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BACKGROUND Targeting kinases as regulators of cellular processes that drive cancer progression is a promising approach to improve patient outcome in GBM management. The glycogen synthase kinase 3 (GSK-3) plays a role in cancer progression and is known for its pro-proliferative activity in gliomas. The anti-proliferative and cytotoxic effects of the GSK-3 inhibitor AZD2858 were assessed in relevant in vitro and in vivo glioma models to confirm GSK-3 as a suitable target for improved single agent or combination treatments. MATERIAL AND METHODS The immortalised cell line U251 and the patient derived cell lines GBM1 and GBM4 were used in in vitro studies including MTT, clonogenic survival, live cell imaging, immunofluorescence microscopy and flow cytometry to assess the cytotoxic and anti-proliferative effects of AZD2858. Observed anti-proliferative effects were investigated by microarray technology for the identification of target genes with known roles in cell proliferation. Clinical relevance of targeting GSK-3 with the inhibitor either for single agent or combination treatment strategies was determined by subcutaneous and orthotopic in vivo modelling. Whole mount mass spectroscopy was used to confirm drug penetration in orthotopic tumour models. RESULTS AZD2858 was cytotoxic at low micromolar concentrations and at sub-micromolar concentrations (0.01 - 1.0 μM) induced mitotic defects in all cell lines examined. Prolonged mitosis, centrosome disruption/duplication and cytokinetic failure leading to cell death featured prominently among the cell lines concomitant with an observed S-phase arrest. No cytotoxic or anti-proliferative effect was observed in normal human astrocytes. Analysis of the RNA microarray screen of AZD2858 treated glioma cells revealed the dysregulation of mitosis-associated genes including ASPM and PRC1, encoding proteins with known roles in cytokinesis. The anti-proliferative and cytotoxic effect of AZD2858 was also confirmed in both subcutaneous and orthotopic in vivo models. In addition, combination treatment with AZD2858 enhanced clinically relevant radiation doses leading to reduced tumour volume and improved survival in orthotopic in vivo models. CONCLUSION GSK-3 inhibition with the small molecule inhibitor AZD2858 led to cell death in glioma stem cells preventing normal centrosome function and promoting mitotic failure. Normal human astrocytes were not affected by treatment with the inhibitor at submicromolar concentrations. Drug penetration was observed alongside an enhanced effect of clinical radiotherapy doses in vivo. The reported aberrant centrosomal duplication may be a direct consequence of failed cytokinesis suggesting a role of GSK-3 in regulation of mitosis in glioma. GSK-3 is a promising target for combination treatment with radiation in GBM management and plays a role in mitosis-associated events in glioma biology.

Purpose: Breast cancer affects both genders, but is understudied in men. Although still rare, male breast cancer (MBC) is being diagnosed more frequently. Treatments are wholly informed by clinical studies conducted in women, based on assumptions that underlying biology is similar. Experimental Design: A transcriptomic investigation of male and female breast cancer was performed, confirming transcriptomic data in silico. Biomarkers were immunohistochemically assessed in 697 MBCs (n = 477, training; n = 220, validation set) and quantified in pre- and posttreatment samples from an MBC patient receiving everolimus and PI3K/mTOR inhibitor. Results: Gender-specific gene expression patterns were identified. eIF transcripts were upregulated in MBC. eIF4E and eIF5 were negatively prognostic for overall survival alone (log-rank P = 0.013; HR = 1.77, 1.12–2.8 and P = 0.035; HR = 1.68, 1.03–2.74, respectively), or when coexpressed (P = 0.01; HR = 2.66, 1.26–5.63), confirmed in the validation set. This remained upon multivariate Cox regression analysis [eIF4E P = 0.016; HR = 2.38 (1.18–4.8), eIF5 P = 0.022; HR = 2.55 (1.14–5.7); coexpression P = 0.001; HR = 7.04 (2.22–22.26)]. Marked reduction in eIF4E and eIF5 expression was seen post BEZ235/everolimus, with extended survival. Conclusions: Translational initiation pathway inhibition could be of clinical utility in MBC patients overexpressing eIF4E and eIF5. With mTOR inhibitors that target this pathway now in the clinic, these biomarkers may represent new targets for therapeutic intervention, although further independent validation is required. Clin Cancer Res; 23(10); 2575–83. ©2016 AACR.

