The proto-oncogene c-KIT is a tyrosine kinase receptor that plays important roles in many pathways. Mutations of the c-KIT gene have been reported in many types of tumors, but it is not clear if the mutant c-KIT isoforms observed can drive tumorigenesis. The mutational spectrum observed in c-KIT in dogs is slightly different than in humans and ranges from deletion mutations to a varying set of tandem repeat mutations in the juxtamembrane domain. In dogs, c-KIT mutations correlate with tumor grade and poor survival. To study the molecular and cellular changes resulting directly from mutations in c-KIT, expression vectors containing normal and mutant canine KIT genes were constructed in the plasmid vector, pACGFP1N1, and transfected into canine primary fibroblasts with the resultant cells and progeny selected for stable expression of the plasmid. The mutations included three deletion mutations and three internal tandem duplications, all within exon 11. As fibroblasts do not constitutively express c-KIT, any effect on phenotype can be attributed to the experimentally induced expression. In the canine primary fibroblast context, ectopic expression of c-KIT mutant isoforms caused the immotalization, malignant transformation, chromosomal instability, and soft tissue sarcoma formation in immunodeficient mice. In the human fibroblast context, the retroviral vectors carrying c-KIT mutations observed in human GISTs, canine GISTs, and canine mast cell tumors were transduced into human primary fibroblasts. Functional consequences derived from ectopic expression showed that mutations identified in GISTs can lead to extended lifespan and immortalization, whereas cells transduced with duplication mutations reported uniquely in canine mast cell tumors resulted in premature senescence. Unlike the results obtained from canine primary fibroblasts, the introduction of a single hit of mutant c-KIT into human primary fibroblasts was demonstrated to have immortalization capacity but not transformation activity, which suggests that additional genetic alteration(s) may be required for c-KIT driven tumorigenesis in the human context. In conclusion, our findings have shown the impact of different c-KIT mutation isoforms in tumorigenesis within the context of primary fibroblasts. Preclinical study models of cells carrying specific genetic alterations that are responsible for diseases are needed to improve the processes of clinical study. As dogs and humans share close similarity in their genome contents and living environments, comparative medicine using canine cells as study models will provide much needed information on tumorigenesis and new cancer therapies in humans.
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