACi treated PCa cells.

<p>a) DU145 and PC3 cells were treated with two different HDACis (vorinostat (SAHA, 1 µM), and VPA (Valproic acid, 1 mM) for incubation periods (2days and 4days). AFA reveals down-regulation of genes involved with DNA damage and response (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011208#pone-0011208-t001" target="_blank">Table 1</a>). AFA results for protein-protein interaction indicate BRCA1 and E2F interacting networks are affected by HDAC inhibition. Color code represents as 10^-n the <i>p-</i>values obtained from the Wilcoxon rank-sum test. b) DNA repair genes downregulated ≥1.3 fold in both PC3 and DU-145 cells upon treatment with both VPA and vorinostat. c) Validation of the AFA was done by a Q-PCR analysis on a subset of genes downregulated upon 1.5 mM VPA treatment. Results are depicted as fold change over untreated control cells.</p

Sensitivity of DU-145 cells to DNA damaging agents upon VPA treatment.

<p>a) Clonogenic assay performed in DU-145 after treatment with different doses of VPA for 48 h before irradiation with different doses of radiation. Top panel shows representative clonogenic plates with 0Gy and 4 Gy radiation graph below depicts surviving fraction after VPA treatments and irradiation. Error bar represents standard deviation of three independent experiments. b) Clonogenic assay performed on DU-145 cells treated with 1.5 mM VPA and cisplatin (100 nM and 250 nM) for 48 h. Error bar represents standard deviation. c) Clonogenic assay performed on DU-145 cells treated with 1.5 mM VPA and hydroxyurea (0.5 mM and 1 mM) for 48 h. Error bar represents standard deviation.</p

Repair protein in prostate cancer cells treated with VPA.

<p>a) Western blot showing acetylation of histone H3 protein in DU-145 cells after treatment with different doses of VPA for 48 h. b) DNAPK and NBS1 protein levels in VPA treated DU-145 cells for 48 h. c) Western blot showing RAD51 and BRCA1 protein levels in LNCaP and DU-145 cells treated with varying dosage of VPA for 48 h. Blot on the right shows DU-145 cells treated with 1.5 mM VPA for varying timepoints probed for BRCA1 protein.</p

Analysis of Functional Annotation results for gene ontology.

<p>The enrichment driven by down-regulation of gene expression showed an overall involvement of processes related to the response of DNA damage. The reports the P-values obtained from the Wilcoxon rank-sum test after correction for multiple testing by the Benjamini-Hochberg method.</p

Downregulation of E2F1 mediates downregulation of DNA damage and response genes.

<p>a) ChIP analysis of VPA (1.5 mM for 48 h) treated DU-145 cells for acetylated histone H3 status in the promoters of repair genes. The bar diagram is a densitometry reading of the agarose gel shown normalized to inputs. b) Activator and repressor E2F protein levels in VPA (1.5 mM for 48 h) treated cells LNCaP and DU-145 cells.E2F levels remained downregulated upon VPA treatment even after irradiation with 4Gy radiation as shown in DU-145 cells, unirradiated (0Gy) cells served as a radiation control. c) Luciferase reporter assays in DU-145 and LNCaP cells, treated with varying concentrations of VPA, using proximal promoter regions, encompassing E2F binding regions of downregulated repair genes. d) ChIP analysis for E2F occupancy in the promoter regions of downregulated genes. ChIP was performed using antibodies against E2Fs (1, 4, and 6) in VPA (1.5 mM for 48 h) treated and control DU-145 cells. The bar diagram represents densitometric readings normalized to respective inputs.</p

Recruitment of HR repair proteins to the damaged site and HR repair in VPA treated PCa cells.

<p>a) Immunofluorescence analysis of DU-145 cells treated with 1.5 mM VPA for 48 h and irradiated with 4Gy of radiation probed for BRCA1 (red) and NBS1 (green). Nuclei were counterstained with DAPI. Cytoplasmic BRCA1 (arrow) seen in VPA treated cells suggest impaired recruitment of BRCA1 in VPA treated cells. b) Immunofluorescence analysis of LNCaP cells treated with 1.5 mM VPA for 48 h and irradiated with 4Gy of radiation probed for H2AX Ser¹³⁹ (red) and RAD51 (green). Nuclei were counterstained with DAPI. Cells having >25 H2AX Ser¹³⁹foci were analyzed. Cytoplasmic RAD51 (arrow) seen in VPA treated cells suggest impaired recruitment of BRCA1 in VPA treated cells. c) Quantification of the number of BRCA1 and RAD51 foci colocalizing with H2AX Ser¹³⁹ foci in DU-145 and LNCaP cells after treatment with 1.5 mM VPA and irradiation with 4Gy of radiation. A total of 100 cells having >25 H2AX Ser¹³⁹foci were counted. Error bars indicate standard deviation from mean. d) FACS analysis depicting a HR repair assay using a plasmid reporter construct in LNCaP cells after treatment with varying concentration of VPA.</p