Final Report Summary - EPIDIACAN (Development of sensitive methodologies for exploitation of early epigenetic marker diagnosis in major types of cancer)
Executive summary:
Cancer is one of the major causes of mortality. Epithelial cells become malignant after accumulating genetic mutations followed by morphological changes in the epithelium. Alterations in DNA include stable genetic changes in oncogenes, tumor suppressor genes and reversible epigenetic changes. Different forms of epigenetic mechanisms have been shown to modify the expression of key genes during tumour progression. Promoter DNA hypermethylation of tumour suppressor genes or DNA repair genes, and covalent histone modifications appear in early stages of neoplasia. Methods to identify early markers in different types of cancer are being developed, although very few are specific and sensitive enough to be applied in the clinic.
The aim of the present consortium EPIDIACAN was to develop sensitive and specific methodologies to identify early epigenetic markers for major types of cancer, like prostate and colorectal cancer. This project is based on recent findings that selected covalent histone modifications and their modifying enzymes can be early markers of tumourigenesis.
For this purpose, the following are applied:
a) selected covalent histone modification like acetylation, methylation, phosphorylation, ubiquitination among others
b) their modifying enzymes, like histone (de)acetylases, (de)methyltransferases
c) appropriate diagnostic methods and tests for detection of selected markers in clinical samples.
During the first period the first focus of EPIDIACAN was on the development of experimental tools and models to detect and study histone modifiers as well as to develop sensitive methodologies for evaluation of their use in preclinical testing. The second EPIDIACAN focus for the first period was on launching non-invasive diagnostic methods for epigenetic modifiers based on technologies developed in the participating organizations to be tested in clinical samples. Appropriately selected clinical samples are utilized according to EU and national ethical procedures. During the second period the first focus of EPIDIACAN was to analyse the biological function of the selected histone modifiers and their target genes in colorectal and prostate cancer. The second EPIDIACAN focus for the second period was on the characterisation, validation of the above epigenetic markers for clinical applications using human prostate and colorectal cancer biopsies. Appropriately selected clinical samples are utilized according to EU and national ethical procedures.
The participants' relevant research activities during the second period resulted in 58 publications in high impact scientific journals. The members of the network also presented the major results in scientific conferences. Interaction between the network participants was intense, which was realized via short-term visits, the annual meetings of the network, bilateral meetings, via ftp site of the network and via electronic communications. The project objectives have been advertised in the press (newspapers).
The project's web site is at:
http://www.eie.gr/nhrf/institutes/ibrb/eu-projects/epidiacan/index-en.html(opens in new window)
Project Context and Objectives:
Concept of the project
In the last few years our understanding of the complex mechanism of tumour progression has been dramatically changed by the identification of new mutated genes, which contribute in the multistep process and clonal expansion of tumours. In particular, genetic alterations of oncogenes and oncosuppressor genes have been closely associated with carcinogenesis. As a consequence, oncogenes/oncosuppressor genes contribute to the development of numerous aspects of the malignant phenotype by promoting cell cycle progression, resistance to apoptotic stimuli, neo-vascularisation and tumour metastasis. On the other hand, in recent years due to the newly developed powerful technologies of sequencing and analysis of gene regulation at the chromatin level, novel epigenetic mechanisms and factors related to cancer tumour progression have been identified. It is of interest that many of these epigenetic events appear early on the onset of cancer development and they can potentially be excellent early tumour markers. Indeed, a small number of epigenetic modifications have been tested already as early tumour markers, with very promising results.
Different forms of epigenetic mechanisms have been shown to modify the expression of key genes during tumour progression. DNA hypermethylation of promoter regions plays a role in silencing tumour suppressor genes or mismatch repair genes during tumourigenesis. Global DNA hypomethylation is observed in cancer and DNA hypomethylation at individual genes activate oncogenes in colorectal cancer. Demethylation in satellite sequences has been described as responsible to chromosomal instability in colorectal cancer. Further, loss of gene imprinting, a process mediated by DNA methylation and covalent histone modifications, is another epigenetic change that appears in the earliest stages of colorectal neoplasia causing abnormal gene expression. Finally, covalent modifications of histones like acetylation, methylation or phosphorylation, among others, distributed along promoters, coding regions and intergenic regions affecting chromosome condensation. Moreover, the cloning and functional characterization of histone modifying enzymes provides valuable information on the role of these epigenetic modifier factors. Although a lot of evidence has been accumulated for the role of epigenetic events and factors early in tumour progression, very few diagnostic applications in the clinic have so far emerged based on epigenetic markers.
The major challenge of this project was the development of new epigenetic markers for major types of cancer, like prostate and colorectal cancer that will allow early detection of tumour cells in patients, and will have important implications for successful therapeutic strategies in individual patients. A better mechanistic understanding of key epigenetically regulated genes involved in tumour progression in vivo will also help to develop tests for later clinical development and use. The key concept of this project was to develop diagnostic tools for the detection of epigenetic modifiers whose function has been implicated in cancer as potential tumour biomarkers.
The focus of the proposed collaborative effort was on:
1. The analysis of Usp22, SMYD3, LSD1, JMJD2c, PRK1, JMJD3 and EzH2 function in newly developed cell culture and animal models.
2. Identification of their downstream target genes to understand their role in tumourigenesis and to discover novel potential tumour biomarkers.
3. Development of efficient technologies for sensitive and specific epigenetic marker detection:
a. characterisation, validation of the above epigenetic markers for clinical applications using human prostate and colorectal cancer biopsies.
b. Characterisation, validation of the above epigenetic markers for clinical applications using circulating cancer cells obtained by non-invasive methods.
EPIDIACAN was carried out in close collaboration between investigators having complementary and multidisciplinary expertise in cell and molecular biology, epigenetics, biochemistry, tumour biology, clinical studies and tumour marker technologies. The studies of this Network have been directed to major aspects of cancer in model organisms, in mammalian systems and in clinical samples. This network composed of such teams provided unique and challenging opportunities for researchers interested in different aspects of epigenetics and cancer by using modern and developing new technologies to achieve their goal. It also provided the participant organisations, especially the small and medium-sized entreprise (SME), with epigenetic markers to be further exploited as industrial applications.
Project objectives
1. Generation of experimental tools (antibodies) and models to study epigenetic modifiers and for use in preclinical testing of epigenetic biomarkers.
a. Development of specific antibodies for the detection of selected histone modifiers (HM)
b. Generation of cell culture and animal models (KO and transgenic) for studying histone modifiers function.
c. Identification of target genes regulated by HM by global gene expression profiling and by ChIP-seq-based global occupancy analysis in the cell culture and animal models.
e. Identification of stage specific and oncogene specific epigenetic alterations in model systems of prostate and colorectal cancer.
2. Development of novel diagnostic tools for the detection of epigenetic modifiers and modifications in clinical samples.
a. Development of diagnostic tools/methods for the detection of selected modifiers including expression in tumour biopsies.
b. Development of diagnostic tools/methods for the detection of selected downstream targets of HM in tumour biopsies.
d. Development of improved non-invasive technologies for the isolation of cancer cells from body fluids that allow early detection of cancer.
e. Evaluation of diagnostic tools/methods detecting modifiers in cancer cells isolated from body fluids.
Project Results:
The major achievements of this period are as follows:
1. Genome-wide occupancy analysis performed by ChIP-sequencing with global expression profiling data and application of comparative analysis, retrieved JMJD2C, LSD1 target genes such as EGFR, CDK1 or the PRK1 target KLK2 and KLK3.
2. In PC3 cells we detected 488 and 1355 genes differentially regulated by LSD1 and PRK1, respectively. In contrast the PRK1 controlled gene set is associated with migration and invasion.
3. SILAC screens were performed for proteins interacting with LSD1 and JMJD2C respectively, and several novel interactors among members of the CoREST and NURD complex have been characterized
4. Our analyses reveal that the protein kinases CDK5 and PKA are able to phosphorylate LSD1 at position T119. Phosphorylation of LSD1 at position T119 does not alter demethylation or complex assembly.
5. An important role of Smyd3 in liver and intestinal cancer was identified. The mechanistic basis of its function involves promotion of epithelial-mesenchymal transition. Smyd3 was not recruited into the regulatory regions of S-phase specific genes. Target genes most relevant to the carcinogenesis process were those regulating EMT. These include members of the matrix metalloprotease family.
6. EZH2 silencing de-represses EMT related genes and affects cell migration and anoikis in colon cancer cells. ITGA2 has been identified as a novel EZH2 target gene and was further validated. ChIP-sequencing analysis has further identified new EZH2 target genes.
7. EzH2 levels are associated with cancer stem cells (CSCs) in our cultures, and EzH2 was down regulated when the CSCs differentiated This has been confirmed by showing that knocking down EzH2 activity using siRNA increases CSC differentiation
8. The tightly controlled deubiquitination activity of the human SAGA complex differentially modifies distinct gene regulatory elements. The structural plasticity of two different SCA7 domains of the SAGA deubiquitination module defines their differential nucleosome-binding properties. Deregulation of H2B ubiquitination may contribute to cancer development.
9. A total of 23 TMAs representing 1.242 tumor tissue samples in duplicate including pairs of primary tumors and metastases was generated in the reported period, from the following tumor indications: prostate, colon, breast, lung, kidney, stomach, head and neck, ovary, cervix and skin.
10. Collection of 240 fresh-frozen samples of prostate cancer tissue, as well as matched bodily fluids (urine and peripheral blood) has been performed, which have been stored at -80ºC. Simultaneously, bodily fluids from 200 healthy donors and patients harboring other urological pathologies have also been collected and stored for further analysis. Collection of fresh-frozen samples of colon cancer tissue and storage for further analysis was performed
11. We found that SMYD3, an H3K4 methyltransferase, was significantly over-expressed in tumor samples mainly in advanced stage prostate cancer
12. The data from our analysis show that LSD1 is predictive marker of prostate cancer superior to PSA.
13. High EZH2 mRNA levels in human colorectal cancer (CRC) specimens mark metastatic disease. Higher EZH2 expression was observed in more invasive CRCs and CRCs with regional lymph node metastases, Higher H3K27me3 expression was observed in more invasive CRCs, CRCs with regional lymph node metastases and in CRCs at advanced stages of disease, as well as in CRCs with lymph and venous vessel invasion, which appears to be associated with the putative role of EZH2 in the progression of CRC.
14. Lower LSD1 expression was observed in more invasive CRCs and in CRCs at advanced stages of disease, suggesting that LSD1 may be associated with less aggressive CRCs. Higher H3K9me3 expression was observed in CRCs with regional lymph node metastases, which appears to be associated with the putative role of EZH2 in the progression of CRC.
15. SETDB1 expression was observed in left-sided CRCs, suggesting a putative role in the carcinogenesis of CRCs with chromosomal instability.
16. Concerning response to treatment, higher survival rates in patients with CRCs treated only with surgery were found for tumours displaying lower SETDB1 expression, suggesting that whenever the expression of SETDB1 occurs, the surgical treatment may not be enough to prolong the patient's survival. Importantly, LSD1 expression and the histone mark H3K9me3 may predict the response of CRC patients treated with Folfiri, and H3K9me3 and H3K27me3 expression may predict CRC patients' response to 5-FU/Leucovorine.
17. F-actin (filamentous actin) is a useful marker of colorectal l (CRC) cell line derived lumens. Lumen formation is a feature of tumours in vivo, and human primary low and high grade colorectal tumours show differences in ezrin/actin and CEA polarisation. Lumens represent enterocyte brush border differentiation, but also contain secretory lineages. Polarised F-Actin is a marker of colonic brush borders in vivo and labels Matrigel grown lumens in vitro.
18. A new approach to establishing primary cultures from fresh CRC patient tumour material was developed.
Potential impact and use
This consortium has assessed epigenetic factors and mechanisms, like histone modifications as well as modifying enzymes involved in these processes. Cancer models have been selected for the most part of the studies and results have been validated in clinical specimens. EPIDIACAN consortium contributes in identification of novel oncogene and tumour stage related epigenetic events and factors, in order to maximize the subsequent clinical exploitation as epigenetic markers. Candidate epigenetic markers as well as marker combinations have been tested in a panel of preclinical models (cell lines and transgenic mice). A tight management plan was adopted in order that the most efficient combination per tumour cell to reach the final evaluation in clinical samples. There is the challenge that all this basic mechanistic analysis of genetic vs. epigenetic pathways will be utilized to improve clinical practice and finally public health.
The benefits on the society of a network focusing on novel early tumour markers are evident, since cancer is a major cause of death in Europe. This network contained groups of scientists from different disciplines like Molecular Biology, Epigenetics, Cell Biology, Tumour Biology, Biochemistry and Clinical Studies.
Bilateral interactions enhanced already successful co-operation by adding a value at the European level. Dissemination of the technologies developed at the individual national level will provide patients all over Europe to have access to new developments concerning cancer treatment, thus enhancing the benefit for the "European Citizen".
Part of the work will therefore have direct implications on colorectal, prostate and breast cancer in humans through novel epigenetic markers for cancer diagnosis and cancer therapeutics. Further characterization of our already existing cell and mouse models for cancer on different genetic backgrounds allowed us to use these mice in preclinical trials and speed up the evaluation of novel markers in the clinic. New knowledge about tumour formation has eventually been translated to an application stage, to the design of new diagnostic tests to detect cancer at an early stage and thus improve medicine in Europe and life quality.
