Correlation between proteasomes activity, clinical behaviour and histopathological grading in feline and canine malignantspontaneous tumours

The proteasome is an abundant multi-enzyme complex that provides the main pathway for degradation of intracellular proteins in eukaryotic cells. As such, it controls the levels of proteins that are important for cell-cycle progression and apoptosis in normal and malignant cells. Furthermore, the proteasome generates the antigenic peptides (epitopes) that are displayed on the cell surface in association with Major Histocompatibility Complex (MHC) class I molecules. By screening for non-self MHC-bound peptides, the immune system identifies and then can eliminate cells that are producing viral or mutant proteins (e.g. products of oncogene). The aim of this research is to evaluate whether alterations in the mechanism of MHC class I antigen presentation in different canine and feline tumors can provide malignant cells with a way to escape the control operated by the immune system. Furthermore, since proteasome inhibitors have shown anticancer activity and are now in clinical use against some human tumors, with our study we expect to obtain indispensable data for a veterinary use of these new therapies. To these purposes, clinical, biochemical and histopathological aspects of tissues obtained from dogs and cats affected by tumors at different developmental stages will be considered. The clinical phase of the study will include the routine clinical evaluation of theanimals, administration of neoadjuvant chemotherapy (when feasible), and the surgical excision of the tumor with wide margins. The tissue samples obtained will be sent to the biochemical and histopathological units. In cases in which chemotherapy will be administered, also part of the incisional biopsy sample (before drug administration) will be sent to the laboratory. All the animals treated will be reevaluated periodically for the presence of local recurrence or metastasis for 24 months. The expression levels and functional activities of proteasomes in tumor and healthy tissues will be evaluated by the biochemical unit. Specifically, the levels of the catalitic subunits of proteasome and immunoproteasome, of the alfa and beta subunits of the proteasome activator PA28 and of the enzyme Leucine Aminopeptidase will be tested by Western Blotting with specific antibodies. Furthermore, the functional activities of these proteins will be measured by spectrofluorimetric assays using specific fluorogenic peptides. Grading, evaluation of the proliferation and apoptotic indexes will be determined by the histopathological unit. Furthermore, immunohistochemical analysis of several components of the proteasome system will be evaluated. Therefore, with this research we'll obtain important data to study tumor immunosurveillance alterations not only in veterinary medicine but also in human oncology and we'll get useful information for a veterinary clinical use of proteasome inhibitors.