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The normal androgen receptor (AR) cistrome and transcriptional program are fundamentally altered in prostate cancer (PCa). Here, we show that SPOP mutations, an early event in prostate tumorigenesis, reshape the chromatin landscape and AR-directed transcriptional program in normal prostate cells. Induction of SPOP mutation results in DNA accessibility and AR binding patterns found in human PCa. Consistent with dependency on this AR reprogramming, castration of SPOP mutant mouse models results in the loss of neoplastic phenotypes. Finally, human SPOP mutant PCa show improved response to AR-targeted therapies. Together, these results show that a single genomic alteration may be sufficient to reprogram the chromatin of normal prostate cells toward oncogenic phenotypes and that SPOP mutant tumors may be preferentially dependent on AR signaling through this mechanism.

BACKGROUND: Molecular characterization of prostate cancer (PCa) has revealed distinct subclasses based on underlying genomic alterations occurring early in the natural history of the disease. However, how these early alterations influence subsequent molecular events and the course of the disease over its long natural history remains unclear. METHODS: We explored the molecular and clinical progression of different genomic subtypes of PCa using distinct tumor lineage models based on human genomic and transcriptomic data. We developed transcriptional classifiers, and defined “early” and “late” categories of molecular subclasses from 8,158 PCa patients. Molecular subclasses were correlated with clinical outcomes and pathologic characteristics using Kaplan-Meier and logistic regression analyses. RESULTS: We identified PTEN and CHD1 alterations as subtype-specific late progression events specifically in ERG-overexpressing (ERG+) and SPOP-mutant tumors, respectively, and 2 distinct progression models consisting of ERG/PTEN (normal to ERG+ to PTEN-deleted) and SPOP/CHD1 (normal to SPOP-mutated to CHD1-deleted) with shared early tumorigenesis but distinct pathways toward progression. We found that within ERG+ and SPOP-mutant subtypes, late events were associated with worse prognosis. Importantly, the clinical and pathologic features associated with distinct late events at radical prostatectomy were strikingly different; PTEN deletions were associated with increased locoregional stage, while CHD1 deletions were only associated with increased grade, despite equivalent metastatic potential. CONCLUSION: These findings suggest a paradigm in which specific subtypes of PCa follow distinct pathways of progression, at both the molecular and clinical levels. Therefore, the interpretation of common clinical parameters such as locoregional tumor stage may be influenced by the underlying tumor lineage, and potentially influence management decisions. FUNDING: Prostate Cancer Foundation, National Cancer Institute, Urology Care Foundation, Damon Runyon Cancer Research Foundation, US Department of Defense, and the AIRC Foundation.

SUMMARY The normal androgen receptor (AR) cistrome and transcriptional program are fundamentally altered in prostate cancer (PCa). Here, we profile the chromatin landscape and AR-directed transcriptional program in normal prostate cells and show the impact of SPOP mutations, an early event in prostate tumorigenesis. In genetically normal mouse prostate organoids, SPOP mutation results in accessibility and AR binding patterns similar to that of human PCa. Consistent with dependence on AR signaling, castration of SPOP mutant mouse models results in the loss of neoplastic phenotypes, and human SPOP mutant PCa shows a favorable response to AR-targeted therapies. Together, these data validate mouse prostate organoids as a robust model for studying epigenomic and transcriptional alterations in normal prostate, provide valuable datasets for further studies, and show that a single genomic alteration may be sufficient to reprogram the chromatin of normal prostate cells toward oncogenic phenotypes, with potential therapeutic implications for AR-targeting therapies., Graphical abstract, In brief Grbesa et al. use multilevel epigenomic and transcriptional profiling to characterize genetically normal prostate organoids and show the impact of an early cancer driver (SPOP mutation). Integrative analysis identifies transcriptional programs that are AR regulated and reprogrammed by SPOP mutation, suggesting a basis for enhanced sensitivity to AR-targeting therapies.

