B: Transcription

B1: Role of NFATc1-NFkB crosstalk interaction in cytokine mediated progression of pancreatic cancer

2009-06-03T11:03:43Z

Pancreatic cancer is characterized by a strong inflammatory reaction with infiltration of immune cells. We have described overexpression and activation of members of NFAT transcription factors in pancreatic cancer and demonstrated that NFATc1 exerts strong oncogenic properties through induction of G1-S phase promoting genes. We are currently investigating how NFAT themselfes are regulated on the transcriptional and posttranslational levels through signalling crosstalk. One focus of our laboratory is the identification and characterization of NFAT partner proteins in promoter binding and regulation.

Among others, we have identified a role of NFkB in NFAT signalling and transcription. We will continue this work to analyse the molecular mechanisms and functional implication of this interaction in pancreatic carcinogenesis with a specific focus on cytokine promoter regulation. For this purpose, we combine in vitro promoter binding studies (ChIP, DNAP, reporter assays, FACS, proliferation assays) with cancer inducing mice models.

B2: Role of the tumor suppressor p73 in inflammation control

2009-06-03T11:03:55Z

The p53 gene family comprises the three transcription factors p53, p63 and p73, which are known as tumor suppressors. p73 knockout mice are characterized by an increased incidence of tumors, infections and signs of chronic inflammation. To better understand the p73 knockout phenotype in molecular terms, we have compared p73-deficient cells to wild-type cells by genome-wide gene expression profiling. Here, the major difference turned out to be a significantly altered expression of inflammatory target genes as well as matrix metalloproteinases. The link between p73 and inflammatory signalling pathways provides an intriguing and potentially unifying explanation for the tumor and inflammation phenotype of the p73 knockout and will be analyzed in this project in molecular detail.

B3: Regulation and function of PPAR-beta/delta in tumor stroma and inflammatory cells

2009-06-03T11:04:04Z

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that modulate target gene expression in response to fatty acid ligands. All three PPAR subtypes (PPARα, PPARβ/δ and PPARγ) activate their target genes through binding to specific DNA elements (PPREs) as heterodimers with the retinoid X receptor (RXR). PPARs can also regulate genes by directly interacting with specific transcription factors, but the underlying mechanisms are not well understood.
PPARβ/δ has a critical role in modulating lipid catabolism, glucose homeostasis and inflammation. Consistent with its function in the regulation of cell differentiation and proliferation, PPARβ/δ has been implicated in tumorigenesis. We have recently shown that the growth of syngeneic Pparb wild-type tumors is severely impaired in Pparb null mice concomitant with a severely altered, hyperplastic tumor stroma. This phenotype apparently arises from the hyperproliferation of endothelial cells and a lack of microvessel maturation differentiation specifically in the inflammatory tumor stroma. Furthermore, we and others have shown that the activation of macrophage in vivo is perturbed in a Pparb background (see Figure). In addition, we have strong experimental evidence for a functionally essential cross-talk of PPARβ/δ and PPARγ in macrophage activation.
Currently, it is largely unclear which genes are regulated by PPARβ/δ and represent critical targets in inflammatory cells. In addition, the molecular mechanisms involved in the interplay of PPARβ/δ and PPARγ are not understood. A main goal of this project is to investigate these questions by combining knockout mouse and genomics technologies, in particular microarrays, chromatin immune precipitation and ChIP-Seq. Based on the results of these studies we will study the chromatin structure of select PPARβ/δ target genes with a focus on how PPARβ and its ligands modulate the assembly and dynamics of PPARβ/δ transcription complexes.

References:

Müller, R., Kömhoff, M., Peters, J.M. and Müller-Brüsselbach, S. (2008). A role for PPARβ/δ in tumor stroma and tumorigenesis. PPAR Res. 2008:534294.

