Allgemeine Pathologie
Pathologie

Institute of Pathology: Group Breuhahn

Topic: Transcriptional Regulation and Signaling Pathways in Hepatocytes and Liver Cancer Cells

With more than 700.000 new cases each year, hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death. Its risk factors are well defined (e.g., chronic viral hepatitis, alcoholic and non-alcoholic steatohepatitis) and are detectable in up to 90% of all cases (Figure 1). However, genetic heterogeneity of HCC severely complicates the development of effective drugs, which is reflected by the current lack of therapeutic options (Breuhahn et al., 2011 Hepatology). 

Figure 1: The multi-step process of hepatocarcinogenesis. Different aetiologies lead to chronic hepatitis characterized by continued cycles of hepatocyte necrosis and regeneration. Stellate cell activation and production of fibrotic tissue mark the stages of fibrosis and cirrhosis. During the ongoing proliferation, hepatocytes acquire somatic mutations and copy-number variations that promote the formation of early HCC. The additional acquisition of epigenetic changes and alterations in molecular signalling pathways lead to HCC progression.

Transcriptional regulators (TRs) such as transcription factors or transcriptional co-activators represent cellular bottlenecks in the process of signal transduction. During carcinogenesis, many TRs as well as upstream regulators have been described as deregulated, leading to uncontrolled tumor cell proliferation and resistance to apoptotic stimuli (Breuhahn et al., 2006 Oncogene). Therefore, TRs have been identified as promising target structures for the development of specific perturbation approaches and innovative therapeutic techniques. In our group, we are focusing on the regulation and function of different TRs critically involved in the regulation of regenerative processes as well as development and progression of HCC (Malz et al., 2012 J Hepatol). In addition, we aim to develop comprehensive mathematical pathway models, which describe dynamic processes at different scales (molecular, cellular, and tissue levels) in liver and lung cancer.

References
  • Malz M, … Breuhahn K. Transcriptional regulators in hepatocarcinogenesis - key integrators of malignant transformation. J Hepatol. 2012 Jul;57(1):186-95. (IF: 10.6)
  • Breuhahn et al., Strategies for hepatocellular carcinoma therapy and diagnostics: lessons learned from high throughput and profiling approaches. Hepatology. 2011 Jun;53(6):2112-21. (IF: 11.7)
  • Breuhahn K et al., Dysregulation of growth factor signaling in human hepatocellular carcinoma. Oncogene. 2006 Jun 26;25(27):3787-800. (IF: 7.9)

The Hippo/yes-associated protein (YAP) signaling pathway

The Hippo signaling pathway is a sensor for cell density and negatively regulates its transcriptional coactivator yes-associated protein (YAP) (Figure 2). Inactivation of Hippo pathway constituents (e.g. Mst1/2) or overexpression of YAP induces liver tumor formation in mice, indicating that the Hippo pathway acts as typical tumor suppressor pathway, while YAP represents an oncogene in hepatocarcinogenesis (Strassburger et al., 2012 Dev Biol; Breuhahn et al., Dev Cell). We showed that YAP is overexpressed in a subgroup of human HCCs and that this factor supports tumor cell proliferation and invasion. Using cross-species comparison of mRNA expression profiles, the Notch receptor ligand Jagged-1 (Jag-1) was identified as central downstream target gene not only in HCC, but also in other gastrointestinal tumor entities (Tschahargeneh et al., 2013). Very recent work demonstrates that YAP induces a gene signature that characterizes HCC patients with chromosomal instability (CIN). Elevated expression of CIN genes is mediated by the transcription factor FOXM1, which is part of the CIN signature (Weiler et al., 2017 Gastroenterology). Ongoing projects are focusing on the molecular 'upstream' mechanisms leading to an aberrant enrichment of YAP in hepatocytes and HCC cells. On the other hand, we aim to identify druggable 'downstream' effector mechanism in liver tumor cells. 