The project extended the service business model and thus increases the competitiveness of the participating Targos Molecular Pathology Gmbh. The company had the opportunity to further explore own biomarker projects and build up own intellectuel property within this project and to profit from the transfer of know-how from the collaboration with outstanding experts from different fields of life sciences and technology. Targos further improved its knowledge in the field of epigenetics on a tumour and patient specific manner and was able to use the network resources in order to improve its competitiveness in the market.
We have also developed a efficient method to grow primary tumours from biopsies and endoscopies and this has the potential to significantly improve diagnosis and tumour classification. The health implications of this approach are potentially large as anti-cancer treatments can now be applied to growing samples derived from a much wide variety of patients. There is scope that our approach may allow some personalised treatments to be tested in vitro for efficacy, for example, antibody mediated immune killing using the patient's own blood.
Furthermore in vitro studies of tumour differentiation may also be used to refine the classical pathological definition of differentiation.
The potential cost benefit for EU healthcare may be substantial as drug treatments can be refined and suitable drug regimes tailored to suit each individual patient.
The prostate is the most common site of malignant transformation in western men. About 30% of men older than 50 years in the western countries develop prostate adenocarcinoma and the lifetime risk of clinical disease is 10%, with the risk of mortality being around 3%. The great variation in clinical behavior of prostate cancer creates a major dilemma in the treatment-decision process, since not all men with microscopic carcinoma require aggressive radical therapy. The major difficulty is the present-day absence of reliable tools to predict which cancers will remain indolent and which are going to kill the patient if left untreated. This project identified epigenetic markers of biological aggressiveness in prostate cancer and this information may have a wide impact on health and socioeconomics, both by reducing over-treatment and by allowing concentration of resources to treat intensively those patients who actually need it.
Participant 1. Alex Pintzas, NHRF.
WP1. Antibodies against EzH2 and JMJD3, as well as against H3K27Me3 were tested for optimal conditions in assays like immunohistochemistry, western blot, immunofluorescence and Chromatin immunoprecipitation (ChIP).
WP2. Generation of colon cells with silenced EzH2. Effect of silenced (inhibited) PIK3CAmut oncogene on EzH2 expression. To determine the contribution of EZH2 over expression to malignant phenotype of CRC cells we silenced EZH2 using small interfering RNA oligonucleotides and a vector to stably over express short hairpin RNA versus EZH2 mRNA (siEZH2 and shEZH2 respectively). As the expression of EZH2 was highest in cell lines with EMT behavior we decided to destroy the EZH2 endogenous protein in HCT116, SW620 and CacoH2 cells with a pool of siEZH2 oligonucleotides. To characterize the phenotype of siEZH2-silenced CRC cells we first evaluated the migratory ability using transwell system. The results showed that transient impairment of EZH2 reduced the migratory ability of CRC cells. In fact, the number of CacoH2- and HCT116-siEZH2 cells compared to parental cells were 69% and 83% respectively. We then concentrated only on HCT116 cell line for stable over expression of shEZH2 vector. The characterization of shEZH2 HCT116 stable clones reveled that EZH2 protein was reduced greater than80% and reduction on global H3K27me3. Furthermore, when shEZH2-HCT116 cells (HCTshEZ-2) were grown in Matrigel, 3D culture, they lost the ability to migrate as demonstrated by wound healing and migration assay (C and D).
Silencing of EZH2 derepresses EMT related genes in Caco-H2 cells. We reported a large list of genes related to EMT phenotype that were deregulated in Caco-H2 compared to parental Caco2 cell line (Joyce et all. 2009) as well as global and local histone post-translational modification pattern in the same cell models proposing that EZH2 might be a possible regulator of EMT (Mazon-Pelaez I. et al., 2010). The outcome of previous works and results obtained with siEZH2 let us to speculate that EZH2 really might be related to the invasiveness and migratory ability of CRC cells. To verify this hypothesis and in order to search for putative EZH2 target genes we decided to compare a group of 30 selected genes, chosen based on previous results, in siEZH2 versus parental EMT cell lines. We initially performed the study using CacoH2 cell line as we had microarray data for this cell system and afterward a subset of the 30 genes was analyzed also in HCT116 and SW620 cell lines. We initially performed the study using CacoH2 cell line as we had microarray data for this cell system and afterward a subset of the 30 genes was analyzed also in HCT116 and SW620 cell lines. As positive control, E-cadherin gene has been identified as an EzH2 target gene. The gene validation is currently in progress. Deliverables 3.1 and 3.2 (scheduled for month 24) have not yet been achieved.
We observed that forced expression of RAS and BRAF oncogenes in Caco2 cells caused hyper activation of RAS-PI3KA-AKT pathways with concomitant over expression of EZH2 (Mazon-Pelaez I. et al., 2010). Silencing of PIK3CA or inhibition of the pathway by small molecule, Wortmannin a PI3KA inhibitor, reduced pAKT and provoked EZH2 down regulation
WP3. Generation of colon cells with silenced EzH2. Effect of silenced (inhibited) PIK3CAmut oncogene on EzH2 expression. ChIP-sequencing analysis of Global EZH2 target genes.
To determine the contribution of EZH2 over expression to malignant phenotype of CRC cells we silenced EZH2 using small interfering RNA oligonucleotides and a vector to stably over express short hairpin RNA versus EZH2 mRNA (siEZH2 and shEZH2 respectively). As the expression of EZH2 was highest in cell lines with EMT behavior we decided to destroy the EZH2 endogenous protein in HCT116, SW620 and CacoH2 cells with a pool of siEZH2 oligonucleotides. To characterize the phenotype of siEZH2-silenced CRC cells we first evaluated the migratory ability using transwell system. The results showed that transient impairment of EZH2 reduced the migratory ability of CRC cells. In fact, the number of CacoH2- and HCT116-siEZH2 cells compared to parental cells were 69% and 83% respectively. We then concentrated only on HCT116 cell line for stable over expression of shEZH2 vector. The characterization of shEZH2 HCT116 stable clones reveled that EZH2 protein was reduced greater than80% as well aw reduction on global H3K27me3 was achieved (A and B). Furthermore, when shEZH2-HCT116 cells (HCTshEZ-2) were grown in Matrigel, 3D culture, they lost the ability to migrate as demonstrated by invasion assay as well as the ability to move as demonstrated by wound healing assay (C and D).
Identification and characterisation of oncogenic signalling pathways that modify EzH2 in colorectal cancer EZH2 is regulated by ERK and AKT pathways via AP-1 transcription factor in colon cancer cells with EMT phenotype. Initially we observed that forced expression of RAS and BRAF oncogenes in Caco2 cells caused hyper activation of RAS-PI3KA-AKT pathways with concomitant over expression of EZH2 (Mazon-Pelaez I. et al., 2010). Silencing of PIK3CA or inhibition of the pathway by small molecule, Wortmannin a PI3KA inhibitor, reduced pAKT and provoked EZH2 down regulation. To better dissect the pathways involved in EZH2 regulation we assessed EZH2 expression before and after blocking ERK and AKT pathways using siRNA and two specific inhibitors, in Caco-2, Caco-H2, HCT116 and SW620 cells. pERK1/2 inhibition by the MEK inhibitor UO126 in Caco-2 cells resulted in 10% reduced EZH2 protein and mRNA levels. In Caco-H2 cells EZH2 mRNA and protein expression (by 49%) were significantly reduced after UO126 treatment. In SW620 cells, a considerable inhibition of pERK1/2 was observed after UO126 treatment with a consequent 30% reduction of EZH2 protein and mRNA levels.
Silencing of EZH2 derepresses EMT related genes.
We reported a large list of genes related to EMT phenotype that were deregulated in Caco-H2 compared to parental Caco2 cell line (Joyce et all. 2009) as well as global and local histone post-translational modification pattern in the same cell models proposing that EZH2 might be a possible regulator of EMT (Mazon-Pelaez I. et al., 2010). The outcome of previous works and results obtained with siEZH2 let us to speculate that EZH2 really might be related to the invasiveness and migratory ability of CRC cells. To verify this hypothesis and in order to search for putative EZH2 target genes we decided to compare a group of 30 selected genes, chosen based on previous results (Joyce et al., 2009), in Caco-H2 siEZH2 versus parental the cell line. A subset of the 30 genes was than analyzed in the other two EMT cell lines available: HCT116 and SW620. Table S1 shows the results of qPCR performed on Caco-H2 cells. ITGA2, SPRY1, CDH17, Coll type II A1 and CCND2 were significantly up-regulated whereas CCND1 was down-regulated after transient reduction of EZH2 in Caco-H2. Downregulation of CCND1 was detected in all cell lines, whereas CCND2 and CDH17 were up regulated in SW620 cells and ITGA2 and Coll Type II A2 were up regulated in HCT116 cells. The analysis indicated that Caco-H2 cell line after EZH2 silencing presented a molecular mark which is a sum of the other two EMT cell lines and that ITGA2 was a potential EZH2 target gene. We compared the ITGA2 mRNA absolute levels in HCT116, SW620 and Caco-H2 before and after EZH2 transient silencing.
ChIP-sequencing analysis of Global EZH2 target genes
In order to best characterize the phenotype of HCTshEZ2-2 cells and search for new EZH2 targets related to cancer, we performed ChIP experiment followed by massive parallel sequencing (ChIP-seq). The template for sequencing was prepared using the anti-EZH2 antibody on sonicated chromatin extracted from HCT116 parental and HCTshEZ2-2 cells. For each cell line we performed sequencing in 2 biological replicates (HCT116_B and HCT116_109; HCTshEZ2-2_A and HCTshEZ2-2_B). The overall quality of sequencing was good even though an optimization of ChIP protocol is needed in order to confirm interesting preliminary results and cover some genomic loci that are poorly resolved.
WP5. EZH2 mRNA levels in human CRC specimens marks metastatic disease.
EZH2 mRNA expression was evaluated in 51 human CRC specimens by qPCR. Over-expression (greater than1.3-fold) was found in 37.2% (19/51) of tumor samples whereas in 29.4% (15/51) EZH2 mRNA was less than 0.7-fold (down regulated) with respect to a pool of normal colon tissues. In 33.3% (17/51) of tumor samples no significant changes were detected. Specimens were grouped based on EZH2 expression and presence of lymph node and/or distant metastasis. Even though the percent-age of specimens positive for metastasis was roughly the same in all three selected groups (68.4%; 62%; 66.7% respectively), in samples with EZH2 over-expression the magnitude of up-regulation was significantly higher in patients presenting metastasis. Fold-change average of EZH2 in this group was 2.83 whereas in samples devoid of metastasis was 1.67 close to the limit of 1.3 fold used for up-regulation. MannWhitney test (p-value = 0.043) performed on the two sub-groups with EZH2 over-expression, indicates that higher EZH2 mRNA levels correlate with presence of lymph node and distant metastasis.
Participant 2. Sir Walter Bodmer, WIMM.
WP2 Development of cell culture and animal models to study epigenetic regulators and modifications
We have analysed the expression data for the 7 genes of special interest to the Epidiacam project on our colorectal cell line panel, using just over 90 of the cell lines. For only one of the genes, PRK1(official name PNK1), is there a clear cut suggestion of two different groups of lines with different levels of expression. In the figure we plot the cell lines in rank order of expression and look for evidence of two or more groups of lines with different levels of expression by testing for non-linearity of the slope of the rank order plot expressed in terms of the expected ranks on the assumption of a normal distribution(a rankit plot). Otherwise the slope of such a plot simply represents the normal pattern of variation in the level of expression, and the greater the slope the more variable the level of expression. It is not ever expected that an unusual level of expression in comparison with normal will be expected in all examples of a given type of cancer. That is the reason we look for such subgrouping of expression levels. JMJD2c shows a possible small subgroup of less than 5%, with one line expressing at a substantially higher level than all the others. The same may be true for EZH2, though here the results are less convincing as the higher levels of expression in the lines overlap those found in some normal samples.
These data, however, do not suggest that the 7 EPIDIACAN genes, with the possible exception of PRK1, are themselves candidates for genes whose expression change has been selected for, at least during colorectal cancer progression. This places the emphasis on looking at their down stream targets using the cell lines, taking into account the observed different levels of expression in different cell lines. Once this is done on a small subset of the lines, then the levels of expression of potentially interesting candidate genes can be further analysed, again looking for clearly defined subgroups with different levels of expression.
Previous studies in our laboratory have suggested that EzH2 levels are associated with CSCs in our cultures, and that EzH2 was down regulated when the CSCs differentiated. This has now been confirmed by showing that knocking down EzH2 activity using siRNA increases CSC differentiation, as measured both by lumen formation and expression of the columnar cell differentiation marker Cytokeratin 20.
We previously gave preliminary evidence that F-actin (filamentous actin) is a useful marker of colorectal l (CRC) cell line derived lumens. We have confirmed the in vivo relevance of this by staining normal human colon cryosections with fluorescent phalloidin, which is specific for F-actin. In normal colon, F-actin is widely expressed in epithelial plasma membranes but is intensely enriched at the apical surface of enterocytes lining the crypt, corresponding to the brush border. This enrichment for F-actin is most probably due to the presence of microvilli on the colonic enterocytes. The intense staining of the muscularismucosum muscle layer was visible under the crypts. We next examined F-actin labelling in single cell derived colonies from a panel of six colon cancer cell lines, all grown under the same conditions in a three dimensional Matrigel matrix. In SW1222, confocal Z-sectioning demonstrated intense F-actin stress fibres were present on the apical cell membranes facing into the lumen.