In the human prostate, expression of prostate-specific genes is known to be directly regulated by the androgen–induced stimulation of the androgen receptor (AR). However, less is known about the expression control of the prostate-restricted TGM4 (hTGP) gene. In the present study we demonstrate that the regulation of the hTGP gene depends mainly on retinoic acid (RA). We provide evidence that the retinoic acid receptor gamma (RAR-G) plays a major role in the regulation of the hTGP gene and that presence of the AR, but not its transcriptional transactivation activity, is critical for hTGP transcription. RA and androgen responsive elements (RARE and ARE) were mapped to the hTGP promoter by chromatin immunoprecipitation (ChIP), which also indicated that the active ARE and RARE sites were adjacent, suggesting that the antagonistic effect of androgen and RA is related to the relative position of binding sites. Publicly available AR and RAR ChIP-seq data was used to find gene potentially regulated by AR and RAR. Four of these genes (CDCA7L, CDK6, BTG1 and SAMD3) were tested for RAR and AR binding and two of them (CDCA7L and CDK6) proved to be antagonistically regulated by androgens and RA confirming that this regulation is not particular of hTGP.

BACKGROUND Prostate development and maintenance in the adult results from an interaction of stromal and glandular components. Androgens can drive this process by direct action on the stroma. We investigated whether there was a direct link between androgens and another key regulator of stromal cells, intracellular Ca2+ ([Ca2+]i). METHODS Prostate stromal cells were freshly obtained and cultures derived from patients with benign prostatic hyperplasia. Gene expression in dihydrotestosterone treated and untreated cells was compared using Affymetrix gene expression arrays and Ca2+ regulated features were identified by Gene Ontology (GO). Changes in [Ca2+]i were determined in Fluo-4 loaded cells. Androgen regulation was confirmed by chromatin immunoprecipitaion. RESULTS Stromal cell cultures were sorted for expression of integrin α1β1, which enriched for cells expressing the androgen receptor (AR). We identified key functional categories, within the androgen-induced gene expression signature, focusing on genes involved in calcium signaling. From this analysis, stromal interaction molecule-1 (STIM1) was identified as a significantly differentially expressed gene with four relevant associated GO terms. DNA sequence analysis showed that the promoter region of STIM1 contained putative androgen response element sequences in which AR binding ability of STIM1 was confirmed. Androgens directly regulated STIM1 expression and STIM1 effects on store-operated calcium entry were inhibited by STIM1 knock-down. Reduced STIM1 expression in prostate stromal cells led to a reduction in basal Ca2+ levels, the amount of Ca2+ released by thapsigargin and a reduction in store filling following TG-induced store depletion. CONCLUSIONS These results indicate that androgens modulate [Ca2+]i through the direct regulation of the STIM1 gene by AR binding to the STIM1 promoter. Prostate 71:1646–1655, 2011. © 2011 Wiley-Liss, Inc.

Background Cancer stem-like cells (CSCs) are responsible for disease progression and relapse (Schatton et al, Bioessays 2009, 31:1038-49). Therefore, the eradication of Ewing's sarcoma (ES)-CSCs is expected to improve outcome for patients. Since the transfer of exosome cargo between cells drives ES progression (Ventura et al, Oncogene, 2016, 35:3944-54) or can be exploited as biomarker (Miller et al, Biology of the Cell 2013, 105:289-303; Tsugita et al, PLoS ONE 2013, 8:e77416) we have characterized primary patient-derived ES, paired ES-CSCs, and the exosomal cargo from these cultures to identify potential prognostic biomarkers of risk and targets for the development of new treatments. Methods ES taken at diagnosis and resection were collected following patient consent (GenoEwing; IRAS-167880). ES-CSCs were isolated using a functional self-renewing assay. EWSR1 translocations were confirmed by FISH using a break-apart probe and RT-PCR. Exosomes were collected after incubation of cultures in serum-free media for 48h, isolated using exoEasy Maxi kit (Qiagen) and characterized by Nanoparticle tracking, Western blot and flow cytometry. The expression of the ES-marker CD99 was investigated by immunocytochemistry, Western blot and flow cytometry. RNA was extracted from ES cells and paired exosomes using the miRNeasy Micro kit (Qiagen) and RNA quality confirmed employing the Agilent Bioanalyser. Total RNA libraries were prepared and sequenced using the HiSeq3000 (Illumina®). Reads were processed using fqtools (Droop, Bioinformatics 2016, 32:1883-4), aligned by STAR (Dobin et al, Bioinformatics 2013, 29:15-21) and differential expression was determined by DESeq2 (Anders et al, Genome Biology 2010, 11:R106). Genes were ranked on adjusted p value and log2 fold change; candidates were validated by RTqPCR. Results ES patient-derived cells expressed CD99 and contained an EWSR1 gene translocation (23/23). The median size of isolated ES exosomes was 84nm (range 32-132nm). Exosomes were enriched for small RNA (53±5% in exosomes vs 12±3% in cells). The optimal protein panel to identify ES exosomes is CD81, CD63 and TSG-101; CD9 was not detected. Consistent with the exosomal cell of origin, expression of CD99 was detected in 71-79% of isolated vesicles. The RNA profile of parental cultures and daughter ES-CSCs was compared and two genes with increased expression in ES-CSCs were validated by RTqPCR (p Conclusions For the first time, we have sequenced paired exosomes and patient-derived ES cells to identify a potential prognostic biomarker and novel target for therapy. Work funded by University of Leeds, Ewing's Sarcoma Research Trust (ESRT) and Bone Cancer Research Trust (BCRT). Citation Format: Mariona Chicón Bosch, Elizabeth A. Roundhill, Alastair P. Droop, Michael Parry, Lee Jeys, Susan A. Burchill. RNAseq of patient-derived cancer stem-like cells and exosomes provides new insights into Ewing's sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3696.

