Bruce Moran

Evaluation of Cysteinyl Leukotriene Signaling as a Therapeutic Target for Colorectal Cancer

Colorectal cancer is the third most common cancer worldwide and is associated with significant morbidity and mortality. Current pharmacotherapy options include cytotoxic chemotherapy, anti-VEGF, and anti-EGFR targeting drugs, but these are limited by toxic side effects, limited responses and ultimately resistance. Cysteinyl leukotriene (CysLT) signaling regulates intestinal homeostasis with mounting evidence suggesting that CysLT signaling also plays a role in the pathogenesis of colorectal cancer. Therefore, CysLT signaling represents a novel target for this malignancy. This review evaluates reported links between CysLT signaling and established hallmarks of cancer in addition to its pharmacological potential as a new therapeutic target.

Fam60a defines a variant Sin3a-Hdac complex in embryonic stem cells required for self-renewal

Sin3a is the central scaffold protein of the prototypical Hdac1/2 chromatin repressor complex, crucially required during early embryonic development for the growth of pluripotent cells of the inner cell mass. Here, we compare the composition of the Sin3a‐Hdac complex between pluripotent embryonic stem (ES) and differentiated cells by establishing a method that couples two independent endogenous immunoprecipitations with quantitative mass spectrometry. We define the precise composition of the Sin3a complex in multiple cell types and identify the Fam60a subunit as a key defining feature of a variant Sin3a complex present in ES cells, which also contains Ogt and Tet1. Fam60a binds on H3K4me3‐positive promoters in ES cells, together with Ogt, Tet1 and Sin3a, and is essential to maintain the complex on chromatin. Finally, we show that depletion of Fam60a phenocopies the loss of Sin3a, leading to reduced proliferation, an extended G1‐phase and the deregulation of lineage genes. Taken together, Fam60a is an essential core subunit of a variant Sin3a complex in ES cells that is required to promote rapid proliferation and prevent unscheduled differentiation.

Transcriptomics of liver and muscle in Holstein cows genetically divergent for fertility highlight differences in nutrient partitioning and inflammation processes

BackgroundThe transition between pregnancy and lactation is a major physiological change for dairy cows. Complex systemic and local processes involving regulation of energy balance, galactopoiesis, utilisation of body reserves, insulin resistance, resumption of oestrous cyclicity and involution of the uterus can affect animal productivity and hence farm profitability. Here we used an established Holstein dairy cow model of fertility that displayed genetic and phenotypic divergence in calving interval. Cows had similar genetic merit for milk production traits, but either very good genetic merit for fertility traits (‘Fert+’; n = 8) or very poor genetic merit for fertility traits (‘Fert-’; n = 8). We used RNA sequencing to investigate gene expression profiles in both liver and muscle tissue biopsies at three distinct time-points: late pregnancy, early lactation and mid lactation (-18, 1 and 147 days relative to parturition, respectively).ResultsWe found 807 and 815 unique genes to be differentially expressed in at least one time-point in liver and muscle respectively, of which 79 % and 83 % were only found in a single time-point; 40 and 41 genes were found differentially expressed at every time-point indicating possible systemic or chronic dysregulation. Functional annotation of all differentially expressed genes highlighted two physiological processes that were impacted at every time-point in the study, These were immune and inflammation, and metabolic, lipid and carbohydrate-binding.ConclusionThese pathways have previously been identified by other researchers. We show that several specific genes which are differentially regulated, including IGF-1, might impact dairy fertility. We postulate that an increased burden of reactive oxidation species, coupled with a chronic inflammatory state, might reduce dairy cow fertility in our model.

Epigenetics of malignant melanoma

Patients with malignant melanoma generally have a good prognosis if the disease presents prior to metastasis. Due to progress with targeted and immunotherapies, the median survival of metastatic melanoma patients is now over 2 years. The disease is characterised by one of the highest somatic mutation rates observed amongst cancer types, with a specific mutational signature based on UV radiation damage evident. Highly prevalent mutations, such as the BRAFV600E, in the MAPK cascade indicate truncal involvement of this pathway in the earliest stage of melanoma. The molecular sub-classification of melanoma based on genetic alterations is now well established. This has paved the way for researchers in epigenetics to investigate specific pathways of known importance, and the involvement of the diverse range of epigenetic mechanisms. Herein, we review the literature to highlight that epigenetic alterations are integrally involved in this malignancy. We focus on the most current evidence around the epigenetic mechanisms: DNA methylation and demethylation including 5-hydroxy-methylcytosine; histone post-translational modifications including variant histones; chromatin remodelling complexes and in particular the polycomb-repressive complex PRC2 and its histone methyltransferase subunit EZH2; and non-coding RNAs. Each mechanism is described generally, studies involving melanoma are assessed and clinical relevance is highlighted where possible.

