Involvement of β-catenin in Androgen-induced Mesenchymal Transition of Breast MDA-MB-453 Cancer Cells

<b>Purpose</b> The cellular and molecular dynamics of DHT-induced EMT in MDA-MB-453 cells were investigated. <b>Methods</b>:PCR arrays were used to examine the expression of EMT-regulatory genes. Immunoblotting was used to detect protein levels and confirm protein-protein interaction following immunoprecipitation. Immunofluorescence was used to observe rearrangement of the actin cytoskeleton and cell morphology. Cell migration was assessed by transwell assay <b>Results:</b> Change of cell morphology was concomitant with increased cell migration after treating cells with DHT. Exposure of cells to DHT for one hour was sufficient to induce changes in cell morphology and actin cytoskeleton after 72 hours indicating altered gene expression. A long-term lasting nuclear translocation of AR was observed after a short exposure of cells to DHT. Investigating the expression of 84 EMT-related genes revealed down-expression of β-catenin, N-cadherin, and TCF-4 and increased expression of Slug, all of which were confirmed at the protein level. Yet, not only early interaction of AR and β-catenin was observed following AR activation, inhibition of β-catenin blocked DHT-induced mesenchymal transition and migration. Wnt signaling was found to be partially important in DHT-induced morphological alteration. The mesenchymal transition of cells could be induced by treating cells with an inhibitor of glycogen synthase kinase-3β, an enzyme that inhibits β-catenin; this morphological transition could be reversed by antagonizing AR suggesting that AR functions downstream of β-catenin. <b>Conclusions:</b> These results suggest that MDA-MB-453 cells undergo partial EMT induced by DHT, β-catenin is critical for this phenotypic change, and AR probably reciprocally mediates the mesenchymal transition of these cells upon activation of GSK-3 β.

研究目的：本研究旨在探究双氢睾酮（Dihydrotestosterone, DHT）诱导MDA-MB-453细胞发生上皮间质转化（Epithelial-Mesenchymal Transition, EMT）的细胞与分子动力学机制。
实验方法：采用PCR芯片检测上皮间质转化调控基因的表达水平；通过免疫印迹法检测蛋白表达水平，并在免疫沉淀后验证蛋白-蛋白相互作用；利用免疫荧光技术观察肌动蛋白细胞骨架的重排与细胞形态变化；采用Transwell实验评估细胞迁移能力。
实验结果：经双氢睾酮处理后的细胞，其形态变化与细胞迁移能力增强同步发生。将细胞暴露于双氢睾酮1小时，即可在72小时后诱导细胞形态与肌动蛋白细胞骨架发生改变，提示基因表达谱发生变化。短暂暴露于双氢睾酮后，可观察到雄激素受体（Androgen Receptor, AR）发生持久的核转位。对84个上皮间质转化相关基因的表达检测发现，β-连环蛋白（β-catenin）、N-钙黏蛋白（N-cadherin）及转录因子4（TCF-4）的表达下调，而Slug的表达上调，上述结果均在蛋白水平得到验证。此外，在雄激素受体激活后，不仅可观察到AR与β-连环蛋白的早期相互作用，抑制β-连环蛋白还可阻断双氢睾酮诱导的间质转化与细胞迁移。研究发现Wnt信号通路在双氢睾酮诱导的形态改变中发挥部分调控作用。使用糖原合成激酶3β（Glycogen Synthase Kinase 3β, GSK-3β）抑制剂处理细胞可诱导细胞发生间质转化，而该酶可抑制β-连环蛋白的活性；通过拮抗AR可逆转该形态转化，提示AR作用于β-连环蛋白的下游通路。
研究结论：上述结果表明，MDA-MB-453细胞可发生双氢睾酮诱导的部分上皮间质转化；β-连环蛋白在该表型改变中发挥关键作用；而在糖原合成激酶3β激活后，AR可能反向介导该类细胞的间质转化过程。