Application of metabolomics and proteomics for biomarker discovery and development of therapies for the transmissible cancer, Tasmanian Devil Facial Tumour Disease

The wild population of Tasmanian devils has declined by more than 80% due to the fatal Devil Facial Tumour Disease (DFTD), one of the few known naturally-occurring, clonally transmissible cancers. There is currently no pre-clinical test or effective treatment for this disease that threatens the species with extinction. Our major goals are to develop a diagnostic test to detect the disease at the latent (pre-clinical) stage and to evaluate immunotherapeutic drugs, such as the approved topical agent imiquimod (R-837), as potential treatments for this aggressive cancer. Biomarker discovery for DFTD was performed using non-targeted metabolomics analysis of serum samples collected from wild Tasmanian devils (35 controls and 35 with tumours) by HPLC/MS. Multivariate models (PLS/DA, random forests) were used to identify potential discriminating features in an age-matched training set. Positive markers for DFTD included amino acids, metabolites related to lipid absorption and a series of overlapping fibrinogen peptides, while cortisol and urea were the most significant health predictors. A support vector machine model utilizing only the major peptide and seven other metabolites was able to classify samples with 94% sensitivity and specificity. Label-free shotgun proteomics was used to study the effects of imiquimod on DFTD tumour cells. Imiquimod is a toll-like receptor 7 (TLR7) agonist that in humans induces immune cell infiltration and cytotoxic immune pathways. Imiquimod may augment this response by direct activation of tumour cell apoptosis, but the underlying mechanism is unknown. Many of the proteins altered by imiquimod were related to ER stress, cell cycle arrest and increased antioxidant activity. Unlike DFTD tumour cells, imiquimod-treated Devil fibroblasts (TLR7-) lacked the hallmarks of ER stress and did not undergo apoptosis. Targeted induction of ER stress is therefore one potential mechanism for the selective effects of imiquimod on DFTD cells and ER stress pathways represent novel targets for DFTD treatment.