Differential gene expression in the endometrium reveals cytoskeletal and immunological genes in lactating dairy cows genetically divergent for fertility traits

Profitable milk production in dairy cows requires good reproductive performance. Calving interval is a trait used to measure reproductive efficiency. Herein we used a novel lactating Holstein cow model of fertility that displayed genetic and phenotypic divergence in calving interval, a trait used to define reproductive performance using a national breeding index in Ireland. Cows had similar genetic merit for milk production traits, but either very good genetic merit for fertility (Fert+; n=7) or very poor genetic merit for fertility (Fert-; n=6). We tested the hypothesis that Fert+ cows would have a corresponding detectable difference in endometrial gene expression compared with the Fert- cows. To do this, we sequenced the transcriptome of endometrial biopsies collected on Day 7 of the oestrous cycle (non-pregnant). This is an important stage for uterine remodelling and initiation of histotroph secretion. Significant differential expression (false discovery rate-adjusted P<0.1) of 403 genes between Fert+ and Fert- cows was found. A novel network-based functional analysis highlighted 123 genes from three physiologically relevant networks of the endometrium: (1) actin and cytoskeletal components; (2) immune function; and (3) ion transportation. In particular, our results indicate an overall downregulation of inflammation-related genes and an upregulation of multiple ion transporters and gated-voltage channels and cytoskeletal genes in Fert+ cows. These three topics, which are discussed in terms of the uterus and in the context of fertility, provide molecular evidence for an association between gene expression in the uterine environment and genetic merit for fertility in dairy cows.

Assessment of concordance between fresh-frozen and formalin-fixed paraffin embedded tumor DNA methylation using a targeted sequencing approach

DNA methylation is altered in many types of disease, including metastatic colorectal cancer. However, the methylome has not yet been fully described in archival formalin-fixed paraffin embedded (FFPE) samples in the context of matched fresh-frozen (FF) tumor material at base-pair resolution using a targeted approach. Using next-generation sequencing, we investigated three pairs of matched FFPE and FF samples to determine the extent of their similarity. We identified a ‘bowing’ pattern specific to FFPE samples categorized by a lower CG proportion at the start of sequence reads. We have found no evidence that this affected methylation calling, nor concordance of results. We also found no significant increase in deamination, measured by C>T transitions, previously considered a result of crosslinking DNA by formalin fixation and a barrier to the use of FFPE in methylation studies. The methods used in this study have shown sensitivity of between 60-70% based on positions also methylated in colorectal cancer cell lines. We demonstrate that FFPE material is a useful source of tumor material for methylation studies using targeted sequencing.

Master Transcriptional Regulators in Cancer: Discovery via Reverse Engineering Approaches and Subsequent Validation

Reverse engineering of transcriptional networks using gene expression data enables identification of genes that underpin the development and progression of different cancers. Methods to this end have been available for over a decade and, with a critical mass of transcriptomic data in the oncology arena having been reached, they are ever more applicable. Extensive and complex networks can be distilled into a small set of key master transcriptional regulators (MTR), genes that are very highly connected and have been shown to be involved in processes of known importance in disease. Interpreting and validating the results of standardized bioinformatic methods is of crucial importance in determining the inherent value of MTRs. In this review, we briefly describe how MTRs are identified and focus on providing an overview of how MTRs can and have been validated for use in clinical decision making in malignant diseases, along with serving as tractable therapeutic targets. Cancer Res; 77(9); 2186-90. ©2017 AACR.