Introduction Bladder cancer (BC) is the most common cancer of the urinary tract and the ninth most common cancer in the world. There are two main forms of the disease; non-muscle-invasive BC (NMIBC) and muscle-invasive BC (MIBC). NMIBC commonly recurs and multiple tumours may be resected from the same patient over many years. This provides a unique opportunity to study the molecular events that occur during disease evolution. Some patients receive courses of intravesical chemotherapy, which may provide a potent selective advantage during disease evolution. To understand chemotherapy-induced or –selected events in bladder cancer, tumours from patients who have undergone intravesical chemotherapy were analysed using next-generation sequencing and patterns of tumour evolution are being assessed through the analysis of DNA copy number and somatic mutations. Material and methods Low-pass whole-genome sequencing was used to generate copy number (CN) profiles for tumour samples from 23 patients with recurrent NMIBC. Whole exome sequencing was performed on paired pre- and post-chemotherapy tumours for a subset of 8 patients. Samples are being analysed for mutational signatures and subclone composition. Results and discussions In total, 67 tumours from 23 patients with NMIBC were analysed for CN alterations (CNAs). Loss of 9 p21.3, a region that includes the tumour suppressor genes CDKN2A and CDKN2B, was the most common event, seen in 64% of the tumours. Of these, two thirds were homozygous deletion events. Complete loss of a copy of chromosome 9 was the most common large-scale event observed in just under half of the tumours. Recurrent tumours from each patient tended to share CNAs and no CN alterations that specifically related to treatment were identified. To examine tumour evolution, exome data will be assessed for mutational burden, mutational signatures and subclone composition. Comparisons between pre- and post- treatment tumours will identify changes potentially associated with chemotherapy treatment. Conclusion Low-pass whole genome sequencing showed that CN changes tend to be shared amongst recurrent tumours from the same patient, suggestive of a monoclonal origin. Exome sequencing data will provide further information on tumour evolution and provide an insight into the influence of chemotherapy on these samples.

Summary: The Sun Grid Engine (SGE) high-performance computing batch queueing system is commonly used in bioinformatics analysis. Creating re-usable scripts for the SGE is a common challenge. The qsubsec template language and interpreter described here allow researchers to easily create generic template definitions that encapsulate a particular computational job, effectively separating the process logic from the specific run details. At submission time, the generic template is filled in with specific values. This system provides an intermediate level between simple scripting and complete workflow management tools. Availability and implementation: Qsubsec is open-source and is available at https://github.com/alastair-droop/qsubsec. Contact: a.p.droop@leeds.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Oral squamous cell carcinoma (OSCC) is a prevalent cancer with poor prognosis. Most OSCC progresses via a non-malignant stage called dysplasia. Effective treatment of dysplasia prior to potential malignant transformation is an unmet clinical need. To identify markers of early disease, we performed RNA sequencing of 19 matched HPV negative patient trios: normal oral mucosa, dysplasia and associated OSCC. We performed differential gene expression, principal component and correlated gene network analysis using these data. We found differences in the immune cell signatures present at different disease stages and were able to distinguish early events in pathogenesis, such as upregulation of many HOX genes, from later events, such as down-regulation of adherens junctions. We herein highlight novel coding and non-coding candidates for involvement in oral dysplasia development and malignant transformation, and speculate on how our findings may guide further translational research into the treatment of oral dysplasia.

