Platform-Independent Classification System to Predict Molecular Subtypes of High-Grade Serous Ovarian Carcinoma.

PURPOSE: Molecular cancer subtyping is an important tool in predicting prognosis and developing novel precision medicine approaches. We developed a novel platform-independent gene expression-based classification system for molecular subtyping of patients with high-grade serous ovarian carcinoma (HGSOC). METHODS: Unprocessed exon array (569 tumor and nine normal) and RNA sequencing (RNA-seq; 376 tumor) HGSOC data sets, with clinical annotations, were downloaded from the Genomic Data Commons portal. Sample clustering was performed by non-negative matrix factorization by using isoform-level expression estimates. The association between the subtypes and overall survival was evaluated by Cox proportional hazards regression model after adjusting for the covariates. A novel classification system was developed for HGSOC molecular subtyping. Robustness and generalizability of the gene signatures were validated using independent microarray and RNA-seq data sets. RESULTS: Sample clustering recaptured the four known The Cancer Genome Atlas molecular subtypes but switched the subtype for 22% of the cases, which resulted in significant (P = .006) survival differences among the refined subgroups. After adjusting for covariate effects, the mesenchymal subgroup was found to be at an increased hazard for death compared with the immunoreactive subgroup. Both gene- and isoform-level signatures achieved more than 92% prediction accuracy when tested on independent samples profiled on the exon array platform. When the classifier was applied to RNA-seq data, the subtyping calls agreed with the predictions made from exon array data for 95% of the 279 samples profiled by both platforms. CONCLUSION: Isoform-level expression analysis successfully stratifies patients with HGSOC into groups with differing prognosis and has led to the development of robust, platform-independent gene signatures for HGSOC molecular subtyping. The association of the refined The Cancer Genome Atlas HGSOC subtypes with overall survival, independent of covariates, enhances the clinical annotation of the HGSOC cohort.

Association of increased primary breast tumor AGR2 with decreased disease-specific survival.

OBJECTIVE: Tumor expression of Anterior Gradient 2 (AGR2), an endoplasmic reticulum protein disulfide isomerase, was associated with decreased breast cancer survival. We aimed to validate the association of tumor AGR2 mRNA expression with disease-specific survival (DSS) and identify differentially expressed signaling pathways between high and low AGR2 expression tumor groups. METHODS: Primary tumor mRNA expression data from the METABRIC study was used to evaluate AGR2 expression as a prognostic factor for DSS while adjusting for survival-determining confounders using Cox proportional-hazards regression. Differentially expressed genes and signaling pathway differences between high and low AGR2 groups were determined by modular enrichment analyses using DAVID and Ingenuity Pathway Analysis. RESULTS: Increased tumor AGR2 mRNA expression was associated with decreased DSS among 1,341 women (per each standard deviation increase of AGR2 expression: HR 1.14, 95% CI: 1.01-1.29, P = 0.03). Pathway analyses supported prior experimental studies showing that estrogen receptor 1 (ESR1) regulated AGR2 expression. Canonical signaling pathways significantly differentially represented between high and low AGR2 groups included those involved in inflammation and immunity. CONCLUSION: Increased primary tumor AGR2 expression was associated with decreased DSS. Pathway analyses suggested that increased AGR2 was associated with endoplasmic reticular homeostasis, possibly allowing tumor cells to overcome hypoxic stress and meet the increased protein demand of tumorigenesis, thereby preventing unfolded protein response-mediated apoptosis.

Low-grade and high-grade endometrial stromal sarcoma: A National Cancer Database study.

OBJECTIVE: To provide refined prognostic information from large cohorts of women with low-grade or high-grade endometrial stromal sarcoma (ESS). METHODS: We performed an observational retrospective cohort analysis of women diagnosed with low-grade or high-grade ESS from the 1998-2013 National Cancer Database. Kaplan-Meier and multivariable accelerated failure time survival analyses were performed to identify prognostic factors after multiple imputation of missing data. Recursive partitioning methods were used to rank prognostic factors in high-grade ESS. Matched cohort analyses were performed to hypothesis-test effects of adjuvant treatments. RESULTS: We identified 2414 and 1383 women with low-grade or high-grade ESS, respectively. Women with high-grade ESS had markedly decreased survival compared to women with low-grade ESS (five-year survival (95% CI): 32.6 (30.1-35.3%) versus 90.5% (89.3-91.8%), P<0.001). Among women with high-grade ESS, median survival (95% CI) was only 19.9 (17.1-22.1) months. Increased age and tumor size were associated with decreased survival in low-grade ESS. In high-grade ESS, additional negative prognostic factors were distant or nodal metastasis, omission of lymphadenectomy, and pathologically-positive surgical margins (all P<0.001). Use of adjuvant chemotherapy (time ratio (TR) (95% CI): 1.36 (1.17-1.58), P<0.001) and radiotherapy (TR (95% CI): 1.57 (1.32-1.87), P<0.001) were associated with increased survival for high-grade ESS. CONCLUSION: The contrasting excellent versus poor prognosis of low-grade versus high-grade ESS, respectively, was confirmed. The best treatment of high-grade ESS is early and complete surgical resection including lymphadenectomy. Adjuvant chemotherapy and radiotherapy may increase survival of women with high-grade ESS.

