Research Interests
Permanently activated signal transduction pathways constitute one of the major driving forces
underlying cancer. Oncogenic signalling mediates aberrant mitogenesis and cell survival, invasion,
angiogenesis and metastasis. Malignancy is intimately coupled with profound alterations of the genetic
program. The Ras pathway is frequently activated by mutations in one of three major Ras isoforms.
The mutations lock the signaling molecule in its activated state and create high levels of GTP-bound Ras
in critical cellular compartments. Alternatively, the Ras pathway is activated by abnormal signals
emanating from receptor tyrosine kinases or by mutations in both negative regulators
(e.g. the GTPase activating protein NF-1) and positive effectors (e.g. the downstream signaling kinase B-Raf).
The central role of activated Ras proteins in triggering malignant phenotypes has been confirmed in numerous
cell culture and animal models. Rational therapies targeting Ras proteins or downstream signalling effectors
have already entered clinical trials. Despite their importance in experimental and clinical cancer research,
the functional relationship between Ras oncogene-mediated cytoplasmic signalling processes and aberrant gene
expression is far from being understood. Ras-responsive genes represent critical factors for cancer diagnosis
and therapy. Therefore, the major aim of the laboratory is to catalogue Ras-responsive genes in various types
of experimental cancer systems and clinical specimens, to understand the modes of their regulation and to unravel
their specific function in executing cancer cell phenotypes.
Ongoing projects:
Molecular profiling of ovarian carcinoma and of colon carcinoma
A growing number of gene expression profiles related to tumor class, stage, histology and clinical parameters are
available in publications and databases. Much less information is available on the relationship between defined
oncogenic lesions affecting oncogenes and tumor suppressor genes and the deregulation of the genetic program.
We have established gene expression profiles in cell culture model systems for ovarian and colorectal cancer.
We have targeted signal transduction pathways essential for proliferation, survival and transformation by pharmacological
inhibitors and studied the effects on the transcriptome. Pathway-dependent target genes are organized in signal-regulated
transcriptional modules suggesting that individual deregulated signaling pathways do no affect gene activity stochastically.
Rather, oncogenic signalling impinges on well-defined regulatory units.
Identification and functional analysis of Ras pathway-regulated genes
We have contrasted gene expression profiles of normal fibroblasts or epithelial cells and ofderivatives transformed by
mutant Ras oncogenes. Suppression subtractive hybridization and microarray technologies were used to establish patterns
of several hundred Ras-regulated genes in cells of human, mouse and rat origin. Thus, the genetic program of cells is
significantly perturbed in conjunction with oncogenic Ras activation. The number of genes up-regulated on Ras-mediated
transformation equals the number of down-regulated target genes. The aim of the project is to find out which target genes
are essential for transformed phenotypes and for malignancy. We apply RNA interference technology for silencing expression
of up-regulated genes in Ras-transformed and for silencing transformation-sensitive genes in normal precursor cells to study
their functional roles in the transformation process.
Characterization of class II tumor suppressor genes
(in cooperation with Dr. C. Sers)
Mutations and deletions in tumor suppressor genes are the hallmark of cancer. While genetic damage leads to an irreversible
functional gene defect in cancer cells, a growing number of genes is prevalently down-regulated in tumors as suggested by gene
expression profiling. Reversible repression of gene activity is due to various mechanisms such as epigenetic silencing or altered
transcription factor binding. The master regulators responsible for suppressor gene silencing provide attractive nodes for therapeutic
intervention. We are in the process of characterizing members of the family of HREV107 proteins, class II tumor suppressors, capable
of blocking proliferation and inducing apoptosis in HRAS-transformed model cells as well as in ovarian carcinoma cells.
HREV107-1 expression is controlled by the interferon signalling pathway, the down-regulation in Ras-tzransformed cells is mediated by
the mitogen-activated protein kinase (MAPK) pathway. A closely related gene family member, TIG3 is induced by retinoic acid.
Dynamic suppression of gene activity in cancer cells by methylation (in cooperation with Dr. C. Sers)
Our group investigates the potential influence of oncogenic RAS-mediated signalling on epigenetic regulation of gene expression.
We focus on regulation of gene expression by silencing of genes by promotor methylation and modulation of histone-acetylation.
For this purpose, we use HRAS-transformed rat and human cells as a model and recently DNMT- (DNA-methyltransferase) knock-out
cells derived from a colon cancer cell line. We successfully identified several genes whose expression is regulated in a Ras-dependent
manner and show a prominent promoter hypermethylation in the RAS-transformed cells. These observations represent an important step
towards understanding the molecular mechanisms by which oncogene-activated pathways contribute to tumorigenesis.
Expression profiling and functional analysis of multi-drug resistance
Multi-drug resistance is a major obstacle for successful cancer therapy. To date, reliable tests for predicting a tumor patient’s
response to chemotherapy are not available. We are developing microarray-based diagnostic techniques for multi-drug resistance.
A predictive gene set for doxorubicin resistance was derived by contrasting expression profiles of various human cancer cell lines
and closely related drug-resistant derivatives. In an independent classification experiment, we applied our model of resistance for
predicting the sensitivity of 44 previously characterized breast cancer samples. The patient group characterized by the gene expression
profile similar to those of doxorubicin-sensitive cell lines exhibited longer survival (49.7+/-26.1 months, n=21, P=0.034) than the
resistant group (32.9+/-18.7 months, n=23). The application of gene expression signatures derived from doxorubicin-resistant
and -sensitive cell lines allowed to predict effectively clinical survival after doxorubicin monotherapy. Our approach demonstrates
the significance of in vitro experiments in the development of new strategies for cancer response prediction. We also study the
direct effects of resistance-associated genes on the expression of resistance as well as that of common signalling inhibitors on
drug resistance.