Rieck, M., Meissner, W., Ries, S., Müller-Brüsselbach, S. and Müller, R. (2008). Ligand-mediated regulation of PPARβ/δ: A comparative analysis of PPAR-selective agonists and all-trans retinoic acid (atRA). Mol Pharm. 74: 1269-1277.   

Müller, R., Rieck, M. and Müller-Brüsselbach, S. (2008). Regulation of cell proliferation and differentiation by PPARβ/δ. PPAR Res. 2008:614852.

Müller-Brüsselbach, S., Kömhoff, M., Rieck, M., Meissner, W., Kaddatz, K., Adamkiewicz, J., Keil, B., Klose, K.J., Moll, R., Burdick, A.D., Peters, J.M. and Müller, R. (2007). Deregulation of tumor angiogenesis and blockade of tumor growth in PPARβ deficient mice. EMBO J. 26: 3686-3698.

Adamkiewicz, J., Kaddatz, K., Rieck, M., Wilke, B., Müller-Brüsselbach, S. and Müller, R. (2007). Proteomic profile of mouse fibroblasts with a targeted disruption of the peroxisome proliferator activated receptor-β/δ gene. Proteomics. 7: 1208-1216.

B4: SUMO-dependent transrepression of inflammation induced gene expression

2009-06-03T11:04:15Z

Transrepression of NFκB-activated proinflammatory genes by peroxysome proliferator-activated receptor-γ (PPARγ) and liver X receptor (LXR) is dependent on SUMOylation. The molecular mechanism by which SUMO-modification of these nuclear receptors effects transrepression is not fully understood.
By using a genome-wide RNA interference screen, we have recently identified SUMO-dependent repression components (1), and described SUMO-modification of transcription factors as a novel mechanism for the initiation and formation of repressive local heterochromatic-like states (2). Guided by the mechanistic clues provided by these findings, we hypothesize that similar chromatin changes could take place upon transrepression of proinflammatory genes by SUMOylated PPARγ and LXR. To address this question, we will perform RNAi and chromatin immuonprecipitation experiments.
PPARγ und LXR are modified by different SUMO paralogs (PPARγ by SUMO1 and LXR by SUMO2/3). Hence, it is very likely that different gene and signal-specific transrepression programs are affected by SUMO1 and SUMO2/3. To identify proflammatory genes that are specifically affected by different SUMO paralogs we will perform RNAi experiments in combination with microarray analyses.

References:

(1) Stielow B, Sapetschnig A, Krüger I, Kunert N, Brehm A, Boutros M., and Suske G (2008) Identification of SUMO-dependent chromatin-associated transcriptional repression components by a genome-wide RNA interference screen. Mol. Cell 29, 742-754.
(2) Stielow B, Sapetschnig A, Wink C., Krüger, I., and Suske G. (2008) SUMO-modified Sp3 represses transcription by provoking local heterochromatic gene silencing. EMBO Rep. 9, 899-906.

B5: Significance of Interferon regulated factors (IRFs) for integration of tumorsuppression and immunosurveillance

2009-06-03T11:04:25Z

Patients with a BCR-ABL positive chronic myeloid leukemia (CML) have also lost expression of the tumor suppressor and transcription factor interferon regulatory factor (IRF)-8 and IRF-4. Since many years, our group is engaged to reveal the role of IRF-8 and IRF-4 for the pathogenesis of CML. Whereas IRF-8 is an essential transcription factor for the maturation of plasmacytoid dendritic cells (pDC) IRF-4 plays an important role for Th17 differentiation.

Using our well established in vitro leukemia modell, we find a strong cooperation of BCR-ABL and IRF-8 during transformation of heamtopoietic progenitor cells. The underlying mechanism of this cooperation will be analysed using primary progenitor cells.

In close cooperation with the group of M. Lohoff (see also subproject B6) an inducible expression system for IRF-8 in BCR-ABL transduced progenitor cells will be established. Using this in vitro model we will ask the question whether IRF-8 will perform its tumorsuppressive function by regulating central gatekeeper genes with transformational capacity like p53 or Arf. To do so, chromatin immunoprecipitation followed by sequencing (ChIP-seq) technique will be used.

