RT‐PCR based Tyrosine Kinase Display Profiling of Canine Melanoma: IGF‐1 Receptor as a Potential Therapeutic Target

Introduction:  There is a wealth of information available regarding tyrosine kinase (TK) expression in human cancer, however there is minimal information regarding the expression and function of TKs in canine melanoma, and no attempt has been made to systematically define the repertoire of TKs expressed. This study employed a molecular technique called RT‐PCR display to simultaneously evaluate the expression of up to 30 different TKs in a canine melanoma cell line.
Materials and Methods:  mRNA was extracted and reverse‐transcribed from the 17CM98 canine melanoma line, then subjected to PCR using degenerate primers coding for highly conserved regions which flank the kinase domains of many receptor and nonreceptor TKs. The resulting product was ligated into a plasmid vector and used to transform E. coli . Multiple colonies were isolated, and the cDNA inserts sequenced.
Results and Conclusions:  Sequencing 46 clones yielded canine homologs of IGF‐1R (50%), JAK1 (17%), PDGFR‐α(11%), FGFR1 (9%), Axl (7%), c‐Abl (4%), and PTK2 (2%). Interestingly, IGF‐1R, JAK1 and Axl were detected using a similar technique in human melanoma, supporting the cross‐species validity of this assay. Given the abundance of IGF‐1R clones, we sought to determine the biologic effect of rhIGF‐1 in 17CM98 cells. IGF‐1 stimulated IGF‐1R phosphorylation, cell proliferation and VEGF production in 17CM98, and protected the cells from serum starvation‐induced apoptosis. Expression of IGF‐1R mRNA was detected in 5 of 5 additional canine melanoma cell lines evaluated, suggesting that IGF‐1R expression may be common in canine melanoma cells and providing a novel target for future therapy.