Ploug Group

Current projects

We are currently studying the interaction between the urokinase type plasminogen activator (uPA) and its receptor (uPAR) from the nano- to the macro-scale levels in biology, i.e. covering protein structure, biochemistry, cell biology, tissue expression, and non-invasive imaging in vivo. The ultimate goal for these studies is to clarify and understand the molecular details governing this interaction, which enables a rational design of the functional properties of uPA and uPAR, and assists development of small molecules suitable for therapeutic intervention and/or non-invasive imaging. A major focus of our investigations is to translate these discoveries into the field of clinical oncology - as illustrated below.



Protein structure-function relationships with a view to rational targeting
Due to the involvement of the uPA•uPAR axis in focalizing plasminogen activation to the cell surface and its role in degradation of the extracellular matrix during cancer invasion and metastasis, we are studying the interactions between uPAR and its natural ligands in great detail. Among other achievements, we have solved the first crystal structures of human and mouse uPAR in complex with an antagonist peptide and the bona fide protease ligand uPA, respectively. Using this information, we have now been able to swap the species specificity of the uPA•uPAR interaction by rational design and use this knowledge to generate a new transgenic mouse strain, which provides important information on the functional role of the uPA•uPAR interaction sensu stricto for fibrin homeostasis under normal physiologic conditions. Presently, we are investigating the role of the different conformations of the receptor that seem to exist for the unoccupied and ligand-bound forms. This difference appears important for the engagement of uPAR in the interplay with matrix-embedded vitronectin and has wide implications for the development of small antagonists targeting the ligand-binding cavity in uPAR.

Another line of research derived from our structural studies on the uPA•uPAR interaction is the development and refinement of high-affinity peptide antagonists, which are useful tools for targeted intervention of cell surface plasminogen activation and for non-invasive imaging of the uPAR in vivo (as outlined in the next section).
To study these interactions, we exploit a number of advanced technologies, which are available either in house or through external collaborations, such as X-ray crystallography, hydrogen-deuterium exchange analyzed by mass spectrometry, SAXS, MALDI-MS, surface plasmon resonance, and CD spectroscopy. Combined with our efficient expression platform for production and purification of recombinant proteins (> 500 individual mutants in our “protein libraries”), this enables us to address these interactions in a highly versatile and efficient manner.




Non-invasive imaging
The ability to detect and localize a certain protein target in vivo by non-invasive imaging is self- evidently an important tool for selection of those patients that may eventually benefit from a given targeted therapy and for the subsequent evaluation of the efficacy of this intervention therapy. We have therefore embarked on the development and refinement of our small peptide antagonists as highly specific beacons for non-invasive imaging of uPAR expression in live animals with a view to future applications in cancer patients. Guided by our knowledge on the structure-functional relationships of the uPA•uPAR interaction described above, we have recently designed highly efficient radionuclide-based imaging probes, which successfully detect uPAR in transplanted cancer models in live mice by positron emission tomography (PET). The versatility of these uPAR-specific targeting probes is being further explored including conjugations to new optimized beacons for improved detection in non-invasive imaging as well as a uPAR-specific targeting principle for nanoparticles. In a well-established collaboration with Dr Andreas Kjær (Cluster for Molecular Imaging & Klinisk Fysiologi, Nuclearmedicin & PET), we are presently exploring the utility of these uPAR-specific imaging probes in various animal model systems with a view to translation into a clinic setting.




Gastric cancer and Helicobacter infection
Gastric cancer is still presenting a high incidence and mortality rate globally, and one of our lines of research focuses on investigating the role of uPAR in the development and progression of this malignancy. We have characterized the expression pattern and localization of uPAR in gastric cancer patient material from Costa Rica and Norway, which are countries with high- and low-risk for this disease, respectively. We have also studied the clinical usefulness of uPAR as a prognostic factor in gastric cancer, emphasizing the contribution to the prognosis of particular uPAR-expressing cell types, as interrogated by semi-quantitative immunohistochemistry. Our localization studies in human non-neoplastic gastric mucosa have shown that uPAR is expressed by epithelial cells that are in close proximity to Helicobacter pylori bacteria, which is the most well-established risk factor of gastric cancer. Given the important implications that this may have for the etiology of gastric cancer, we are currently systematically investigating the potential link between these two parameters in experimental Helicobacter-based mouse models of gastric carcinogenesis.




C4.4A expression and prognostic significance in non-small cell lung cancer
In another line of research, we focus on a structural uPAR homologue denoted C4.4A. Through prognostic and descriptive studies using immunohistochemistry, we investigate the expression of C4.4A in non-small cell lung cancer. We have shown that high levels of C4.4A correlate to a very poor survival of patients with adenocarcinoma, the most common histologic subtype of non-small cell lung cancer. On the contrary, in the other major subtype, squamous cell carcinoma, C4.4A does not provide any prognostic information. Reasons for this have been revealed in another study on precursor lesions of malignant pulmonary carcinomas, where C4.4A consistently shows an intense expression in very early changes (bronchial metaplasia) in the progression to squamous cell carcinoma. This indicates that C4.4A could be a marker of early squamous differentiation, a hypothesis that we are presently looking into. C4.4A also appears at early stages (atypical alveolar hyperplasia) of the progression to adenocarcinoma, but in this case in only a fraction of these lesions. This might be connected to the fraction of adenocarcinoma patients having high levels of C4.4A, thus making C4.4A a possible marker for the detection of more malignant adenocarcinomas. Accordingly, we are currently interrogating the clinical potential of C4.4A. On a broader level, we are pursuing studies on the as yet unknown biological function of C4.4A, which could explain the prognostic impact of C4.4A in pulmonary adenocarcinoma.




C4.4A and Haldisin in benign and malignant skin lesions
We have recently found that the expression of two GPI-anchored structural homologues of uPAR (C4.4A and Haldisin) define distinct differentiation states of the squamous epithelium. We have therefore initiated a project aiming at comparing their expression patterns during various pathological conditions of the skin, where the lesions compromise the functional and structural integrity of the squamous epithelium. This will include various inherited and acquired blistering diseases such as pemphigus, pemphigoid, icthyosis, epidermolysis bullosa, and Nethertons syndrome. We will also put emphasis on benign and malignant invasive conditions such as the pre-cancerous keratoacanthomas and actinic keratosis lesions, the low-malignant basal cell carcinoma as well as the highly malignant squamous cell carcinoma and melanoma.



National and international collaborators:
This research is executed by the collaborative effort of a well-established national and international network including:

Drs. Mingdong Huang & Lin Lin (BDIMC, Harvard, Boston, US & FIRMS, Fuzhou, China).
Dr. Thomas Bugge (NIH, Bethesda, US).
Dr. Thomas Muley (Section for Translational Research, Thoraxklinik, Heidelberg, Germany).
Dr. Ole Petter Clausen (Dept. Pathol., Oslo, Norway).

Dr. Andreas Kjær (Cluster for Molecular Imaging & Klinisk Fysiologi, Nuclearmedicin & PET, Rigshospitalet, Copenhagen, Denmark).
Dr. Thomas D. J. Jørgensen (Dept. Biochem. Mol Biol., SDU, Odense, Denmark).
Drs. Peter Andreasen & Ernst-Martin Füchtbauer (Dept. Mol Biol., Aarhus University, Denmark).
Dr. Eric Santoni-Rugiu (Dept. Pathol., Rigshospitalet, Copenhagen, Denmark).
Dr. Masood Hosseini (Dept. Chem., Life, Frederiksberg, Denmark).
Dr. Søren Østergaard (DPPC, NOVO Nordic, Måløv, Denmark).