Kliniken & Institute ... Institute Pathologisches Institut... Allgemeine Pathologie ... Forschung Arbeitsgruppen AG Breuhahn

Breuhahn Group

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 complicates the development of effective drugs, which is reflected by the current lack of therapeutic options.

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. In carcinogenesis, many TRs as well as upstream regulators have been described as deregulated, which Leads to uncontrolled tumor cell proliferation and resistance to apoptotic stimuli. 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 involved in the regulation of regenerative processes as well as development and progression of HCC. In collaboration with systems biologists, we aim to develop comprehensive mathematical pathway models, which describe dynamic processes at different scales (molecular, cellular, and tissue levels).

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 (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, 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). 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 (Wei et al., 2019 Oncogene; Wan et al.  2018 Hepatology; 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

  • Wei T, ... Breuhahn K. YAP-dependent induction of UHMK1 supports nuclear enrichment of the oncogene MYBL2 and proliferation in liver cancer cells. Oncogene. 2019 Jul;38(27):5541-5550. (IF: 6.6)
  • Wan S, ... Breuhahn K. Cytoplasmic localization of the cell polarity factor scribble supports liver tumor formation and tumor cell invasiveness. Hepatology. 2018 May;67(5):1842-1856. (IF: 15.0)
  • Weiler SME, ... Breuhahn K. Induction of Chromosome Instability by Activation of Yes-Associated Protein and Forkhead Box M1 in Liver Cancer. Gastroenterology. 2017Jun;152(8):2037-2051. (IF: 20.8)
  • 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: 13.9)
  • 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)

Connecting of signaling pathways during liver regeneration and carcinogenesis

Previous data illustrate an intense cross talk between different signaling pathways under physiological and patho-physiological conditions (Strassburger et al., 2012 Dev Biol). 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 3). 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 tumor growth. 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 3: Mathematical pathway modelling. Exemplary scheme of multi-scale integrative model topology describing NF-kB activation upon TNF stimulation. Liver sinusoidal endothelial 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

  • Pinna F, ... Breuhahn K. A20/TNFAIP3 Discriminates Tumor Necrosis Factor (TNF)-Induced NF-κB from JNK Pathway Activation in Hepatocytes. Front Physiol. 2017 Aug 23;8:610. (IF: 3.2)
  • 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: 3.6)
  • 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: 3.2)

Currently not in the lab

Group Members

Active collaborations

  • Prof. N. Gretz, Zentrum für Medizinische Forschung, Mannheim
  • 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. Hanno Glim, Clinical and Translational Research Program - Applied Stem Cell Biology, Nationales Centrum für Tumorerkrankungen (NCT) Heidelberg
  • Prof. Mathias Heikenwälder (Deutsches Krebsforschungszentrum, DKFZ)
  • Prof. Ahmed Abdelaziz Fahmy (NewGiza University, Cairo, Egypt)

Our work is currently supported by

  • Deutsche Forschungsgemeinschaft (DFG)
  • The Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology (HBIGS)
  • The China Scholarship Council (CSC) for Higher Education Exhibition

Former group members

  • Maria Knaub (PhD student)
  • Dr. Shan Wan (PhD student)
  • Dr. Margarita González-Vallinas Garrachón (Postdoc)
  • Dr. Federico Pinna
  • Dr. Jana Samarin (diploma student)
  • Dr. Mona Malz (PhD student, Postdoc)
  • Dr. Sabrina Schmitt (Postdoc)
  • Dr. Benedikt Müller ()
  • Dr. Antje Brauckhoff (PhD student)
  • Dr. Tanja Nussbaum (PhD student)
  • Dr. Stephanie Schnickman (PhD student)
  • Dr. Sebastian Vreden (PhD student)
  • Dr. Teng Wei (Dr. sc. hum. student)
  • Simone Marquard (medical 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)
  • Georg Beresin (trainee)
  • Christina Bär (trainee)