Summary: Many Next Generation Sequencing analyses involve the basic manipulation of input sequence data before downstream processing (e.g. searching for specific sequences, format conversion or basic file statistics). The rapidly increasing data volumes involved in NGS make any dataset manipulation a time-consuming and error-prone process. I have developed fqtools; a fast and reliable FASTQ file manipulation suite that can process the full set of valid FASTQ files, including those with multi-line sequences, whilst identifying invalid files. Fqtools is faster than similar tools, and is designed for use in automatic processing pipelines. Availability and implementation: fqtools is open source and is available at: https://github.com/alastair-droop/fqtools. Supplementary information: Supplementary data are available at Bioinformatics online. Contact: a.p.droop@leeds.ac.uk

INTRODUCTION Glioblastoma multiforme remains one of the most difficult to treat cancers due to the highly infiltrative nature of cancer cells. Targeting the invasive properties of glioblastoma presents a novel treatment avenue. Recently, specific GSK-3 beta (GSK3b) inhibitors such as Bio-Indirubin (BIO), have been described that inhibit the migratory activity of glioma cells. In this study, the GSK-3b downstream targets were investigated.

MicroRNA expression can be exploited to define tumor prognosis and stratification for precision medicine. It remains unclear whether prognostic microRNA signatures are exclusively tumor grade and/or molecular subtype-specific, or whether common signatures of aggressive clinical behavior can be identified. Here, we defined microRNAs that are associated with good and poor prognosis in grade III and IV gliomas using data from The Cancer Genome Atlas. Pathway analysis of microRNA targets that are differentially expressed in good and poor prognosis glioma identified a link to oligodendrocyte development. Notably, a microRNA expression profile that is characteristic of a specific oligodendrocyte precursor cell type (OP1) correlates with microRNA expression from 597 of these tumors and is consistently associated with poor patient outcome in grade III and IV gliomas. Our study reveals grade-independent and subtype-independent prognostic molecular signatures in high-grade glioma and provides a framework for investigating the mechanisms of brain tumor aggressiveness.

Summary Human epithelia are organized in a hierarchical structure, where stem cells generate terminally differentiated cells via intermediate progenitors. This two-step differentiation process is conserved in all tissues, but it is not known whether a common gene set contributes to its regulation. Here, we show that retinoic acid (RA) regulates early human prostate epithelial differentiation by activating a tightly coexpressed set of 80 genes (e.g., TMPRSS2). Response kinetics suggested that some of these genes could be direct RA targets, whereas others are probably responding indirectly to RA stimulation. Comparative bioinformatic analyses of published tissue-specific microarrays and a large-scale transcriptomic data set revealed that these 80 genes are not only RA responsive but also significantly coexpressed in many human cell systems. The same gene set preferentially responds to androgens during terminal prostate epithelial differentiation, implying a cell-type-dependent interplay between RA and tissue-specific transcription factor-mediated signaling in regulating the two steps of epithelial differentiation., Graphical Abstract, Highlights • Four sets of coexpressed genes mark primary human prostate stem cell differentiation • These gene sets are also tightly coexpressed in >150 human cell types • Retinoic acid induces early differentiation while upregulating one of the gene sets • Androgens preferentially regulate the same gene set during terminal differentiation, Maitland and colleagues demonstrate that a set of 80 genes could be important for the regulation of adult human epithelial differentiation, irrespective of tissue of origin. They also show that the identified gene set responds to retinoic acid during early differentiation while preferentially responding to a tissue-specific transcription factor during terminal differentiation.

Prostate cancer (CaP) is mostly composed of luminal-like differentiated cells, but contains a small subpopulation of basal cells (including stem-like cells), which can proliferate and differentiate into luminal-like cells. In cancers, CpG island hypermethylation has been associated with gene downregulation, but the causal relationship between the two phenomena is still debated. Here we clarify the origin and function of CpG island hypermethylation in CaP, in the context of a cancer cell hierarchy and epithelial differentiation, by analysis of separated basal and luminal cells from cancers. For a set of genes (including GSTP1) that are hypermethylated in CaP, gene downregulation is the result of cell differentiation and is not cancer specific. Hypermethylation is however seen in more differentiated cancer cells and is promoted by hyperproliferation. These genes are maintained as actively expressed and methylation-free in undifferentiated CaP cells, and their hypermethylation is not essential for either tumour development or expansion. We present evidence for the causes and the dynamics of CpG island hypermethylation in CaP, showing that, for a specific set of genes, promoter methylation is downstream of gene downregulation and is not a driver of gene repression, while gene repression is a result of tissue-specific differentiation.

