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

Abstract

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.

Estrogen receptor-α, progesterone receptor, and c-erbB/HER-family receptor mRNA detection and phenotype analysis in spontaneous canine models of breast cancer

“…Continuing characterization of genetic defects in these cell lines has focused on obtaining evidence of selection for atypical clones that are not representative of the primary tumor types. Although many of these cell lines have been in culture for multiple passages and no original primary tumor biopsy samples exist, comparison to fresh biopsy specimens from canine patients has confirmed the conserved nature of many of the primary genetic defects, especially those associated with the cyclin-dependent kinase tumor suppressor gene p16/INK4A [1819]. The data presented here demonstrate that this similarity can be extended beyond the tumor suppressor genes to include an array of cell surface receptors, including those reported to promote breast cancer in humans.…”

“…Combining such analyses with known expression risk factors, such as those reported here, should promote more precise and personalized diagnosis of breast cancer in both species. Our previous discoveries of common tumor suppressor cyclin-dependent kinase inhibitor defects in canine mammary cancers, especially in the combined p16/INK4A and p14ARF locus, are important examples of approaches that can provide enhanced diagnostic power and point the way toward effective therapeutic targets [212181920]. …”

“…CMT cell lines and canine mammary epithelial cells (CMECs) were cultured in L-15 medium (Gibco, USA) with antibiotics (Sigma, USA), and 10% FBS (fetal bovine serum; HyClone, USA) in tissue culture flasks (Corning, USA) at 37℃ (95% air; 5% CO 2 ) as previously described [61213182030]. All use of animal cell cultures was under the guidance and review of the Auburn University Institutional Animal Care and Use Committee (protocols PRN 2009-1633, PRN 2007-1155, PRN 2005-0826).…”

“…Spontaneously occurring canine mammary cancers have been used to model human breast cancer in terms of causative mutations as well as for development of new treatment strategies for both species [271820]. A more rapid and quantitative assay, based on quantitative reverse transcriptase polymerase chain reaction (QrtPCR), of canine mammary cancer transcripts would promote more precise mRNA-based phenotype determination of canine breast cancer subtypes, promote utilization of more effective and precise treatments, and promote development or adaptation of new therapies designed to target-specific breast cancer subtypes in both species.…”

“…Investigation of canine mammary cancer genetics has been a long-standing focus of our laboratory both for the purpose of understanding genetic defects that promote these tumors and of providing a more comparative perspective on mammary cancers in dog and human patients [261213181920]. Such immune-intact models provide the opportunity for development of advanced immune-modulatory therapies and represent a more similar pathology than murine models [7].…”