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Inhibitors of the plasminogen cascade - a possible treatment for the epithelial adhesion defect and tumor formation in JEB patients

Completed
Project lead Dr Laurent Gagnoux-Palacios
Organisation A.D.S.M. Association pour le Développement des Sciences Médiacles
Project budget EUR 163,800.00
Start date / Duration 01. Nov 2009 / 36 months
Funder(s) / Co-Funder(s) DEBRA Austria, MSAP/EBEP Recommended
Research area Skin cancer & fibrosis

Project details

Short lay summary

Uncontrolled degradation of the extracellular matrix leads to cell detachment, migration and tumor formation. Recent results of our group show that absence of Lm-332 impairs the matricial deposition of another important molecule called Perlecan. Absence of Perlecan deposition results in the activation of an enzymatic degradation process of the cutaneous extracellular matrix, which involves the protease uPA. We show that the enhanced uPA activity and the absence of Perlecan observed in JEB keratinocytes participate in the disadhesive phenotype and may favour the development of squamous cell carcinoma. Accordingly, transgenic animals with constitutive uPA activation develop a JEB like phenotype characterized by extensive alopecia and sub-epidermal blisters. Interestingly, we could also demonstrate that inhibition of the protease uPA in JEB keratinocyte cultures by specific inhibitors significantly improves cell adhesion. This suggests a potential therapeutic effect of uPA inhibitors for the treatment of the disadhesive phenotype associated with hampered expression of Lm-332. Interstingly, several uPA inhibitors are already on the market or in advanced preclinical studies for the treatment of various cancers.

In this context, we aim to develop clinical applications of uPA inhibitors for the prevention of blisters and SSC formation in patients suffering of EB.

Scientific summary

Uncontrolled degradation of the extracellular matrix leads to cell detachment, migration and tumor formation. We show that absence of Laminin-332 impairs the matricial deposition of another important molecule called perlecan. Absence of perlecan deposition results in the activation of an enzymatic degradation process of the cutaneous extracellular matrix, which involves the protease uPA. We also demonstrate that the enhanced uPA activity and absence of perlecan observed in JEB keratinocytes participate in the disadhesive phenotype and may favor the development of squamous cell carcinoma (SCC). Accordingly, transgenic animals with constitutive uPA activation develop a JEB like phenotype characterized by extensive alopecia and sub-epidermal blisters. Interestingly, we could also demonstrate that inhibition of the protease uPA in JEB keratinocyte cultures by specific inhibitors significantly improves cell adhesion. This suggests a potential therapeutic effect of uPA inhibitors for the treatment of the disadhesive phenotype associated with hampered expression of Laminin-332 and the development of squamous cell carcinoma. Interestingly, several uPA inhibitors are in advanced preclinical studies for the treatment of various cancers.

What did this project achieve?

During the grant period, we try to progress on the molecular mechanisms involved in the regulation of uPA proteolytic activity in EB and non-EB keratinocytes. We obtained data that allow us to propose a detailed mechanism of regulation of the uPA-dependent proteolysis in the skin. Considering the important role of perlecan in the regulation of uPA proteolytic activity, we study the mechanism of stabilization of this molecule in the ECM. We demonstrate that interaction of perlecan with a specific domain of Laminin-332 is critical for the efficient deposition of perlecan in the ECM. We also show that ECM deposition of perlecan and inhibition of uPA proteolytic activity in HK required maturation of intercellular junction. Intercellular junctions consist of multiprotein complexes that provide contact between neighboring cells. These junctions hold cells together and provide tissues with structural cohesion. We demonstrate that establishment of intercellular junctions is required to promote the ECM deposition of perlecan and to inhibit uPA-dependent proteolysis. Using carcinomas derived cell lines, we demonstrate that cancer cells failed to deposit perlecan in the ECM and display a sustained uPA proteolytic activity. We demonstrate that uncontrolled uPAdependent proteolysis in carcinoma is due to an alteration of intercellular junctions. These observations suggest for the first time that alteration of cell-cell junction lead to uncontrolled uPA-dependent proteolysis and extracellular matrix structuration. Finally, we demonstrate that sustained activation of the uPA-dependent proteolysis in both non-EB and DEB-derived carcinoma favors tumor cell invasion. Moreover, we demonstrate that inhibition of uPA enzymatic activity efficiently reduced cell invasion. Our data support the hypothesis that the use of uPA inhibitors may reduce SSC invasion capacity in patients suffering of EB.

In summary, our work allows us to propose a detailed mechanism of regulation of the uPA dependent proteolytic cascade in the skin. This model supports an essential role of perlecan-laminin-332 complex in the down-regulation of the proteolytic activity, suggesting that a defect in the deposition of one of this molecule result in an increase activity of uPA. We also demonstrate that establishment of intercellular junctions is crucial to regulate uPA proteolytic activity and deposition of extracellular matrix molecules. Interestingly, we demonstrate that in both non-EB and EB-derived SCC defect of intercellular junction stability correlates with an absence of perlecan deposition and a sustained uPA-activity. This uncontrolled proteolytic activity favors invasion of SCC in the surrounded matrix. In both non-EB and EB-derived SCC, inhibition of uPA activity using chemical inhibitors strongly reduces carcinoma cell invasion. Overall, this work clearly demonstrates that uPA inhibitors may be promising drugs to reduce the local invasion of cancer cells in DEB patients as well as a possible treatment to improve the epithelial adhesion defect in non-Herlitz JEB patients. Indeed, uPA inhibitors are considered as a promising approach in various cancer therapies to specifically block tumour metastasis in solid cancers.


Keywords

SSC
Gagnoux
INSERM
JEB
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