Figure 2: The Hippo/YAP signalling in hepatocarcinogenesis. (A.) Activation of the evolutionary conserved Hippo pathway is associated with YAP phosphorylation, their cytoplasmic retention, and proteasomal degradation. In contrast, dephosphorylated YAP is efficiently transported into the nucleus. Since both transcriptional co-activators do not contain DNA-binding domains, their interaction with transcription factors (e.g. TEADs) is necessary to induce the expression of genes involved in the regulation of cell proliferation and anti-apoptosis. TF: transcription factor. (B.) The previously described CIN25 signature was identified in primary HCCs and defines patients with poor clinical outcome. (C.) Administration of Thiostrepton for 2 weeks reduced YAP-induced hepatomegaly in mice 8 weeks after YAPS127A induction. (D.) Thiostrepton injection reduced hepatocytic aneuploidy in YAP transgenic mice.

References  
  • Weiler SM, ... Breuhahn K. Induction of Chromosome Instability by Activation of Yes Associated Protein and Forkhead box M1 in Liver Cancer. Gastroenterology. 2017 (Epub ahead of print) (IF: 18.2)
  • Tschaharganeh DF, ... Breuhahn K. Yes-Associated Protein Up-regulates Jagged-1 and Activates the NOTCH Pathway in Human Hepatocellular Carcinoma. Gastroenterology. 2013 Jun;144(7):1530-1542. (IF: 18.2)
  • Straßburger K, ... Breuhahn K, Teleman AA. Insulin/IGF signaling drives cell proliferation in part via Yorkie/YAP. Dev Biol. 2012 Jul 15;367(2):187-96. (IF: 3.2)
  • Breuhahn K, Schirmacher P. A cellular view of Nf2 in liver homeostasis and tumorigenesis. Dev Cell. 2010 Sep 14;19(3):363-4. (IF: 9.3)


Far-upstream element (FUSE)-binding proteins (FBPs)

Based on comprehensive expression profiling of primary human HCC (Breuhahn et al., 2004 Cancer Res), we identified far-upstream element (FUSE)-binding factors (FBPs) as highly expressed in HCC cells (Figure 3). The FBP family consists of three family members (FBP-1, FBP-2, FBP-3), which exclusively recognize and bind single-stranded DNA structures (called FUSE) in promoters of target genes (e.g. the proto-oncogene c-Myc). We showed that nuclear FBP was detectable in about 60-70% of all HCC cases while low-level expression was observed in normal human hepatocytes (Malz et al., 2009, Hepatology). The gene-specific knockdown of FBPs in HCC cells revealed that FBP-1 and FBP-3 predominantly affected tumor cell proliferation, while inhibition of FBP-2 diminished growth factor-dependent tumor cell migration. These biological effects are partly mediated through the FBP-dependent activation of microtubule-interacting proteins such as Stathmin in HCC (Singer et al., 2007 Hepatology). Upstream regulation of FBP accumulation in HCC involves overexpression and nuclear translocation of E3 ubiquitin ligases such as seven in absentia homologue (Siah)-1 (Brauckhoff et al., 2011 J Hepatol; Malz et al., 2012 Int J Cancer). Our data also illustrates that the FBP-interacting repressor (FIR) is losing its inhibitory properties and stimulates oncogenic FBP expression (Malz et al., 2014 Hepatology). Very recent data shows that tumor-supporting FBP expression is mediated through the activation of the PI3K/Akt pathway in HCC cells (Samarin et al., 2016 Hepatology). Interestingly, all major findings were confirmed in other solid tumor types such as lung cancer, suggesting that these mechanisms are relevant for many human cancer types (Singer et al., 2009 Cancer Res, Müller et al., 2015 J Pathol).

Figure 3: FBP family members in hepatocarcinogenesis. (A.) The high degree of homology in the DNA binding domain indicates similar nucleic acid binding properties. In contrast, low amino acid homology in the transactivation and repressor domains suggests varying biological activity. The degree of homology for each FBP is indicated for each region. (B.) Immunohistochemical analysis of FBPs on TMAs containing human non-neoplastic liver tissues, premalignant lesions (DN), and HCC (grading: G1, G2, and G3). Arrows indicate FBP-1/2 staining in non-parenchymal cells (magnification x200). (C.) Correlation of FBP overexpression in HCC tissues with patient survival. (D.) FIR positively regulates FBP abundance in HCC cells. Immunoblot showing reduced FBP levels after FIR inhibition by siRNA (E.) FIR silencing slows down tumor growth in a subcutaneous murine xenograft transplantation model with HCC cells lacking different FIR isoforms.

