Proteolytic processing of the cellular prion protein (PrPC) in canine cancer cells and cancer stem cells in response to DNA damage
Cancer is a group of diseases associated with abnormal cell growth, space occupation and metastasis. Tumours consist of a heterogeneous mix of cells including a small population of cells, called cancer stem cells (CSCs) that are able to self-renew and differentiate into all cell types of the tumour, and are proposed to drive tumourigenesis. Traditional chemotherapy kills most of differentiated cancer cells, but CSCs are inherently resistant and are able to reinitiate tumour growth. Accumulating evidence indicates that targeting both cancer cells and CSCs is required to elicit a cure. Therefore, it is a necessity to understand the biology of all the cells within a tumour. Cellular prion protein (PrPC) is an extracellular membrane glycoprotein that is mainly known for its association with transmissible spongiform encephalopathies. Several types of human cancer express high levels of PrPC including: breast, gastric, glioblastoma and colorectal. Notably, PrPC has roles in self-renewal, anti-apoptosis and differentiation. However, the role and proteolytic processing of PrPC in canine cancer cells and CSCs remain unclear. In this project, we aimed to investigate the expression levels and relative amounts of different isoforms of PrPC in a panel of different canine cancer cell lines; to study the proteolytic processing of PrPC in response to doxorubicin, a commonly used chemotherapy; and to develop and optimise an assay to determine the relative amount of secreted PrPC in cell culture medium. We have found that PrPC is detected in several canine cancer cell lines by western blot and that different cancer cell lines have different PRNP expression patterns. In response to DNA damage by doxorubicin, both a canine mammary inflammatory carcinoma (Lily) and a canine glioma (J3T) cell line, showed increases at the protein and the mRNA level of PrPC. PrPC is subject to proteolytic processes, including: α- cleavage, β-cleavage and shedding, which produce C1/N1, C2/N2 and full-length isoforms respectively. We have demonstrated that the ratio of full-length PrPC to C1 increases after DNA damage treatment. Conversely, the ratio of full-length PrPC to C1 decreased in CSCs. We also identified that different isoforms are degraded independently: the half-life of C1 becomes longer under the stress of chemotherapy, whereas, the half-life of full-length becomes shorter. Together these data indicate that the different isoforms may have independent biological significance in cancer cells. PrPC is also cleaved after the GPI anchor and secreted into the cell culture medium. To investigate the secreted isoforms of PrPC we developed and optimised an immunoprecipitation protocol. In conclusion, PrPC and its isoforms are detectable in different canine cancer cells and is expressed at different levels. The role of PrPC in cancer cells and CSCs may differ when stressed under chemotherapy, and we present evidence that C1 may act distinctively from full-length PrPC in response to chemotherapy. Future studies are needed to investigate the role of different isoforms of PrPC in canine cancer cells and CSCs in specific pathways relating to cell survival, apoptosis and metastatic potential, and to evaluate PrPC and its isoforms as a therapeutic target in oncotherapy.