WP5 Clinical validation of selected epigenetic markers
To determine if lumen formation or lack thereof had relevance to tumours in vivo, we first examined actin and ezrin polarisation in murine xenografts derived from the injection of 500 cells of HT29, HCT116 or SW1222 into the flanks of NOD/SCID mice. Mice were sacrificed after one month and resulting tumours removed and processed for FFPE tissue sections which were stained by hematoxylin and eosin or immunolabelled with anti-actin/ezrin. Hematoxylin and eosin staining of these xenograft tumours demonstrated poorly differentiated high grade tumour morphology for HCT116 and HT29 tumours that lacked lumens or actin/ezrin polarisation. SW1222 tumours exhibited a well differentiated phenotype with numerous lumens surrounded by polarised cells.
WP6 Development of epigenomic biomarker tests for diagnosis prognosis and therapy prediction
We have continued our evaluation of variations in the procedures for handling patient blood samples for CTC detection. We are particularly concerned to assess the best approaches for both short and long term storage for freshly collected blood samples and to reassess the comparison between filtration and Ficoll-Hypaque partial purification of epithelial cells from the blood. We have also been testing various procedures for multiple antibody staining using our routine monoclonal antibodies, AUA1, an anti EpCAM and CAM5.2 against cytokeratins7/8 , together with other antibodies , for example against the differentiation control marker CDX1 and the prostate specific markers PSA and PSMA, as well as FISH for the common translocations found in prostate cancers.
Participant 3. Carmen Jeronimo, IPOPFG.
The major achievements are as follows: In summary
Regarding prostate cancer:1-We have explored the role of HMTs altered expression in PCa onset and progression and from the 37 genes explored we found that SMYD3 plays an important role in prostate carcinogenesis and therefore it may be useful as a therapeutic target in aggressive human PCa. 2- We did not find prognostic value in PCa patients for EZH2 and H3K18Ac immunoexpression in biopsies, contrarily to what has been previously reported.
In more detail:
WP3. Identification of downstream targets of epigenetic modifiers and proof of their role in prostate and colorectal cancer models. Deliverables 3.2 (Month 24) have been achieved.
We have developed engineered prostate cancer cell lines lacking SMYD3 in order to characterize the biological function of SMYD3. (WP3; Deliverable 3.2) To clarify the role of HMTs altered expression in PCa onset and progression and to translate those findings into clinically useful tools for management of PCa patients. To achieve this goal, epigenetic gene expression of 37 HMTs was investigated by analysis of TaqMan® Arrays Plates in 10 primary PCa and 5 Morphological Normal Prostate Tissue (MNPT).
WP4. Work performed for Deliverable D4.1 and 4.2 (scheduled for Month 24) and D4.3 (scheduled for Month 36). Collect and store biological samples of prostate cancer patients and controls, as well as relevant clinical data.From month one to 36th and after informed consent of the patients we were able to collect 240 fresh-frozen samples of prostate cancer tissue, as well as matched bodily fluids (urine and peripheral blood), which have been stored at -80ºC. Simultaneously, bodily fluids from 200 healthy donors and patients harboring other urological pathologies have also been collected and stored for further analysis.
WP5. (cont.) The parts of work corresponding to Deliverables 5.2 (scheduled for Month 30), 5.3 (scheduled for Month 30), 5.4 (Scheduled for Month 30) and 5.5 (Scheduled for Month 30) are being pursued and have not been achieved yet. Work performed for Deliverable D5.2 (scheduled for Month 30). Establish the clinical value of factors including JMJD2C, LSD1, and PRK1 as a predictive parameter for aggressive and hormone-refractory prostate cancers. (Delivery month 30)
WP6. Development of epigenomic biomarker tests for diagnosis prognosis and therapy prediction
The parts of work corresponding to Deliverables 6.1 (scheduled for Month 36) and 6.2 (scheduled for Month 36) are being pursued and have not been achieved yet. Work performed for Deliverable D6.2 (scheduled for Month 36).Evaluation of epigenetic markers in cancer cells from body fluids (Delivery Month 36). We have identified several epigenetic markers for prostate cancer in WP5. However, we were unable to detect these surrogate markers either at RNA or protein level in human body fluids such as urine.
Participant 4. Laszlo Tora, GIE-CERBM.
S&T results on USP22 and the deubiquitination module from GIE-CERBM
The tightly controlled deubiquitination activity of the human SAGA complex differentially modifies distinct gene regulatory elements.
The multisubunit SAGA (Spt-Ada-Gcn5 acetyltransferase) coactivator complex facilitates access of general transcription factors to DNA through histone acetylation mediated by GCN5. USP22 (ubiquitin-specific protease 22) was recently described as a subunit of the human SAGA complex that removes ubiquitin from monoubiquitinated histone H2B and H2A in vitro. We discibed an allosteric regulation of USP22 through multiple interactions with different domains of other subunits of the SAGA deubiquitination module (ATXN7, ATXN7L3, and ENY2). Downregulation of ATXN7L3 by short hairpin RNA (shRNA) specifically inactivated the SAGA deubiquitination activity, leading to a strong increase of global H2B ubiquitination and a moderate increase of H2A ubiquitination. Thus, SAGA is the major H2Bub deubiquitinase in human cells, and this activity cannot be fully compensated by other deubiquitinases.
Role for H2Bub in DNA repair and carcinogenesis
While enzyme-mediated epigenetic control of gene transcription is a critical aspect of embryonic development and cellular differentiation, this same mechanism is often deregulated in human diseases, where aberrant gene expression or repression is a hallmark of cancer and other diseases. There is growing evidence that amplification, mutation and other alterations of epigenetic enzymes are molecular causes of certain cancers and several human diseases. A number of observations suggest that deregulation of histone H2B ubiquitination and the resulting effect on transcription may be key events in cellular transformation and metastasis. First, gene expression analysis of RNF20 depleted cells suggested that RNF20 acts as a putative tumor suppressor gene. Indeed, the expression of several proto-oncogenes and proliferation-related genes was increased after reduction of H2Bub via RNF20 depletion. In contrast RNF20 positively regulates p53 and different studies showed that RNF20 and WAC regulate p53-dependent genes in response to DNA damage.
1. Generation of antibodies against SMYD3. (WP1; Deliverable D1.1)
Initially we have tested several commercially available Smyd3 antibodies (Abcam, Santa Cruz, Sigma) using western blots from liver extracts of wild type and Smyd3 transgenic (Smyd3-Tg) animals and by immunohistochemistry in frozen or paraffin-embedded liver sections. The antibodies did not perform well in any of the applications. Next we generated a polyclonal antibody by immunizing 2 rabbits with a KLH-conjugated N-terminal peptide of Smyd3. The collected sera recognized recombinant Smyd3 as well as overexpressed Smyd3 in extracts prepared from CMV-Smyd3 transfected cells. The peptide antibodies did not recognize endogenous Smyd3. After this we immunized rabbits with recombinant full-length Smyd3 protein. The sera of these mice were tested in different applications: They gave a good signal in western blots of extracts containing overexpressed proteins. The antibody also gave good result in detecting endogenous Smyd3 by western blots and immunoprecipitations.
2. Validation of SMYD3 as biomarker in human colon cancer biopsies. (WP3, WP5; Deliverable 3.6 and 5.5)
The antibody was extensively tested in immunohistochemistry applications in mouse colon cancer sections and human colon cancer. Parallel measurements of RNA levels by RT-PCR were performed. The histochemical signals correlated well with the expression of Smyd3.
Deliverables 3.6 and 5.5 has been achieved.
3. Generation and analysis of animal models lacking Smyd3 and overexpressing Smyd3 in intestine. (WP2; Deliverable 2.3)
4. Generation and analysis of experimentally induced colon cancer. (WP2; Deliverable 2.5).
To generate Smyd3 KO mice we used a gene-trap ES cell clone to inject mouse blastocysts. Chimeric mice and germ-line transmission was obtained before the initiation of the program. During the reporting period we obtained a good number of homozygous mice to perform experimental work. The mice look overall normal. Histological analysis of livers and intestines did not show major morphological alterations. We have performed drug-induced liver and colon cancer induction programs using DEN only and DEN-TC (10 and 7 months treatment, respectively) and DMH (3 months treatment) protocol in 2 groups of mice. Each group consists of 10 animals.
a. To induce colon cancer, 2 months old mice were injected with DMH, followed by treatment with DSS in drinking water for 1 week. The mice were then left for 2 weeks with normal drinking water. The DSS treatment was repeated two more times and the mice were sacrificed.
Smyd3 KO mice exhibited inflammatory response similar to wild type mice, but essentially no formation of adenomas.
b. To induce liver cancer, at day 14 after birth each litter received a single intraperitoneal injection of the DNA-damaging tumor initiator diethylnitrosamine (DEN) and analyzed at 10 months age (DEN only protocol). In DEN-TC protocol, two weeks after DEN treatment, and every two weeks afterwards, the animals were given intraperitoneal injection of the hepatotoxic nuclear receptor CAR ligand TCPOBOP (TC) for 4 to 6 months. Using this protocol full-blown liver tumor was detectable at 4 months after treatment in wild type mice.
Smyd3 Tg mice:
Smyd3 Tg mice were generated using a construct containing the full-length Smyd3 cDNA under the control of the liver-specific TTR promoter/enhancer. The transgenic animals express 5'HA and 3'Flag tagged version of full-length Smyd3, specifically in hepatocytes. We have expanded 2 transgenic lines, where transgene expression was monitored by RT-PCR and western blot analysis. HA-tag antibody detected a 52 kD band only in whole cell extracts, while Flag antibody detected a 52 and 48 kD band in whole cell extracts and a 48 kD band in nuclear extracts. This suggests that in the nuclear form of Smyd3 the N-terminal region is cleaved.
Smyd3 Tg mice did not develop tumors spontaneously (at least within 10 months of age) but, as expected, somewhat accelerated DEN-induced carcinogenesis.
Deliverables 2.3 and 2.5 have been achieved.
5. Global expression profiling and occupancy analysis of Smyd3-regulated genes.
(WP 3, Deliverable 3.3)
We attempted to identify genome wide occupancy patterns of Smyd3 using several antibodies (including those developed by us) recognizing endogenous Smyd3. None of them worked in ChIP assays. As an alternative, we took advantage of our transgenic mice, where the nuclear form of Smyd3 can be detected by Flag antibodies. ChIP-seq analysis revealed several (more than 8000) locations of the genome bound by Smyd3. About 20% of them were located in promoters. While several interesting target genes were identified, it was interesting to note that Smyd3 was not recruited into the regulatory regions of S-phase specific genes. As mentioned above, target genes most relevant to the carcinogenesis process were those regulating EMT. These include members of the matrix metalloprotease family, which have also been detected in human colon cancer.
Chromosome-wide (chr12) distribution of peaks in Smyd3 ChIP-seq experiment. 2 biological replicates show similar binding pattern.
Deliverable 3.3 has been achieved.
6. Provide experimental samples from animal models to the partners of the network.
Several exchanges of scientific materials between EPIDIACAN members have been realized during the project.
7. Other activities
Another line of our research in this program was to identify the lysine specificity of Smyd3. Previous studies established that Smyd3 is a histone 3 lysine 4 methylase. We performed several in vitro studies using bacterially or baculovirus expressed Smyd3 recombinant proteins. While in the presence of HSP90 we could observe some methylation at H3K4, this activity was very low compared to other enzymes. On the other hand we identified Smyd3 as an H4K20 methylase, which seems to be the main specificity of the enzyme. We have also been searching for novel, non-histone substrates of Smyd3. We made use of our transgenic mice to perform immunoprecipitations followed by quantitative mass-spectrometry. This effort led to the identification of several cytoplasmic proteins including Cyld. Although, Cyld methylations still needs verification, we hypothesized that the mechanism by which Smyd3 modulates hepatocarcinogenesis may involve regulation of NFkB signalling. While the roles of the components of NFkB signalling in cancer have been studied before, no information about the potential role of the deubiquitinase Cyld was available.
Conclusion
Our studies revealed an important role of Smyd3 in liver and intestinal cancer. The mechanistic basis of its function involves promotion of epithelial-mesenchymal transition. The possibility of regulating tumorigenic signalling pathways was also raised, which opens new avenues for future research. It was also shown that Smyd3 could serve as a good biomarker for colon cancer. Future studies may be directed towards developing and testing drugs specifically modulating the activities of the enzyme.
Participant 6: Roland Schuele, UKL-FR
WP1: We generate several new antibodies with the goal of increased specificity for histological, ChIP and other applications. These include antibodies against JMJD2A-D, LSD1, and PRK1. For immunization purified recombinant proteins were used. For the production of polyclonal antibodies, New Zealand White rabbits were immunized by a standard, 70-day immunization program. Test bleeds were collected and assayed in western blots of cellular extracts and with purified recommbinand target proteins. Then the antibodies were purified by affinity chromatography and their performance was veified in western blots, ChIP assays and immunohistological applications. In summary, we met deliverables 1.1 and 1.2 in full and deliver superior antibodies for JMJD2A-D, LSD1, and PRK1 to the consortium.
WP2: Cell culture models
We established and optimized siRNA-mediated knock-down strategies to reduce the levels of JMJD2C, LSD1, and PRK1in human prostate cancer cell lines such as LNCaP. for the purpose of generating cell lines with reduced. For inducible expression we developed the tetracycline-controlled approach to conditionally overexpress shRNAs using a lentiviral gene silencing system. For overexpression we also used lentiviral gene delivery system of the various wild type and mutant proteins. Bona fide expression of the corresponding gene was veryfied by RT-PCR and western blot analysis.