Non-muscle-invasive bladder cancers (NMIBC) and muscle-invasive bladder cancers (MIBC) show distinct molecular and clinical features and are considered to follow different pathogenesis pathways. MIBC commonly metastasise (>50%) and have poor prognosis whereas NMIBC, particularly low-stage low-grade tumors, rarely progress to invade muscle (T transitions, followed by C>G transversions (24.8%). Similar to MIBC, APOBEC mutagenesis was the strongest source of mutation in NMIBC with 75% of samples showing up to 5-fold enrichment of the APOBEC mutation signature. Significantly mutated genes included genes implicated in epigenetic regulation and several genes that have not previously been reported as significantly mutated in bladder cancer. Comparison of mutation frequencies in NMIBC with those found in MIBC revealed both known differences (absence of TP53 mutations and high frequency of FGFR3, PIK3CA and STAG2 mutations in NMIBC) and novel findings including a higher frequency of mutations in chromatin-state regulators in NMIBC. Notably CDKN1A, RB1, ERCC2, ERBB3 and FBXW7, which are mutated in >10% of MIBC were not significantly mutated in TaG2 tumors, further delineating the distinct pathogenesis pathways of NMIBC and MIBC. Whole-genome expression array profiling and immunohistochemistry using a panel of markers were carried out to further examine the molecular features of NMIBC samples and define potentially clinically relevant subtypes. These data provide a detailed view of the genomic landscape of NMIBC that highlights chromatin modification as a key area for consideration in the development of potential future therapeutic approaches to the treatment of patients with non-muscle-invasive disease. Citation Format: Carolyn D. Hurst, Olivia Alder, Fiona M. Platt, Alastair Droop, Lucy F. Stead, Julie E. Burns, George J. Burghel, Sunjay Jain, Leszek J. Klimczak, Helen Lindsay, Jo-An Roulson, Claire F. Taylor, Helene Thygesen, Angus J. Cameron, Anne J. Ridley, Helene R. Mott, Dmitry A. Gordenin, Margaret A. Knowles. The genomic landscape of non-muscle-invasive bladder cancer: implications for molecular classification and treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-323.

Open-ended evolutionary systems offer us the tantalising prospect of creating artificial life from simple precursors. One of the issues in designing open ended systems is that there exist few metrics for measuring their evolutionary activity. Current measures of evolutionary activity are only applicable to systems in which the fitness of a single component is defined by an explicit fitness function. However, this is not guaranteed in systems where a significant part of the fitness is intrinsic (for example caused by interactions between components). In this paper, we evaluate a new approach to the problem of measuring evolutionary activity that is applicable to systems exhibiting both explicit and intrinsic fitness pressures. To evaluate this measure, we ran 22,000 grid-based simulations of two automata chemistries, Tierra and Stringmol. Both of these systems have strong intrinsic fitness pressures. We examine the effect of varying the mutation rate in both systems, and demonstrate that the new measure identifies an optimal mutation rate.

We report a study of networks constructed from mutation patterns observed in biology. These networks form evolutionary trajectories, which allow for both frequent substitution of closely related structures, and a small evolutionary distance between any two structures. These two properties define the small-world phenomenon. The mutation behavior between tokens in an evolvable artificial chemistry determines its ability to explore evolutionary space. This concept is underrepresented in previous work on string-based chemistries. We argue that small-world mutation networks will confer better exploration of the evolutionary space than either random or fully regular mutation strategies. We calculate network statistics from two data sets: amino acid substitution matrices, and codon-level single point mutations. The first class are observed data from protein alignments; while the second class is defined by the standard genetic code that is used to translate RNA into amino acids. We report a methodology for creating small-world mutation networks for artificial chemistries with arbitrary node count and connectivity. We argue that ALife systems would benefit from this approach, as it delivers a more viable exploration of evolutionary space.

INTRODUCTION: There is a pressing need for new therapeutic approaches for the treatment of glioblastoma (GBM). MicroRNAs are single-stranded 22-24nt non-coding RNAs which function by reducing translation or causing degradation of target mRNAs. MiR-1300 was identified in a high-throughput miRNA mimic library screen as a promising therapeutic candidate, inducing cell death. METHOD: To validate the cytotoxic effect of miR-1300, a panel of four established cell lines (U251,LN229,U373,KNS42)and two patient-derived stem-like cell lines were utilised for RTqPCR, imaging and flow-cytometry to assess endogenous expression levels, cellular morphology, apoptosis, and cell cycle delay respectively. Bioformatic approaches identified potential miR-1300 targets as mediators of this effect and validation of this involvement in the cytotoxic phenotype using siRNA knock-down is being investigated. Validation of the direct targeting by miR-1300 is being performed using 3' UTR Luciferase assays. RESULTS: Expression, of miR-1300, by transfection of oligonucleotide mimics, in our panel of GBM cells consistently led to G2M arrest followed by apoptosis within 72h. The associated phenotype is binucleation, indicative of cytokinesis failure.Preliminary data suggest this may be mediated through ECT2, a predicted target of miR-1300 known to be involved in formation of the cleavage furrow. CONCLUSION: We have identified a highly potent pro-apoptotic microRNA that may be exploitable for GBM therapy. Importantly, there is no detectable endogenous miR-1300 expression in any GBM samples or cell lines. On-going work will confirm the targets of miR-1300 that drive this effect as well as exploring potential approaches to deliver this as a biotherapeutic agent.

