Epigenetics in Canine Mammary Tumors: Upregulation of miR-18a and miR-18b Oncogenes Is Associated with Decreased ERS1 Target mRNA Expression and ERα Immunoexpression in Highly Proliferating Carcinomas

SIMPLE SUMMARY: Canine mammary neoplasms commonly lose ERα expression along with an increase in stage and grade, negatively correlating with patient prognosis. The ESR1 gene encodes ERα and represents an important target mRNA for miR-18a and miR-18b, described as overexpressed oncogenes in canine mammary carcinomas. In this study, we demonstrate a negative correlation between the expression of miR-18a and miR-18b and their target ESR1 gene. Notably, a significant overexpression of oncogenic miRNAs was observed in malignant canine mammary tumors (CMTs) compared with the non-neoplastic mammary gland and benign CMTs. In contrast, the expression of target ESR1 mRNA was significantly downregulated along with the increase in tumor grade, and was associated with the progressive loss of ERα immunoexpression. It is noteworthy that the overexpression of miR-18a and miR-18b was observed in malignant tumors with increased proliferation of tumor cells. The results suggest a central role of miR-18a and miR-18b in the pathophysiology of canine mammary tumors, also representing promising biomarkers with predictive and prognostic value. ABSTRACT: The expression of miRNAs is one of the main epigenetic mechanisms responsible for the regulation of gene expression in mammals, and in cancer, miRNAs participate by regulating the expression of protein-coding cancer-associated genes. In canine mammary tumors (CMTs), the ESR1 gene encodes for ERα, and represents a major target gene for miR-18a and miR-18b, previously found to be overexpressed in mammary carcinomas. A loss in ERα expression in CMTs is commonly associated with poor prognosis, and it is noteworthy that the downregulation of the ESR1 would appear to be more epigenetic than genetic in nature. In this study, the expression of ESR1 mRNA in formalin-fixed, paraffin-embedded (FFPE) canine mammary tumors (CMTs) was evaluated and compared with the expression levels of miR18a and miR18b, both assessed via RT-qPCR. Furthermore, the possible correlation between the miRNA expression data and the immunohistochemical prognostic factors (ERα immunoexpression; Ki67 proliferative index) was explored. A total of twenty-six FFPE mammary samples were used, including 22 CMTs (7 benign; 15 malignant) and four control samples (three normal mammary glands and one case of lobular hyperplasia). The obtained results demonstrate that miR-18a and miR-18b are upregulated in malignant CMTs, negatively correlating with the expression of target ESR1 mRNA. Of note, the upregulation of miRNAs strictly reflects the progressive loss of ERα immunoexpression and increased tumor cell proliferation as measured using the Ki67 index. The results suggest a central role of miR-18a and miR-18b in the pathophysiology of canine mammary tumors as potential epigenetic mechanisms involved in ERα downregulation. Moreover, as miRNA expression reflects ERα protein status and a high proliferative index, miR-18a and miR-18b may represent promising biomarkers with prognostic value. More detailed investigations on a larger number of cases are needed to better understand the influence of these miRNAs in canine mammary tumors.