References
  • Samarin J, ... Breuhahn K. PI3K/AKT/mTOR-dependent stabilization of oncogenic far-upstream element binding proteins in hepatocellular carcinoma cells. Hepatology. 2016 Mar;63(3):813-26. (IF: 11.7)
  • Müller B, ... Breuhahn K. Concomitant expression of far upstream element (FUSE) binding protein (FBP) interacting repressor (FIR) and its splice variants induce migration and invasion of non-small cell lung cancer (NSCLC) cells. J Pathol. 2015 Nov;237(3):390-401. (IF: 7.4)
  • Malz M, ... Breuhahn K. Nuclear accumulation of seven in absentia homologue-2 supports motility and proliferation of liver cancer cells. Int J Cancer. 2012 Nov 1;131(9):2016-26. (IF: 5.5)
  • Brauckhoff A, ... Breuhahn K. Nuclear expression of the ubiquitin ligase seven in absentia homolog (SIAH)-1 induces proliferation and migration of liver cancer cells. J Hepatol. 2011 Nov;55(5):1049-57. (IF: 10.6)
  • Malz M, ... Breuhahn K. Overexpression of far upstream element binding proteins: a mechanism regulating proliferation and migration in liver cancer cells. Hepatology. 2009 Oct;50(4):1130-9. (IF: 11.7)
  • Singer S, ... Breuhahn K. Coordinated expression of stathmin family members by far upstream sequence element-binding protein-1 increases motility in non-small cell lung cancer. Cancer Res. 2009 Mar 15;69(6):2234-43. (IF: 8.6)
  • Singer S, ... Breuhahn K. Protumorigenic overexpression of stathmin/Op18 by gain-of-function mutation in p53 in human hepatocarcinogenesis. Hepatology. 2007 Sep;46(3):759-68. (IF: 11.7)
  • Breuhahn K, et al., Molecular profiling of human hepatocellular carcinoma defines mutually exclusive interferon regulation and insulin-like growth factor II overexpression. Cancer Res. 2004 Sep 1;64(17):6058-64. (IF: 8.6)


Connecting of signaling pathways during liver regeneration and carcinogenesis

Previous data clearly indicate an intense cross talk between different signaling pathways under physiological and patho-physiological conditions (Strassburger et al., 2012 Dev Biol; Breuhahn 2006 Oncogene). Therefore, a deeper understanding of the spatio-temporal interplay between pathways is pivotal and may give important insights relevant for our understanding of impaired regeneration and the establishment of cancer therapeutics. In this setting, systems biology and systems medicine have emerged as viable tools to increase our knowledge of highly complex processes and systems such as tissue regeneration and tumors development. One aspect of our work is focussing on TNF-induced pathway activation in hepatic cells. Using freshly isolated murine hepatocytes and different immortalized HCC cell lines we have established a first mathematical model for TNF-induced NF-kB signalling (Beuke et al., 2017 FEBS J, Pinna et al., 2012 Front Physiol) (Figure 4). In collaboration with systems biologists, different signalling pathways, which are relevant for physiological liver regeneration and liver cancer, are currently integrated in a comprehensive network.
In this context, we are collaborating with different clinical, experimental, and theoretical groups to establish a multi-scale model for the growth and the early spread of non-small lung cancer cells (NSCLCs) (Singer et al., 2009 Cancer Res). This approach integrates mathematical models and information of different scales ranging from molecular (e.g. pathway models), cellular (e.g., models for tumor cell invasion), tissue (3D reconstructions) as well as clinical data (e.g., MRT and CT) in order to define the behaviour of tumor vascularization, growth and tumor cell dissemination with and without therapeutic perturbation.

Figure 4: Mathematical pathway modelling. Exemplary scheme of multi-scale integrative model topology describing NF-kB activation upon TNF stimulation. Endothelial liver cells (LSECs) and liver-resident macrophages (MCs) represent the major source for secreted TNF. The experimental data-based model consists of five compartments, with 33 computational species (molecules changing over time) described by 33 non-linear ordinary differential equations with a total of 67 parameters of which 35 were fitted during parameter estimations.

