Novel frameshift mutation in the p16/INK4A tumor suppressor gene in canine breast cancer alters expression from the p16/INK4A/p14ARF locus.

The INK4 family of cyclin-dependent kinase inhibitors (CKI) encode important cell cycle regulators that tightly control cell cycle during G1 to S phase. These related genes are considered tumor suppressors as loss of function contributes to the malignant phenotype. Expression of CKIs p16, p14ARF, or p15 were defective in six different canine mammary tumor (CMT) cell lines compared to normal thoracic canine fibroblasts. This suggests CKI defects are frequently responsible for neoplastic transformation in canine mammary carcinomas. p16 and p14ARF are two alternatively spliced products derived from the canine p16/INK4A/p14ARF gene locus. Despite omissions in the published p16 transcript and canine genome and the presence of GC-rich repeats, we determined the complete coding sequence of canine p16 revealing a deletion and frameshift mutation in p16 exon 1α in CMT28 cells. In addition, we determined canine p14ARF mRNA and protein sequences. Mapping of these mutations uncovered important aspects of p16 and p14ARF expression and defects in CMT28 cells shifting the p16 reading frame into p14ARF making a fusion protein that was predicted to be truncated, unstable and devoid of structural and functional integrity. This data describes an important neoplastic mechanism in the p16/INK4A/p14ARF locus in a spontaneous canine model of breast cancer.

Frequent genetic defects in the p16/INK4A tumor suppressor in canine cell models of breast cancer and melanoma.

The cyclin-dependent kinase inhibitors (CKIs) belong to a group of key cell cycle proteins that regulate important cancer drug targets such as the cyclin/CDK complexes. Gene defects in the INK4A/B CKI tumor suppressor locus are frequently associated with human cancers and we have previously identified similar defects in canine models. Many of the cancer-associated genetic alterations, known to play roles in mammary tumor development and progression, appear similar in humans and dogs. The objectives of this study were to characterize expression defects in the INK4 genes, and the encoded p16 family proteins, in spontaneous canine primary mammary tumors (CMT) as well as in canine malignant melanoma (CML) cell lines to further develop these models of spontaneous cancers. Gene expression profiles and characterization of p16 protein were performed by rtPCR assay and immunoblotting followed by an analysis of relevant sequences with bioinformatics. The INK4 gene family were expressed differentially and the genes encoding the tumor suppressor p16, p14, and p15 proteins were often identified as defective in CMT and CML cell lines. The altered expression profiles for INK4 locus encoded tumor suppressor genes was also confirmed by the identification of similar gene defects in primary canine mammary tumor biopsy specimens which were also comparable to defects found in human breast cancer. These data strongly suggest that defects identified in the INK4 locus in canine cell lines are lesions originating in spontaneous canine cancers and are not the product of selection in culture. These findings further validate canine tumor models for use in developing a clear understanding of the gene defects present and may help identify new therapeutic cancer treatments that restore these tumor suppressor pathways based on precision medicine in canine cancers.