Pathologic signal transduction in cancer with emphasis on JAK/STAT signaling
Signalaustausch zwischen Krebszellen und mit Zellen der Tumor-Mikroumgebung
Signal transduction is a central aspect of life. Well organized distribution of information throughout the organism is crucial for its integrity and homeostasis. The body constitutes an extremely complex network of cells which release, perceive and process chemical signals. The work of our research group is concerned with molecular mechanisms of cellular signal transduction and with dysfunction of these processes in the course of disease. A wide spectrum of techniques and methods is being applied, ranging from DNA cloning to protein biochemistry, cell culture and in vitro and in vivo disease models. We strive for a better understanding of how cells and molecules "talk to each other" and attempt, by doing so, to contribute to novel developments in molecular medicine.
Members of the research group come from different fields (e.g. Biochemistry, Biotechnology, (Molecular) Medicine) and different countries and pursue their research interests in a particularly cooperative manner. Intense collaboration is maintained with academic and industrial partners both on a national and international scale. Frequently there are opportunities for master or doctoral theses or for participation in different projects in the frame of research stays or internships. In case of interest please get in touch () – “we can talk about it…”
Research during recent years was supported by grants from:
Recently, we have become involved in a German-Tunisian collaborative project (funded by the Ministries of Research of both countries) aiming at the development of a flow cytometry-based novel diagnostic technique for early indications of solid cancers. In this context, a network of academic and industrial partners has been initiated which promotes exchange of ideas, materials and researchers with their know how. Please find more information here
Elucidation of mechanisms underlying aberrant cytokine receptor activation, signal transduction/gene regulation and intercellular communication in allergic disorders. Development of approaches to specifically inhibit disease-relevant cytokine receptor function
Background
Allergic diseases of the immediate type such as allergic Asthma bronchiale are characterized by elevated serum levels of IgE, infiltration of the affected tissue by inflammatory cells, and a Th2-dominated type of immune reaction. Dysregulation of cytokine function is crucial for both acute and chronic symptoms of the disorder. We have recently concentrated on two cytokines which both signal via heterodimeric cytokine receptors and JAK/STAT pathways, and play central roles in Asthma: Interleukin-13 and Thymic Stromal Lymphopoietin (TSLP).
Figure A
IL-13 is a cytokine with multiple functions in normal immune processes and inflammatory diseases. It is mainly produced by activated T cells, but also by various other cell types such as mast cells, basophils, eosinophils, and dendritic cells. IL-13 is structurally and functionally closely related to the prime Th2 cytokine IL-4 and shares its property to induce immunoglobulin class switching to IgE. However, it has specific critical functions in the development of allergic asthma and its symptoms (hyperresponsiveness to specific antigens, elevated IgE levels, mucus hypersecretion and subepithelial fibrosis).
TSLP is an interleukin-7-like cytokine which emerged in the last years as a central player in the development of allergic symptoms, especially in the airways, and is a prime regulatory cytokine at the interface of virus- or antigen-exposed epithelial cells and dendritic cells. Since TSLP links contact of allergen with the airway epithelium to the onset and maintainance of the asthmatic syndrome, defining the signal transduction underlying TSLP expression and function is of profound interest for a better understanding of the disease and for the development of new therapeutics. The TSLP receptor consists of a specific TSLPR a-chain and the IL-7 receptor a-chain. Upon ligand-induced activation, it activates JAK/STAT pathways and influences cell proliferation as well as specific gene regulation (see Fig. A).
We have expressed recombinant cytokine receptors in lymphoid cell lines to study IL-13- and TSLP-induced intracellular signal transduction by Western blot and reporter gene experiments. This led e.g. to the identification of Janus kinases and STAT factors involved in TSLP signaling. Moreover, effects of both cytokines on primary epithelial and fibroid cells of the airways were studied to better understand disease-relevant differentiation processes, e.g. expression of pro-apoptotic genes and genes related to fibrosis. These investigations, for instance, revealed a role of IL-13 in the development of fibrosis, a long-term consequence of chronic asthma.
