Altered microRNA Expression Profiles and Regulation of INK4A/CDKN2A Tumor Suppressor Genes in Canine Breast Cancer Models

Abstract

microRNA (miRNA) expression profiling of cancer versus normal cells may reveal the characteristic regulatory features that can be correlated to altered gene expression in both human and animal models of cancers. In this study, the comprehensive expression profiles of the 277 highly characterized miRNAs from the canine genome were evaluated in spontaneous canine mammary tumor (CMT) models harboring defects in a group of cell cycle regulatory and potent tumor suppressor genes of INK4/CDKN2 family including p16/INK4A, p14ARF, and p15/INK4B. A large number of differentially expressed miRNAs were identified in three CMT cell lines to potentially target oncogenes, tumor suppressor genes and cancer biomarkers. A group of the altered miRNAs were identified by miRNA target prediction tools for regulation of the INK4/CDKN2 family tumor suppressor genes. miRNA-141 was experimentally validated for INK4A 3'-UTR target binding in the CMT cell lines providing an essential mechanism for the post-transcriptional regulation of the INK4A tumor suppressor gene in CMT models. A well-recognized group of miRNAs including miR-21, miR-155, miR-9, miR-34a, miR-143/145, and miR-31 were found to be altered in both CMTs and human breast cancer. These altered miRNAs might serve as potential targets for advancing the development of future therapeutic reagents. These findings further strengthen the validity and use of canine breast cancers as appropriate models for the study of human breast cancers.

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

“…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).…”

“… * Originally derived by Dr. L. Wolfe or were derived by the authors [62030]. All CMT cell lines, as well as the primary canine mammary epithelial cells (CMEC), were derived and cultured as previously described [13].…”

“…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].…”