The Ntr1 and Ntr2 proteins of Saccharomyces cerevisiae have been reported to interact with proteins involved in pre-mRNA splicing, but their roles in the splicing process are unknown. We show here that they associate with a postsplicing complex containing the excised intron and the spliceosomal U2, U5, and U6 snRNAs, supporting a link with a late stage in the pre-mRNA splicing process. Extract from cells that had been metabolically depleted of Ntr1 has low splicing activity and accumulates the excised intron. Also, the level of U4/U6 di-snRNP is increased but those of the free U5 and U6 snRNPs are decreased in Ntr1-depleted extract, and increased levels of U2 and decreased levels of U4 are found associated with the U5 snRNP protein Prp8. These results suggest a requirement for Ntr1 for turnover of the excised intron complex and recycling of snRNPs. Ntr1 interacts directly or indirectly with the intron release factor Prp43 and is required for its association with the excised intron. We propose that Ntr1 promotes release of excised introns from splicing complexes by acting as a spliceosome receptor or RNA-targeting factor for Prp43, possibly assisted by the Ntr2 protein.

MicroRNAs are short non-coding RNAs that act as micro-managers of cellular function. Their dysregulation is well documented in many cancers. TCGA microRNA expression data is available to identify the prognostic potential of microRNAs in malignant glioma. Using the LASSO statistical approach for 475 glioblastomas in the TCGA dataset we identified a nine-microRNA signature that predicts overall (p = 2.26e-09) and progression-free survival (p = 9.91e-08) with superior power than conventional molecular markers, including MGMT methylation status. This signature has significance in all molecular subtypes of glioblastoma with the exception of the non-G-CIMP proneural group and validated in a separate validation cohort (p = 4.50E-02). The signature also predicts survival in grade II and III disease (p = 5.20e-03). The great majority of microRNAs in the signature have previously been shown to play a role in glioma biology. In addition, when we compared patient groups at the extremes of survival in grade III astrocytoma (poor prognosis n = 10, survival 48 months) and glioblastoma (poor prognosis n = 14, survival 48 months), we found that microRNA profiles aligned with specific neuro-developmental pathways were associated with outcomes. Specifically, poorer prognosis disease was associated with profiles aligned towards glial-restricted and oligodendrocyte precursor (OP) cells. Furthermore, correlation of microRNA expression patterns of OPs with expression of 39 grade III astrocytomas and 558 glioblastomas predicts survival with high significance (p= 5.50e-06), indicating that the more tightly a grade III or IV tumor correlates with an OP profile, the poorer the outcome. These observations indicate that microRNAs have predictive power in malignant glioma. MicroRNA expression patterns may also reveal important aspects of underlying tumor biology. This data, coupled with the superior stability of microRNAs over mRNAs in clinical specimens, suggests they may represent attractive candidates as novel biomarkers for the disease.

Prostate cancer (CaP) is mostly composed of differentiated luminal cells, but contains a small subpopulation of undifferentiated basal cells (including stem-like cells), which is thought to be the driving force of CaP. In cancers, CpG island hypermethylation has been associated with gene downregulation, but the causal relationship between the two phenomena is still debated. This study aimed to understand the function and origin of CpG island hypermethylation in CaP, in the context of cancer hierarchy and differentiation. We have analyzed separately primary prostate basal and luminal cells derived from BPH and CaP. This allowed us to dissect the intra-tumor heterogeneity, and to understand the origin of gene downregulation and hypermethylation in CaP. We report that a set of genes commonly hypermethylated in CaP (including GSTP1) is: (i) downregulated as a result of prostate-specific epithelial differentiation in both CaP and benign prostatic hyperplasia (BPH); (ii) selectively hypermethylated in differentiated (luminal) cancer cells, process promoted by the hyperproliferating phenotype of these cells; (iii) actively expressed and methylation free in undifferentiated (basal) CaP cells. Downregulation and hypermethylation of these genes is not essential for tumor development or tumor expansion. Moreover, for all these genes, downregulation induced by prostate-specific differentiation, is independent of DNA hypermethylation, and is associated with detachment of RNA PolII from their promoter and reduction in histone marks associated with active transcription. Citation Format: Davide Pellacani, Dimitra Kestoras, Alastair P. Droop, Fiona M. Frame, Paul A. Berry, Mitchell G. Lawrence, Michael J. Stower, Matthew S. Simms, Vincent M. Mann, Anne T. Collins, Gail P. Risbridger, Norman J. Maitland. DNA hypermethylation in prostate cancer is a consequence of aberrant epithelial differentiation and hyperproliferation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B25.