Cellular test systems reflecting IL-13- and TSLP-dependent signal transduction on the basis of murine lymphocytes have been established. Expression of the cytokine receptors is monitored by cytometry and Western blot studies. The use of STAT-specific reporter gene constructs allows for the quantification of cytokine effects and analysis of intracellular reactions involved. Ongoing studies are aiming at the development of inhibitory substances for asthma-related cellular signaling. In particular, we have employed different methods (“classical” hybridoma techniques for the generation of monoclonal antibodies as well as recombinant antibody development) to isolate antibodies that efficiently block allergy-relevant signaling through the receptors for IL-13 and TSLP. These reagents serve as potential lead structures for the establishment of drugs that can interfere with disease-relevant activities of IL-13 or TSLP.
In the course of our characterization of an inhibitory antibody to the TSLP receptor a-chain, we could (as a side project) also show that this reagent can reduce TSLP-dependent proliferation of malignant B cells which occur in a special type of acute lymphoid leukemia (ALL).
Throughout our studies, we have progressively developed the heterodimeric TSLP receptor into a generally adaptable platform for the determination of activities of cytokine/growth factor activities (see Fig. B). This system, which rests on the establishment of cells expressing customized hybrid receptors has proven very useful in projects aiming at the development of specific inhibitors of cytokines/growth factors with relevance in disease.
Selected References
Molecular analysis of aberrant signal transduction processes in cancer cells and correlation with malignant cell behavior and tumor progression
Signal transduction through Janus kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs) is essential for a multitude of fundamental cell functions such as proliferation and differentiation. Best characterized is the triggering of JAK/STAT signaling by cytokine receptors. JAKs, upon receptor dimerization, tyrosine phosphorylate the receptor and, thus, generate docking sites for signaling proteins comprising SH-2 domains, e.g. STATs. In the course of interaction with the cytokine receptor complex, STATs themselves become phosphorylated. Subsequently, they dimerize, translocate to the cell nucleus and, upon binding to cognate DNA elements, act as transcriptional modulators of cytokine target genes. Aberrant activation of JAK/STAT pathways has been observed in various human cancers.
A particularly important aspect of tumor malignancy is the ability of tumor cells to grow in an invasive manner and to ultimately form metastases at distant locations in the organism. STAT3 is a critical determinant of tumor-stroma interactions and of cellular invasiveness. STAT1, on the other hand, is believed to have tumor-suppressive properties. It is, thus, important to study the contribution of STATs to the invasive behavior of tumor cells in vitro and in vivo.
We analyzed the roles of dysregulated JAK/STAT signaling in different types of malignant human tumors (e.g. colorectal, lung and bladder carcinoma). Experiments addressed in how far aberrant STAT activity is operative in cancer tissue, if it is of importance for the malignancy of the tumor, and what signaling mechanisms functionally connect the activities of STATs and other molecules with pathogenic properties.
Constitutive tyrosine phosphorylation and DNA binding activity of STAT3 and STAT1 was observed in biopsies from the majority of malignant colorectal and lung tumors. Fig. A shows specific STAT-DNA interactions and tyrosine phosphorylation in colon tumor tumor tissue analyzed by Electrophoretic Mobility Shift Assay (EMSA) and immunohistochemistry using an antibody to pTyr-STAT3 (fluorescence signals).
Studies on various tumor cell lines revealed that STAT3 activity directly drives cell proliferation and invasiveness. In xenograft experiments, these cell lines formed tumors upon implantation into immunodeficient mice. These results indicate that STAT3 is immediately associated with the pathogenesis of solid cancers such as CRC and constitutes an attractive target for therapeutic intervention. Similar experimental and statistical studies have been performed on STAT1 and STAT5 and pointed to individual and combined roles of these factors in cancer development and progression. Importantly, we could show that not only the respective activities of STAT3 or STAT1 alone were relevant for CRC malignancy. Instead, the ratio of STAT3 or STAT1 activities proved an independent predictive parameter for cancer outcome both in human patients and in a mouse disease model. These findings are of potential clinical relevance and also provide novel insight into the molecular interplay between the STAT factors in tumorigenicity (Fig. B).
Another aspect of work pursued in recent years was the investigation of STAT-dependent alteration of gene expression. We found an association of STAT1 and STAT3 activities with altered expression of matrix metalloproteinases (MMPs) and studied molecular mechanisms underlying transcriptional regulation of the MMP-1 promoter.