Prognosis and treatment of uterine leiomyosarcoma: A National Cancer Database study.

OBJECTIVE: To determine overall survival and factors associated with survival of women with uterine leiomyosarcoma. METHODS: We performed an observational cohort study of women with uterine leiomyosarcoma (n=7455) from the 1998-2013 National Cancer Database. Kaplan-Meier and multivariable accelerated failure time survival analyses were performed to investigate predictors of survival. Sensitivity and matched cohort analyses were performed to evaluate the roles of oophorectomy, lymphadenectomy, and chemotherapy in early leiomyosarcoma and chemotherapy in metastatic leiomyosarcoma. RESULTS: Median (interquartile range) age at diagnosis was 54 (48-63) years. Older age, higher comorbidity, black race, higher stage or grade, larger tumor size, lymph node involvement, metastasis at diagnosis, positive surgical margin, adjuvant chemotherapy, and brachytherapy were independently associated with decreased survival by unmatched cohort analyses. Private insurance was associated with increased survival. By matched cohort analyses, omitting oophorectomy was not associated with survival among women≤51years old at diagnosis (event time ratio (ETR) (95% CI) 1.06 (0.90-1.25), P=0.48). Omitting lymphadenectomy was not associated with survival (ETR (95% CI) 1.02 (0.94-1.10), P=0.60). Among women with stage I leiomyosarcoma, adjuvant chemotherapy was not associated with increased survival (ETR (95% CI) 0.91 (0.78-1.05), P=0.18). Chemotherapy was associated with increased survival of women with metastatic leiomyosarcoma (median survival (95% CI) 19.4 (16.4-23.0) versus 10.9 (7.7-14.3) months, ETR (95% CI) 1.66 (1.46-1.90), P<0.001). CONCLUSION: Early and complete resection is the best-evidenced treatment for uterine leiomyosarcoma. Oophorectomy and lymphadenectomy may be safely omitted for clinically uterus-confined leiomyosarcoma. Chemotherapy increases survival of women with metastatic leiomyosarcoma.

Identification of Genetic and Epigenetic Variants Associated with Breast Cancer Prognosis by Integrative Bioinformatics Analysis.

INTRODUCTION: Breast cancer being a multifaceted disease constitutes a wide spectrum of histological and molecular variability in tumors. However, the task for the identification of these variances is complicated by the interplay between inherited genetic and epigenetic aberrations. Therefore, this study provides an extrapolate outlook to the sinister partnership between DNA methylation and single-nucleotide polymorphisms (SNPs) in relevance to the identification of prognostic markers in breast cancer. The effect of these SNPs on methylation is defined as methylation quantitative trait loci (meQTL). MATERIALS AND METHODS: We developed a novel method to identify prognostic gene signatures for breast cancer by integrating genomic and epigenomic data. This is based on the hypothesis that multiple sources of evidence pointing to the same gene or pathway are likely to lead to reduced false positives. We also apply random resampling to reduce overfitting noise by dividing samples into training and testing data sets. Specifically, the common samples between Illumina 450 DNA methylation, Affymetrix SNP array, and clinical data sets obtained from the Cancer Genome Atlas (TCGA) for breast invasive carcinoma (BRCA) were randomly divided into training and test models. An intensive statistical analysis based on log-rank test and Cox proportional hazard model has established a significant association between differential methylation and the stratification of breast cancer patients into high- and low-risk groups, respectively. RESULTS: The comprehensive assessment based on the conjoint effect of CpG-SNP pair has guided in delaminating the breast cancer patients into the high- and low-risk groups. In particular, the most significant association was found with respect to cg05370838-rs2230576, cg00956490-rs940453, and cg11340537-rs2640785 CpG-SNP pairs. These CpG-SNP pairs were strongly associated with differential expression of ADAM8, CREB5, and EXPH5 genes, respectively. Besides, the exclusive effect of SNPs such as rs10101376, rs140679, and rs1538146 also hold significant prognostic determinant. CONCLUSIONS: Thus, the analysis based on DNA methylation and SNPs have resulted in the identification of novel susceptible loci that hold prognostic relevance in breast cancer.