S-adenosylhomocysteine hydrolase (AHCY) is thought to be located at the sites of ongoing AdoMet-dependent methylation, presumably in the cell nucleus. Endogenous AHCY is located both in cytoplasm and the nucleus. Little is known regarding mechanisms that drive its subcellular distribution, and even less is known on how mutations causing AHCY deficiency affect its intracellular dynamics. Using fluorescence microscopy and GFP-tagged AHCY constructs we show significant differences in the intensity ratio between nuclei and cytoplasm for mutant proteins when compared with wild type AHCY. Interestingly, nuclear export of AHCY is not affected by leptomycin B. Systematic deletions showed that AHCY has two regions, located at both sides of the protein, that contribute to its nuclear localization, implying the interaction with various proteins. In order to evaluate protein interactions in vivo we engaged in bimolecular fluorescence complementation (BiFC) based studies. We investigated previously assumed interaction with AHCY-like-1 protein (AHCYL1), a paralog of AHCY. Indeed, significant interaction between both proteins exists. Additionally, silencing AHCYL1 leads to moderate inhibition of nuclear export of endogenous AHCY.

The transcription factor glucocorticoid receptor (GR) is a key mediator of stress response and a broad range of physiological processes. How can GR rapidly activate the expression of some genes while repress others, remains an open question due to the challenge to associate GR binding sites (GBSs) to their distant gene targets. Mapping the full 3D scope of GR-responsive promoters using high-resolution 4C-seq unravelled spatial separation between chromatin interaction networks of GR-activated and repressed genes. Analysing GR binding sites and other regulatory loci in their functional 3D context revealed that GR sequesters the co-activator Ep300 from active non-GBS enhancers in both activated and repressed gene compartments. While this is sufficient for rapid gene repression, gene activation is countered by productive recruitment of Ep300 to GBS. Importantly, in GR-activated compartments Klf4 binding at non-GBS regulatory elements cluster in 3D with GBS and antagonizes GR activation. In addition, we revealed ROR and Rev-erb transcription factors as novel co-regulators for GR-mediated gene expression.

Glucocorticoids and their receptor (GR) have been an important area of research because of their pleiotropic physiological functions and extensive use in the clinic. In addition, the association between GR and glucocorticoids, which is highly specific, leads to rapid nuclear translocation where GR associates with chromatin to regulate gene transcription. This simplified model system has been instrumental for studying the complexity of transcription regulation processes occurring at chromatin. In this review we discuss our current understanding of GR action that has been enhanced by recent developments in genome wide measurements of chromatin accessibility, histone marks, chromatin remodeling and 3D chromatin structure in various cell types responding to glucocorticoids.

Finkel-Biskis-Reilly murine sarcoma virus (FBR- MuSV) ubiquitously expressed (FAU) gene is down-regulated in human prostate, breast and ovarian cancers. Moreover, its dysregulation is associated with poor prognosis in breast cancer. Sponges (Porifera) are animals without tissues which branched off first from the common ancestor of all metazoans. A large majority of genes implicated in human cancers have their homologues in the sponge genome. Our study suggests that FAU gene from the sponge Suberites domuncula reflects characteristics of the FAU gene from the metazoan ancestor, which have changed only slightly during the course of animal evolution. We found pro-apoptotic activity of sponge FAU protein. The same as its human homologue, sponge FAU increases apoptosis in human HEK293T cells. This indicates that the biological functions of FAU, usually associated with “higher” metazoans, particularly in cancer etiology, possess a biochemical background established early in metazoan evolution. The ancestor of all animals possibly possessed FAU protein with the structure and function similar to evolutionarily more recent versions of the protein, even before the appearance of true tissues and the origin of tumors and metastasis. It provides an opportunity to use pre-bilaterian animals as a simpler model for studying complex interactions in human cancerogenesis.

Recently, functional connections between S-adenosylhomocysteine hydrolase (AHCY) activity and cancer have been reported. As the properties of AHCY include the hydrolysis of S-adenosylhomocysteine and maintenance of the cellular methylation potential, the connection between AHCY and cancer is not obvious. The mechanisms by which AHCY influences the cell cycle or cell proliferation have not yet been confirmed. To elucidate AHCY-driven cancer-specific mechanisms, we pursued a multi-omics approach to investigate the effect of AHCY-knockdown on hepatocellular carcinoma cells. Here, we show that reduced AHCY activity causes adenosine depletion with activation of the DNA damage response (DDR), leading to cell cycle arrest, a decreased proliferation rate and DNA damage. The underlying mechanism behind these effects might be applicable to cancer types that have either significant levels of endogenous AHCY and/or are dependent on high concentrations of adenosine in their microenvironments. Thus, adenosine monitoring might be used as a preventive measure in liver disease, whereas induced adenosine depletion might be the desired approach for provoking the DDR in diagnosed cancer, thus opening new avenues for targeted therapy. Additionally, including AHCY in mutational screens as a potential risk factor may be a beneficial preventive measure. This work was supported by FP7-REGPOT-2012-2013-1, grant agreement number 316289-InnoMol, and the FP7 PRIME-XS- project - grant no. 262067. The CRG/UPF Proteomics Unit is part of the “Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed)” supported by grant PT13/0001 of the Instituto de Salud Carlos III (ISCIII) and the Spanish Ministry of Economy and Competitiveness. We acknowledge support from the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013–2017”, SEV-2012-0208, and “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya” (2014SGR678). JHP is supported by the grant NRF 2016R1A2A1A05005295. We acknowledge the kind support of staff at the University Clinic Freiburg, Germany, Zentrum für Kinder- und Jugendmedizin, Labor für Klinische Biochemie und Stoffwechsel (LKBS), especially B.Sc. Sidney Behringer, and Dr. Malkanthi Fernando, for performing adenosine measurements. We also thank Dr. Marko Marjanović for help with the flow cytometry analysis.