In a side project, we have also worked on strategies to specifically inhibit oncogenetic signaling of constitutively active Ras proteins. A promising approach was developed which rests on the application of multimerized protein domains that bind and inactivate oncogenenic Ras in tumor cells.
Unraveling communication processes among cancer cells and with cells of the tumor microenvironment to better understand the role of tumor stroma in cancer progression
Recent research has generated novel insight into mechanisms by which tumor cells interact with each other as well as with activated fibroblasts, endothelial cells, inflammatory and immune cells and the extracellular matrix. All these signal exchange processes functionally involve secreted factors such as cytokines and transmembrane receptors.
Cancer cells and cells of the stroma intensely communicate and, consequently, co-evolve throughout tumorigenesis. The tumor stroma is now known as an essential contributor to tumor establishment, progression and dissemination. Moreover, so called stroma niches have been recognized as important determinant of metastasis. However, disease-relevant communication between tumor cells and the tumor stroma is as yet poorly understood. Thus, progress in defining specific roles of the microenvironment in tumor biology renders the tumor stroma and its signal exchange with cancer cells attractive potential future target for advanced cancer therapies. To proceed in this direction, much effort is currently being devoted to the development of co-culture protocols for cancer and stromal cells which mimick in vivo interactions.
Signals from stromal cells, e.g. from tumor associated fibroblasts contribute to epithelial-mesenchymal transition (EMT), a hallmark of cellular de-differentiation and acquisition of cancerous properties. EMT is driven by a group of transcription factors (Snail, Slug, Zeb etc.) which favor loosening of cell-cell contacts, e.g. by repression of E-cadherin expression.
Cancer cell malignancy is also triggered by interactions between tumor cells via transmembrane proteins. An important molecule with central importance for cell-cell communication (among cancer cells and also with fibroblasts) is EMMPRIN (Extracellular matrix metalloproteinase inducer), which can exist both in a membrane associated and a soluble form (the latter resulting from specific shedding off of the extracellular domain). The detailed roles of EMMPRIN in genesis and development of tumors are largely obscure.
Our work has recently concentrated (i) on the crosstalk between cancer cells and tumor-associated fibroblasts and the underlying functions of cytokines and intracellular signal mediators such as transcription factors and (ii) on the roles of the cell surface molecule EMMPRIN in the development of malignant properties of cancer cells.
We have analyzed the communication between tumor cells and different types of fibroblasts. Bladder cancer cell lines were used as a tumor cell model. They were grown separately or in co-culture with e.g. tumor-associated fibroblasts. Migratory and invasive cell properties of tumor cells and fibroblasts were quantified by wound healing and other assays. Cytokine production of cells was measured by intracellular staining followed by cytometry or by subjecting conditioned medium to ELISA and Western blot analysis. Factors secreted by tumor cells and by fibroblasts were identified and shown to mutually evoke changes in the expression of cancer-relevant genes, e.g. genes for matrix metalloproteinases (MMPs). As particularly important secreted soluble factors promoting cellular malignancy we could identify IL-1a (Interleukin-1a) and HGF (Hepatocyte Growth Factor) (Fig. A).
We have studied the effects of EMMPRIN on malignancy parameters in breast cancer cells. As tools for this purpose, a recombinant form of soluble EMMPRIN and engeneered cells with knocked-down EMMPRIN expression have been generated. Analyzing the activities of soluble EMMPRIN we obsered a stimulatory effect on cell proliferation, however, the protein proved repressive for cell migration (as quantified in a “wound healing” or “scratch” assay, Fig. B). Obviously, EMMPRIN can change the malignancy profile of tumor cells in a specific manner and is, thus, a determinant of cellular tumorigenicity. This interpretation is supported by findings that demonstrated crosstalk of EMMPRIN signaling with other cancer-related pathways such as the Wnt- and JAK/STAT cascades.
Recently, we have become involved in a German-Tunisian collaborative project (funded by the Ministries of Research of both countries) aiming at the development of a flow cytometry-based novel diagnostic technique for early indications of solid cancers. In this context, a network of academic and industrial partners has been initiated which promotes exchange of ideas, materials and researchers with their know how. Please find more information here