DNA copy number variation (CNV) has been identified in many cancers. Recent studies have shown that CNV data, in combination with other high-throughput techniques, can provide novel biological insight to tumor biology. Although a number of CNV whole-genome studies have been performed on melanoma using cell lines and frozen tumors, very few have used formalin fixed paraffin embedded (FFPE)-derived material. Melanoma tumors are small and rarely frozen, which means that FFPE tumor blocks would be a very valuable source of genomic material to be used for CNV analysis if the methodologies were adequately developed. It has been shown that reliable next generation sequencing data can be produced from FFPE-derived DNA in lung cancers (Wood et al., 2010). Here, we report a study showing that DNA from FFPE melanoma tumor blocks can be used for CNV detection on a next generation sequencing platform. DNA from 20 FFPE melanoma samples was used to prepare genomic libraries, which were sequenced on the Illumina GAIIx instrument. Successful library preparation occurred at DNA levels of at least 30ng, while current protocols generally suggest 50ng with high quality material and 1μg generally. The use of SPRIworks (Beckman Coulter) and Bravo (Agilent) robots was compared with the manual method for library preparation with input DNA mass of 30-150ng. Libraries prepared manually were always of better quality and yield. We also investigated means of improving library preparation. Melanin co-purifies with DNA and inhibits downstream reactions, and indeed lower coverage was seen in our samples if they were heavily pigmented. To overcome the adverse effects of melanin, 10 DNA samples (5 pairs) were used for library preparation with and without bovine serum albumin (BSA). BSA has been shown to bind to melanin, and as a result the melanin does not inhibit the DNA polymerase reaction. All 5 libraries with BSA were of higher quality and yield when compared to those prepared without BSA. All comparisons were made after running the libraries on an Agilent DNA chip. Currently available data from the GAIIx were used to develop the analysis pipeline. The first 10 samples were multiplexed together and run twice, while the remaining 10 were multiplexed at 5 samples per lane and run once. The average coverage obtained was 0.10x with a range of 0.03x-0.11x. We observed a strong effect on mapping quality based upon the end-cycle quality scores, leading us to adopt an aggressive data trimming procedure. More samples from the Leeds Melanoma Cohort will soon be prepared and sequenced on the Illumina HiSeq2000 instrument. We aim to identify if tumors with driver BRAF mutations have different genomic signatures when compared to those with NRAS mutations. Finally, genomic data will be combined with clinical data and we will check for the prognostic significance of CNV changes identified in our samples. Citation Format: Anastasia Filia, Alastair Droop, Mark Harland, Tim Bishop, Julia Newton-Bishop. Using formalin-fixed paraffin-embedded melanoma tumors for the detection of copy number variation by next generation sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3140. doi:10.1158/1538-7445.AM2013-3140

