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

为了阐明上皮-间充质转化(EMT)在犬乳腺肿瘤恶变中的意义，进行了一系列实验。对手术切除的自发性cMGT患者的组织标本进行EMT相关因子检测。免疫组织化学分析显示，提示EMT的蛋白表达模式仅见于腺癌，而在腺瘤中未见。为了进一步的体外研究，我们获得了来自同一cMGT患者的两个克隆细胞系，它们在培养条件和移植小鼠模型中表现出不同的恶性程度。恶性程度高的克隆细胞株CHMp5b具有高表达波形蛋白和N-钙粘蛋白的间质特征，而低度恶性的克隆细胞株CHMp13a具有高表达E-钙粘蛋白和β-连环蛋白的上皮性特征。在跨孔实验中，CHMp5b表现出明显高于CHMp13a的运动能力和侵袭潜能。这些结果提示EMT状态与cMGT细胞的恶性程度有关。在转化生长因子-β的刺激下，波形蛋白的表达被诱导，并且这种表达与Smad2的磷酸化平行。在使用xCELigence系统的实验中，EMT相关蛋白表达的变化与这些细胞的运动性和侵袭性动态相关。从这些实验结果来看，EMT可能与cMGT的恶性转化动态相关，Smad2级联可能是导致EMT状态转变的关键因素。我们继续用RNAi方法进行对照实验，进一步研究EMT在犬MGT恶性肿瘤中的意义，并在临床cMGT病例中证实了这些研究结果。

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.