JMJD2c, LSD1, PRK1 KO, transgenic mice
To investigate the biological and pathophysiological functions of JMJD2C, LSD1, and PRK1and their contribution to the development of cancer we cloned targeting constructs that allow the Cre-loxP mediated deletion of PRK1 exon 1 of PRK1 and LSD1 respectively. We generated homozygous floxed animals (LSD1 and PRK1) and breeding colonies of LSD1 floxed animals are established while the breeding colony of PRK1 floxed animals is expanding. Next, we generated animals that have prostate specific gene knockout by breading LSD1flox/flox animals with transgenic ARR2Probasin-Cre (PB-Cre4) and ARR2Probasin-CreERt (PB-CreERt) mice that express Cre recombinase specifically in the prostate epithelium in a constitutive or Tamoxifen inducible manner, respectively.
WP3: To characterize the biological function of the demethylases JMJD2C, LSD1 and the kinase PRK1 we combined a genome-wide occupancy analysis performed by ChIP-sequencing with global expression profiling data derived from Affimetrix platform, applied comparative analysis and retrieved JMJD2C, LSD1 target genes such as EGFR, CDK1 or the PRK1 target KLK2 and KLK3. The physiological role of the various target genes will be analysed in detail.
WP3: D 3.2: To investigate the biological and pathophysiological functions of JMJD2C, LSD1, and PRK1and their contribution to the development of cancer we generated homozygous floxed animals (LSD1 and PRK1). A breeding colony of PRK1 floxed animals is expanding. So far we have obtained three PRK1 null animals. The first physiological and histological analysis revealed no obvious phenotype of the PRK1 knockout animals. Further detailed analyses are ongoing. To characterize the functions of LSD1 we generated prostate specific gene knockout by breading LSD1flox/flox animals with transgenic ARR2Probasin-Cre (PB-Cre4) mice that express Cre recombinase specifically in the prostate epithelium. The detailed biochemical and histological analysis revealed no obvious phenotype of the prostate specific LSD1knockout animals. Further detailed analyses are ongoing. Next, we ubiquitously deleted LSD1 using a Rosa26-Cre deleter. Homozygous LSD1 knockout animals stop development at around E7.5-8.0 due to impaired trophoblats stem cell development. Furthermore, we produce knockout mice for the demethylase JMJD2C by using gene-trap technology. The detailed biochemical, histological, and functional assaying for altered performance and physiology revealed no obvious phenotype of the knockout animals.
D 3.3: To identify LSD1and PRK1 regulated genes we performed global analysis of gene expression by RNA-seq analysis. In PC3 cells we detected 488 and 1355 genes differentially regulated by LSD1 and PRK1, respectively. About half of the genes are either up-or downregulated. The bioinformatic analysis uncovered that LSD1 controls a gene set regulating transcription proliferation and metabolism. In contrast the PRK1 controlled gene set is associated with migration and invasion. In addition, we used the antibodies develop in WP1 to establish LSD1 Chip-seq methodology. The genome-wird chromatin binding profile of LSD1 in LNCaP and PC3 cells was established. The bioinformatic analysis uncovered that LSD1 is enriched a promoters, defined by 2000bp around the transcription start site. LSD1 locations overlap with active histone marks such as H3K4me3 but not with the repressive mark H3K27ac. HOMER analysis identified AR, REST, and FOX binding motifs as significantly enriched motifs.
D 3.4: To identify LSD1 interacting proteins we used the antibodies develop in WP1 to immunoprecipitate endogenously expressed LSD1 in LNCaP and PC3 cells. The immunoprecipitated LSD1-associated proteins were identified by mass-spectrometry. Interestingly, we were able to identify the CoREST complex as interaction partner. However, we did not detect any member of the NuRD complex. Interestingly, we identified novel signalling molecules such as dynactin 1, dynactin 2, or protein phosphatase 12 as interactin partners. The functional analyses of these novel LSD1 associated proteins in ongoing.
D 3.5: We characterize by an unbiased genome wide screening strategy, kinases that modify LSD1 and analyzed the consequences of this modification on biological function. In vivo, LSD1 is modified by post-translational modifications such as acetylation and phosphorylation. We expressed and purified a pool of modifiers i.e. enzymatic active forms of all known acetylases and 180+ kinases covering a large percentage of all cytoplasmic and nuclear kinases present in the human genome. LSD1, expressed either in E. coli or insect cells, was tested for modification by the modifiers. Phosphorylation of LSD1 at position 119 was identified by mass-spectrometry. We developed phospho-specific antibodies (anti LSD1Tph119) and used the modification-specific antibodies to confirm the data obtained by mass-spectrometry. Phosporylation was verified by mutagenesis (LSD1T119A). Our analyses reveal that the protein kinases CDK5 and PKA are able to phosphorylate LSD1 at position T119.
WP5: D 5.2 and: D 5.2:To establish the clinical value of JMJD2C, LSD1, and PRK1 as a predictive marker of prostate cancer we initiated to characterized a study cohort of 500+ patients, which underwent radical prostatectomy for locally limited diseases within a single surgical center and were followed-up between 3 to 9 years. All prostate specimens were classified according to TNM stage and Gleason score following defined procedures and used to generate tissue microarrays (TMAs). Patients were be grouped into two categories according to the following tumour parameters: Group 1 (high-risk tumours): pT3 or higher, all pN0M0, Gleason score 8 or higher. Group 2 (low-risk tumours): pT2c or lower, all pN0M0, Gleason score 7 or lower. So far we have use the TMAs for immunostaining a panel of antigens such as LSD1 PRK1, p53, Ki67 etc.
Participant 7. Thomas Henkel, Targos.
The cooperation with the Clinical Research Group (KFO179) at the University of Göttingen was continued to get access to tumor samples from two prospective rectal cancer trials in which patients have preoperatively been treated by chemo-radiotherapy (CRT). In the first trial (from 1995 to 2002) follow-up data covered at least five years (CAO/ARO/AIO-94), the second trial is still on-going (CAO/ARO/AIO-04). The trials were approved by the medical ethics committees of all participating hospitals.
WP 4.1 In cooperation with the Clinical Research Group (KFO179) at the University of Göttingen tumor samples of two prospective rectal cancer trials were collected, in which patients have preoperatively been treated by chemo-radiotherapy (CRT). In the first trial (from 1995 to 2002) follow-up data covered at least five years (CAO/ARO/AIO-94), the second trial is still on-going (CAO/ARO/AIO-04). The trials were approved by the medical ethics committees of all participating hospitals.
WP 4.2 The planned SEAL trials, which was planned to be a prospective randomized phase III trial of limited versus extended lymph node dissection in patients with intermediate/high risk prostate cancer (Trial number AP49/08) was finally not funded and thus was not started as planned in 2010. A second attempt for funding was not approved in 2011/2012. Thus no prospectively collected prostate cancer samples were available for analysis. These samples were replaced by retrospectively collected tumor bank samples (see WP 5.1) and commercially available tissues.
WP 5.1 During the reported period Targos generated further multiple tissue micro-arrays (TMA) of high quality (Multiblock melting technology) from anonymized tumor bank samples including clinical data, mainly derived from the tumor bank of the comprehensive Cancer Care Center Cologne/Bonn (Prof. Büttner)
WP 5.2. As a first marker LSD1 was analysed by means of IHC in the Targos lab on n=148 FFPE blocks of biopsies from colorectal cancer patients prior to neoadjuvant CRT and n=42 FFPE blocks from colorectal resection specimens post-treatment derived from the CAO/ARO/AIO-04 trial. As controls, n=40 resection specimens of patients not undergoing neo-adjuvant treatment have been stained by Targos technicians on the automated platforms Benchmark XT and Symphony
WP 5.3. A spreadsheet data base for all available reagents within the consortium was generated to allow all consortium members to scan for interesting reagents relevant to their respective work. Further technical and clinical validation of any marker assays for diagnostic purpose was not performed for lack of scientific justification.
Potential Impact:
Tumour markers tests have been recently developed based on genomic technologies which have been shown by researchers and clinicians to have an important role in tumour progression. The work of several laboratories from USA and from Europe, including members of the EPIDIACAN consortium has elucidated mechanisms of epigenetic gene regulation, which can be exploited by the tumour biomarker field. Indeed, participant Laboratories of this network have analysed epigenetic markers in colorectal tumour (models) (Participants 1, 2) as well as in prostate and breast cancer (Participants 3, 6, 7). Other participant Labs have been contributing in basic knowledge and mechanistic analysis of epigenetic pathways and factors (Participants 4, 5). This consortium has assessed epigenetic factors and mechanisms, like histone modifications as well as modifying enzymes involved in these processes. Cancer models have been selected for the most part of the studies and results have been validated in clinical specimens.
Contribution to wider societal objectives
The benefits on the society of a network focusing on novel early tumour markers are evident, since cancer is a major cause of death in Europe. This network contained groups of scientists from different disciplines like Molecular Biology, Epigenetics, Cell Biology, Tumour Biology, Biochemistry and Clinical Studies.
Contribution to policy objectives of Health
Cancer is the second major cause of mortality in Europe. A new era in human biology has been opened by the sequencing of the human genome offering opportunities to improve human health and to stimulate industrial activity. Our project represents a basic and applied research programme aimed to exploit the potential of genomic approaches to decipher mechanisms underlying genetic vs epigenetic mechanisms in oncogenesis. The goals of this proposed project were in accordance with the relevant objectives of the Health priority laid down in the 7th EU framework programme, because our research is focused on major fundamental biological processes concerning cell proliferation which can be exploited for novel tumour markers. Part of the work will therefore have direct implications on colorectal, prostate and breast cancer in humans through novel epigenetic markers for cancer diagnosis and cancer therapeutics. Further characterization of our already existing cell and mouse models for cancer on different genetic backgrounds allowed us to use these mice in preclinical trials and speed up the evaluation of novel markers in the clinic. New knowledge about tumour formation has eventually been translated to an application stage, to the design of new diagnostic tests to detect cancer at an early stage and thus improve medicine in Europe and life quality.
Reinforcing European competitiveness
EPIDIACAN has aspects in very important and competitive areas on preclinical models of cancer, cancer genetics and genomics, as well in imaging technologies. While a number of leading scientists within the field are active in Europe today, judging from the published literature it is evident that European research output lags behind that of the United States in production of efficient novel cancer diagnostics. The domination of US laboratories of the field has as a consequence the continuous drain of talented young European researchers towards the US. This consortium has established world leadership in the field, mainly due to the competitive edge provided by EU support for intensive collaboration of a critical mass of researchers from different disciplines. The integrated approach has neutralized the problem of fragmentation of relevant expertise in Europe, thus translating the potential success of the project into tangible benefits for European science.
Transnational cooperation
The complexity and the quantity of the proposed work demanded the collaboration of many laboratories possessing different expertise. Some of the participants (1, 4, 5) and (6 ,7) had already initiated bilateral collaborations studying related topics. Although they had built up fruitful collaborations, these activities were fragmented and addressed specific aspects only. This project combined and expanded these individual activities to a more comprehensive collaboration. There were no chances to carry out such an ambitious project at the level of individual laboratories, or to gather the diverse but complementary expertise needed at the national level. Besides the combination of different expertise, the multidisciplinary nature of the program required the integration of resources available at the individual laboratories.
Impact on participating companies
The project extended the service business model and thus increases the competitiveness of the participating Targos Molecular Pathology Gmbh. The company had the opportunity to further explore own biomarker projects and build up own intellectuel property within this project and to profit from the transfer of know-how from the collaboration with outstanding experts from different fields of life sciences and technology. Targos further improved its knowledge in the field of epigenetics on a tumour and patient specific manner and was able to use the network resources in order to improve its competitiveness in the market.
Unique training opportunities
The participating laboratories had continuously open calls for positions for young European researchers. Although, training was not the prime objective of this application, given the cutting-edge scientific level of research to be performed and the opportunity to carry-out research projects in collaboration with laboratories of a different discipline, the network provided a competitive alternative to US laboratories for training young researchers interested in the field of cancer epigenetics. Via short-term visits, the young fellows had an opportunity to work in more than one laboratory. The opportunity to meet and collaborate with several network members and to be exposed to the continuous information exchange of the whole network has set up a precedent of a novel, highly productive training method highly attractive to young researchers interested in the field.
WIMM
The main dissemination for us was publication and presentations at various scientific meetings and seminars. The potential impact is to improved diagnosis and treatment of colorectal cancer. At the stage we have no immediate plans for exploitation of results, though there may be an opportunity for doing something with the development of effective cultures from fresh tumour material. We have also obtained additional information on the colorectal cancer cell line panel which is useful for assessing in vitro drug response assay results in relation to the properties of a cancer.
IPOFG
The findings are expected to originate at least six articles in the course of the project, which will be submitted for publication in international, peer-reviewed journals. More publications are expected when the long-term objectives are accomplished. Furthermore, the findings will be presented in local seminars, and in several national and international conferences in which the team investigators participate, including: Workshop on Molecular Genetics of Human Solid Tumors, Meetings of the European Association for Cancer Research and of the Portuguese Society of Human Genetics, Congress of the Portuguese Oncology Society, European Congress of Pathology and the Workshop on Medical Oncology organized by our Institution.