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Background Mass spectrometry (MS) are a group of a high-throughput techniques used to increase knowledge about biomolecules. They produce a large amount of data which is presented as a list of hundreds or thousands of proteins. Filtering those data efficiently is the first step for extracting biologically relevant information. The filtering may increase interest by merging previous data with the data obtained from public databases, resulting in an accurate list of proteins which meet the predetermined conditions. Results In this article we present msBiodat Analysis Tool, a web-based application thought to approach proteomics to the big data analysis. With this tool, researchers can easily select the most relevant information from their MS experiments using an easy-to-use web interface. An interesting feature of msBiodat analysis tool is the possibility of selecting proteins by its annotation on Gene Ontology using its Gene Id, ensembl or UniProt codes. Conclusion The msBiodat analysis tool is a web-based application that allows researchers with any programming experience to deal with efficient database querying advantages. Its versatility and user-friendly interface makes easy to perform fast and accurate data screening by using complex queries. Once the analysis is finished, the result is delivered by e-mail. msBiodat analysis tool is freely available at http://msbiodata.irb.hr Electronic supplementary material The online version of this article (doi:10.1186/s13040-016-0104-6) contains supplementary material, which is available to authorized users.

The gene for insulin-like growth factor two, IGF2 is maternally imprinted. Fifteen heterozygous samples were analyzed for the IGF2 imprinting status and promoter usage. IGF2 LOI was detected in four non-tumorous tissues and in six laryngeal squamous cell carcinoma (LSCC) tumors. There was no clear pattern of specific promoter activity in LSCC tumors and the adjacent normal tissues. P1 promoter usage was active in eight LSCCs, among them four with LOI. As it was activated in four tumors with maintenance of imprinting (MOI) and four non-tumors, we concluded that P1 promoter is not exclusively connected with IGF2 LOI in LSCC.

BACKGROUND:Sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) are NAD+-dependent protein deacetylases involved in the regulation of key cancer-associated genes. In this study we evaluated the relevance of these deacetylases in lung cancer biology. MATERIAL AND METHODS:Protein levels of SIRT1 and SIRT2 were determined in non-small cell lung cancer (NSCLC) cell lines and primary tumors from 105 patients. Changes in proliferation were assessed after SIRT1 and SIRT2 downregulation in lung cancer cell lines using siRNA-mediated technology or tenovin-1, a SIRT1 and SIRT2 inhibitor. RESULTS:High SIRT1 and SIRT2 protein levels were found in NSCLC cell lines compared with non-tumor lung epithelial cells. The expression of SIRT1 and SIRT2 proteins was also significantly higher in lung primary tumors than in normal tissue (P

BORIS is a paralog of a highly conserved, multi-functional chromatin factor CTCF. Unlike CTCF, which has been shown to possess tumor-suppressive properties, BORIS belongs to the "cancer/testis antigen" family normally expressed only in germ cells and aberrantly activated in a variety of tumors. The consequences of BORIS expression, relative abundance of its isoforms, and its role in carcinogenesis have not been completely elucidated. It activates transcription of hTERT and MYC, genes relevant for laryngeal carcinoma progression. In this study, BORIS expression has been analyzed at the transcriptional level by RT-PCR and protein level by semi-quantitative immunohistochemistry in 32 laryngeal squamous cell carcinomas and adjacent non-tumorous tissue. BORIS was detected in 44 % (14/32) laryngeal squamous cell carcinoma samples, while it was detected only in one normal, tumor-adjacent tissue sample. Tree based survival analysis, using the recursive partitioning algorithm mvpart, extracted the ratio of relative abundance of BORIS transcript variants containing exon 7 (BORIS 7+) and those lacking exon 7 (BORIS 7-) as an independent prognostic factor associated with disease relapse during a 5-year follow-up period. Patients having BORIS 7+/BORIS 7- ratio ≥1 had a higher rate of disease relapse than patients with BORIS 7+/BORIS 7- ratio

