Molecular biomarcers of canine and feline primary osteosarcoma

In the dog the osteosarcoma accounts for more than 85% of skeletal tumours. Amputation is the method of choice for controlling a primary appendicular tumour, however, this generally has little effect on long term survival except in the feline species. The analysis of canine and feline osteosarcoma tumour cells has a major advantage over other experimental designs which aim to identify novel biomarkers with crucial function in tumorigenesis and tumour behaviour. Examining the cat ostoesarcoma offers the possibility to study a species that is affected by osteosarcoma, however, does not develop metastases as often, whereas in contrast osteosarcomas in dogs commomly metastasize. Based on this biological conspicuty, we expect a difference in the biomarker profile (matrixmetalloproteinases (MMPs), ezrin, growth hormone (GR) and growth hormone receptor (GRR), insulin- like-growth factor (IGF), insulin-like-growth- factor binding protein (IGFBP), vascular endothelial growth factors (VEGF), platelet-derived growth factor (PDGF) between cat and dog helping us to identify key factors that contribute to tumor dissemination and the formation of metastases. Using immunohistochemistry, western blotting, zymography and gene expression profiling on tumour cells, should lead to better diagnostic, prognostic and therapeutic tools for the disease. In addition, miRNAs which are known to regulate gene expression post- transcriptional, are supposed to contribute to cancer. Therefore, we will compare canine and feline mRNA and miRNA profiles in healthy and neoplastic tissue (inlcuding cell lines) to contribute to the knowledge of the role miRNAs in osteosarcoma. RNA analyisis will be performed by the next generation sequencing method. Moreover, proteome analysis by MALDI-TOF should help to identify proteins that are involved in the metastasizing process. In the future, these informations should lead to a more targeted, individualized therapy of each patient. The VetBioBank, a facility that is engaged in standardized collecting and archiving animal tissue samples (in liquid nitrogen and paraffin) offers the opportunity to apply all histological and molecular biological methods on samples from one patient. In parallel, canine and human osteosarcoma cell lines will be examined with the same panel of methods as described above as they are frequently used for experimental applications. Our results will bring a detailed comparison of biomarkers of the osteosarcoma cells in vivo and in vitro. As the osteosarcoma in dogs is a recognized model for the human osteosarcoma, the results are of interest for the human species and may provide information for diagnostic and therapeutic purposes in clinical oncology as well.

Primary bone tumours (osteosarcomas) are the most frequent malignant bone tumours in human, dog and cat. Due to an impressive similarity of the human (children and young adults) and canine osteosarcoma the dog is an accepted comparative model for this disease. In both species the osteosarcoma is an aggressive tumour with a poor prognosis in case of metastasation. The dissemination of tumour cells occurs mainly to the lung via the bloodstream but also other bones are affected. The majority of canine patients die due to the fast developing distant metastases. This progressive course is in contrast to the feline species. In the cat, the development of metastases is rare. The reason behind this difference is unknown and has not been studied until today. Goal of our project was to test a number of biomarkers in osteosarcomas of the feline and canine species and to find out if one or more biomarkers are significantly different between these two species. These differential biomarkers could be involved in the diverse clinical behaviour of the tumours and open new possibilities for therapeutic approaches. Several biomarkers were addressed on the RNA and protein level and we were able to identify species-specific differences of the following factors: matrix metalloproteinase-2 (MMP-2), ezrin/radixin/moesin (ERM) protein family, erythropoietin receptor, and stem cell factor receptor (KIT). Matrix metalloproteinases are involved in migration processes and extravasation of tumor cells as they are capable to degrade the basement membrane matrix. Applying gelatine zymography we found in canine osteosarcomas significant more active MMP-2 compared to the cat. Moreover, in the canine osteosarcoma the amount of phosphorylated ezrin and moesin was higher than in the feline counterpart. Ezrin and moesin link the actin cytoskeleton to the cell membrane when they are activated and were shown to be necessary for tumor cell migration and survival of metastasis cells. This role of ezrin and moesin has been demonstrated for the dog and human species before, however, no data about the feline osteosarcoma existed so far. The stem cell factor receptor (KIT) was differentially expressed between cat and dog osteosarcoma. KIT mRNA was higher expressed in canine tumour samples compared to cat. Applying immunohistochemistry KIT was beyond the detection limit in feline osteosarcomas. In contrast EPO-receptor was higher expressed in feline osteosarcomas; interestingly, this was also the case for control tissues such as bone marrow and kidney. The biological meaning of this finding needs to be addressed in further studies. The identified differentially expressed biomarkers could potentially be involved in the metastasising behaviour of the osteosarcoma in cats and dogs. Moreover, if they can be verified in follow-up studies new therapeutic approaches could be opened that might also be relevant for human medicine as the dog osteosarcoma is an accepted model for the human paediatric osteosarcoma.