List of Websites:
http://www.eie.gr/nhrf/institutes/ibrb/eu-projects/epidiacan/index-en.html(opens in new window)
Cancer is one of the major causes of mortality. Epithelial cells become malignant after accumulating genetic mutations followed by morphological changes in the epithelium. Alterations in DNA include stable genetic changes in oncogenes, tumor suppressor genes and reversible epigenetic changes. Different forms of epigenetic mechanisms have been shown to modify the expression of key genes during tumour progression. Promoter DNA hypermethylation of tumour suppressor genes or DNA repair genes, and covalent histone modifications appear in early stages of neoplasia. Methods to identify early markers in different types of cancer are being developed, although very few are specific and sensitive enough to be applied in the clinic.
The aim of the present consortium EPIDIACAN was to develop sensitive and specific methodologies to identify early epigenetic markers for major types of cancer, like prostate and colorectal cancer. This project is based on recent findings that selected covalent histone modifications and their modifying enzymes can be early markers of tumourigenesis.
For this purpose, the following are applied:
a) selected covalent histone modification like acetylation, methylation, phosphorylation, ubiquitination among others
b) their modifying enzymes, like histone (de)acetylases, (de)methyltransferases
c) appropriate diagnostic methods and tests for detection of selected markers in clinical samples.
During the first period the first focus of EPIDIACAN was on the development of experimental tools and models to detect and study histone modifiers as well as to develop sensitive methodologies for evaluation of their use in preclinical testing. The second EPIDIACAN focus for the first period was on launching non-invasive diagnostic methods for epigenetic modifiers based on technologies developed in the participating organizations to be tested in clinical samples. Appropriately selected clinical samples are utilized according to EU and national ethical procedures. During the second period the first focus of EPIDIACAN was to analyse the biological function of the selected histone modifiers and their target genes in colorectal and prostate cancer. The second EPIDIACAN focus for the second period was on the characterisation, validation of the above epigenetic markers for clinical applications using human prostate and colorectal cancer biopsies. Appropriately selected clinical samples are utilized according to EU and national ethical procedures.
The participants' relevant research activities during the second period resulted in 58 publications in high impact scientific journals. The members of the network also presented the major results in scientific conferences. Interaction between the network participants was intense, which was realized via short-term visits, the annual meetings of the network, bilateral meetings, via ftp site of the network and via electronic communications. The project objectives have been advertised in the press (newspapers).
The project's web site is at:
http://www.eie.gr/nhrf/institutes/ibrb/eu-projects/epidiacan/index-en.html(opens in new window)
Project Context and Objectives:
Concept of the project
In the last few years our understanding of the complex mechanism of tumour progression has been dramatically changed by the identification of new mutated genes, which contribute in the multistep process and clonal expansion of tumours. In particular, genetic alterations of oncogenes and oncosuppressor genes have been closely associated with carcinogenesis. As a consequence, oncogenes/oncosuppressor genes contribute to the development of numerous aspects of the malignant phenotype by promoting cell cycle progression, resistance to apoptotic stimuli, neo-vascularisation and tumour metastasis. On the other hand, in recent years due to the newly developed powerful technologies of sequencing and analysis of gene regulation at the chromatin level, novel epigenetic mechanisms and factors related to cancer tumour progression have been identified. It is of interest that many of these epigenetic events appear early on the onset of cancer development and they can potentially be excellent early tumour markers. Indeed, a small number of epigenetic modifications have been tested already as early tumour markers, with very promising results.
Different forms of epigenetic mechanisms have been shown to modify the expression of key genes during tumour progression. DNA hypermethylation of promoter regions plays a role in silencing tumour suppressor genes or mismatch repair genes during tumourigenesis. Global DNA hypomethylation is observed in cancer and DNA hypomethylation at individual genes activate oncogenes in colorectal cancer. Demethylation in satellite sequences has been described as responsible to chromosomal instability in colorectal cancer. Further, loss of gene imprinting, a process mediated by DNA methylation and covalent histone modifications, is another epigenetic change that appears in the earliest stages of colorectal neoplasia causing abnormal gene expression. Finally, covalent modifications of histones like acetylation, methylation or phosphorylation, among others, distributed along promoters, coding regions and intergenic regions affecting chromosome condensation. Moreover, the cloning and functional characterization of histone modifying enzymes provides valuable information on the role of these epigenetic modifier factors. Although a lot of evidence has been accumulated for the role of epigenetic events and factors early in tumour progression, very few diagnostic applications in the clinic have so far emerged based on epigenetic markers.
The major challenge of this project was the development of new epigenetic markers for major types of cancer, like prostate and colorectal cancer that will allow early detection of tumour cells in patients, and will have important implications for successful therapeutic strategies in individual patients. A better mechanistic understanding of key epigenetically regulated genes involved in tumour progression in vivo will also help to develop tests for later clinical development and use. The key concept of this project was to develop diagnostic tools for the detection of epigenetic modifiers whose function has been implicated in cancer as potential tumour biomarkers.
The focus of the proposed collaborative effort was on:
1. The analysis of Usp22, SMYD3, LSD1, JMJD2c, PRK1, JMJD3 and EzH2 function in newly developed cell culture and animal models.
2. Identification of their downstream target genes to understand their role in tumourigenesis and to discover novel potential tumour biomarkers.
3. Development of efficient technologies for sensitive and specific epigenetic marker detection:
a. characterisation, validation of the above epigenetic markers for clinical applications using human prostate and colorectal cancer biopsies.
b. Characterisation, validation of the above epigenetic markers for clinical applications using circulating cancer cells obtained by non-invasive methods.
EPIDIACAN was carried out in close collaboration between investigators having complementary and multidisciplinary expertise in cell and molecular biology, epigenetics, biochemistry, tumour biology, clinical studies and tumour marker technologies. The studies of this Network have been directed to major aspects of cancer in model organisms, in mammalian systems and in clinical samples. This network composed of such teams provided unique and challenging opportunities for researchers interested in different aspects of epigenetics and cancer by using modern and developing new technologies to achieve their goal. It also provided the participant organisations, especially the small and medium-sized entreprise (SME), with epigenetic markers to be further exploited as industrial applications.
Project objectives
1. Generation of experimental tools (antibodies) and models to study epigenetic modifiers and for use in preclinical testing of epigenetic biomarkers.
a. Development of specific antibodies for the detection of selected histone modifiers (HM)
b. Generation of cell culture and animal models (KO and transgenic) for studying histone modifiers function.
c. Identification of target genes regulated by HM by global gene expression profiling and by ChIP-seq-based global occupancy analysis in the cell culture and animal models.
e. Identification of stage specific and oncogene specific epigenetic alterations in model systems of prostate and colorectal cancer.
2. Development of novel diagnostic tools for the detection of epigenetic modifiers and modifications in clinical samples.
a. Development of diagnostic tools/methods for the detection of selected modifiers including expression in tumour biopsies.
b. Development of diagnostic tools/methods for the detection of selected downstream targets of HM in tumour biopsies.
d. Development of improved non-invasive technologies for the isolation of cancer cells from body fluids that allow early detection of cancer.
e. Evaluation of diagnostic tools/methods detecting modifiers in cancer cells isolated from body fluids.
Project Results:
The major achievements of this period are as follows:
1. Genome-wide occupancy analysis performed by ChIP-sequencing with global expression profiling data and application of comparative analysis, retrieved JMJD2C, LSD1 target genes such as EGFR, CDK1 or the PRK1 target KLK2 and KLK3.
2. In PC3 cells we detected 488 and 1355 genes differentially regulated by LSD1 and PRK1, respectively. In contrast the PRK1 controlled gene set is associated with migration and invasion.
3. SILAC screens were performed for proteins interacting with LSD1 and JMJD2C respectively, and several novel interactors among members of the CoREST and NURD complex have been characterized
4. Our analyses reveal that the protein kinases CDK5 and PKA are able to phosphorylate LSD1 at position T119. Phosphorylation of LSD1 at position T119 does not alter demethylation or complex assembly.
5. An important role of Smyd3 in liver and intestinal cancer was identified. The mechanistic basis of its function involves promotion of epithelial-mesenchymal transition. Smyd3 was not recruited into the regulatory regions of S-phase specific genes. Target genes most relevant to the carcinogenesis process were those regulating EMT. These include members of the matrix metalloprotease family.
6. EZH2 silencing de-represses EMT related genes and affects cell migration and anoikis in colon cancer cells. ITGA2 has been identified as a novel EZH2 target gene and was further validated. ChIP-sequencing analysis has further identified new EZH2 target genes.
7. EzH2 levels are associated with cancer stem cells (CSCs) in our cultures, and EzH2 was down regulated when the CSCs differentiated This has been confirmed by showing that knocking down EzH2 activity using siRNA increases CSC differentiation
8. The tightly controlled deubiquitination activity of the human SAGA complex differentially modifies distinct gene regulatory elements. The structural plasticity of two different SCA7 domains of the SAGA deubiquitination module defines their differential nucleosome-binding properties. Deregulation of H2B ubiquitination may contribute to cancer development.
9. A total of 23 TMAs representing 1.242 tumor tissue samples in duplicate including pairs of primary tumors and metastases was generated in the reported period, from the following tumor indications: prostate, colon, breast, lung, kidney, stomach, head and neck, ovary, cervix and skin.
10. Collection of 240 fresh-frozen samples of prostate cancer tissue, as well as matched bodily fluids (urine and peripheral blood) has been performed, which have been stored at -80ºC. Simultaneously, bodily fluids from 200 healthy donors and patients harboring other urological pathologies have also been collected and stored for further analysis. Collection of fresh-frozen samples of colon cancer tissue and storage for further analysis was performed
11. We found that SMYD3, an H3K4 methyltransferase, was significantly over-expressed in tumor samples mainly in advanced stage prostate cancer
12. The data from our analysis show that LSD1 is predictive marker of prostate cancer superior to PSA.
13. High EZH2 mRNA levels in human colorectal cancer (CRC) specimens mark metastatic disease. Higher EZH2 expression was observed in more invasive CRCs and CRCs with regional lymph node metastases, Higher H3K27me3 expression was observed in more invasive CRCs, CRCs with regional lymph node metastases and in CRCs at advanced stages of disease, as well as in CRCs with lymph and venous vessel invasion, which appears to be associated with the putative role of EZH2 in the progression of CRC.
14. Lower LSD1 expression was observed in more invasive CRCs and in CRCs at advanced stages of disease, suggesting that LSD1 may be associated with less aggressive CRCs. Higher H3K9me3 expression was observed in CRCs with regional lymph node metastases, which appears to be associated with the putative role of EZH2 in the progression of CRC.
15. SETDB1 expression was observed in left-sided CRCs, suggesting a putative role in the carcinogenesis of CRCs with chromosomal instability.
16. Concerning response to treatment, higher survival rates in patients with CRCs treated only with surgery were found for tumours displaying lower SETDB1 expression, suggesting that whenever the expression of SETDB1 occurs, the surgical treatment may not be enough to prolong the patient's survival. Importantly, LSD1 expression and the histone mark H3K9me3 may predict the response of CRC patients treated with Folfiri, and H3K9me3 and H3K27me3 expression may predict CRC patients' response to 5-FU/Leucovorine.
17. F-actin (filamentous actin) is a useful marker of colorectal l (CRC) cell line derived lumens. Lumen formation is a feature of tumours in vivo, and human primary low and high grade colorectal tumours show differences in ezrin/actin and CEA polarisation. Lumens represent enterocyte brush border differentiation, but also contain secretory lineages. Polarised F-Actin is a marker of colonic brush borders in vivo and labels Matrigel grown lumens in vitro.
18. A new approach to establishing primary cultures from fresh CRC patient tumour material was developed.
Potential impact and use
This consortium has assessed epigenetic factors and mechanisms, like histone modifications as well as modifying enzymes involved in these processes. Cancer models have been selected for the most part of the studies and results have been validated in clinical specimens. EPIDIACAN consortium contributes in identification of novel oncogene and tumour stage related epigenetic events and factors, in order to maximize the subsequent clinical exploitation as epigenetic markers. Candidate epigenetic markers as well as marker combinations have been tested in a panel of preclinical models (cell lines and transgenic mice). A tight management plan was adopted in order that the most efficient combination per tumour cell to reach the final evaluation in clinical samples. There is the challenge that all this basic mechanistic analysis of genetic vs. epigenetic pathways will be utilized to improve clinical practice and finally public health.
The benefits on the society of a network focusing on novel early tumour markers are evident, since cancer is a major cause of death in Europe. This network contained groups of scientists from different disciplines like Molecular Biology, Epigenetics, Cell Biology, Tumour Biology, Biochemistry and Clinical Studies.
Bilateral interactions enhanced already successful co-operation by adding a value at the European level. Dissemination of the technologies developed at the individual national level will provide patients all over Europe to have access to new developments concerning cancer treatment, thus enhancing the benefit for the "European Citizen".
Part of the work will therefore have direct implications on colorectal, prostate and breast cancer in humans through novel epigenetic markers for cancer diagnosis and cancer therapeutics. Further characterization of our already existing cell and mouse models for cancer on different genetic backgrounds allowed us to use these mice in preclinical trials and speed up the evaluation of novel markers in the clinic. New knowledge about tumour formation has eventually been translated to an application stage, to the design of new diagnostic tests to detect cancer at an early stage and thus improve medicine in Europe and life quality.