Insights into the molecular interactions of thymoquinone with histone deacetylase: evaluation of the therapeutic intervention potential against breast cancer.

Many HDAC inhibitors have passed through the gateway of clinical trials. However, they have limited therapeutic implications due to their pleiotropic pharmaceutical properties and off-target effects. In view of this, dietary active phytochemicals were evaluated. Based upon the chemical and structural insights of HDAC active pockets, thymoquinone (TQ) was investigated to uncover its active participation in HDAC inhibition. The synergistic analysis of docking and molecular dynamics simulation disclosed the elementary interaction and stability of TQ with human HDACs. The in silico findings were corroborated with an in vitro analysis, demonstrating the efficient role of TQ in the attenuation of global HDAC activity. Furthermore, TQ also elicited downstream effects of HDAC inhibition: reactivation of HDAC target genes (p21 and Maspin), induction of the pro-apoptotic gene Bax, down regulation of the anti-apoptotic gene Bcl-2 and arrest of the cell cycle at the G2/M phase. Finally, the result of a higher cytotoxicity of TQ towards MCF-7 breast cancer cells in comparison to normal cells indicates the potential of TQ to be an anticancer drug.

Clusterin gene is predominantly regulated by histone modifications in human colon cancer and ectopic expression of the nuclear isoform induces cell death.

Clusterin (CLU) is an important glycoprotein involved in various cellular functions. Different reports have mentioned that the two isoforms of CLU; secretary (sCLU) and nuclear (nCLU) have opposite (paradoxical) roles in cancer development. sCLU provides pro-survival signal, whereas nCLU is involved in pro-apoptotic signaling. However, the molecular mechanism of CLU gene regulation is not clear as of yet. We hypothesize that CLU gene is regulated by DNA methylation and histone modifications and clusterin plays an important role in colon cancer. To evaluate the hypothesis, we investigated CLU expression in colon cancer tissues and DNA methylation and histone modification status of CLU gene promoter. It is apparent from immonohistology data that both benign and cancerous (primary and metastasis) formalin fixed paraffin embedded (FFPE) tissue samples exhibit CLU expression. However and interestingly only noncancerous tissue samples show nCLU expression. Ectopic expression of nCLU either by epigenetic modulators or by nCLU transfection is responsible for colon cancer cell death. To clarify the molecular mechanisms for regulation of expression of CLU isoforms, we have analyzed DNA methylation and histone modifications, such as histone H3K9me3, H3K27me3, H3K4me3, and H3K9AcS10P patterns around the CLU promoter. There is no remarkable change in the DNA methylation status upon treatment of the cells by AZA, TSA and SAM. Our findings clearly show that promoter histone H3K9me3 and H3K27me3 marks are elevated in comparison to H3K4me3 and H3K9AcS10P marks in colon cancer cell lines.

Mechanisms of DNA methyltransferase-inhibitor interactions: Procyanidin B2 shows new promise for therapeutic intervention of cancer.

DNA methyltransferases (DNMTs) is a key epigenetic enzyme for pharmacological manipulation and is employed in cancer reprogramming. During past few years multiple strategies have been implemented to excavate epigenetic compounds targeting DNMTs. In light of the emerging concept of chemoinformatics, molecular docking and simulation studies have been employed to accelerate the development of DNMT inhibitors. Among the DNMT inhibitors known till date, epigallocathechin-3-gallate (EGCG) was identified to be effective in reducing DNMT activity. However, the broad spectrum of EGCG to other diseases and variable target enzymes offers some limitations. In view of this, 32 EGCG analogues were screened at S-Adnosyl-L-homocysteine (SAH) binding pocket of DNMTs and procyanidin B2-3, 3'-di-O-gallate (procyanidin B2) was obtained as potent inhibitor having medicinally relevant chemical space. Further, in vitro analysis demonstrates the efficiency of procyanidin B2 in attenuating DNMT activity at IC50 of 6.88±0.647 µM and subsequently enhancing the expression of DNMT target genes, E-cadherin, Maspin and BRCA1. Moreover, the toxic property of procyanidin B2 towards triple negative breast cancer cells to normal cells offers platform for pre-clinical trial and an insight to the treatment of cancer.

Expression profiling of DNA methylation-mediated epigenetic gene-silencing factors in breast cancer.

