Functional Genome Analysis  (B070)
Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580
D-69120 Heidelberg, Germany.
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Archive      Functional Tumour Analyses

 

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FINISHED PROJECT:
COST Action Pancreatic Cancer

Large-scale international collaboration is essential to decipher relevant information in the context of omics-scale interrogations in cancer research. This is even more important for relatively rare but very fatal diseases like pancreas cancer. The COST Action Pancreatic Cancer facilitated the collaboration of a broad range of European and international research groups on pancreatic cancer in order to: (1) integrate knowledge and experience in a multidisciplinary way ‘from molecule via cell to society’, (2) promote the application of uniform study tools and protocols, (3) foster their optimal use by early-stage researchers, (4) enhance the mobility and training of researchers, and (5) disseminate the results produced to society.
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The Action developed novel tools that improved our understanding of pancreatic cancer and its prevention, diagnosis and treatment. It also aimed at answering questions related to the tumours’ etiology, early detection, evidence-based and personalised treatment, as well as to aspects of health management. We aimed at attracting young scholars across a range of disciplines, who worked in collaboration with more experienced researchers. This enhanced active European participation in the international research efforts on pancreatic cancer, with the objective of reducing disease mortality.




Milne et al. (2014) Public Health Genomics 16, 305-312.  pdf icon



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scheme of genome-wide shRNA assay

Scheme of genome-wide shRNA knockdown experiments. Meanwhile, the read-out is done by next-generation sequencing instead of microarray analysis as depicted.
   
FINISHED PROJECT:
Functional screens by means of lentiviral shRNA libraries

RNA interference (RNAi) has become a popular and important tool for the analysis of gene function. Loss-of-function studies, commonly performed by transfection of short interfering RNAs (siRNAs), have greatly facilitated functional analyses of the human transcriptome. However, there are major downsides to siRNA experiments, most importantly the transient inhibition of gene expression as well as their inefficient transfection into non-dividing cells. Overcoming those limitations, short hairpin RNA (shRNA) expression vectors are available, which stably integrate into a target cell's genome via retro- or lentiviral gene transfer. Intracellular processing of shRNAs results in short duplex RNAs with siRNA-like properties. Viral integration ensures a broad range of infectable target cell types and a stable expression of specific shRNAs, resulting in the permanent reduction of the targeted gene product. Complex shRNA expression libraries allow the targeted knockdown of thousands of different genes in a single experiment.
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Using such lentiviral vector shRNA libraries and initially barcode arrays and meanwhile next-generation sequencing analysis for decoding of the pooled RNAi screens, we are able to quantify the abundance of individual shRNAs and thus determine in a complex pool the number of cells infected with an individual shRNA construct. We used the technique to predict anti-proliferative effects of individual shRNAs from pooled negative selection screens, for example, identified synthetic-lethal activities toward combination therapies, defined genes which are required for a stem-cell like phenotype and found tumour suppressor genes by in vivo studies. For more details, see below.
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Further studies are under way, both for the elucidation of basic regulative processes associated to cancer and for the identification of pathways that are affected by particular drugs or compounds. In particular, we use the technique for obtaining more detailed information on the functional effects of particularly potentially druggable gene products.
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More recently, CRISPR-Cas constructs have been used for similar analyses and other purposes.



Wolf et al. (2014) Oncogene 33, 4273-4278. pdf icon
Fredebohm et al. (2013) J. Cell Sci. 126, 3380-3389. pdf icon

Böttcher et al. (2014) BMC Genomics 15, 158. pdf icon
Böttcher et al. (2010) BMC Genomics 11, 7. pdf icon

Wolf et al. (2013) Breast Cancer Res. 15, R109. pdf icon
Böttcher & Hoheisel (2010) Curr. Genom. 11, 162-167.