Beside the strong significance of IRF-8 deficiency in BCR-ABL induced transformation processes, at time it is not clear, if IRF-8 deficiency is also responsible for the maintenance of the malignant phenotype. For this, it will be necessary to induce in vivo IRF-8 in BCR-ABL positive/IRF8-negative leukemia cells and to analyze wether a leukemia phenotype can be reverted. For this experiments not only IRF-8- but also IRF-4 ko mice may be used as:

I) IRF-4 deficiency is also observed in CML patients, demonstrating the significance of the IRF-4 pathway for leukemia and

II) the significance of T cell subpopulations (Th17, NK cells and CD8-positive T cells) for leukemia induction may be answered, as these mice show defects in differentiation of T cell populations. IRF-4 mice will be provided by the group of Lohoff.

The role of pDC during leukemogenesis and leukemia maintenance is not yet clear, but we observed an IRF-8 dependent loss of pDCs in CML patients. The adoptive add-back of pDCs into IRF-8-deficient mice and treatment with TLR-9 specific CpG-Oligos will allow to reveal the importance of pDCs in leukemia manifestation and therapy response.

References:

Wang, Y., Cai, D., Brendel, C., Barett, C., Erben, P., Manley, P.W., Hochhaus, A., Neubauer, A., Burchert, A. (2007). Adaptive secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates imatinib and nilotinib resistance in BCR/ABL+ progenitors via JAK-2/STAT-5 pathway activation. Blood, 109, 2147-2155.

Ortmann CA, Burchert A, Hölzle K, Nitsche A, Wittig B, Neubauer A, Schmidt M. (2005). Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region. Nucl. Acids Res, 33(21):6895-905.

Burchert, A., Cai, D., Hofbauer, L.C., Samuelsson, M.K., Slater, E.P., Duyster, J., Ritter, M., Hochhaus, A., Müller, R., Eilers, M., Schmidt, M., Neubauer, A. (2004). Interferon consensus sequence binding protein (ICSBP; IRF-8) antagonizes BCR/ABL and down-regulates bcl-2. Blood, 103, 3480-3489.

B6: Role of interferon regulated factor (IRF) 4 for Th17 differentiation

2009-06-03T11:42:47Z

CD4+ T cells can be classified into Th1- Th2-, Th17- and regulatory T (Treg) cells according to their specific cytokine profile upon stimulation (see also corresponding figure). During differentiation from naive CD4+ cells to the different subgroups, cytokine-induced specific transcription factors play a dominant role. Our group was the first one who could demonstrate that Interferon-regulator-factor (IRF) transcription factors are responsible for the formation of the different T cell subtypes. IRF1 and IRF2 are important for Th1 differentiation and play also a dominant role during murine leishmaniosis. IRF4 was identified as the ‚master-transcription factor’ for Th2 development and its absence is associated with a dramatically enhanced activation-induced cell death (AICD). IRF4 is the central transcription factor not only for Th2- but also for Th17-differentiation with significance also for the development of experimental autoimmune encephalomyelitis (EAE), the murine disease model for multiple sclerosis (MS).

Project description

Beside STAT3 and RORγt, IRF4 is the central transcription factor for Th17 differentiation, albeit IRF4 is expressed by all CD4+ subtypes upon T-cell activation. IRF4-expression by itself is therefore not sufficient to direct T-cell differentiation into a particular subtype, but additional IRF4-binding-‚partners’ are necessary and/or modifications at the transcription factor itself. A still open question is also how IRF4 induces Th17 differentiation in the presence of TGF-ß plus IL-6, but does not play any role for Treg differention in the presence of TGF-ß alone.

The central question to be answered in our project is therefore to specify the role of IRF4 during Th17 differentiation. Chromatin precipitation of IRF4 and ChIP-Seq technique will allow to define IRF4-binding sites genomewide.

