Does Valproic Acid Induce Neuroendocrine Differentiation in Prostate Cancer?

Valproic Acid (VPA) is a histone deacetylase inhibitor that holds promise for cancer therapy. Here, we investigate whether VPA treatment induces neuroendocrine differentiation of Prostate Cancer (PCa). A tissue microarray of VPA-treated and untreated tumor xenografts and cell lines of human PCa (LNCaP, C4-2, DU145, and PC-3) were generated and were analyzed by immunohistochemical analysis (IHC) for NE markers chromogranin A (CgA), synaptophysin, and NCAM (neural cell adhesion molecule). Western blot analysis for CgA was performed to confirm the results of the TMA. IHC analysis did not reveal any induction of CgA, synaptophysin, or NCAM in any xenograft after VPA treatment in vivo. In vitro, VPA treatment induced little synaptophysin expression in C4-2 and PC-3 cells and NCAM expression in LNCaP and PC-3 cells. In the case of CgA, VPA treatment decreased its expression in vitro in a dose-dependent manner, as determined by western blot analysis. Thus our data demonstrates that VPA does not induce NE differentiation of PCa cells in the physiologically relevant in vivo setting

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

Pre-processing Agilent microarray data

<p>Abstract</p> <p>Background</p> <p>Pre-processing methods for two-sample long oligonucleotide arrays, specifically the Agilent technology, have not been extensively studied. The goal of this study is to quantify some of the sources of error that affect measurement of expression using Agilent arrays and to compare Agilent's Feature Extraction software with pre-processing methods that have become the standard for normalization of cDNA arrays. These include log transformation followed by loess normalization with or without background subtraction and often a between array scale normalization procedure. The larger goal is to define best study design and pre-processing practices for Agilent arrays, and we offer some suggestions.</p> <p>Results</p> <p>Simple loess normalization without background subtraction produced the lowest variability. However, without background subtraction, fold changes were biased towards zero, particularly at low intensities. ROC analysis of a spike-in experiment showed that differentially expressed genes are most reliably detected when background is not subtracted. Loess normalization and no background subtraction yielded an AUC of 99.7% compared with 88.8% for Agilent processed fold changes. All methods performed well when error was taken into account by t- or z-statistics, AUCs ≥ 99.8%. A substantial proportion of genes showed dye effects, 43% (99%<it>CI </it>: 39%, 47%). However, these effects were generally small regardless of the pre-processing method.</p> <p>Conclusion</p> <p>Simple loess normalization without background subtraction resulted in low variance fold changes that more reliably ranked gene expression than the other methods. While t-statistics and other measures that take variation into account, including Agilent's z-statistic, can also be used to reliably select differentially expressed genes, fold changes are a standard measure of differential expression for exploratory work, cross platform comparison, and biological interpretation and can not be entirely replaced. Although dye effects are small for most genes, many array features are affected. Therefore, an experimental design that incorporates dye swaps or a common reference could be valuable.</p

The N-Myc Down Regulated Gene1 (NDRG1) Is a Rab4a Effector Involved in Vesicular Recycling of E-Cadherin

Cell to cell adhesion is mediated by adhesion molecules present on the cell surface. Downregulation of molecules that form the adhesion complex is a characteristic of metastatic cancer cells. Downregulation of the N-myc down regulated gene1 (NDRG1) increases prostate and breast metastasis. The exact function of NDRG1 is not known. Here by using live cell confocal microscopy and in vitro reconstitution, we report that NDRG1 is involved in recycling the adhesion molecule E-cadherin thereby stabilizing it. Evidence is provided that NDRG1 recruits on recycling endosomes in the Trans Golgi network by binding to phosphotidylinositol 4-phosphate and interacts with membrane bound Rab4aGTPase. NDRG1 specifically interacts with constitutively active Rab4aQ67L mutant protein and not with GDP-bound Rab4aS22N mutant proving NDRG1 as a novel Rab4a effector. Transferrin recycling experiments reveals NDRG1 colocalizes with transferrin during the recycling phase. NDRG1 alters the kinetics of transferrin recycling in cells. NDRG1 knockdown cells show a delay in recycling transferrin, conversely NDRG1 overexpressing cells reveal an increase in rate of transferrin recycling. This novel finding of NDRG1 as a recycling protein involved with recycling of E-cadherin will aid in understanding NDRG1 role as a metastasis suppressor protein

Mechanism of Growth Inhibition of Prostate Cancer Xenografts by Valproic Acid

Valproic Acid (VPA), a histone deacetylase inhibitor, has been demonstrated to cause a marked decrease in proliferation of prostate cancer (PCa) cells in vitro and a significant reduction in tumor volume in vivo. The goal of this study is to better understand the VPA-induced growth inhibition in vivo, by studying expression of various markers in PCa xenografts. Methods. For in vitro experiments, PCa cells were treated with 0, 0.6, and 1.2 mM VPA for 14 days. For in vivo models, experimental animals received 0.4% VPA in drinking water for 35 days. Tissue microarray was generated using cell pellets and excised xenografts. Results. VPA treatment causes cell cycle arrest in PCa cells in vivo, as determined by increase in p21 and p27 and decrease in cyclin D1 expression. Increased expression of cytokeratin18 was also seen in xenografts. LNCaP xenografts in treated animals had reduced androgen receptor (AR) expression. While decreased proliferation was found in vitro, increase in apoptosis was found to be the reason for decreased tumor growth in vivo. Also, an anti-angiogenic effect was observed after VPA treatment. Conclusion. VPA inhibits tumor growth by multiple mechanisms including cell cycle arrest, induction of differentiation, and inhibition of growth of tumor vasculature