The project extended the service business model and thus increases the competitiveness of the participating Targos Molecular Pathology Gmbh. The company had the opportunity to further explore own biomarker projects and build up own intellectuel property within this project and to profit from the transfer of know-how from the collaboration with outstanding experts from different fields of life sciences and technology. Targos further improved its knowledge in the field of epigenetics on a tumour and patient specific manner and was able to use the network resources in order to improve its competitiveness in the market.
We have also developed a efficient method to grow primary tumours from biopsies and endoscopies and this has the potential to significantly improve diagnosis and tumour classification. The health implications of this approach are potentially large as anti-cancer treatments can now be applied to growing samples derived from a much wide variety of patients. There is scope that our approach may allow some personalised treatments to be tested in vitro for efficacy, for example, antibody mediated immune killing using the patient's own blood.
Furthermore in vitro studies of tumour differentiation may also be used to refine the classical pathological definition of differentiation.
The potential cost benefit for EU healthcare may be substantial as drug treatments can be refined and suitable drug regimes tailored to suit each individual patient.
The prostate is the most common site of malignant transformation in western men. About 30% of men older than 50 years in the western countries develop prostate adenocarcinoma and the lifetime risk of clinical disease is 10%, with the risk of mortality being around 3%. The great variation in clinical behavior of prostate cancer creates a major dilemma in the treatment-decision process, since not all men with microscopic carcinoma require aggressive radical therapy. The major difficulty is the present-day absence of reliable tools to predict which cancers will remain indolent and which are going to kill the patient if left untreated. This project identified epigenetic markers of biological aggressiveness in prostate cancer and this information may have a wide impact on health and socioeconomics, both by reducing over-treatment and by allowing concentration of resources to treat intensively those patients who actually need it.
Participant 1. Alex Pintzas, NHRF.
WP1. Antibodies against EzH2 and JMJD3, as well as against H3K27Me3 were tested for optimal conditions in assays like immunohistochemistry, western blot, immunofluorescence and Chromatin immunoprecipitation (ChIP).
WP2. Generation of colon cells with silenced EzH2. Effect of silenced (inhibited) PIK3CAmut oncogene on EzH2 expression. To determine the contribution of EZH2 over expression to malignant phenotype of CRC cells we silenced EZH2 using small interfering RNA oligonucleotides and a vector to stably over express short hairpin RNA versus EZH2 mRNA (siEZH2 and shEZH2 respectively). As the expression of EZH2 was highest in cell lines with EMT behavior we decided to destroy the EZH2 endogenous protein in HCT116, SW620 and CacoH2 cells with a pool of siEZH2 oligonucleotides. To characterize the phenotype of siEZH2-silenced CRC cells we first evaluated the migratory ability using transwell system. The results showed that transient impairment of EZH2 reduced the migratory ability of CRC cells. In fact, the number of CacoH2- and HCT116-siEZH2 cells compared to parental cells were 69% and 83% respectively. We then concentrated only on HCT116 cell line for stable over expression of shEZH2 vector. The characterization of shEZH2 HCT116 stable clones reveled that EZH2 protein was reduced greater than80% and reduction on global H3K27me3. Furthermore, when shEZH2-HCT116 cells (HCTshEZ-2) were grown in Matrigel, 3D culture, they lost the ability to migrate as demonstrated by wound healing and migration assay (C and D).
Silencing of EZH2 derepresses EMT related genes in Caco-H2 cells. We reported a large list of genes related to EMT phenotype that were deregulated in Caco-H2 compared to parental Caco2 cell line (Joyce et all. 2009) as well as global and local histone post-translational modification pattern in the same cell models proposing that EZH2 might be a possible regulator of EMT (Mazon-Pelaez I. et al., 2010). The outcome of previous works and results obtained with siEZH2 let us to speculate that EZH2 really might be related to the invasiveness and migratory ability of CRC cells. To verify this hypothesis and in order to search for putative EZH2 target genes we decided to compare a group of 30 selected genes, chosen based on previous results, in siEZH2 versus parental EMT cell lines. We initially performed the study using CacoH2 cell line as we had microarray data for this cell system and afterward a subset of the 30 genes was analyzed also in HCT116 and SW620 cell lines. We initially performed the study using CacoH2 cell line as we had microarray data for this cell system and afterward a subset of the 30 genes was analyzed also in HCT116 and SW620 cell lines. As positive control, E-cadherin gene has been identified as an EzH2 target gene. The gene validation is currently in progress. Deliverables 3.1 and 3.2 (scheduled for month 24) have not yet been achieved.
We observed that forced expression of RAS and BRAF oncogenes in Caco2 cells caused hyper activation of RAS-PI3KA-AKT pathways with concomitant over expression of EZH2 (Mazon-Pelaez I. et al., 2010). Silencing of PIK3CA or inhibition of the pathway by small molecule, Wortmannin a PI3KA inhibitor, reduced pAKT and provoked EZH2 down regulation
WP3. Generation of colon cells with silenced EzH2. Effect of silenced (inhibited) PIK3CAmut oncogene on EzH2 expression. ChIP-sequencing analysis of Global EZH2 target genes.
To determine the contribution of EZH2 over expression to malignant phenotype of CRC cells we silenced EZH2 using small interfering RNA oligonucleotides and a vector to stably over express short hairpin RNA versus EZH2 mRNA (siEZH2 and shEZH2 respectively). As the expression of EZH2 was highest in cell lines with EMT behavior we decided to destroy the EZH2 endogenous protein in HCT116, SW620 and CacoH2 cells with a pool of siEZH2 oligonucleotides. To characterize the phenotype of siEZH2-silenced CRC cells we first evaluated the migratory ability using transwell system. The results showed that transient impairment of EZH2 reduced the migratory ability of CRC cells. In fact, the number of CacoH2- and HCT116-siEZH2 cells compared to parental cells were 69% and 83% respectively. We then concentrated only on HCT116 cell line for stable over expression of shEZH2 vector. The characterization of shEZH2 HCT116 stable clones reveled that EZH2 protein was reduced greater than80% as well aw reduction on global H3K27me3 was achieved (A and B). Furthermore, when shEZH2-HCT116 cells (HCTshEZ-2) were grown in Matrigel, 3D culture, they lost the ability to migrate as demonstrated by invasion assay as well as the ability to move as demonstrated by wound healing assay (C and D).
Identification and characterisation of oncogenic signalling pathways that modify EzH2 in colorectal cancer EZH2 is regulated by ERK and AKT pathways via AP-1 transcription factor in colon cancer cells with EMT phenotype. Initially we observed that forced expression of RAS and BRAF oncogenes in Caco2 cells caused hyper activation of RAS-PI3KA-AKT pathways with concomitant over expression of EZH2 (Mazon-Pelaez I. et al., 2010). Silencing of PIK3CA or inhibition of the pathway by small molecule, Wortmannin a PI3KA inhibitor, reduced pAKT and provoked EZH2 down regulation. To better dissect the pathways involved in EZH2 regulation we assessed EZH2 expression before and after blocking ERK and AKT pathways using siRNA and two specific inhibitors, in Caco-2, Caco-H2, HCT116 and SW620 cells. pERK1/2 inhibition by the MEK inhibitor UO126 in Caco-2 cells resulted in 10% reduced EZH2 protein and mRNA levels. In Caco-H2 cells EZH2 mRNA and protein expression (by 49%) were significantly reduced after UO126 treatment. In SW620 cells, a considerable inhibition of pERK1/2 was observed after UO126 treatment with a consequent 30% reduction of EZH2 protein and mRNA levels.
Silencing of EZH2 derepresses EMT related genes.
We reported a large list of genes related to EMT phenotype that were deregulated in Caco-H2 compared to parental Caco2 cell line (Joyce et all. 2009) as well as global and local histone post-translational modification pattern in the same cell models proposing that EZH2 might be a possible regulator of EMT (Mazon-Pelaez I. et al., 2010). The outcome of previous works and results obtained with siEZH2 let us to speculate that EZH2 really might be related to the invasiveness and migratory ability of CRC cells. To verify this hypothesis and in order to search for putative EZH2 target genes we decided to compare a group of 30 selected genes, chosen based on previous results (Joyce et al., 2009), in Caco-H2 siEZH2 versus parental the cell line. A subset of the 30 genes was than analyzed in the other two EMT cell lines available: HCT116 and SW620. Table S1 shows the results of qPCR performed on Caco-H2 cells. ITGA2, SPRY1, CDH17, Coll type II A1 and CCND2 were significantly up-regulated whereas CCND1 was down-regulated after transient reduction of EZH2 in Caco-H2. Downregulation of CCND1 was detected in all cell lines, whereas CCND2 and CDH17 were up regulated in SW620 cells and ITGA2 and Coll Type II A2 were up regulated in HCT116 cells. The analysis indicated that Caco-H2 cell line after EZH2 silencing presented a molecular mark which is a sum of the other two EMT cell lines and that ITGA2 was a potential EZH2 target gene. We compared the ITGA2 mRNA absolute levels in HCT116, SW620 and Caco-H2 before and after EZH2 transient silencing.
ChIP-sequencing analysis of Global EZH2 target genes
In order to best characterize the phenotype of HCTshEZ2-2 cells and search for new EZH2 targets related to cancer, we performed ChIP experiment followed by massive parallel sequencing (ChIP-seq). The template for sequencing was prepared using the anti-EZH2 antibody on sonicated chromatin extracted from HCT116 parental and HCTshEZ2-2 cells. For each cell line we performed sequencing in 2 biological replicates (HCT116_B and HCT116_109; HCTshEZ2-2_A and HCTshEZ2-2_B). The overall quality of sequencing was good even though an optimization of ChIP protocol is needed in order to confirm interesting preliminary results and cover some genomic loci that are poorly resolved.
WP5. EZH2 mRNA levels in human CRC specimens marks metastatic disease.
EZH2 mRNA expression was evaluated in 51 human CRC specimens by qPCR. Over-expression (greater than1.3-fold) was found in 37.2% (19/51) of tumor samples whereas in 29.4% (15/51) EZH2 mRNA was less than 0.7-fold (down regulated) with respect to a pool of normal colon tissues. In 33.3% (17/51) of tumor samples no significant changes were detected. Specimens were grouped based on EZH2 expression and presence of lymph node and/or distant metastasis. Even though the percent-age of specimens positive for metastasis was roughly the same in all three selected groups (68.4%; 62%; 66.7% respectively), in samples with EZH2 over-expression the magnitude of up-regulation was significantly higher in patients presenting metastasis. Fold-change average of EZH2 in this group was 2.83 whereas in samples devoid of metastasis was 1.67 close to the limit of 1.3 fold used for up-regulation. MannWhitney test (p-value = 0.043) performed on the two sub-groups with EZH2 over-expression, indicates that higher EZH2 mRNA levels correlate with presence of lymph node and distant metastasis.
Participant 2. Sir Walter Bodmer, WIMM.
WP2 Development of cell culture and animal models to study epigenetic regulators and modifications
We have analysed the expression data for the 7 genes of special interest to the Epidiacam project on our colorectal cell line panel, using just over 90 of the cell lines. For only one of the genes, PRK1(official name PNK1), is there a clear cut suggestion of two different groups of lines with different levels of expression. In the figure we plot the cell lines in rank order of expression and look for evidence of two or more groups of lines with different levels of expression by testing for non-linearity of the slope of the rank order plot expressed in terms of the expected ranks on the assumption of a normal distribution(a rankit plot). Otherwise the slope of such a plot simply represents the normal pattern of variation in the level of expression, and the greater the slope the more variable the level of expression. It is not ever expected that an unusual level of expression in comparison with normal will be expected in all examples of a given type of cancer. That is the reason we look for such subgrouping of expression levels. JMJD2c shows a possible small subgroup of less than 5%, with one line expressing at a substantially higher level than all the others. The same may be true for EZH2, though here the results are less convincing as the higher levels of expression in the lines overlap those found in some normal samples.
These data, however, do not suggest that the 7 EPIDIACAN genes, with the possible exception of PRK1, are themselves candidates for genes whose expression change has been selected for, at least during colorectal cancer progression. This places the emphasis on looking at their down stream targets using the cell lines, taking into account the observed different levels of expression in different cell lines. Once this is done on a small subset of the lines, then the levels of expression of potentially interesting candidate genes can be further analysed, again looking for clearly defined subgroups with different levels of expression.
Previous studies in our laboratory have suggested that EzH2 levels are associated with CSCs in our cultures, and that EzH2 was down regulated when the CSCs differentiated. This has now been confirmed by showing that knocking down EzH2 activity using siRNA increases CSC differentiation, as measured both by lumen formation and expression of the columnar cell differentiation marker Cytokeratin 20.
We previously gave preliminary evidence that F-actin (filamentous actin) is a useful marker of colorectal l (CRC) cell line derived lumens. We have confirmed the in vivo relevance of this by staining normal human colon cryosections with fluorescent phalloidin, which is specific for F-actin. In normal colon, F-actin is widely expressed in epithelial plasma membranes but is intensely enriched at the apical surface of enterocytes lining the crypt, corresponding to the brush border. This enrichment for F-actin is most probably due to the presence of microvilli on the colonic enterocytes. The intense staining of the muscularismucosum muscle layer was visible under the crypts. We next examined F-actin labelling in single cell derived colonies from a panel of six colon cancer cell lines, all grown under the same conditions in a three dimensional Matrigel matrix. In SW1222, confocal Z-sectioning demonstrated intense F-actin stress fibres were present on the apical cell membranes facing into the lumen.