Further we will also analyse the impact of the different transcription factors and their mutual interaction during T cell differentiation at different time points. For this we will establish an inducible retroviral Tet-On-system. Hereby we will able to answer the following question:

how do the different transcription factors interact
at what time point do they interact
which are the critical time points during T cell subgroup differentiation

Our findings will also allow to analyse the significance of the tumor microenvironment on the generation of T cell subpopulations with pro- and/or anti-tumoral properties.

TP13: Control of inflammation induced gene expression by cell cycle dependent kinases of the IL-1 and TNF pathway

2010-12-07T09:51:02Z

The potent proinflammatory cytokine Interleukin-1 (IL-1) is produced at sites of tumors by tumor-associated macrophages but also by tumor cells and by endothelial or epithelial stroma cells. Depending on the cellular context, IL-1 can have tumor-promoting but also tumor-suppressing effects. The underlying molecular mechanisms are not clear. Gene amplification, overexpression, loss of CDK inhibitors or overexpression of D-type cyclins activates interphase kinases (CDK2, CDK4, CDK6) which promote uncontrolled tumor cell proliferation. Hence, small molecules directed against CDK4/6 (e.g. PD0293391) are being tested for treatment of several human tumors. Our unpublished data reveal that CDK6 specifically affects tumor cell-associated inflammatory gene expression unravelling an unexpected function of this kinase whose contribution to tumor pathology is unknown. These results have widespread biological implications as they define a novel cross talk mechanism between cytokine signaling and cell cycle. Accordingly, aberrant CDK6 expression or activation that is frequently observed in human tumors contributes through NF-κB to chronic inflammation and neoplasia. We postulate that interphase kinases such as CDK4 or CDK6 exploite the IL-1 signaling network to establish a pro-tumorigenic microenvironment by affecting signaling “thresholds” and feedback loops in the NF-κB and possibly other pathways. With the help of LOEWE colleagues we aim to systematically characterize and identify the molecular links between activated interphase kinases, their regulators and major IL-1-induced signaling pathways and assess the relevance of these observations for tumors by establishing appropriate genetic models in mice.

TP14: The role of the proinflammatory protein kinases IKKε and TBK1 for cell proliferation and tumor development

2010-12-07T09:43:30Z

There is increasing evidence for a critical role of the NF-κB transcription factor network for initiation and promotion of tumors. The central step of NF-kB activation is the IKK-mediated phosphorylation of IκB, which is the prerequisite for subsequent proteasome/ubiquitin-mediated degradation of Iκ B. Cells do not only contain the canonical IKK complex, but also the so-called non-canonical IKK complex which consists of the two kinases IKKε and TBK1 (TANK-binding kinase) as well as adapter proteins such as TANK. The non-canonical IKKs are dispensable for IkB phosphorylation but instead are important for the control of nuclear NF-κB activity and for the activation of transcription factors IRF3/7, as schematically shown in the figure. Recent evidence points to an important role of the non-canonical IKK complex for the development of solid cancers. The expression of both kinases is frequently deregulated in solid tumors such as breast cancer (IKKε) and KRAS-driven cancers (TBK1).

The research plan consists of three different parts. In the first part it is planned to investigate the molecular mechanisms regulating the function of IKKε and TBK1. The activating signals, interacting proteins and phosphorylation substrates of both kinases are identified in unbiased screens. This part will generate important experimental tools and informations that will feed in another experimental part. The wildtype kinases, adequate mutants and phosphorylation substrates are tested for their contribution to oncogenic transformation of cells and their relevance for the pro-proliferative functions of IKKε and TBK1. We will also determine the expression levels and intracellular localization of IKKε and TBK1 in tumor samples. In the third part we will develop adequate mouse models to study the functional consequences of IKKε/TBK1 overexpression for the induction and promotion of tumors.