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FINISHED PROJECT:
Breast cancer
An in vivo RNAi screen identifies SALL1 as a tumour suppressor in human breast cancer with a role in CDH1 regulation
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The gold standard for determining the tumorigenic potential of human cancer cells is a xenotransplantation into immunodeficient mice. Higher tumorigenicity of cells is associated with earlier tumour onset.
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We employed xenotransplantation to assess the tumorigenic potential of human breast cancer cells following RNAi-mediated inhibition of over 5,000 genes. We identified sixteen candidate tumor suppressors, one of which is the zinc finger transcription factor SALL1. Analysing this particular molecule in more detail, we showed that inhibition of SALL1 correlated with reduced levels of CDH1, an important contributor to epithelial-to-mesenchymal (EMT) transition. Furthermore, SALL1 expression led to increased migration and more than twice as many cells expressing a cancer stem cell signature. Also, SALL1 expression correlated with the survival of breast cancer patients. These findings cast new light on a gene, which has previously been described to be relevant during embryogenesis, but not carcinogenesis.
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Wolf et al. (2014) Oncogene 33, 4273-4278. pdf icon



FINISHED PROJECT:
Breast cancer
A mammosphere formation RNAi screen reveals genes that promote a breast cancer stem-like phenotype
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Breast cancer stem cells are suspected to be responsible for tumour recurrence, metastasis formation as well as chemoresistance. Consequently, great efforts are made to understand the molecular mechanisms underlying cancer stem cell maintenance. In order to study these rare cells in vitro, they are typically enriched via mammosphere culture. We developed a mammosphere-based negative selection shRNAi screening system suitable to analyse the involvement of thousands of genes in the survival of cells with cancer stem cell properties. We used a sub-population with cancer stem cell properties of cell line SUM149 that were enriched in mammospheres. Identified candidates were validated in mammosphere and soft agar colony formation assays. Further, we evaluated the influence of their expression on the stem cell sub-population. Also, the tumorigenic potential of SUM149 after up- or down-regulation of candidates was examined by xenograft experiments.
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Using this approach, Jak-STAT as well as cytokine signalling were identified to be involved in mammosphere formation. Furthermore, the autophagy regulator ATG4A was found to be essential for maintenance of the cancer stem cell sub-population and regulation of breast cancer cell tumourigenicity in vivo.
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Wolf et al. (2013) Breast Cancer Res. 15, R109. pdf icon












FINISHED PROJECT:
Synthetic-lethal screens:
Depletion of Rad17 sensitises pancreatic cancer cells to gemcitabine

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Chemotherapy of advanced pancreatic cancer has mainly been gemcitabine-based, but with only limited effect. Recently, combination therapy that also targets checkpoint kinase 1 (CHK1) has become an attractive option. The central role of CHK1 in many DNA damage response pathways, however, may result in undesired cytotoxicity in normal cells. We were searching for other target molecules that may be more specific and thus better suited for combination therapy.
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A negative selection RNAi screen was performed, targeting over 10,000 genes. Genes that were found to be synthetically lethal with gemcitabine and whose proteins are acting upstream of CHK1 were characterised in more detail. The inhibition of RAD17 potentiated gemcitabine cytotoxicity particularly, leading to forced mitotic entry of cells arrested in S-phase by gemcitabine treatment, resulting in asymmetric DNA distribution during anaphase followed by DNA fragmentation and finally cell death by mitotic catastrophe.
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Our data suggest RAD17 for gemcitabine combination therapy supplementing or complementing inhibition of checkpoint kinase 1. As opposed to CHK1, RAD17 knockdown by itself does not inhibit cell proliferation and does not lead to abnormal DNA segregation, suggesting a more specific action.
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Fredebohm et al. (2013) J. Cell Sci. 126, 3380-3389. pdf icon

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FINISHED PROJECT:
NGFNplus Translational Genome Research Network Pancreatic Cancer
          logo NGFN
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Genome projects have generated knowledge and technology with great potential to contribute to the understanding of the molecular pathogenesis in the pancreas and to provide molecular targets. However, even though multiple genome scale screening approaches of pancreatic tumours and their preneoplastic lesions have been conducted, only few targets have reached the level of preclinical or clinical applications.
  
The PaCaNet consortium was an Integrated Genome Research Network comprising groups who (i) set the standards of clinical care and histopathology of pancreatic cancer and its precursor lesions, (ii) have pioneered the use of high-throughput genome technology in pancreatic research, (iii) have generated in-vitro and in-vivo models of the disease and (iv) were among the first to transfer individual target genes or groups of target genes into preclinical and clinical applications. These German centers of excellence in pancreatic cancer research were joined by genome research groups and partners from the pharmaceutical industry in an integrated approach for an efficient characterisation and exploitation of genome project candidate genes for pancreatic cancer. The prime objective was to foster the rapid development and transfer of novel genome-based, molecular targeted therapeutic and diagnostic approaches from basic research, over preclinical testing into clinical applications. The workplan was conducted at several levels of research: patforms: data, patients, resources, models; functional characterisation in human in vitro and mouse in vitro and in vivo models; preclinical testing in mouse models of pancreatic cancer; and clinical testing.
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FINISHED PROJECT:
Pancreatic cancer susceptibility loci and their role in survival...

