Clinical significance of the Epithelial-Mesenchymal Transition in the malignant transformation of canine mammary gland tumors.

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To clarify the significance of epithelial-mesenchymal transition(EMT) in the malignant transformation of canine mammary gland tumors(cMGTs), serial experiments were intended. EMT related factors were examined on tissue specimens collected by the surgical resection from clinical cases of spontaneous cMGTs. Immunohistochemical analysis revealed that the protein expression pattern indicating EMT was observed only in the adenocarcinoma cases, but not in the adenoma cases. For further in vitro studies, we obtained two clonal cell lines derived from the same cMGT patient, which show the different malignancy in the culture condition and the transplanted mouse model. Highly malignant clonal cell line, CHMp5b, was found to have the mesencymal characteristics with high expressions of vimentin and N-cadherin, whereas low malignant cell line, CHMp13a, showed the epithelial characteristics with high expressions of E-cadherin and β-catenin. In the trans-well assay, CHMp5b showed significantly higher motility and invasive potential than CHMp13a. These results suggested the relationship between the EMT status and the malignancy of cMGT cells. By the stimulus of TGF-β on these cell lines, vimentin expression was induced and this expression was paralleled with the phosphorylation of Smad2. In the experiment using xCELLigence system, changes of expressions of EMT related proteins were correlated with motility and invasive potential of these cells dynamically. From the results of these experiments, EMT might relate to the malignant transformation of cMGT dynamically and Smad2 cascades might be a crucial factor in converting the EMT status. We continue the further study on the significance of EMT in canine MGT malignancy with the controlled experiment by RNAi method, and confirm these findings obtained from these studies in the clinical cMGT cases.