Subcutaneous 5-azacitidine treatment of naturally occurring canine urothelial carcinoma: a novel epigenetic approach to human urothelial carcinoma drug development.

PURPOSE: We determined the efficacy, biological activity, pharmacokinetics and safety of the hypomethylating agent 5-azacitidine (Celgene Corp., Summit, New Jersey) in dogs with naturally occurring invasive urothelial carcinoma. MATERIALS AND METHODS: We performed a preclinical phase I trial in dogs with naturally occurring invasive urothelial carcinoma to examine once daily subcutaneous administration of 5-azacitidine in 28-day cycles at doses of 0.10 to 0.30 mg/kg per day according to 2 dose schedules, including days 1 to 5 (28-day cohort) or days 1 to 5 and 15 to 19 (14-day cohort). Clinical efficacy was assessed by serial cystosonography, radiography and cystoscopy. Urinary 5-azacitidine pharmacokinetic analysis was also done. Pretreatment and posttreatment peripheral blood mononuclear cell and invasive urothelial carcinoma DNA, respectively, was analyzed for global and gene specific [CDKN2A (p14ARF)] methylation changes. RESULTS: Enrolled in the study were 19 dogs with naturally occurring invasive urothelial carcinoma. In the 28-day cohort the maximum tolerated dose was 0.20 mg/kg per day with higher doses resulting in grade 3 or 4 neutropenia in 4 of 6 dogs. In the 14-day cohort the maximum tolerated dose was 0.10 mg/kg per day with grade 3 or 4 neutropenia seen in 2 of 3 dogs treated at higher doses. No grade 3 or 4 nonhematological toxicity was observed during either dosing schedule. Of 18 dogs evaluable for tumor response partial remission, stable disease and progressive disease were observed in 4 (22.2%), 9 (50.0%) and 4 (22.2%), respectively. Consistent 5-azacitidine levels (205 to 857 ng/ml) were detected in urine. Pretreatment and posttreatment methylation analysis revealed no significant correlation with clinical response. CONCLUSIONS: Subcutaneous 5-azacitidine showed promising clinical activity in a canine invasive urothelial carcinoma model, thus meriting further development in humans with urothelial carcinoma.

Role of epigenomics in ovarian and endometrial cancers.

Ovarian cancer is the most lethal gynecologic malignancy and while constituting only 3% of all female cancers, it causes 14,600 deaths in the USA annually. Endometrial cancer, the most diagnosed and second-most fatal gynecologic cancer, afflicts over 40,000 US women annually, causing an estimated 7780 deaths in 2009. In both advanced ovarian and endometrial carcinomas, the majority of initially therapy-responsive tumors eventually evolve to a fully drug-resistant phenotype. In addition to genetic mutations, epigenetic anomalies are frequent in both gynecologic malignancies, including aberrant DNA methylation, atypical histone modifications and dysregulated expression of distinct microRNAs, resulting in altered gene-expression patterns favoring cell survival. In this article, we summarize the most recent hypotheses regarding the role of epigenetics in ovarian and endometrial cancers, including a possible role in tumor 'stemness' and also evaluate the possible therapeutic benefits of reversal of these oncogenic chromatin aberrations.

The role of chromatin, microRNAs, and tumor stem cells in ovarian cancer.

SUMMARY: The term "epigenetics" refers to DNA sequence-independent events that regulate gene expression, including DNA methylation, modifications to histone proteins, and microRNA-associated post-transcriptional control of protein translation. Epigenetic aberrations are now considered a defining characteristic of numerous human pathologies, including cancer. However, in contrast to DNA sequence mutations, many oncogenic epigenetic modifications are potentially reversible, with studies now showing numerous tumor cell types to possess remarkable phenotypic "plasticity," strongly supporting possible reversion of their malignant status. The "epigenome" of one such malignancy, ovarian cancer, has now been studied in depth, with specific epigenetic alterations correlated with tumor initiation, progression, metastasis, and therapy resistance. Ovarian cancer is the most lethal malignancy of the female reproductive tract, largely due to lack or diagnoses of early-stage disease and the eventual develoment of drug resistance; thus, reliable early detection biomarkers and alternative therapies are urgently needed. In this chapter, we discuss the epigenetics of ovarian cancer, including its likely role in the establishment of "cancer stem cells," i.e., highly tumorigenic progenitor cells believed to be largely or solely responsible for the propagation of malignant, chemotherapy-resistant and metastatic disease. Improved understanding of these gene-regulatory phenomena could likely result in improved detection and survival of this highly destructive malignancy.

An integrative ChIP-chip and gene expression profiling to model SMAD regulatory modules.

