Shabana Shabbeer

Targeting Tumor Angiogenesis with Histone Deacetylase Inhibitors: the Hydroxamic Acid Derivative LBH589

Purpose: Angiogenesis is required for tumor progression and represents a rational target for therapeutic intervention. Histone deacetylase (HDAC) inhibitors have been shown to have activity against various tumor cell types by inhibiting proliferation and inducing apoptosis both in vitro and in vivo. HDAC inhibitors have also been reported to inhibit angiogenesis. The goal of this study was to characterize the antiangiogenic and antitumor activity of a recently developed HDAC inhibitor, the hydroxamic derivative LBH589. Materials and Methods: To evaluate the antiangiogenesis activity of LBH589, we did cell cycle analysis, cell proliferation, tube formation, invasion assays in vitro, and Matrigel plug assay in vivo. To determine the antitumor activity of LBH589, we established human prostate carcinoma cell PC-3 xenografts in vivo. To evaluate the effect of LBH589 on endothelial signaling pathways, gene expression, and protein acetylation, we did Western blots and reverse transcription-PCR in human umbilical vein endothelial cells (HUVEC). Immunohistochemical analysis was done to evaluate new blood vessel formation in vivo. Results: LBH589 induced acetylation of histone H3 and α-tubulin protein in HUVECs. Histone and nonhistone protein acetylation correlated with induction of G2-M cell cycle arrest, inhibition of HUVEC proliferation, and viability. Noncytotoxic concentrations of LBH589 inhibited endothelial tube formation, Matrigel invasion, AKT, extracellular signal-regulated kinase 1/2 phosphorylation, and chemokine receptor CXCR4 expression. In vivo dosing of mice with LBH589 (10 mg/kg/d) reduced angiogenesis and PC-3 tumor growth. Conclusion: This study provides evidence that LBH589 induces a wide range of effects on endothelial cells that lead to inhibition of tumor angiogenesis. These results support the role of HDAC inhibitors as a therapeutic strategy to target both the tumor and endothelial compartment and warrant the clinical development of these agents in combination with angiogenesis inhibitors.

Pre-processing Agilent microarray data.

BackgroundPre-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 Agilents 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.ResultsSimple 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%CI : 39%, 47%). However, these effects were generally small regardless of the pre-processing method.ConclusionSimple 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 Agilents 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.

Chronic administration of valproic acid inhibits prostate cancer cell growth in vitro and in vivo

Valproic acid (VPA) is an established drug in the long-term therapy of seizure disorders. Recently, VPA has been associated with anticancer activity, an effect thought to be mediated through the inhibition of cellular histone deacetylase 1. We investigated the effect of various doses of VPA (0, 1.2, and 5.0 mmol/L) administered either acutely or chronically on histone acetylation, p21 gene expression, androgen receptor expression, prostate-specific antigen (PSA) expression, and cell survival and proliferation in prostate cancer cell lines. We also studied the effect of chronic VPA on tumor xenograft growth in vivo. Our results show that acute treatment (3 days) VPA can increase net histone H3 acetylation and up-regulate p21, AR, and cytosolic PSA expression. Interestingly, the effects on AR and PSA are reversed with chronic treatment. In addition, acute VPA reduces cell survival but has no effect on the subsequent proliferation of surviving cells following drug withdrawal. However, when VPA is chronically administered (10-14 days) to prostate cancer cells, even lower doses of VPA result in marked decreases in the net proliferation rate, correlating with increased caspase-2 and caspase-3 activation. These effects are evident in both androgen receptor-positive (LNCaP and C4-2) and androgen receptor-negative (DU145 and PC3) prostate cancer cells. Moreover, chronic VPA treatment results in statistically significant reduction of tumor xenograft growth in vivo. We conclude that acute treatment has nominal effects on prostate cancer cell survival and proliferation, but chronic VPA results in profound decreases in proliferation, independently of androgen regulation.

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

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

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.

Fenugreek: a naturally occurring edible spice as an anticancer agent

In recent years, various dietary components that can potentially be used for the prevention and treatment of cancer have been identified. In this study, we demonstrate that extract (FE) from the seeds of the plant Trigonella foenum graecum, commonly called fenugreek, are cytotoxic in vitro to a panel of cancer but not normal cells. Treatment with 10-15 ug/mL of FE for 72h was growth inhibitory to breast, pancreatic and prostate cancer cell lines (PCa). When tested at higher doses (15-20 ug/mL), FE continued to be growth inhibitory to PCa cell lines but not to either primary prostate or htert-immortalized prostate cells. At least part of the growth inhibition is due to induction of cell death, as seen by incorporation of Ethidium Bromide III into cancer cells exposed to FE. Molecular changes induced in PCa cells are: in DU-145 cells: down regulation of mutant p53, and in PC-3 cells up regulation of p21 and inhibition of TGF-β induced phosphorylation of Akt. The surprising finding of our studies is that death of cancer cells occurs despite growth stimulatory pathways being simultaneously up regulated (phosphorylated) by FE. Thus, these studies add another biologically active agent to our armamentarium of naturally occurring agents with therapeutic potential.

