EBV Associated Breast Cancer Whole Methylome Analysis Reveals Viral and Developmental Enriched Pathways

Background: Breast cancer (BC) ranks among the most common cancers in Sudan and worldwide with hefty toll on female health and human resources. Recent studies have uncovered a common BC signature characterized by low frequency of oncogenic mutations and high frequency of epigenetic silencing of major BC tumor suppressor genes. Therefore, we conducted a pilot genome-wide methylome study to characterize aberrant DNA methylation in breast cancer.Results: Differential methylation analysis between primary tumor samples and normal samples from healthy adjacent tissues yielded 20,188 differentially methylated positions (DMPs), which is further divided into 13,633 hypermethylated sites corresponding to 5339 genes and 6,555 hypomethylated sites corresponding to 2811 genes. Moreover, bioinformatics analysis revealed epigenetic dysregulation of major developmental pathways including hippo signaling pathway. We also uncovered many clues to a possible role for EBV infection in BC.Conclusion: Our results clearly show the utility of epigenetic assays in interrogating breast cancer tumorigenesis, and pinpointing specific developmental and viral pathways dysregulation that might serve as potential biomarkers or targets for therapeutic interventions

From Metabolism to Genetics and Vice Versa: The Rising Role of Oncometabolites in Cancer Development and Therapy

Over the last decades, the study of cancer metabolism has returned to the forefront of cancer research and challenged the role of genetics in the understanding of cancer development. One of the major impulses of this new trend came from the discovery of oncometabolites, metabolic intermediates whose abnormal cellular accumulation triggers oncogenic signalling and tumorigenesis. These findings have led to reconsideration and support for the long-forgotten hypothesis of Warburg of altered metabolism as oncogenic driver of cancer and started a novel paradigm whereby mitochondrial metabolites play a pivotal role in malignant transformation. In this review, we describe the evolution of the cancer metabolism research from a historical perspective up to the oncometabolites discovery that spawned the new vision of cancer as a metabolic disease. The oncometabolites' mechanisms of cellular transformation and their contribution to the development of new targeted cancer therapies together with their drawbacks are further reviewed and discussed

Integrative Analysis of Prostate Cancer Methylome and Smoking-induced Transgenerational Epigenomic Reprogramming

Epigenetic factors such as DNA methylation, histone modification and noncoding RNAs are highly associated with early developmental processes, later environmental adaption and diseases development such as cancer. With the availability of current high throughput assays (microarray and next generation sequencing), one can already produce comprehensive picture of the epigenetic profile, especially the DNA methylome, in normal and tumor/diseased cells. However, managing and analyzing such vast datasets is challenging. In addition, interpretation of the observations from (epi)genetic information is also a limiting factor due to the lack of understanding epigenetic mechanisms and the interactions between genetic and epigenetic factors under environmental selection. Thus, during my PhD studies, two pipelines were developed to process genome-wide methylation data generated by Methyl-CpG-immunoprecipitation sequencing (MCIP-seq) for the ICGC early onset prostate project and whole genome bisulfite sequencing (WGBS) for the environment induced transgenerational epigenetic remodeling project. The WGBS pipeline was adjusted later for a modified WGBS protocol, tagementaion-based WGBS, which allows to investigate the whole methylome (around 27 million CpGs) at single base resolution by using only 10-20 ng of input DNA compared to 3-5 ug required for traditional WGBS. Developing these computational tools, provided an opportunity to look closely at methylation changes in prostate cancers. With an integrative meta-analysis of public prostate (epi)genomic data and a large cohort of 7682 prostate cancer specimens, BAZ2A was found to be overexpressed in a large subset of prostate tumors that are characterized by early post-operative PSA recurrence and high tumor grades. In multivariate analyses, BAZ2A was found to be an independent factor predicting recurrence. Furthermore, high levels of BAZ2A were tightly associated with a distinct molecular subtype demarked by aberrant genome-wide DNA methylation and elevated numbers of genetic alterations suggesting a CpG island-methylator phenotype (CIMP) to selectively occur in BAZ2A-upregulated tumors. In summary, this study showed the clinical impact of BAZ2A as a key epigenetic regulator linking aberrant DNA methylation and outcome in prostate cancer. In addition, epigenetic changes is not only important for the diseased individuals including cancer, but also for the healthy individuals to adapt the external environmental stimulus such as smoking. In order to investigate the interaction between the methylome and environmental factor in a human prospective mother-child study at single base resolution, tobacco smoke-induced changes to epigenetic programming during the prenatal period was studied by WGBS and targeted methylation analysis. In mothers and children a distinct, genome-wide epigenetic response is induced. While mothers showed a genome-wide hypomethylation profile, children revealed tobacco-smoke induced hyper- and hypomethylation. By focusing on chromatin regulators, differential DNA methylation with functionally deregulated histone modifiers was linked, which together induce epigenetic reprogramming upon exposure to tobacco smoking. Together with the observed deregulation of a number of disease related pathways, the identified aberrant DNA methylation was suggested as a possible molecular mechanism linking between prenatal exposure and disease outcomes later in life. In summary, comprehensive epigenomic analyses were performed on both diseased and healthy individuals in order to shed a light on how epigenetic factors influence the tumor development and interact with external environmental stimulus