BACKGROUND: The TGF-beta/SMAD pathway is part of a broader signaling network in which crosstalk between pathways occurs. While the molecular mechanisms of TGF-beta/SMAD signaling pathway have been studied in detail, the global networks downstream of SMAD remain largely unknown. The regulatory effect of SMAD complex likely depends on transcriptional modules, in which the SMAD binding elements and partner transcription factor binding sites (SMAD modules) are present in specific context. RESULTS: To address this question and develop a computational model for SMAD modules, we simultaneously performed chromatin immunoprecipitation followed by microarray analysis (ChIP-chip) and mRNA expression profiling to identify TGF-beta/SMAD regulated and synchronously coexpressed gene sets in ovarian surface epithelium. Intersecting the ChIP-chip and gene expression data yielded 150 direct targets, of which 141 were grouped into 3 co-expressed gene sets (sustained up-regulated, transient up-regulated and down-regulated), based on their temporal changes in expression after TGF-beta activation. We developed a data-mining method driven by the Random Forest algorithm to model SMAD transcriptional modules in the target sequences. The predicted SMAD modules contain SMAD binding element and up to 2 of 7 other transcription factor binding sites (E2F, P53, LEF1, ELK1, COUPTF, PAX4 and DR1). CONCLUSION: Together, the computational results further the understanding of the interactions between SMAD and other transcription factors at specific target promoters, and provide the basis for more targeted experimental verification of the co-regulatory modules.

MicroRNA and mRNA integrated analysis (MMIA): a web tool for examining biological functions of microRNA expression.

MicroRNAs (miRNAs) are small (19-24 nt), nonprotein-coding nucleic acids that regulate specific 'target' gene products via hybridization to mRNA transcripts, resulting in translational blockade or transcript degradation. Although miRNAs have been implicated in numerous developmental and adult diseases, their specific impact on biological pathways and cellular phenotypes, in addition to miRNA gene promoter regulation, remain largely unknown. To improve and facilitate research of miRNA functions and regulation, we have developed MMIA (microRNA and mRNA integrated analysis), a versatile and user-friendly web server. By incorporating three commonly used and accurate miRNA prediction algorithms, TargetScan, PITA and PicTar, MMIA integrates miRNA and mRNA expression data with predicted miRNA target information for analyzing miRNA-associated phenotypes and biological functions by gene set analysis, in addition to analysis of miRNA primary transcript gene promoters. To assign biological relevance to the integrated miRNA/mRNA profiles, MMIA uses exhaustive human genome coverage, including classification into various disease-associated genes as well as conventional canonical pathways and Gene Ontology. In summary, this novel web server (cancer.informatics.indiana.edu/mmia) will provide life science researchers with a valuable tool for the study of the biological (and pathological) causes and effects of the expression of this class of interesting protein regulators.

A mixture model-based discriminate analysis for identifying ordered transcription factor binding site pairs in gene promoters directly regulated by estrogen receptor-alpha.

MOTIVATION: To detect and select patterns of transcription factor binding sites (TFBSs) which distinguish genes directly regulated by estrogen receptor-alpha (ERalpha), we developed an innovative mixture model-based discriminate analysis for identifying ordered TFBS pairs. RESULTS: Biologically, our proposed new algorithm clearly suggests that TFBSs are not randomly distributed within ERalpha target promoters (P-value < 0.001). The up-regulated targets significantly (P-value < 0.01) possess TFBS pairs, (DBP, MYC), (DBP, MYC/MAX heterodimer), (DBP, USF2) and (DBP, MYOGENIN); and down-regulated ERalpha target genes significantly (P-value < 0.01) possess TFBS pairs, such as (DBP, c-ETS1-68), (DBP, USF2) and (DBP, MYOGENIN). Statistically, our proposed mixture model-based discriminate analysis can simultaneously perform TFBS pattern recognition, TFBS pattern selection, and target class prediction; such integrative power cannot be achieved by current methods. AVAILABILITY: The software is available on request from the authors. CONTACT: lali@iupui.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Dominant-negative histone H3 lysine 27 mutant derepresses silenced tumor suppressor genes and reverses the drug-resistant phenotype in cancer cells.

Histone modifications and DNA methylation are epigenetic phenomena that play a critical role in many neoplastic processes, including silencing of tumor suppressor genes. One such histone modification, particularly at H3 and H4, is methylation at specific lysine (K) residues. Whereas histone methylation of H3-K9 has been linked to DNA methylation and aberrant gene silencing in cancer cells, no such studies of H3-K27 have been reported. Here, we generated a stable cell line overexpressing a dominant-negative point mutant, H3-K27R, to examine the role of that specific lysine in ovarian cancer. Expression of this construct resulted in loss of methylation at H3-K27, global reduction of DNA methylation, and increased expression of tumor suppressor genes. One of the affected genes, RASSF1, was shown to be a direct target of H3-K27 methylation-mediated silencing. By increasing DNA-platinum adduct formation, indicating increased access of the drug to target DNA sequences, removal of H3-K27 methylation resensitized drug-resistant ovarian cancer cells to the chemotherapeutic agent cisplatin. This increased platinum-DNA access was likely due to relaxation of condensed chromatin. Our results show that overexpression of mutant H3-K27 in mammalian cells represents a novel tool for studying epigenetic mechanisms and the Histone Code Hypothesis in human cancer. Such findings show the significance of H3-K27 methylation as a promising target for epigenetic-based cancer therapies.

Prognostic DNA methylation biomarkers in ovarian cancer.