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.

Acetylation and Histone Deacetylase Inhibitors in Cancer

In recent years, the role of epigenetics in the development and treatment of cancer has gained interest and the effects of internal and external factors on the epigenetic profile are under investigation. The term “epigenetics” refers to modifications that influence phenotype without altering genotype. Epigenetic changes are potentially reversible but generally stably maintained during the cell cycle. Since Feinberg et al. described differences in DNA methylation in human cancer in 1983 [66], several mechanisms of epigenetic control have been identified such as DNA methylation and histone modifications including acetylation, methylation and phosphorylation. Recently Seligson et al. reported that specific histone modification patterns are predictive of the risk of prostate cancer recurrence [183] and Fraga et al. showed that a profound disruption in histone modification patterns is a common feature of cancer [70]. Epigenetic abnormalities can be acquired during fetal development and during the course of a life contributing to common cancer risk in adults [65]. Recently Fraga et al. reported that, although monozygotic twins are epigenetically identical during the first years of life, the patterns of global and locus specific epigenetic modifications and gene expression patterns in monozygotic twin pairs diverge as they become older [70]. These differences could be explained by the influence of external factors such as smoking, physical activity and diet [17,61] as well as by accumu-

A Multiple-Loop, Double-Cube Microarray Design Applied to Prostate Cancer Cell Lines with Variable Sensitivity to Histone Deacetylase Inhibitors

Purpose: Although microarray technology has been widely adopted by the scientific community, analysis of the ensuing data remains challenging. In this article, we present our experience with a complex design microarray experiment on resistance mechanisms of histone deacetylase inhibitors (HDACI). Experimental Design: To improve our understanding of the underlying mechanism of HDACI resistance in prostate cancer cells, we designed a novel “multiple-loop, double-cube” cDNA microarray experiment. In the experiment of 22 arrays, DU145 and PC3 cells were treated with two different HDACIs (vorinostat and valproic acid) and incubation periods (48 and 96 h). Preprocessing included exploratory analyses of the quality of the arrays and intensity-dependent within-array Loess normalization. An ANOVA model was used for inference. The results were validated by Western blot analysis of known treatment targets. Results: Treatment of PC3 and DU145 cells with HDACIs caused 2.8% to 10% (P < 0.001) differential expression across conditions; 51% to 73% of these genes were up-regulated and 28% to 49% were down-regulated. The extent of differential expression was associated with cell line (DU145 > PC3), HDACI (valproic acid ≥ vorinostat), and duration of treatment (96 > 48 h). We identified known and new treatment targets involved in cell cycle and apoptosis. Conclusion: A multiple-loop, double-cube microarray design can be used to identify HDACI-induced changes in gene expression possibly related to drug resistance.

Progression of Prostate Carcinogenesis and Dietary Methyl Donors: Temporal Dependence

Insufficient dose of dietary methyl groups are associated with a host of conditions ranging from neural tube defects to cancer. On the other hand, it is not certain what effect excess dietary methyl groups could have on cancer. This is especially true for prostate cancer, a disease that is characterized by increasing DNA methylation changes with increasing grade of the cancer. In this three-part study in animals, we look at (i) the effect of excess methyl donors on the growth rate of prostate cancer in vivo, (ii) the ability of 5-aza-2′-deoxycytidine (AdC), a demethylating agent, to demethylate in the presence of excess dietary methyl donors, and (iii) the effect of in utero feeding of excess methyl donors to the later onset of prostate cancer. The results show that when mice are fed a dietary excess of methyl donors, we do not see (i) an increase in the growth rate of DU-145 and PC-3 xenografts in vivo, or (ii) interference in the ability of AdC to demethylate the promoters of androgen receptor or Reprimo of prostate cancer xenografts but (iii) a protective effect on the development of higher grades of prostate cancer in the “Hi-myc” mouse model of prostate cancer which were fed the increased methyl donors in utero. We conclude that the impact of dietary methyl donors on prostate cancer progression depends upon the timing of exposure to the dietary agents. When fed before the onset of cancer, that is, in utero, excess methyl donors can have a protective effect on the progression of cancer. Cancer Prev Res; 5(2); 229–39. ©2011 AACR.