Development and validation of methods for genome-wide epigenetic analyses of human myogenic cells

Epigenetics is subjected to a pressing attention from the scientific community, because of its potential to explain the mechanisms of gene activation or repression. In this thesis I present a discovery-driven project aimed to the investigation of the epigenetic role in human myogenesis (and in particular the differentiation of myoblasts in myotubes). Studying epigenetics still presents significant hurdles, both experimental and computational. Therefore my first task was the establishment of robust protocols for investigating the role of epigenetics players during skeletal muscle differentiation. In particular, I focused on setting up the tools for studying DNA methylation and protein-DNA interactions, through bisulfite sequencing and chromatin immunoprecipitation. In this thesis, together with the description of the protocols that I developed, I report the first results that were obtained on myogenic cells. DNA methylation was investigated with bisulfite treatment of the DNA coupled with next generation sequencing. A novel method for studying the whole methylome was conceived and applied on one myoblast sample using SOLiD 5500xl platform giving reliable results. However, since the cost of methylome sequencing is still very high, instead of producing data for the whole methylome, I decided to focus on selected regions that could be relevant in methylation studies. For this reason I used the SureSelect MethylSeq Target Enrichment kit (Agilent) that effectively captures more than 2,700,000 CpG sites in the human genome. We identified less than 600 differentially methylated sites (DMS) in myoblasts compared to myotubes, however we observed that the activation of muscle specific genes seem to be poorly correlated with DNA methylation changes. Therefore, we argue that DNA methylation does not show a major role in the control of muscle specific genes. Interestingly, further analysis revealed that a high percentage of differentially methylated regions (DMR) localizes near novel non-coding RNA genes. On the one hand, this observation suggests a role for novel regulatory RNAs in the epigenetics of muscle differentiation; on the other hand, DNA methylation might have a role in the regulation of these RNAs. Together with DNA methylation, chromatin compaction is a major epigenetics player. In order to describe the epigenetic landscape of muscle differentiation, I optimized the ChIP-seq approach to define the localization of specific histone modifications on DNA. After setting-up the protocol for ChIP-seq, H3K4me3, H3K27me3 and H3K9ac histone modifications were mapped on myoblast DNA. As I started integrating gene expression and ChIP-seq data, I verified that a great concordance exists between gene expression and ChIP-seq results. In particular, euchromatin-associated histone modifications are found in transcription start sites of active genes, and heterochromatic signature spans promoters and bodies of inactive genes. Further investigation show that genes for non-coding RNAs have an euchromatic signature. This observation, together with the findings of DMRs in novel non-coding RNA genes, endorses the hypothesis of a role for novel regulatory RNAs in myogenic differentiation. In conclusion, the integration of BS-seq, ChIP-seq and RNA-seq data are opening interesting scenarios concerning the involvement of regulatory RNAs, while recent reports are suggesting to extend our investigation even to DNA hydroxymethylation in the epigenetics of muscle differentiation

DNMT gene expression and methylome in Marek’s disease resistant and susceptible chickens prior to and following infection by MDV

