Abstract
BackgroundCanine mast cell tumour proliferation depends to a large extent on the activity of KIT, a tyrosine kinase receptor. Inhibitors of the KIT tyrosine kinase have recently been introduced and successfully applied as a therapeutic agent for this tumour type. However, little is known on the downstream target genes of this signaling pathway and molecular changes after inhibition.ResultsTranscriptome analysis of the canine mast cell tumour cell line C2 treated for up to 72 hours with the tyrosine kinase inhibitor masitinib identified significant changes in the expression levels of approximately 3500 genes or 16% of the canine genome. Approximately 40% of these genes had increased mRNA expression levels including genes associated with the pro-proliferative pathways of B- and T-cell receptors, chemokine receptors, steroid hormone receptors and EPO-, RAS and MAP kinase signaling. Proteome analysis of C2 cells treated for 72 hours identified 24 proteins with changed expression levels, most of which being involved in gene transcription, e.g. EIA3, EIA4, TARDBP, protein folding, e.g. HSP90, UCHL3, PDIA3 and protection from oxidative stress, GSTT3, SELENBP1.ConclusionsTranscriptome and proteome analysis of neoplastic canine mast cells treated with masitinib confirmed the strong important and complex role of KIT in these cells. Approximately 16% of the total canine genome and thus the majority of the active genes were significantly transcriptionally regulated. Most of these changes were associated with reduced proliferation and metabolism of treated cells. Interestingly, several pro-proliferative pathways were up-regulated, which may represent attempts of masitinib treated cells to activate alternative pro-proliferative pathways. These pathways may contain hypothetical targets for a combination therapy with masitinib to further improve its therapeutic effect.
Highlights
Canine mast cell tumour proliferation depends to a large extent on the activity of KIT, a tyrosine kinase receptor
There was an approximately 60% reduction in WST-1 conversion of masitinib treated cells at all indicated time points when compared to the initial activity of the untreated cells
Lactate dehydrogenase (LDH)-leakage assay identified a significant increase of 11.4% after 72 hours but no changes after 24 and 48 hours, Figure 1 Effect of masitinib on WST-1 conversion
Summary
Canine mast cell tumour proliferation depends to a large extent on the activity of KIT, a tyrosine kinase receptor. Canine mast cell tumours (MCT) are currently treated with one or a combination of four different therapeutic approaches: surgical, radiation, classical chemotherapy and the recently introduced tyrosine kinase inhibitors (TKI) [1,2,3]. The latter mainly act by inhibiting the stem. Tandem duplications in the juxtamembrane subunit of KIT have been identified in up to 12% of all canine MCT and 40% of malignant grade III MCT [6,7,8] This mutation results in a constitutively activated KIT tyrosine kinase and leads to increased malignant behaviour in most affected tumours [1]. The activity of non-KIT tyrosine kinases appear much lesser relevant for canine mast cell proliferation and the principle of TKI action in canine MCT is thought to mostly rely on KIT inhibition [1,13]
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