Oral cancer is the 6th most common form of cancer worldwide. The mechanism behind oral carcinogenesis is poorly understood and current treatment strategies are ineffective. Survival rates drop significantly once metastasis develops and predicting which cancers will go on to metastasise is currently not possible. Further mechanistic studies are thus required to improve patient outcomes. Legumain is a cysteine protease that has previously been reported to contribute to the carcinogenesis of several cancers. We hypothesised that legumain may also contribute to other features of oral carcinogenesis.
Using the activity-based probe LE28, we measured legumain activity in human oral squamous cell carcinomas (OSCC) and patient-matched normal oral mucosa. We found that legumain activity was strongly upregulated in tumours of all patients examined compared to normal oral mucosa. We further validated this finding using tumour samples from two murine models of oral cancer: orthotopic xenografts of HSC-3 human OSCC cells and 4NQO carcinogen-induced OSCC. Immunohistological analysis of samples from these mice revealed that legumain is expressed predominantly at the invasive edge of tumours.
Using CRISPR-Cas9, we generated legumain-deficient HSC-3 cells and analysed them for features of carcinogenesis in vitro and in vivo. Compared to wild-type cells, legumain knockout cells exhibited reduced proliferation and invasion. When legumain-deficient HSC-3 cells were inoculated subcutaneously in mice, the resulting tumours exhibited >90% reduction in volume compared to tumours generated from wild-type cells. Thus, it appears that tumour-derived legumain promotes oral cancer growth.
Collectively, these results demonstrate that legumain is highly activated in the oral cancer microenvironment and may contribute to tumour growth and invasion. Future studies aim to distinguish the proteolytic and non-proteolytic functions of legumain in the oral cancer microenvironment as well as its cell-specific roles. Ultimately, we aim to determine whether legumain inhibitors will have therapeutic value, resulting in improved outcomes for patients.