The aim of present investigation is to elucidate the complex stem cell dynamics within prostate cancer, which can be utilized to design novel diagnostic and therapeutic strategies for the management of prostate cancer. In order to determine precise transcriptional and microRNA regulatory mechanisms modulating stem cell self-renewal and differentiation, unique cellular assays have been developed in our lab that utilize homogeneous fractionated cell populations enriched from primary patient prostate cultures. Using a prospective bioinformatic analysis of gene expression data from Birnie et. al., 2008, we have identified LCN2, CEACAM6, and S100p as candidate genes for regulation of prostate stem cell differentiation. Their over-expression in differentiated cells, as compared to stem cells, was validated in respective cells enriched from cultures obtained from BPH, cancer and castration resistant prostate cancer samples and from primary human prostate cancer xenografts. Interestingly, the analysis of 25,000 published human Affymetrix microarray chips revealed that LCN2, CEACAM6, and S100p have a more similar expression pattern than that of any other genes in the entire human genome, suggesting that they may have common function and are co-regulated. Indeed, the promoter analysis showed that the promoters (1kb from TSS) of all these genes have common binding sites for 40 transcription factors with very high affinity and P < 0.001. Most of these transcription factors have a well-documented role in cell differentiation (RA, AR, and NANOG) and prostate carcinogenesis (NF-kB). Significant up-regulation in the expression of these genes in prostate cell lines after treatment with all-trans retinoic acid and androgen analogue R1881 further suggested the role of AR and RA in prostate differentiation. Along with transcriptional regulation, Agilent v3 miRNA microarray data revealed obviously distinct miRNA expression profiles in stem and differentiated prostate epithelial cells, confirming crucial role of miRNA in main taining epithelial hierarchies, in prostate. We anticipate that evaluation of integrative transcriptional (LCN2-CEACAM6-S100p)-microRNA regulatory network, with further functional studies, will comprehensively establish a detailed knowledge base for potential regulatory mechanisms involved in prostate stem cell and prostate cancer stem cell differentiation. These insights will be valuable to formulate efficient ‘differentiation therapy’ for the management of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5198. doi:1538-7445.AM2012-5198

Prostate cancer is phenotypically, genetically and epigenetically heterogeneous. In the present study we have dissected the epigenetic heterogeneity of prostate cancer by analyzing DNA methylation patterns in pure subpopulations of primary cancer cells, on the basis of their differentiation status. GSTP1, an enzyme involved in intracellular detoxification, is highly expressed in prostate basal epithelial cells but is down-regulated in luminal cells by a mechanism independent of DNA methylation. The GSTP1 promoter becomes frequently hypermethylated in prostate cancer, where the majority of cells bear a luminal-like phenotype. However, a small subpopulation of basal cells ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5209. doi:1538-7445.AM2012-5209

The mechanism of the anti-colorectal cancer (CRC) activity of the omega-3 fatty acid eicosapentaenoic acid (EPA) is not understood. We tested the hypothesis that EPA reduces expression of chemokine C-C motif ligand 2 (CCL2), a pro-inflammatory chemokine with known roles in metastasis. We measured CCL2 in clinical samples from a randomized trial of EPA in patients undergoing liver surgery for CRC liver metastasis (LM) and preclinical models. Genome-wide transcriptional profiling of tumors from EPA-treated patients was performed. EPA decreased CCL2 synthesis by CRC cells in a dose-dependent manner. CCL2 was localized to malignant epithelial cells in human CRCLM. EPA did not reduce CCL2 content in human or mouse tumors compare to control. However, EPA treatment was associated with decreased plasma CCL2 levels compared with controls (P=0.04). Reduction in plasma CCL2 following EPA treatment predicted improved disease-free survival (HR 0.32; P=0.003). Lack of ‘CCL2 response’ was associated with a specific CRCLM gene expression signature. In conclusion, reduction in plasma CCL2 in patients with CRCLM treated with EPA predicts better clinical outcome and a specific tumor gene expression profile. Further work is needed to validate CCL2 as a therapeutic response biomarker for omega-3 fatty acid treatment of CRC patients.

An expression signature of human prostate cancer stem cells identifies 581 differentially expressed genes and suggests that the JAK-STAT pathway and focal adhesion signaling are important., Background The tumor-initiating capacity of many cancers is considered to reside in a small subpopulation of cells (cancer stem cells). We have previously shown that rare prostate epithelial cells with a CD133+/α2β1hi phenotype have the properties of prostate cancer stem cells. We have compared gene expression in these cells relative to their normal and differentiated (CD133-/α2β1low) counterparts, resulting in an informative cancer stem cell gene-expression signature. Results Cell cultures were generated from specimens of human prostate cancers (n = 12) and non-malignant control tissues (n = 7). Affymetrix gene-expression arrays were used to analyze total cell RNA from sorted cell populations, and expression changes were selectively validated by quantitative RT-PCR, flow cytometry and immunocytochemistry. Differential expression of multiple genes associated with inflammation, cellular adhesion, and metastasis was observed. Functional studies, using an inhibitor of nuclear factor κB (NF-κB), revealed preferential targeting of the cancer stem cell and progenitor population for apoptosis whilst sparing normal stem cells. NF-κB is a major factor controlling the ability of tumor cells to resist apoptosis and provides an attractive target for new chemopreventative and chemotherapeutic approaches. Conclusion We describe an expression signature of 581 genes whose levels are significantly different in prostate cancer stem cells. Functional annotation of this signature identified the JAK-STAT pathway and focal adhesion signaling as key processes in the biology of cancer stem cells.