There are strong epidemiologic evidences indicating that genetic common variability could be implicated in the risk developing of pancreatic cancer and various risk loci have been proposed. Genome-wide association studies (GWAS) have been performed worldwide, and resulted in several loci associated with risk of pancreatic cancer.
  
In the context of the PANcreatic Disease ReseArch (PANDoRA) consortium, coordinated by Federico Canzian (DKFZ), we replicated the associations found by others in two additional, independent populations (from Germany and the UK) and also evaluated the possible impact of these SNPs on patient survival. For the latter, we focused particularly on the ABO locus. Moreover, we performed stratified analyses considering the tumor stage in order to verify whether genetic variability could be involved in the disease prognosis. Also, we attempted to replicate novel risk loci identified in studies conducted in Japan and China in individuals of European descent.






Campa et al. (2013) Can. Epidem. Biomarkers Prev. 22, 320-322.




Rizzato et al. (2013) Oncol. Report 29, 1637-1644.




Rizzato et al. (2011) PLoS ONE 6, e27921. pdf icon




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FINISHED PROJECT:
Mutation spectrum and prognostic significance of K-RAS and CDKN2A in exocrine pancreatic tumours ...

K-RAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. Therefore, we screened tumors from 171 pancreatic cancer patients for mutations in K-RAS, CDKN2A, BRAF and GNAS genes. Mutations in K-RAS were detected in 134 tumors, with 131 in codon 13 and 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors; GNAS mutations were present in two tumors.

Analysis showed that K-RAS mutations were associated with reduced patient survival in all sub-categories. Patients with malignant exocrine tumors that had K-RAS mutations showed a median survival of 17 months compared 30 months for those without mutations. Patients with a G12D mutation showed a median survival of 16 months. Although the association of survival in pancreatic cancer patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant K-RAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13 months compared 30 months without mutation. Our results clearly show an association between mutational status and survival in pancreatic cancer patients.

Rachakonda et al. (2013) PLoS ONE 8, e60870.  pdf icon



Kaplan-Meier survival curves showing difference in overall survival in PDAC patients with and without mutations.










FINISHED PROJECT:
Breast Cancer
Epigenetically de-regulated miR-375 is involved in a positive feedback loop with Estrogen Receptor alpha in breast cancer cells


     logo NGFN                  logo BMBF

Breast cancer is the leading cause of cancer death in women worldwide. Although it is a heterogeneous disease, two-thirds of breast cancers share the common feature of being dependent on the presence and interaction of estrogen with the nuclear Estrogen Receptor α (ERα) protein. Approximately 70% of invasive breast cancers express ERα in actively proliferating cells. It has become evident that ERα is up-regulated in luminal mammary epithelial cells during early stages of tumorigenesis and its overexpression is an important stimulatory factor for proliferation of mammary cells, leading to cell division and eventually to tumor development. The obvious role of ERα signalling in orchestrating the expression of genes involved in growth-related pathways, has established ERα as an important therapeutic target in breast cancer treatment. However, our understanding of the molecular mechanisms underlying deregulation of this signaling pathway is scarce.
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We identified a high expression of microRNA 375 (miR-375) in ERα-positive cell lines. miR-375 overexpression is mainly caused by the loss of epigenetic marks, such as H3K9me2 and local DNA hypomethylation, which, in turn, triggers the dissociation of the transcriptional repressor CTCF from the promoter and enables interactions of ERα with regulatory regions of miR-375. Inhibition of miR-375 in ERα positive MCF-7 cells results in reduced ERα activation and cell proliferation. A combination of expression profiling from tumour samples and miRNA target prediction identified RASD1 as a potential miR-375 target. Our findings show that miR-375 regulates RASD1 through targeting its 3' UTR. In addition, we demonstrated that RASD1 negatively regulates ERα expression. Our data indicate the existence of a positive regulation between ERα and miR-375 and suggest new strategies for the treatment of ER-positive invasive breast tumours.

de Soza Rocha Simonini et al. (2010) Cancer Res. 70, 9175-9184.  pdf icon









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FINISHED PROJECT:
Pancreatic Cancer
Developing novel molecular tools for the prevention and diagnosis of pancreatic cancer

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logo MolDiagPaCa

While project funding has ceased, the collaboration between many partners involved in this consortium continues nevertheless.
  