WP5 Clinical validation of selected epigenetic markers
To determine if lumen formation or lack thereof had relevance to tumours in vivo, we first examined actin and ezrin polarisation in murine xenografts derived from the injection of 500 cells of HT29, HCT116 or SW1222 into the flanks of NOD/SCID mice. Mice were sacrificed after one month and resulting tumours removed and processed for FFPE tissue sections which were stained by hematoxylin and eosin or immunolabelled with anti-actin/ezrin. Hematoxylin and eosin staining of these xenograft tumours demonstrated poorly differentiated high grade tumour morphology for HCT116 and HT29 tumours that lacked lumens or actin/ezrin polarisation. SW1222 tumours exhibited a well differentiated phenotype with numerous lumens surrounded by polarised cells.
WP6 Development of epigenomic biomarker tests for diagnosis prognosis and therapy prediction
We have continued our evaluation of variations in the procedures for handling patient blood samples for CTC detection. We are particularly concerned to assess the best approaches for both short and long term storage for freshly collected blood samples and to reassess the comparison between filtration and Ficoll-Hypaque partial purification of epithelial cells from the blood. We have also been testing various procedures for multiple antibody staining using our routine monoclonal antibodies, AUA1, an anti EpCAM and CAM5.2 against cytokeratins7/8 , together with other antibodies , for example against the differentiation control marker CDX1 and the prostate specific markers PSA and PSMA, as well as FISH for the common translocations found in prostate cancers.
Participant 3. Carmen Jeronimo, IPOPFG.
The major achievements are as follows: In summary
Regarding prostate cancer:1-We have explored the role of HMTs altered expression in PCa onset and progression and from the 37 genes explored we found that SMYD3 plays an important role in prostate carcinogenesis and therefore it may be useful as a therapeutic target in aggressive human PCa. 2- We did not find prognostic value in PCa patients for EZH2 and H3K18Ac immunoexpression in biopsies, contrarily to what has been previously reported.
In more detail:
WP3. Identification of downstream targets of epigenetic modifiers and proof of their role in prostate and colorectal cancer models. Deliverables 3.2 (Month 24) have been achieved.
We have developed engineered prostate cancer cell lines lacking SMYD3 in order to characterize the biological function of SMYD3. (WP3; Deliverable 3.2) To clarify the role of HMTs altered expression in PCa onset and progression and to translate those findings into clinically useful tools for management of PCa patients. To achieve this goal, epigenetic gene expression of 37 HMTs was investigated by analysis of TaqMan® Arrays Plates in 10 primary PCa and 5 Morphological Normal Prostate Tissue (MNPT).
WP4. Work performed for Deliverable D4.1 and 4.2 (scheduled for Month 24) and D4.3 (scheduled for Month 36). Collect and store biological samples of prostate cancer patients and controls, as well as relevant clinical data.From month one to 36th and after informed consent of the patients we were able to collect 240 fresh-frozen samples of prostate cancer tissue, as well as matched bodily fluids (urine and peripheral blood), which have been stored at -80ºC. Simultaneously, bodily fluids from 200 healthy donors and patients harboring other urological pathologies have also been collected and stored for further analysis.
WP5. (cont.) The parts of work corresponding to Deliverables 5.2 (scheduled for Month 30), 5.3 (scheduled for Month 30), 5.4 (Scheduled for Month 30) and 5.5 (Scheduled for Month 30) are being pursued and have not been achieved yet. Work performed for Deliverable D5.2 (scheduled for Month 30). Establish the clinical value of factors including JMJD2C, LSD1, and PRK1 as a predictive parameter for aggressive and hormone-refractory prostate cancers. (Delivery month 30)
WP6. Development of epigenomic biomarker tests for diagnosis prognosis and therapy prediction
The parts of work corresponding to Deliverables 6.1 (scheduled for Month 36) and 6.2 (scheduled for Month 36) are being pursued and have not been achieved yet. Work performed for Deliverable D6.2 (scheduled for Month 36).Evaluation of epigenetic markers in cancer cells from body fluids (Delivery Month 36). We have identified several epigenetic markers for prostate cancer in WP5. However, we were unable to detect these surrogate markers either at RNA or protein level in human body fluids such as urine.
Participant 4. Laszlo Tora, GIE-CERBM.
S&T results on USP22 and the deubiquitination module from GIE-CERBM
The tightly controlled deubiquitination activity of the human SAGA complex differentially modifies distinct gene regulatory elements.
The multisubunit SAGA (Spt-Ada-Gcn5 acetyltransferase) coactivator complex facilitates access of general transcription factors to DNA through histone acetylation mediated by GCN5. USP22 (ubiquitin-specific protease 22) was recently described as a subunit of the human SAGA complex that removes ubiquitin from monoubiquitinated histone H2B and H2A in vitro. We discibed an allosteric regulation of USP22 through multiple interactions with different domains of other subunits of the SAGA deubiquitination module (ATXN7, ATXN7L3, and ENY2). Downregulation of ATXN7L3 by short hairpin RNA (shRNA) specifically inactivated the SAGA deubiquitination activity, leading to a strong increase of global H2B ubiquitination and a moderate increase of H2A ubiquitination. Thus, SAGA is the major H2Bub deubiquitinase in human cells, and this activity cannot be fully compensated by other deubiquitinases.
Role for H2Bub in DNA repair and carcinogenesis
While enzyme-mediated epigenetic control of gene transcription is a critical aspect of embryonic development and cellular differentiation, this same mechanism is often deregulated in human diseases, where aberrant gene expression or repression is a hallmark of cancer and other diseases. There is growing evidence that amplification, mutation and other alterations of epigenetic enzymes are molecular causes of certain cancers and several human diseases. A number of observations suggest that deregulation of histone H2B ubiquitination and the resulting effect on transcription may be key events in cellular transformation and metastasis. First, gene expression analysis of RNF20 depleted cells suggested that RNF20 acts as a putative tumor suppressor gene. Indeed, the expression of several proto-oncogenes and proliferation-related genes was increased after reduction of H2Bub via RNF20 depletion. In contrast RNF20 positively regulates p53 and different studies showed that RNF20 and WAC regulate p53-dependent genes in response to DNA damage.
1. Generation of antibodies against SMYD3. (WP1; Deliverable D1.1)
Initially we have tested several commercially available Smyd3 antibodies (Abcam, Santa Cruz, Sigma) using western blots from liver extracts of wild type and Smyd3 transgenic (Smyd3-Tg) animals and by immunohistochemistry in frozen or paraffin-embedded liver sections. The antibodies did not perform well in any of the applications. Next we generated a polyclonal antibody by immunizing 2 rabbits with a KLH-conjugated N-terminal peptide of Smyd3. The collected sera recognized recombinant Smyd3 as well as overexpressed Smyd3 in extracts prepared from CMV-Smyd3 transfected cells. The peptide antibodies did not recognize endogenous Smyd3. After this we immunized rabbits with recombinant full-length Smyd3 protein. The sera of these mice were tested in different applications: They gave a good signal in western blots of extracts containing overexpressed proteins. The antibody also gave good result in detecting endogenous Smyd3 by western blots and immunoprecipitations.
2. Validation of SMYD3 as biomarker in human colon cancer biopsies. (WP3, WP5; Deliverable 3.6 and 5.5)
The antibody was extensively tested in immunohistochemistry applications in mouse colon cancer sections and human colon cancer. Parallel measurements of RNA levels by RT-PCR were performed. The histochemical signals correlated well with the expression of Smyd3.
Deliverables 3.6 and 5.5 has been achieved.
3. Generation and analysis of animal models lacking Smyd3 and overexpressing Smyd3 in intestine. (WP2; Deliverable 2.3)
4. Generation and analysis of experimentally induced colon cancer. (WP2; Deliverable 2.5).
To generate Smyd3 KO mice we used a gene-trap ES cell clone to inject mouse blastocysts. Chimeric mice and germ-line transmission was obtained before the initiation of the program. During the reporting period we obtained a good number of homozygous mice to perform experimental work. The mice look overall normal. Histological analysis of livers and intestines did not show major morphological alterations. We have performed drug-induced liver and colon cancer induction programs using DEN only and DEN-TC (10 and 7 months treatment, respectively) and DMH (3 months treatment) protocol in 2 groups of mice. Each group consists of 10 animals.
a. To induce colon cancer, 2 months old mice were injected with DMH, followed by treatment with DSS in drinking water for 1 week. The mice were then left for 2 weeks with normal drinking water. The DSS treatment was repeated two more times and the mice were sacrificed.
Smyd3 KO mice exhibited inflammatory response similar to wild type mice, but essentially no formation of adenomas.
b. To induce liver cancer, at day 14 after birth each litter received a single intraperitoneal injection of the DNA-damaging tumor initiator diethylnitrosamine (DEN) and analyzed at 10 months age (DEN only protocol). In DEN-TC protocol, two weeks after DEN treatment, and every two weeks afterwards, the animals were given intraperitoneal injection of the hepatotoxic nuclear receptor CAR ligand TCPOBOP (TC) for 4 to 6 months. Using this protocol full-blown liver tumor was detectable at 4 months after treatment in wild type mice.
Smyd3 Tg mice:
Smyd3 Tg mice were generated using a construct containing the full-length Smyd3 cDNA under the control of the liver-specific TTR promoter/enhancer. The transgenic animals express 5'HA and 3'Flag tagged version of full-length Smyd3, specifically in hepatocytes. We have expanded 2 transgenic lines, where transgene expression was monitored by RT-PCR and western blot analysis. HA-tag antibody detected a 52 kD band only in whole cell extracts, while Flag antibody detected a 52 and 48 kD band in whole cell extracts and a 48 kD band in nuclear extracts. This suggests that in the nuclear form of Smyd3 the N-terminal region is cleaved.
Smyd3 Tg mice did not develop tumors spontaneously (at least within 10 months of age) but, as expected, somewhat accelerated DEN-induced carcinogenesis.
Deliverables 2.3 and 2.5 have been achieved.
5. Global expression profiling and occupancy analysis of Smyd3-regulated genes.
(WP 3, Deliverable 3.3)
We attempted to identify genome wide occupancy patterns of Smyd3 using several antibodies (including those developed by us) recognizing endogenous Smyd3. None of them worked in ChIP assays. As an alternative, we took advantage of our transgenic mice, where the nuclear form of Smyd3 can be detected by Flag antibodies. ChIP-seq analysis revealed several (more than 8000) locations of the genome bound by Smyd3. About 20% of them were located in promoters. While several interesting target genes were identified, it was interesting to note that Smyd3 was not recruited into the regulatory regions of S-phase specific genes. As mentioned above, target genes most relevant to the carcinogenesis process were those regulating EMT. These include members of the matrix metalloprotease family, which have also been detected in human colon cancer.
Chromosome-wide (chr12) distribution of peaks in Smyd3 ChIP-seq experiment. 2 biological replicates show similar binding pattern.
Deliverable 3.3 has been achieved.
6. Provide experimental samples from animal models to the partners of the network.
Several exchanges of scientific materials between EPIDIACAN members have been realized during the project.
7. Other activities
Another line of our research in this program was to identify the lysine specificity of Smyd3. Previous studies established that Smyd3 is a histone 3 lysine 4 methylase. We performed several in vitro studies using bacterially or baculovirus expressed Smyd3 recombinant proteins. While in the presence of HSP90 we could observe some methylation at H3K4, this activity was very low compared to other enzymes. On the other hand we identified Smyd3 as an H4K20 methylase, which seems to be the main specificity of the enzyme. We have also been searching for novel, non-histone substrates of Smyd3. We made use of our transgenic mice to perform immunoprecipitations followed by quantitative mass-spectrometry. This effort led to the identification of several cytoplasmic proteins including Cyld. Although, Cyld methylations still needs verification, we hypothesized that the mechanism by which Smyd3 modulates hepatocarcinogenesis may involve regulation of NFkB signalling. While the roles of the components of NFkB signalling in cancer have been studied before, no information about the potential role of the deubiquitinase Cyld was available.
Conclusion
Our studies revealed an important role of Smyd3 in liver and intestinal cancer. The mechanistic basis of its function involves promotion of epithelial-mesenchymal transition. The possibility of regulating tumorigenic signalling pathways was also raised, which opens new avenues for future research. It was also shown that Smyd3 could serve as a good biomarker for colon cancer. Future studies may be directed towards developing and testing drugs specifically modulating the activities of the enzyme.
Participant 6: Roland Schuele, UKL-FR
WP1: We generate several new antibodies with the goal of increased specificity for histological, ChIP and other applications. These include antibodies against JMJD2A-D, LSD1, and PRK1. For immunization purified recombinant proteins were used. For the production of polyclonal antibodies, New Zealand White rabbits were immunized by a standard, 70-day immunization program. Test bleeds were collected and assayed in western blots of cellular extracts and with purified recommbinand target proteins. Then the antibodies were purified by affinity chromatography and their performance was veified in western blots, ChIP assays and immunohistological applications. In summary, we met deliverables 1.1 and 1.2 in full and deliver superior antibodies for JMJD2A-D, LSD1, and PRK1 to the consortium.
WP2: Cell culture models
We established and optimized siRNA-mediated knock-down strategies to reduce the levels of JMJD2C, LSD1, and PRK1in human prostate cancer cell lines such as LNCaP. for the purpose of generating cell lines with reduced. For inducible expression we developed the tetracycline-controlled approach to conditionally overexpress shRNAs using a lentiviral gene silencing system. For overexpression we also used lentiviral gene delivery system of the various wild type and mutant proteins. Bona fide expression of the corresponding gene was veryfied by RT-PCR and western blot analysis.