Marek’s disease (MD) is characterized as a T cell lymphoma induced by a cell-associated α-herpesvirus, Marek’s disease virus type 1 (MDV1). As with many viral infectious diseases, DNA methylation variations were observed in the progression of MD; these variations are thought to play an important role in host-virus interactions. We observed that DNA methyltransferase 3a (DNMT3a) and 3b (DNMT3b) were differentially expressed in chicken MD-resistant line 6(3) and MD-susceptible line 7(2) at 21 d after MDV infection. To better understand the role of methylation variation induced by MDV infection in both chicken lines, we mapped the genome-wide DNA methylation profiles in each line using Methyl-MAPS (methylation mapping analysis by paired-end sequencing). Collectively, the data sets collected in this study provide a more comprehensive picture of the chicken methylome. Overall, methylation levels were reduced in chickens from the resistant line 6(3) after MDV infection. We identified 11,512 infection-induced differential methylation regions (iDMRs). The number of iDMRs was larger in line 7(2) than in line 6(3), and most of iDMRs found in line 6(3) were overlapped with the iDMRs found in line 7(2). We further showed that in vitro methylation levels were associated with MDV replication, and found that MDV propagation in the infected cells was restricted by pharmacological inhibition of DNA methylation. Our results suggest that DNA methylation in the host may be associated with disease resistance or susceptibility. The methylation variations induced by viral infection may consequentially change the host transcriptome and result in diverse disease outcomes

Integrated analysis of epigenetic and genetic changes during MDS progression

DNA methylation is important during development of vertebrate organisms as well as for sustaining genome integrity and gene expression. Alterations of DNA methylation patterns are often associated with different diseases, for instance myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). Consequences of aberrant DNA methylation are the silencing of tumor suppressor genes due to hypermethylation as well as the hypomethylation‐mediated weakening of transcriptional repression, reactivation of retrotransposons and genomic instability. The major aim of this thesis was the integrated analysis of epigenetic and genetic changes during disease progression to identify target genes that could be involved in development or progression of myelodysplastic syndromes. To address this issue, DNA methylation analysis was performed in pediatric and adult MDS patients using the methyl-CpG-immunoprecipitation sequencing approach (MCIp-seq) and the targeted bisulfite sequencing of the myeloid regulome, respectively. It could be demonstrated that adult MDS patients show largely private DNA methylation changes and almost no common differentially methylated regions. Identified DMRs include RUNX1, FOXO3 and ZFPM1, which show methylation sensitivity in in vitro reporter gene assays. Another observation was made in this patient cohort in which DNA methylation changes only occur with alterations in clonal architecture. In cases of a genetically stable disease, no differences in DNA methylation patterns were observed over time. In pediatric MDS patients global DNA methylation analysis revealed a correlation of DNA methylation changes with germline GATA2 mutations and refractory cytopenia (RC). In detail, a patient cluster with lower DNA methylation degree exhibited the mentioned two features, while the other cluster of patients was associated with more advanced subtypes and higher DNA methylation. DMRs identified between these two patient groups are ZIC5, VILL and TRIM45, possibly playing a role in cancer. Methylation sensitivity of these regions has to be tested with in vitro reporter gene assays and will give information about a possible role for the development or progress of MDS. Regarding longitudinal studies in pediatric MDS patients, we could show the same result like in adult patients where DNA methylation changes correlate with alterations in genetic landscape. One potential epigenetic target gene found to be methylation sensitive and already described to play an important role in AML is the VENTX promoter region. In summary, our data suggest a tight correlation of epigenetic changes with clonal architecture of the diseased hematopoiesis, but the chronological order of appearance is still an open issue

Colorectal Cancers: From Present Problems to Future Solutions

The scientific community has made significant progress in our molecular understanding of sporadic and hereditary colorectal carcinogenesis and progression. Thie pertains to, e.g., the discovery of (mutated) oncogenes and tumor suppressor genes, microsatellite instabilities, modifications in DNA repair, cellular aging, signaling cascades, genomic, epigenetic, transcriptional, translational, and protein modifications, as well as microbiotic factors and further parameters. Progression and metastasis have been more intensively studied, especially during recent years, leading to an intensified knowledge on molecular protagonists and microenvironmental interactions contributing to invasion, dissemination, and metastasis; still, more concerted efforts need to be made to better understand issues such as metastasis to different sites or the metastatic heterogeneity of single cells. Nevertheless, based on actual discoveries, personalized medicine, together with highly interdisciplinary therapeutic strategies combining advanced levels of surgical techniques, oncology, and radiation in neoadjuvant, adjuvant, or palliative settings, has started to improve the clinical prognosis of individual patients with colorectal cancer. The present Special Issue features articles of excellent international experts with the latest data in the fields mentioned. With this Special Issue, we aim to deepen discussions amongst colleagues in all kinds of disciplines working on this disease and to intensify interdisciplinary collaborations aimed at an ultimate understanding of strategies to defeat and prevent, colorectal cancer, and its progression