PO-045 Evaluating liquid biopsies for methylomic profiling of prostate cancer

Introduction Epigenetic modifications, particularly DNA methylation, are centrally involved in prostate cancer (PCa) initiation and progression. Yet, how these alterations unfold and interplay in the progression to the lethal castration resistant phenotype is poorly understood. One reason for this is the difficulty in accessing metastatic tumour deposits for study. Recently, the analysis of liquid biopsies has emerged as a useful and minimally invasive method to study tumour characteristics. The aim of this study is to explore and compare how accurately the DNA methylation patterns of liquid biopsies reflect those of the primary tumour. Material and methods We identified 4 metastatic treatment-naïve PCa patients for whom matched biopsy cores (tumour and histologically matched normal), pre-biopsy urine (≤50 ml), and peripheral blood plasma (3 ml) were available. DNA was isolated from all sample types and quantified using the Qubit Fluorometer. DNA methylation was profiled using the Infinium MethylationEPIC BeadChip (Illumina), and analysed using RnBeads software. Absolute β-values were used to filter the data into probes of interest, with cut-offs for hyper- and hypo-methylation of >0.8,<0.2, respectively. Results and discussions We first considered whether matched normal tissue was epigenetically distinct from tumour by comparing the methylation patterns of several genes (i.e. RARB), for which hypermethylation is considered a hallmark of PCa. Focusing next on the methylation extremes (β >0.8 or β <0.2), we observed that hypermethylation was consistently more prevalent than hypomethylation in both tissue and liquid biopsies. Enumerating these methylation extreme probes revealed that the liquid biopsies contained a higher absolute abundance of hypo- and hypermethylation than the tissue biopsies. We also found that we could detect more hyper- and hypomethylated tumour-specific probes in urine than in plasma (80% vs. 62% and 69% vs. 64%, respectively). Conclusion Liquid biopsies are excellent surrogates for profiling tumour-specific DNA methylation, with urine demonstrating superior sensitivity over blood. Further analysis of differentially methylated regions in the liquid and tissue biopsies, and their relevance is PCa biology, is underway.

PO-090 Expression of the cocaine- and amphetamine-regulated transcript (CART) recruits SWI/SNF chromatin remodelling complexes to the oestrogen receptor

Introduction Cocaine- and amphetamine-regulated transcript (CART) peptides are neuropeptides involved in regulating physiological processes, such as feeding and drug reward. Recent studies have associated high CART expression with worse overall survival in patients with small-bowel carcinoid tumours and oestrogen receptor-positive (ER+), lymph node-negative breast cancer. CART was also shown to be associated with poor patient response to tamoxifen, suggesting CART may play a role in conferring tamoxifen resistance. Material and methods We have previously demonstrated that CART can impact the transcriptional activity of ERα through the use of western blotting and qPCR for specific ERα gene targets. RNA sequencing was carried out using a stable CART-inducible cell line model to identify genes which are upregulated/downregulated in cells expressing CART. Further, using our stable CART-inducible cell line model, we preformed ERα-Immunoprecipitation followed by in-solution mass spectrometry to identify differentially recruited protein complexes+/-CART expression. Results and discussions RNA sequencing revealed 156 significantly downregulated, and 100 significantly upregulated, genes in cells expressing CART (p<0.05). Through mining of publicly available ERα ChIP-seq data sets, both upregulated and downregulated gene sets were found to contain genes which have previously been shown to contain ERα binding events within their promotor regions. Mass spectrometry analysis revealed that the majority of proteins recruited to ERα in the presence of CART were members of the SWI/SNF (BAF) chromatin remodelling complex. The identification of SMARCD1 within this complex was of particular interest to this study, as this protein has previously been reported to be a critical mediator of nuclear receptor function. Further in silico analysis demonstrated high expression of SMARCD1 correlates with poor overall survival (OS) (p<0.00001) and distant metastasis free survival (DMFS) (p=0.00708) in a cohort of ER +breast cancer patients. Intriguingly, SMARCD1 expression did not correlate with poor OS or DMFS in a cohort of ER- breast cancer patients, suggesting that this negative impact on survival is potentially related to ERα. Conclusion In conclusion, we suggest that CART expression results in the recruitment of chromatin remodelling complexes to ERα in order to facilitate the regulation of receptor function.

Assessing DNA Methylation in Cancer Stem Cells

Many cancer-associated epigenetic signatures are also commonly observed in stem cells, just as epigenetic stem cell patterns are in cancer cells. DNA methylation is recognized as a hallmark of cancer development and progression. Herein, we describe two approaches to analyze DNA methylation, which can be applied to study or discover DNA methylation aberrations throughout the genome, as well as a more targeted investigation of regions of interest in cancer stem cells.