BACKGROUND: DNA methylation mediates gene silencing primarily by inducing repressive chromatin architecture via a common theme of interaction involving methyl-CpG binding (MBD) proteins, histone modifying enzymes and chromatin remodelling complexes. Hence, targeted inhibition of MBD protein function is now considered a potential therapeutic alternative for thwarting DNA hypermethylation prompted neoplastic progress. We have analyzed the gene and protein expression level of the principal factors responsible for gene silencing, that is, DNMT and MBD proteins in MCF-7 and MDA-MB-231 breast cancer cell lines after treatment with various epigenetic drugs. RESULTS: Our study reveals that the epigenetic modulators affect the expression levels at both transcript and protein levels as well as encourage growth arrest and apoptosis in MCF-7 and MDA-MB-231 cells. AZA, TSA, SFN, and SAM inhibit cell growth in MCF-7 and MDA-MB-231 cell lines in a dose-dependent manner, that is, with increasing concentrations of drugs the cell viability gradually decreases. All the epigenetic modulators promote apoptotic cell death, as is evident form increased chromatin condensation which is a distinct characteristic of apoptotic cells. From FACS analysis, it is also clear that these drugs induce G2-M arrest and apoptosis in breast cancer cells. Further, transcript and protein level expression of MBDs and DNMTs is also affected - after treatment with epigenetic drugs; the level of transcripts/mRNA of MBDs and DNMTs has consistently increased in general. The increase in level of gene expression is substantiated at the protein level also where treated cells show higher expression of DNMT1, DNMT3A, DNMT3B, and MBD proteins in comparison to untreated cells. In case of tissue samples, the expression of different DNMTs is tissue stage-specific. DNMT1 exhibits significantly higher expression in the metastatic stage, whereas, DNMT3A and DNMT3B have higher expression in the primary stage in comparison to the metastatic samples. CONCLUSION: The epigenetic modulators AZA, TSA, SFN, and SAM may provide opportunities for cancer prevention by regulating the components of epigenetic gene-silencing machinery especially DNMTs and MBDs.

Elucidation of caveolin 1 both as a tumor suppressor and metastasis promoter in light of epigenetic modulators.

Caveolin-1 (CAV1) is an integral part of plasma membrane protein playing a vital role in breast cancer initiation and progression. CAV1 acts both as a tumor suppressor as well as an oncogene, and its activity is thus highly dependent on cellular environment. Keeping this fact in mind, the recent work is designed to reveal the role of CAV1 in inhibiting cancer cell progression in presence of epigenetic modulators like 5-aza-2'-deoxycytidine (AZA), trichostatin A (TSA), S-adenosyl methionine (SAM) and sulforaphane (SFN). Forced expression of CAV1 by AZA, TSA, and SFN is correlated to induction of apoptosis and inhibition of cell migration in breast cancer. In breast cancer along with promoter DNA methylation, other epigenetic mechanisms are also involved in CAV1 expression. These observations clearly provide a new scenario regarding the role of CAV1 in cancer and as a possible therapeutic target in breast cancer.

Chromatin dynamics: H3K4 methylation and H3 variant replacement during development and in cancer.

The dynamic nature of chromatin and its myriad modifications play a crucial role in gene regulation (expression and repression) during development, cellular survival, homeostasis, ageing, and apoptosis/death. Histone 3 lysine 4 methylation (H3K4 methylation) catalyzed by H3K4 specific histone methyltransferases is one of the more critical chromatin modifications that is generally associated with gene activation. Additionally, the deposition of H3 variant(s) in conjunction with H3K4 methylation generates an intricately reliable epigenetic regulatory circuit that guides transcriptional activity in normal development and homeostasis. Consequently, alterations in this epigenetic circuit may trigger disease development. The mechanistic relationship between H3 variant deposition and H3K4 methylation during normal development has remained foggy. However, recent investigations in the field of chromatin dynamics in various model organisms, tumors, cancer tissues, and cell lines cultured without and with therapeutic agents, as well as from model reconstituted chromatins reveal that there may be different subsets of chromatin assemblage with specific patterns of histone replacement executing similar functions. In this light, we attempt to explain the intricate control system that maintains chromatin structure and dynamics during normal development as well as during tumor development and cancer progression in this review. Our focus is to highlight the contribution of H3K4 methylation-histone variant crosstalk in regulating chromatin architecture and subsequently its function.

Epigenetic choreography of stem cells: the DNA demethylation episode of development.