The overall aim of this EU Framework Programme 6 Integrated Project was to make use of genetic profiles of pancreatic cancer and precursor lesions to improve the outcome of pancreatic cancer patients by providing novel and highly efficient molecular diagnostic tools. One of the major prerequisites in order to achieve this ambitious aim was an integrated multidisciplinary research approach, which enabled a strong interaction between technology, biology and medicine to translate genome data into practical, clinical applications.
  
The consortium included molecular biologists, bioinformaticians, pathologists, epidemiologists, molecular oncologists, surgical and medical oncologists, radiologists and nuclear medicine physicians. Since we expected to generate molecular diagnostic tools that will be ready for clinical applications, a number of companies was involved that have a particular interest in developing molecular diagnostic tools, and one partner from pharmaceutical industry.
The scientific objectives of the project were defined in seven workpackages:
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Level 1:  Data, patients, resources:
WP1:  Epidemiology, patients at risk, patient resources, coordinated by J.P. Neoptolemos, W. Greenhalf and N. Malats
WP2:  Molecular alterations of preneoplastic lesions, early and advanced tumours: genomic and proteomic profiles, coordinated by N. Lemoine & T. Jurcevic
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Level 2:  Development of novel molecular diagnostic tools
WP3:  RNA-based diagnostics, coordinated by T.M. Gress
WP4:  Proteome based diagnostics, coordinated by E. Costello and S. Hahn
WP5:  Epigenetics, coordinated by J.D. Hoheisel
WP6:  Novel Molecular Imaging tools based on single proteins identified in high-throughput approaches, coordinated by S. Hahn
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Level 3:  Clinical trials of novel molecular diagnostic tools:
WP7:  Prospective clinical trials of novel molecular diagnostic tools, coordinated by J.P. Neoptolemos

The project used clinical samples such as serum, urine, fine needle aspirates and surgically resected materials of pancreatic cancer patients collected in large multinational European trials such as ESPAC or EUROPAC. During the last phase prospective clinical trials of novel diagnostic tools developed in the integrated project were designed and started.
For more information see MolDiagPaCa webpage.
 
map of MolDiagPaCa partners








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FINISHED PROJECTS:
Identification and characterisation of disease genes
by Representational Difference Analysis

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Complementary and parallel to differential hybridisation techniques, Representational Difference Analysis (RDA) was employed as an alternative method for the detection of differentially expressed genes. The technique was adapted from the original protocol of Hubank and Schatz (1994) so that analyses are possible even with small amounts of starting material. In various projects, this technique was applied for the isolation of genes related with and potentially causative to diseases. In collaborations with the companies Merck, Hoffman la Roche and Knoll (now Abbott), analyses were carried out for the identification of disease-related genes on a wide variety of tissues. Large number of cancer-related transcriptional differences were identified and some of the relevant genes and gene products analysed in more detail. Also, antibodies were generated on the basis of such studies and are being used in actual protein expression analyses.

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RDA was performed on larynx carcinoma tissue versus normal larynx tissue. Total difference products were cloned and and individula clones were picked. Redundancy of the library was minimised by iterative hybridisations of clones back to the library. PCR-products were spotted on filters, with the clones representing overexpression in normal and cancer tissue, respectively, being spotted in different orientation. Upon hybridisation of the respective starting material, all but one signal were of the expected orientation.









Schütz et al. (2006).J. Mol. Biol. 358, 997-1009. pdf icon
Frohme et al..(2002) Biochem. Biophys. Res. Commun. 293, 1377-1382.




Steinberg et al. (2006).J. Periodontal. Res. 41, 426-446. pdf icon
Frohme et al..(2000) Ann. N.Y. Acad. Sci. 910, 85-104.





Frohme & Hoheisel (2006).Cell Biology 3rd ed., Elsevier, 113-120.

Geng et al..(1998) Biotechniques 25, 434-438. pdf icon




Malanchi et al..(2004).J. Virol. 78, 13769-13778.
Gress et al. (1997) Genes Chrom. Cancer 19, 97-103.




Zubakov et al..(2003).FEBS Lett. 547, 51-57. pdf icon
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other publications and patents






Scheideler & Hoheisel (2002) Screening 5/02, 22-25.

















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