JMJD2c, LSD1, PRK1 KO, transgenic mice
To investigate the biological and pathophysiological functions of JMJD2C, LSD1, and PRK1and their contribution to the development of cancer we cloned targeting constructs that allow the Cre-loxP mediated deletion of PRK1 exon 1 of PRK1 and LSD1 respectively. We generated homozygous floxed animals (LSD1 and PRK1) and breeding colonies of LSD1 floxed animals are established while the breeding colony of PRK1 floxed animals is expanding. Next, we generated animals that have prostate specific gene knockout by breading LSD1flox/flox animals with transgenic ARR2Probasin-Cre (PB-Cre4) and ARR2Probasin-CreERt (PB-CreERt) mice that express Cre recombinase specifically in the prostate epithelium in a constitutive or Tamoxifen inducible manner, respectively.
WP3: To characterize the biological function of the demethylases JMJD2C, LSD1 and the kinase PRK1 we combined a genome-wide occupancy analysis performed by ChIP-sequencing with global expression profiling data derived from Affimetrix platform, applied comparative analysis and retrieved JMJD2C, LSD1 target genes such as EGFR, CDK1 or the PRK1 target KLK2 and KLK3. The physiological role of the various target genes will be analysed in detail.
WP3: D 3.2: To investigate the biological and pathophysiological functions of JMJD2C, LSD1, and PRK1and their contribution to the development of cancer we generated homozygous floxed animals (LSD1 and PRK1). A breeding colony of PRK1 floxed animals is expanding. So far we have obtained three PRK1 null animals. The first physiological and histological analysis revealed no obvious phenotype of the PRK1 knockout animals. Further detailed analyses are ongoing. To characterize the functions of LSD1 we generated prostate specific gene knockout by breading LSD1flox/flox animals with transgenic ARR2Probasin-Cre (PB-Cre4) mice that express Cre recombinase specifically in the prostate epithelium. The detailed biochemical and histological analysis revealed no obvious phenotype of the prostate specific LSD1knockout animals. Further detailed analyses are ongoing. Next, we ubiquitously deleted LSD1 using a Rosa26-Cre deleter. Homozygous LSD1 knockout animals stop development at around E7.5-8.0 due to impaired trophoblats stem cell development. Furthermore, we produce knockout mice for the demethylase JMJD2C by using gene-trap technology. The detailed biochemical, histological, and functional assaying for altered performance and physiology revealed no obvious phenotype of the knockout animals.
D 3.3: To identify LSD1and PRK1 regulated genes we performed global analysis of gene expression by RNA-seq analysis. In PC3 cells we detected 488 and 1355 genes differentially regulated by LSD1 and PRK1, respectively. About half of the genes are either up-or downregulated. The bioinformatic analysis uncovered that LSD1 controls a gene set regulating transcription proliferation and metabolism. In contrast the PRK1 controlled gene set is associated with migration and invasion. In addition, we used the antibodies develop in WP1 to establish LSD1 Chip-seq methodology. The genome-wird chromatin binding profile of LSD1 in LNCaP and PC3 cells was established. The bioinformatic analysis uncovered that LSD1 is enriched a promoters, defined by 2000bp around the transcription start site. LSD1 locations overlap with active histone marks such as H3K4me3 but not with the repressive mark H3K27ac. HOMER analysis identified AR, REST, and FOX binding motifs as significantly enriched motifs.
D 3.4: To identify LSD1 interacting proteins we used the antibodies develop in WP1 to immunoprecipitate endogenously expressed LSD1 in LNCaP and PC3 cells. The immunoprecipitated LSD1-associated proteins were identified by mass-spectrometry. Interestingly, we were able to identify the CoREST complex as interaction partner. However, we did not detect any member of the NuRD complex. Interestingly, we identified novel signalling molecules such as dynactin 1, dynactin 2, or protein phosphatase 12 as interactin partners. The functional analyses of these novel LSD1 associated proteins in ongoing.
D 3.5: We characterize by an unbiased genome wide screening strategy, kinases that modify LSD1 and analyzed the consequences of this modification on biological function. In vivo, LSD1 is modified by post-translational modifications such as acetylation and phosphorylation. We expressed and purified a pool of modifiers i.e. enzymatic active forms of all known acetylases and 180+ kinases covering a large percentage of all cytoplasmic and nuclear kinases present in the human genome. LSD1, expressed either in E. coli or insect cells, was tested for modification by the modifiers. Phosphorylation of LSD1 at position 119 was identified by mass-spectrometry. We developed phospho-specific antibodies (anti LSD1Tph119) and used the modification-specific antibodies to confirm the data obtained by mass-spectrometry. Phosporylation was verified by mutagenesis (LSD1T119A). Our analyses reveal that the protein kinases CDK5 and PKA are able to phosphorylate LSD1 at position T119.
WP5: D 5.2 and: D 5.2:To establish the clinical value of JMJD2C, LSD1, and PRK1 as a predictive marker of prostate cancer we initiated to characterized a study cohort of 500+ patients, which underwent radical prostatectomy for locally limited diseases within a single surgical center and were followed-up between 3 to 9 years. All prostate specimens were classified according to TNM stage and Gleason score following defined procedures and used to generate tissue microarrays (TMAs). Patients were be grouped into two categories according to the following tumour parameters: Group 1 (high-risk tumours): pT3 or higher, all pN0M0, Gleason score 8 or higher. Group 2 (low-risk tumours): pT2c or lower, all pN0M0, Gleason score 7 or lower. So far we have use the TMAs for immunostaining a panel of antigens such as LSD1 PRK1, p53, Ki67 etc.
Participant 7. Thomas Henkel, Targos.
The cooperation with the Clinical Research Group (KFO179) at the University of Göttingen was continued to get access to tumor samples from two prospective rectal cancer trials in which patients have preoperatively been treated by chemo-radiotherapy (CRT). In the first trial (from 1995 to 2002) follow-up data covered at least five years (CAO/ARO/AIO-94), the second trial is still on-going (CAO/ARO/AIO-04). The trials were approved by the medical ethics committees of all participating hospitals.
WP 4.1 In cooperation with the Clinical Research Group (KFO179) at the University of Göttingen tumor samples of two prospective rectal cancer trials were collected, in which patients have preoperatively been treated by chemo-radiotherapy (CRT). In the first trial (from 1995 to 2002) follow-up data covered at least five years (CAO/ARO/AIO-94), the second trial is still on-going (CAO/ARO/AIO-04). The trials were approved by the medical ethics committees of all participating hospitals.
WP 4.2 The planned SEAL trials, which was planned to be a prospective randomized phase III trial of limited versus extended lymph node dissection in patients with intermediate/high risk prostate cancer (Trial number AP49/08) was finally not funded and thus was not started as planned in 2010. A second attempt for funding was not approved in 2011/2012. Thus no prospectively collected prostate cancer samples were available for analysis. These samples were replaced by retrospectively collected tumor bank samples (see WP 5.1) and commercially available tissues.
WP 5.1 During the reported period Targos generated further multiple tissue micro-arrays (TMA) of high quality (Multiblock melting technology) from anonymized tumor bank samples including clinical data, mainly derived from the tumor bank of the comprehensive Cancer Care Center Cologne/Bonn (Prof. Büttner)
WP 5.2. As a first marker LSD1 was analysed by means of IHC in the Targos lab on n=148 FFPE blocks of biopsies from colorectal cancer patients prior to neoadjuvant CRT and n=42 FFPE blocks from colorectal resection specimens post-treatment derived from the CAO/ARO/AIO-04 trial. As controls, n=40 resection specimens of patients not undergoing neo-adjuvant treatment have been stained by Targos technicians on the automated platforms Benchmark XT and Symphony
WP 5.3. A spreadsheet data base for all available reagents within the consortium was generated to allow all consortium members to scan for interesting reagents relevant to their respective work. Further technical and clinical validation of any marker assays for diagnostic purpose was not performed for lack of scientific justification.
Potential Impact:
Tumour markers tests have been recently developed based on genomic technologies which have been shown by researchers and clinicians to have an important role in tumour progression. The work of several laboratories from USA and from Europe, including members of the EPIDIACAN consortium has elucidated mechanisms of epigenetic gene regulation, which can be exploited by the tumour biomarker field. Indeed, participant Laboratories of this network have analysed epigenetic markers in colorectal tumour (models) (Participants 1, 2) as well as in prostate and breast cancer (Participants 3, 6, 7). Other participant Labs have been contributing in basic knowledge and mechanistic analysis of epigenetic pathways and factors (Participants 4, 5). This consortium has assessed epigenetic factors and mechanisms, like histone modifications as well as modifying enzymes involved in these processes. Cancer models have been selected for the most part of the studies and results have been validated in clinical specimens.
Contribution to wider societal objectives
The benefits on the society of a network focusing on novel early tumour markers are evident, since cancer is a major cause of death in Europe. This network contained groups of scientists from different disciplines like Molecular Biology, Epigenetics, Cell Biology, Tumour Biology, Biochemistry and Clinical Studies.
Contribution to policy objectives of Health
Cancer is the second major cause of mortality in Europe. A new era in human biology has been opened by the sequencing of the human genome offering opportunities to improve human health and to stimulate industrial activity. Our project represents a basic and applied research programme aimed to exploit the potential of genomic approaches to decipher mechanisms underlying genetic vs epigenetic mechanisms in oncogenesis. The goals of this proposed project were in accordance with the relevant objectives of the Health priority laid down in the 7th EU framework programme, because our research is focused on major fundamental biological processes concerning cell proliferation which can be exploited for novel tumour markers. Part of the work will therefore have direct implications on colorectal, prostate and breast cancer in humans through novel epigenetic markers for cancer diagnosis and cancer therapeutics. Further characterization of our already existing cell and mouse models for cancer on different genetic backgrounds allowed us to use these mice in preclinical trials and speed up the evaluation of novel markers in the clinic. New knowledge about tumour formation has eventually been translated to an application stage, to the design of new diagnostic tests to detect cancer at an early stage and thus improve medicine in Europe and life quality.
Reinforcing European competitiveness
EPIDIACAN has aspects in very important and competitive areas on preclinical models of cancer, cancer genetics and genomics, as well in imaging technologies. While a number of leading scientists within the field are active in Europe today, judging from the published literature it is evident that European research output lags behind that of the United States in production of efficient novel cancer diagnostics. The domination of US laboratories of the field has as a consequence the continuous drain of talented young European researchers towards the US. This consortium has established world leadership in the field, mainly due to the competitive edge provided by EU support for intensive collaboration of a critical mass of researchers from different disciplines. The integrated approach has neutralized the problem of fragmentation of relevant expertise in Europe, thus translating the potential success of the project into tangible benefits for European science.
Transnational cooperation
The complexity and the quantity of the proposed work demanded the collaboration of many laboratories possessing different expertise. Some of the participants (1, 4, 5) and (6 ,7) had already initiated bilateral collaborations studying related topics. Although they had built up fruitful collaborations, these activities were fragmented and addressed specific aspects only. This project combined and expanded these individual activities to a more comprehensive collaboration. There were no chances to carry out such an ambitious project at the level of individual laboratories, or to gather the diverse but complementary expertise needed at the national level. Besides the combination of different expertise, the multidisciplinary nature of the program required the integration of resources available at the individual laboratories.
Impact on participating companies
The project extended the service business model and thus increases the competitiveness of the participating Targos Molecular Pathology Gmbh. The company had the opportunity to further explore own biomarker projects and build up own intellectuel property within this project and to profit from the transfer of know-how from the collaboration with outstanding experts from different fields of life sciences and technology. Targos further improved its knowledge in the field of epigenetics on a tumour and patient specific manner and was able to use the network resources in order to improve its competitiveness in the market.
Unique training opportunities
The participating laboratories had continuously open calls for positions for young European researchers. Although, training was not the prime objective of this application, given the cutting-edge scientific level of research to be performed and the opportunity to carry-out research projects in collaboration with laboratories of a different discipline, the network provided a competitive alternative to US laboratories for training young researchers interested in the field of cancer epigenetics. Via short-term visits, the young fellows had an opportunity to work in more than one laboratory. The opportunity to meet and collaborate with several network members and to be exposed to the continuous information exchange of the whole network has set up a precedent of a novel, highly productive training method highly attractive to young researchers interested in the field.
WIMM
The main dissemination for us was publication and presentations at various scientific meetings and seminars. The potential impact is to improved diagnosis and treatment of colorectal cancer. At the stage we have no immediate plans for exploitation of results, though there may be an opportunity for doing something with the development of effective cultures from fresh tumour material. We have also obtained additional information on the colorectal cancer cell line panel which is useful for assessing in vitro drug response assay results in relation to the properties of a cancer.
IPOFG
The findings are expected to originate at least six articles in the course of the project, which will be submitted for publication in international, peer-reviewed journals. More publications are expected when the long-term objectives are accomplished. Furthermore, the findings will be presented in local seminars, and in several national and international conferences in which the team investigators participate, including: Workshop on Molecular Genetics of Human Solid Tumors, Meetings of the European Association for Cancer Research and of the Portuguese Society of Human Genetics, Congress of the Portuguese Oncology Society, European Congress of Pathology and the Workshop on Medical Oncology organized by our Institution.
List of Websites:
http://www.eie.gr/nhrf/institutes/ibrb/eu-projects/epidiacan/index-en.html(opens in new window)