Cancer has long been considered primarily a genetic disease, caused by different mutations throughout the genome. In 1983., Feinberg and Vogelstein discovered that the level of DNA methylation significantly varies between primary human malignant tumors and their normal counterparts (Feinberg & Vogelstein, 1983). Before this publication, there had been a paper describing changes in DNA methylation in cancer cell cultures, including the influence of N-methyl-N-nitrosourea on the level of DNA methylation in Raji cells (Boehm & Drahovsky, 1981). Currently, we are presented with much experimental data showing multilevel changes in cancer cells. In this context, two major areas of epigenomic research DNA methylation and histone modifications appear most promising in understanding the multistep nature of carcinogenesis. Additionally, they seem to have the potential for being cancer biomarkers, useful in early detection, in predicting the biological behavior of tumors and for therapy monitoring, as recently reviewed (Rodriguez-Paredes & Esteller, 2011; Baylin & Jones, 2011). Finally, epigenetic changes are well-recognized targets for cancer therapy, alone, or in combination with various cytostatics (Ren et al, 2011). Epigenetic changes are also of the greatest importance in chemoprevention, as there is increasing data relating to possibly reversing epigenetic changes in the earliest phase of carcinogenesis, when genetic changes have yet to develop (reviewed, Huang et al., 2011). It is not easy to understand the particular rules applicable to epigenomic processes. If one specific epigenetic change, relating to a specific gene/its promoter, exists in a majority of tumors of a specific type, it does not necessarily mean that the same change exists in another type of tumor. This is consequential, and represents one reason for obvious differences in responses to epigenomic therapy. Recently, we wrote a review article on some aspects of epigenomic changes in which we used the term „epigenetic networking“. If we imagine each of our living cells as an orchestra performing the symphony of life, then each player (a gene) of the orchestra needs to play in concert with 30,000 other players. The communication that produces the network of our epi-genome is established at many levels: transcriptionally, post-transcriptionally, through protein translation, at the level of post-translational protein modifications, through their orchestrated interactions and, finally, their interaction with the DNA that can be modified in order to accept or reject the protein partner. This is the way of

Za razliku od genomike, koja se temelji na proučavanju građe – anatomije gena, epigenomika se temelji na izučavanju nasljednih varijacija u aktivnosti gena, dakle njihovoj fiziologiji. Osnovni epigenetički procesi, regulatori aktivnosti gena su metilacija molekule DNA i post translacijske modifikacije histona. Ova dva procesa međusobno se nadopunjuju pri čemu stvaraju epigenetičku mrežu događaja koja u konačnici regulira aktivnost pojedinih gena. Uspostava određenog tipa epigenetičke mreže ovisi o anatomiji gena i njegovog promotora te stalnom međudjelovanju egzogenih i endogenih čimbenika koji dovode do stvaranja karakterističnog epigenetičkog biljega. Sve se više uviđa važnost reverzibilnosti uspostave i uklanjanja epigenetičkih molekularnih biljega u svim, a naročito u zloćudnim bolestima. Istraživanja u području epigenomike, primjene novih, epigenomskih pristupa u liječenju, a posebno u području razvoja “pametnih”, epigenetičkih lijekova, u uzlaznoj su putanji koja još uvijek nije dosegla svoj zenit., Epigenetics is focused on gene physiology, analyzing inherited variations in gene expression, while genomics is focused on gene anatomy, analyzing gene structure. DNA methylation and histone post-translational modifications are the basic epigentic mechanisms regulating gene activity. These two processes complement each other, creating an epigenetic network of events which regulates specifi c gene activity. Establishing a particular type of epigenetic network depends on the anatomy of both the gene and its promoter, as well as the permanent interaction of exogenic and endogenic factors, which result in a particular epigenetic mark. As a result of new data, the importance of epigenetic marks as a reversible process, is becoming increasingly relevant to disease development, especially for cancer. One looks forward to the promise, still to be fully realized, of research in epigenetics, including new approaches to therapy and the development of “smart” epigenetic drugs.