 References    
  • Y. Yin, ... Breuhahn K*, Vignon-Clementel I*, Drasdo D*. Tumor cell load and heterogeneity estimation from diffusion-weighted MRI calibrated with histological data: an example from lung cancer. IEEE Transactions on Medical Imaging 2017 (accepted for publication) (*shared senior authorship) (IF: 3.8)
  • Beuke K, ... Breuhahn K*, Sahle S*. Quantitative and integrative analysis of paracrine hepatocyte activation by nonparenchymal cells upon lipopolysaccharide induction. FEBS J. 2017 Mar;284(5):796-813. (*shared senior authorship) (IF: 4.2)
  • Pinna F, ... Kummer U, Breuhahn K. A Systems Biology Study on NFκB Signaling in Primary Mouse Hepatocytes. Front Physiol. 2012;3:466. (IF: 8.6)
  • Straßburger K, ... Breuhahn K, Teleman AA. Insulin/IGF signaling drives cell proliferation in part via Yorkie/YAP. Dev Biol. 2012 Jul 15;367(2):187-96. (IF: 4.0)
  • Singer S, ... Breuhahn K. Coordinated expression of stathmin family members by far upstream sequence element-binding protein-1 increases motility in non-small cell lung cancer. Cancer Res. 2009 Mar 15;69(6):2234-43. (IF: 8.6)
  • Breuhahn K et al., Dysregulation of growth factor signaling in human hepatocellular carcinoma. Oncogene. 2006 Jun 26;25(27):3787-800. (IF: 7.9)


Active collaborations
  • Prof. N. Gretz, Zentrum für Medizinische Forschung, Mannheim
  • Prof. V. Kalinichenko, Division of Pulmonary Biology at Cincinnati Children's Hospital Medical Center, Cincinnati
  • Prof. U. Kummer, Bioquant (Modeling of Biological Processes), Heidelberg
  • Prof. J. Marquardt, I. Department of Internal Medicine, University Medical Center, Johannes Gutenberg University Mainz
  • Prof. F. Matthäus, Center for Modeling and Simulation in the Biosciences (BIOMS) and Interdisciplinary Center for Scientific Computing, Heidelberg
  • Prof. Hanno Glim, Clinical and Translational Research Program - Applied Stem Cell Biology, Nationales Centrum für Tumorerkrankungen (NCT) Heidelberg
  • Prof. Eduard Ryschich, Department of Surgery, University Hospital Heidelberg
  • Alessandro Ori, Leipniz Institute on Ageing, Fritz Lipmann Institute, Jena
  • Dr. D. Drasdo, Institut National de Recherche en Informatique et aen Automatique (INRIA), Paris

Our work is supported by
  • The German Cancer Aid (Deutsche Krebshilfe e.V.)
  • The Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology (HBIGS)
  • The Alfonso Martin Escudero Foudation Fellowship
  • The China Scholarship Council (CSC) for Higher Education Exhibition

 

Former group members
  • Dr. Federico Pinna (Postdoc)
  • Dr. Jana Samarin (diploma student, Posdoc)
  • Dr. Mona Malz (PhD student, Postdoc)
  • Dr. Sabrina Schmitt (PhD student, Postdoc)
  • Dr. Benedikt Müller (PhD student)
  • Dr. Antje Brauckhoff (PhD student)
  • Dr. Tanja Nussbaum (PhD student)
  • Dr. Stephanie Schnickman (PhD student)
  • Dr. Sebastian Vreden (PhD student)
  • Dr. Charlotte Jankovitz (medical student)
  • Teresa Lutz (medical student)
  • Dr. Philipp Latzko (medical student)
  • Dr. Michael Bovet (medical student)
  • Dr. Pia Moinzadeh (medical student)
  • Anne-Sophie Meyer (physician)
  • Dr. Christian Rupp (physician)
  • Dr. Darjus Tschaharganeh (physician)
  • Dr. Stephan Singer (physician)
  • Vera Riehmer (diploma student)
  • Yelena Burda (diploma student)
  • Ute Müller (technician)
  • Petra Hubbe (technician)
  • Martina Keith (technician)
Print Diese Seite per E-Mail weiterempfehlen