Poster Presentation 31st Lorne Cancer Conference 2019

Investigating the contribution of legumain to oral cancer pain (#118)

Bethany M Anderson 1 , Brian L Schmidt 2 , Laura E Edgington-Mitchell 1 2 3
  1. Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria, Australia
  2. Department of Maxillofacial Surgery, College of Dentistry, New York University, New York, USA
  3. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia

Oral cancer is the 6th most common form of cancer worldwide. Oral cancer is extremely painful, and patients suffer from profound difficulties in talking, eating, and drinking. Previous studies have demonstrated that this pain is at least partially mediated by activation of protease-activated receptor 2 (PAR2), a G protein-coupled receptor (GPCR) found on the surface of primary sensory neurons; however, the PAR2-activating proteases within the oral cancer microenvironment have not yet been identified.

 

Using activity-based probes, we measured the activity of cysteine and serine proteases in human oral squamous cell carcinomas (SCC) and patient-matched normal oral mucosa. We found that the activity of legumain, an asparginyl endopeptidase, was strongly upregulated in the tumours of all patients examined. We validated this finding in two murine models of oral cancer: HSC-3 orthotopic xenografts and 4NQO carcinogen-induced SCC. Immunohistological analysis revealed that legumain is expressed predominantly at the invasive edge of tumours.

 

We next investigated the relationship between legumain, PAR2 and pain. Using mass spectrometry, we found that legumain could cleave peptides corresponding to the N terminus of human PAR2 at Asn30. Using FLAG-tagged PAR2, we also demonstrated that legumain could cleave the intact receptor at the surface of HEK cells. When applied to cultured trigeminal neurons, recombinant legumain invoked a decrease in rheobase that could be prevented by pre-treatment with a PAR2 antagonist. Furthermore, legumain induced calcium mobilisation in wildtype, but not PAR2-deficient, neurons. Both of these results indicate that legumain can activate neurons in a PAR2-dependent manner. When applied directly to the mouse tongue, legumain induced orofacial dysfunction in wildtype, but not PAR2-deficient mice. Finally, administration of a legumain inhibitor to mice with xenografted oral cancer resulted in reduced mechanical sensitivity. Collectively, these results suggest that legumain is a novel activator of PAR2 and that its activity may contribute to oral cancer pain. Future studies will carefully dissect the signalling pathways invoked by legumain.