Mathematical modelling of Ras-induced signalling cascades, an interdisciplinary project (in cooperation with Prof. Herzel)
To study general aspects of intracellular signaling such as switch-like behaviour, bistability, feed-back, cross-talk and single-cell
dynamics, we implement mathematical models permitting simulations of Ras/mitogen-activated kinase signalling cascades.
To obtain experimental parameters for the model simulations, we determine the kinetics of signalling kinase activation downstream
of Ras, measure the transcriptional end points of signalling cascades, and perturb signal transduction by inhibiting effector kinases.
For this purpose we have established cell culture systems with conditionally expressed Ras genes, customized oligonucleotide microarrays
representing major Ras targets (“RASTA”, Ras target array) and optimized the conditions for gene silencing by RNA interference as well
as pharmacological inhibition of kinase activity.
Selected Recent Publications
Previous work:
Schwartz, A.C. and Schäfer, R. (1973)
New amino acids and heterocyclic compounds participating in the Stickland reaction of Clostridium sticklandii.
Archiv für Mikrobiologie 93: 267-276
Schäfer, R., Doehmer, J., Drüge, P.M., Rademacher, I. and Willecke, K. (1981)
Genetic analysis of transformed and malignant phenotypes in somatic cell hybrids between tumorigenic Chinese hamster cells and diploid mouse fibroblasts.
Cancer Research 41: 1214-1221
Schäfer, R., Griegel, S., Dubbert, M.-A. and Willecke, K. (1984)
Unstable transformation of mouse 3T3 cells by transfection with DNA from human lymphocytes.
EMBO Journal 3: 659-663
Schäfer, R., Griegel, S., Schwarte, I., Geisse, S., Traub, O. and Willecke, K. (1985)
Transforming activity of DNA fragments from normal human lymphocytes results from spontaneous activation of a c-Ha-ras1 gene.
Molecular and Cellular Biology 5: 3617-3620
Schäfer, R., Iyer, J., Iten, E. and Nirkko, A.C. (1988)
Partial reversion of the transformed phenotype in HRAS-transfected tumorigenic cells by transfer of a human gene.
Proceedings of the National Academy of Sciences USA 85: 1590-1594
Klemenz, R., Fröhli, E., Aoyama, A., Hoffmann, S., Simpson, R.J., Moritz, R.L. and Schäfer, R. (1991)
αB crystallin accumulation is a specific response to Ha-ras and v-mos oncogene expression in mouse NIH 3T3 fibroblasts.
Molecular and Cellular Biology 11: 803-812
Hajnal, A., Klemenz, R. and Schäfer, R. (1994)
Subtraction cloning of H-rev107, a gene specifically expressed in H-ras resistant fibroblasts.
Oncogene 9, 479-490
Sers, C., Husmann, K., Andres, A.-C., Bucher, K., Emmenegger, U. and Schäfer, R. (1997)
Down-regulation and growth-inhibitory activity of the class II tumor suppressor gene H-rev107 in tumor cell lines and experimental tumors,
Journal of Cell Biology 136, 935-944
Recent work:
Zuber, J., Tchernitsa, O.I., Hinzmann, B., Schmitz, A.C., Grips, M., Hellriegel, M., Sers, C., Rosenthal, A., and Schäfer, R. (2000)
A genome-wide survey of Ras transformation targets.
Nature Genetics, 24, 144-152
Sers, C., Husmann, K., Nazarenko, I., Reich, S., Wiechen K., Zhumabayeva, B., Adhikari, P., Schröder, K., Gontarewicz, A., and Schäfer, R. (2002)
The class II tumour suppressor gene HREV107-1 is a target of interferon-regulatory factor-1 and is involved in IFN?-induced cell death in human ovarian carcinoma cells.
Oncogene 25, 2829-2839
Tchernitsa, O.I., Sers, C., Zuber, J., Hinzmann, B., Grips, M., Schramme, A., Lund, P., Schwendel, A., Rosenthal, A. and Schäfer, R. (2004)
The transcriptional basis of KRAS oncogene-mediated transformation in ovarian epithelial cells.
Oncogene 23, 4536-4555
Györffy, B., Serra, V., Jürchott, K., Garber, M., Stein, U., Petersen, I., Lage, H., Dietel, M. and Schäfer, R. (2005)
Prediction of doxorubicin sensitivity in breast tumors based on gene expression profiles of drug resistant cell lines correlates with patient survival.
Oncogene, 24, 7542-7551
Legewie, S., Blüthgen, N., Schäfer, R., Herzel, H. (2005)
Supersensitization. Switch-like behavior of cellular signalling by transcriptional induction.
PloS Computational Biology, 1 (5), e54
Abdul-Ghani, R., Serra, V., Györffy, B., Jürchott, K., Solf, A., Dietel, M. and Schäfer, R. (2006)
Inhibition of the PI3K pathway blocks drug export from resistant colon carcinoma cells overexpressing MRP1.
Oncogene, 25, 1743-1752
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Prof
Dr. Reinhold Schäfer