Reversible DNA methylation is a fundamental epigenetic manipulator of the genomic information in eukaryotes. DNA demethylation plays a very significant role during embryonic development and stands out for its contribution in molecular reconfiguration during cellular differentiation for determining stem cell fate. DNA demethylation arbitrated extensive make-over of the genome via reprogramming in the early embryo results in stem cell plasticity followed by commitment to the principal cell lineages. This article attempts to highlight the sequential phases and hierarchical mode of DNA demethylation events during enactment of the molecular strategy for developmental transition. A comprehensive knowledge regarding the pattern of DNA demethylation during embryogenesis and organogenesis and study of the related lacunae will offer exciting avenues for future biomedical research and stem cell-based regenerative therapy.

Histone deacetylases: a saga of perturbed acetylation homeostasis in cancer.

In the current era of genomic medicine, diseases are identified as manifestations of anomalous patterns of gene expression. Cancer is the principal example among such maladies. Although remarkable progress has been achieved in the understanding of the molecular mechanisms involved in the genesis and progression of cancer, its epigenetic regulation, particularly histone deacetylation, demands further studies. Histone deacetylases (HDACs) are one of the key players in the gene expression regulation network in cancer because of their repressive role on tumor suppressor genes. Higher expression and function of deacetylases disrupt the finely tuned acetylation homeostasis in both histone and non-histone target proteins. This brings about alterations in the genes implicated in the regulation of cell proliferation, differentiation, apoptosis and other cellular processes. Moreover, the reversible nature of epigenetic modulation by HDACs makes them attractive targets for cancer remedy. This review summarizes the current knowledge of HDACs in tumorigenesis and tumor progression as well as their contribution to the hallmarks of cancer. The present report also describes briefly various assays to detect histone deacetylase activity and discusses the potential role of histone deacetylase inhibitors as emerging epigenetic drugs to cure cancer.

An insight into the various regulatory mechanisms modulating human DNA methyltransferase 1 stability and function.

DNA methylation is one of the principal epigenetic signals that participate in cell specific gene expression in vertebrates. DNA methylation plays a quintessential role in the control of gene expression, cellular differentiation and development. It also plays a central role in the preservation of chromatin structure and chromosomal integrity, parental imprinting, X-chromosome inactivation, aging and carcinogenesis. The foremost contributor in the mammalian methylation scheme is DNMT1, a maintenance methyltransferase that faithfully copies the pre-existing methyl marks onto hemimethylated daughter strands during DNA replication to maintain the established methylation patterns across successive cell divisions. The ever-changing cellular physiology and the significant part that DNA methylation plays in genome regulation necessitate rigid management of this enzyme. In mammalian cells, a host of intrinsic and extrinsic mechanisms regulate the expression, activity and stability of DNMT1. Transcriptional regulation, post-transcriptional auto-inhibitory controls and post-translational modifications of the enzyme are responsible for the efficient inheritance of DNA methylation patterns. Also, a large number of intra- and intercellular signaling cascades and numerous interactions with other modulator molecules that affect the catalytic activity of the enzyme at multiple levels function as major checkpoints of the DNMT1 control system. An in-depth understanding of the DNMT1 enzyme, its targeting and function is crucial for comprehending how DNA methylation is coordinated with other critical developmental and physiological processes. This review aims to provide a comprehensive account of the various regulatory mechanisms and interactions of DNMT1 so as to elucidate its function at the molecular level and understand the dynamics of DNA methylation at the cellular level.

Intricacies of hedgehog signaling pathways: a perspective in tumorigenesis.

The hedgehog (HH) signaling pathway is a crucial negotiator of developmental proceedings in the embryo governing a diverse array of processes including cell proliferation, differentiation, and tissue patterning. The overall activity of the pathway is significantly curtailed after embryogenesis as well as in adults, yet it retains many of its functional capacities. However, aberration in HH signaling mediates the initiation, proliferation and continued sustenance of malignancy in different tissues to varying degrees through different mechanisms. In this review, we provide an overview of the role of constitutively active aberrant HH signaling pathway in different types of human cancer and the underlying molecular and genetic mechanisms that drive tumorigenesis in that particular tissue. An insight into the various modes of anomalous HH signaling in different organs will provide a comprehensive knowledge of the pathway in these tissues and open a window for individually tailored, tissue-specific therapeutic interventions. The synergistic cross talking of HH pathway with many other regulatory molecules and developmentally inclined signaling pathways may offer many avenues for pharmacological advances. Understanding the molecular basis of abnormal HH signaling in cancer will provide an opportunity to inhibit the deregulated pathway in many aggressive and therapeutically challenging cancers where promising options are not available.