MicroRNA-145 Antagonism Reverses TGF-ß Inhibition of F508del CFTR Correction in Airway Epithelia.

RATIONALE: MicroRNAs (miRNAs) destabilize mRNA transcripts and inhibit protein translation. miR-145 is of particular interest in cystic fibrosis (CF) as it has a direct binding site in the 3'-untranslated region of CFTR (cystic fibrosis transmembrane conductance regulator) and is upregulated by the CF genetic modifier TGF (transforming growth factor)-ß. OBJECTIVES: To demonstrate that miR-145 mediates TGF-ß inhibition of CFTR synthesis and function in airway epithelia. METHODS: Primary human CF (F508del homozygous) and non-CF airway epithelial cells were grown to terminal differentiation at the air-liquid interface on permeable supports. TGF-ß (5 ng/ml), a miR-145 mimic (20 nM), and a miR-145 antagonist (20 nM) were used to manipulate CFTR function. In CF cells, lumacaftor (3 µM) and ivacaftor (10 µM) corrected mutant F508del CFTR. Quantification of CFTR mRNA, protein, and function was done by standard techniques. MEASUREMENTS AND MAIN RESULTS: miR-145 is increased fourfold in CF BAL fluid compared with non-CF (P < 0.01) and increased 10-fold in CF primary airway epithelial cells (P < 0.01). Exogenous TGF-ß doubles miR-145 expression (P < 0.05), halves wild-type CFTR mRNA and protein levels (P < 0.01), and nullifies lumacaftor/ivacaftor F508del CFTR correction. miR-145 overexpression similarly decreases wild-type CFTR protein synthesis (P < 0.01) and function (P < 0.05), and eliminates F508del corrector benefit. miR-145 antagonism blocks TGF-ß suppression of CFTR and enhances lumacaftor correction of F508del CFTR. CONCLUSIONS: miR-145 mediates TGF-ß inhibition of CFTR synthesis and function in airway epithelia. Specific antagonists to miR-145 interrupt TGF-ß signaling to restore F508del CFTR modulation. miR-145 antagonism may offer a novel therapeutic opportunity to enhance therapeutic benefit of F508del CFTR correction in CF epithelia.

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

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.

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.

Autologous hybrid cell fusion vaccine in a spontaneous intermediate model of breast carcinoma.

Breast cancer is among the most common malignancies affecting women and reproductively intact female dogs, resulting in death from metastatic disease if not treated effectively. To better manage the disease progression, canine mammary tumor (CMT) cells derived from malignant canine mammary cancers were fused to autologous dendritic cells (DCs) to produce living hybrid-cell fusion vaccines for canine patients diagnosed with spontaneous mammary carcinoma. The high-speed sorting of rare autologous canine patient DCs from the peripheral blood provides the autologous component of fusion vaccines, and fusion to major histocompatibility complex-unmatched CMT cells were produced at high rates. The vaccinations were delivered to each patient following a surgical resection 3 times at 3-week intervals in combination with immuno-stimulatory oligonucleotides and Gemcitabine adjunct therapy. The immunized patient animals survived 3.3-times longer (median survival 611 days) than the control patients (median survival 184 days) and also appeared to exhibit an enhanced quality of life. A comparison of vaccinated patients diagnosed with inflammatory mammary carcinoma resulted in a very short median survival (42 days), suggesting no effect of vaccination. The data showed that the development of autologous living DC-based vaccine strategies in patient animals designed to improve the management of canine mammary carcinoma can be successful and may allow an identification of the antigens that can be translatable to promote effective immunity in canine and human patients.

Canine Mammary Carcinomas: A Comparative Analysis of Altered Gene Expression.

Breast cancer represents the second most frequent neoplasm in humans and sexually intact female dogs after lung and skin cancers, respectively. Many similar features in human and dog cancers including, spontaneous development, clinical presentation, tumor heterogeneity, disease progression and response to conventional therapies have supported development of this comparative model as an alternative to mice. The highly conserved similarities between canine and human genomes are also key to this comparative analysis, especially when compared to the murine genome. Studies with canine mammary tumor (CMT) models have shown a strong genetic correlation with their human counterparts, particularly in terms of altered expression profiles of cell cycle regulatory genes, tumor suppressor and oncogenes and also a large group of non-coding RNAs or microRNAs (miRNAs). Because CMTs are considered predictive intermediate models for human breast cancer, similarities in genetic alterations and cancer predisposition between humans and dogs have raised further interest. Many cancer-associated genetic defects critical to mammary tumor development and oncogenic determinants of metastasis have been reported and appear to be similar in both species. Comparative analysis of deregulated gene sets or cancer signaling pathways has shown that a significant proportion of orthologous genes are comparably up- or down-regulated in both human and dog breast tumors. Particularly, a group of cell cycle regulators called cyclin-dependent kinase inhibitors (CKIs) acting as potent tumor suppressors are frequently defective in CMTs. Interestingly, comparative analysis of coding sequences has also shown that these genes are highly conserved in mammals in terms of their evolutionary divergence from a common ancestor. Moreover, co-deletion and/or homozygous loss of the INK4A/ARF/INK4B (CDKN2A/B) locus, encoding three members of the CKI tumor suppressor gene families (p16/INK4A, p14ARF and p15/INK4B), in many human and dog cancers including mammary carcinomas, suggested their important conserved genetic order and localization in orthologous chromosomal regions. miRNAs, as powerful post-transcriptional regulators of most of the cancer-associated genes, have not been well evaluated to date in animal cancer models. Comprehensive expression profiles of miRNAs in CMTs have revealed their altered regulation showing a strong correlation with those found in human breast cancers. These genetic correlations between human and dog mammary cancers will greatly advance our understanding of regulatory mechanisms involving many critical cancer-associated genes that promote neoplasia and contribute to the promising development of future therapeutics.