Imprinting analyses of IGF2 and H19, loss of heterozygosity (LOH) analyses of IGF2R and CTCF and Helicobacter pylori detection, were performed on 35 human laryngeal squamous cell carcinomas (LSCC). Forty-six percent of the tumors were heterozygous for IGF2, and 54% were informative for the H19. Biallelic expression of IGF2 was observed in 33% (5 out of 15) of the tumors and in 27% (4 out of 15) of adjacent non-tumorous laryngeal tissues. H19 loss of imprinting (LOI) was observed in 24% (4 out of 17) of the tumors. For IGF2R and CTCF, 71% (25 out of 35) and 50% (17/34), respectively, of the samples were heterozygous, and LOH was detected in 12% (3 out of 25) and 6% (1 out of 17), respectively, of the tumors. H. pylori was found in 26% (9/35) of these tumors. Among them, four were informative for the imprinting analysis. The presence of H. pylori had no effect on IGF2/H19 imprinting. Only the H. pylori detection was further broadened with an additional 47 laryngeal tumors, resulting in a total final positivity of close to 16% (13 out of 82). This study represents the largest comprehensive IGF2/H19 imprinting study done to date on well-defined samples of human laryngeal carcinomas and corresponding non-tumorous tissue. For the first time, the analyses of IGF2/H19 imprinting have been broadened with LOH analyses of IGF2R and CTCF, with both of these genes acting as modulators of IGF2 and H19 activity. Although there were indications that H. pylori may be present in LSCC, we are the first to show its presence in LSCC by two direct techniques: Giemsa staining and nested-PCR.

Curcumin (diferuloymethane), a natural compound used in traditional medicine, exerts an antiproliferative effect on various tumor cell lines by an incompletely understood mechanism. It has been shown that low doses of curcumin downregulate DNA topoisomerase II alpha (TOP2A) which is upregulated in many malignances. The activity of TOP2A is required for RNA polymerase II transcription on chromatin templates. Recently, it has been reported that CTCF, a multifunctional transcription factor, recruits the largest subunit of RNA polymerase II (LS Pol II) to its target sites genome-wide. This recruitment of LS Pol II is more pronounced in proliferating cells than in fully differentiated cells. As expression of imprinted genes is often altered in tumors, we investigated the potential effect of curcumin treatment on transcription of the imprinted H19 gene, located distally from the CTCF binding site, in human tumor cell lines HCT 116, SW 620, HeLa, Cal 27, Hep-2 and Detroit 562. Transcription of TOP2A and concomitantly H19 was supressed in all tumor cell lines tested. Monoallelic IGF2 expression was maintained in curcumin-treated cancer cells, indicating the involvement of mechanism/s other than disturbance of CTCF insulator function at the IGF2/H19 locus. Curcumin did not alter H19 gene transcription in primary cell cultures derived from normal human tissues. J. Cell. Biochem. 104: 1781–1792, 2008. © 2008 Wiley-Liss, Inc.

Liver is a unique mammalian organ with a great capacity of regeneration related to its function. After surgical resection or injury, hepatic cells, especially hepatocytes, can proliferate rapidly to repair the damage and to regenerate the structure without affecting the function of the liver. Loss of catalase activity during regeneration indicates that oxidative stress is present in the liver not only in pathological conditions but also as a 'physiological' factor during regeneration. As we have shown in our previous work, liver stem cell-like cells treated with 4-hydroxynonenal (HNE), a cytotoxic and growth regulating lipid peroxidation product, recover in the presence of spleen cells. In the current study we characterized this novel cell line as liver-derived progenitor/oval-like cells, (LDP/OCs), i.e. functional liver stem-like cells. We showed that LDP/OC were OV6 positive, with abundant glycogen content in the cytoplasm and expressed alpha-fetoprotein, albumin, biliverdin reductase and gamma-glutamyl transferase. Also, we compared their growth in vitro with the growth of cultured primary hepatocytes stressed with HNE and co-cultured with autologous spleen cells. The influence of spleen cells on HNE-treated primary hepatocytes and on LDP/OCs showed that spleen cells support in a similar manner the recovery of both types of liver cells indicating their important role in regeneration. Hence, LDP/OC cells may provide a valuable tool to study cell interactions and the role on HNE in liver regeneration.