PURPOSE: Aberrant DNA methylation, now recognized as a contributing factor to neoplasia, often shows definitive gene/sequence preferences unique to specific cancer types. Correspondingly, distinct combinations of methylated loci can function as biomarkers for numerous clinical correlates of ovarian and other cancers. EXPERIMENTAL DESIGN: We used a microarray approach to identify methylated loci prognostic for reduced progression-free survival (PFS) in advanced ovarian cancer patients. Two data set classification algorithms, Significance Analysis of Microarray and Prediction Analysis of Microarray, successfully identified 220 candidate PFS-discriminatory methylated loci. Of those, 112 were found capable of predicting PFS with 95% accuracy, by Prediction Analysis of Microarray, using an independent set of 40 advanced ovarian tumors (from 20 short-PFS and 20 long-PFS patients, respectively). Additionally, we showed the use of these predictive loci using two bioinformatics machine-learning algorithms, Support Vector Machine and Multilayer Perceptron. CONCLUSION: In this report, we show that highly prognostic DNA methylation biomarkers can be successfully identified and characterized, using previously unused, rigorous classifying algorithms. Such ovarian cancer biomarkers represent a promising approach for the assessment and management of this devastating disease.

Antimitogenic and chemosensitizing effects of the methylation inhibitor zebularine in ovarian cancer.

Deoxycytosine methylation within CpG islands of tumor suppressor genes plays a prominent role in the development and progression of drug-resistant ovarian cancer. Consequently, epigenetic therapies directed toward tumor suppressor demethylation/reexpression could potentially reverse malignant phenotypes and chemosensitize recalcitrant tumors. In this report, we examined the demethylating agent zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one], in comparison with the well-known methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), for its ability to inhibit ovarian cancer cell proliferation and to demethylate and induce tumor suppressor genes. Zebularine exerted significant (>5-aza-dC) antiproliferative effects against the ovarian cancer cell lines Hey, A2780, and the cisplatin-resistant A2780/CP in a dose-dependent manner (65% versus 35% inhibition at 48 hours, zebularine versus 5-aza-dC). Moreover, 48-hour treatment with 0.2 mmol/L zebularine significantly induced demethylation of the tumor suppressors ras-associated domain family 1A and human MutL homologue-1. RASSF1A gene reexpression was also observed, as was reexpression of two other tumor suppressors, ARHI and BLU, although levels differed from those induced by 5-aza-dC. Global analyses of DNA methylation revealed similar overall demethylation (2.5- to 3-fold) by 5-aza-dC and zebularine as determined by methyl acceptance assay. However, differences in demethylation of individual loci were observed as determined by differential methylation hybridization. Finally, we found that zebularine could resensitize the drug-resistant cell line A2780/CP to cisplatin, with a 16-fold reduction in the IC50 of that conventional agent. In summary, zebularine seems to be a promising clinical candidate, singly or combined with conventional regimens, for the therapy of drug-resistant ovarian cancer.

New anti-cancer strategies: epigenetic therapies and biomarkers.

Epigenetics is the study of chromatin modifications that affect gene expression without altering DNA nucleotide sequences. This review highlights a prominent role for epigenetic therapies, particularly those that reverse aberrant DNA methylation and histone acetylation, in the potential treatment of cancer. Administration of such therapies to reverse epigenetic "silencing" of tumor suppressors, including genes involved in chemotherapy responses, could prove useful in the management of cancer patients. In this review, we summarize recent advances in the use of methyltransferase and histone deacetylase inhibitors and possible synergistic combinations of these to achieve maximal tumor suppressor gene re-expression. Moreover, when used in combination with conventional chemotherapeutic agents, epigenetic-based therapies may provide a means to resensitize drug-resistant tumors to established treatments. As specific, aberrant epigenetic modifications are frequently associated with distinct cancer types, and likely occur early in tumorigenesis, these have potential utility as biomarkers. Finally, future directions are addressed, including alternative epigenetic targets, gene-specific modifications, and the use of bioinformatics.

The epigenetics of ovarian cancer drug resistance and resensitization.

Ovarian cancer is the most lethal of all gynecologic neoplasms. Early-stage malignancy is frequently asymptomatic and difficult to detect and thus, by the time of diagnosis, most women have advanced disease. Most of these patients, although initially responsive, eventually develop and succumb to drug-resistant metastases. The success of typical postsurgical regimens, usually a platinum/taxane combination, is limited by primary tumors being intrinsically refractory to treatment and initially responsive tumors becoming refractory to treatment, due to the emergence of drug-resistant tumor cells. This review highlights a prominent role for epigenetics, particularly aberrant DNA methylation and histone acetylation, in both intrinsic and acquired drug-resistance genetic pathways in ovarian cancer. Administration of therapies that reverse epigenetic "silencing" of tumor suppressors and other genes involved in drug response cascades could prove useful in the management of drug-resistant ovarian cancer patients. In this review, we summarize recent advances in the use of methyltransferase and histone deacetylase inhibitors and possible synergistic combinations of these to achieve maximal tumor suppressor gene re-expression. Moreover, when used in combination with conventional chemotherapeutic agents, epigenetic-based therapies may provide a means to resensitize ovarian tumors to the proven cytotoxic activities of conventional chemotherapeutics.