Altered Expression Profiles and Defects in a Group of Cell Cycle Regulators and Tumor Suppressor Genes (INK4) and Evaluation of Comprehensive Expression Profiles of Canine miRNAs in Spontaneous Canine Breast Cancer Models

Abstract

The cyclin-dependent kinase inhibitors (CKIs) act as powerful cell cycle regulators and endogenous tumor suppressors. Defects in members of the INK4A/B CKI tumor suppressors have been associated with human and canine cancers. Most of the cancer-associated genetic alterations that are known to play roles in mammary tumor development and progression are similar in both species. The objectives of this study were to evaluate genetic regulation and defects in INK4 genes as well as the comprehensive expression profiles and altered regulation of miRNAs in spontaneous canine mammary tumor (CMT) and malignant melanoma (CML) models. Gene expression profiles and sequencing of INK4 genes have been evaluated by RT-PCR, rapid amplification of cDNA ends (RACE)-PCR, and touchdown-PCR assays as well as subsequent cloning experiments. The sequences were analyzed by Vector NTI and bioinformatics tools. The comprehensive miRNA expression profile was evaluated by miRNA qPCR arrays. Members of the INK4 genes are differentially expressed while genes encoded by the p16/INK4A/B locus (p16, p14 and p15) have been found most frequently defective in CMT and CML models. A novel frameshift mutation has been discovered in p16 exon1α resulting in altered mRNA and protein expression in the CMT28 cell line derived from canine mammary adenocarcinoma. These altered expression profiles for the INK4 tumor suppressor genes were also demonstrated in CML cell lines and primary canine mammary tumors and found to be highly correlated with those found in common human breast cancer cell lines. For the first time, the 277 most abundantly expressed miRNAs in the canine genome have been screened in three CMT cell lines (CMT12, CMT27 and CMT28) that were characterized for INK4 gene defects. Several miRNAs that were altered in the CMT cell lines could potentially target these INK4 genes and also correlated to orthologous miRNAs identified in human breast cancer. Particularly, the miR-141 was validated by functional 3’-UTR reporter assay for binding of the INK4A 3’-UTR target sequence. These altered miRNAs and their target genes may represent unique and critical regulatory features in CMT models that can be used to better understand the conserved cell cycle regulatory mechanisms in human breast cancer.