转录因子Goosecoid诱导乳腺癌转移的分子机制研究

Tumor metastasis is the most common cause of death in cancer patients.The mechanism of tumor metastasis is poorly understood. Epithelial-Mesenchymal Transition (EMT) is a critical cellular process for tumor cells to acquire the ability to migrate and invade extracellular matrix (ECM). EMT is a key event for the invasion and metastasis of many carcinomas. In cancer cells, the reversible EMT/MET process suggests that transcriptional regulation is involved. Functional loss of E-cadherin in epithelial cell has been considered a hallmark of EMT. The dominant transcriptional repression is mainly responsible for the transient loss of E-cadherin expression. Several transcription factors have been implicated in the transcriptional regulation of E-cadherin. EMT-inducing transcription factors may serve as major signaling mediators of EMT program to promote metastasis. In our previous study, we have found the repression of EMT by inhibition of Src activity in breast cancer cells. The molecular mechanism underlying the transcriptional regulation of EMT remains unclear. Transcription factor Goosecoid is involved in EMT and breast carcinoma metastasis.In this project,we will prisiely identify Goosecoid-binding specific DNA sequence by Chip-exo and deep sequencing. Further more, we'd like to analyze the effect of transcription factor Goosecoid signaling pathway on EMT process in breast cancer cells. We will identify the potential proteins that interact with Goosecoid by overexpression of dual-tagged Goosecoid in HEK 293T cells, affinity purification, separation by SDS-PAGE, silver staining and mass spectrometry analysis. We will confirm the interaction by Co-IP, Western Blot analysis， GST Pull Down and immunofluorescence colocalization analysis. The functional interaction of Goosecoid with the target protein during EMT will be characterized through in vitro breast cancer cell culture and the tumour formation experiments in nude mice. The results will be important for better understanding the transcriptional regulation of EMT in tumour formation, progression and metastasis.

肿瘤转移是恶性肿瘤患者死亡的首要因素，但转移机制还不清楚。EMT是上皮细胞来源的肿瘤细胞获得迁移侵袭性的一个关键过程。我们前期研究证明抑制Src激酶活性，能抑制乳腺癌细胞的EMT，抑制细胞迁移，逆转高转移性乳腺癌细胞表型。可逆的EMT/MET的转录调控在癌转移中起重要作用。转录因子Goosecoid是重要的EMT调节因子并参与乳腺癌转移过程，Goosecoid有多个DNA结合位点，本项目创新性地利用Chip-exo技术和深度测序技术检测Goosecoid及其突变体与DNA结合的序列特异性，分析相应的功能。利用蛋白质组学和生物信息学方法鉴定Goosecoid的相互作用蛋白，分析Goosecoid及其相互作用蛋白的功能，研究相应的信号通路。用体外培养的乳腺癌细胞和裸鼠体内成瘤实验研究Goosecoid信号通路诱导EMT发生的分子机制，研究结果将为恶性肿瘤转移的预防和靶向治疗提供新靶点。

上皮-间质转化(Epithelial-mesenchymal transition, EMT) 的重要特征表现在细胞形态改变，上皮和间质细胞标记表达改变并使细胞获得运动和迁移能力。EMT使癌细胞获得运动和迁移能力涉及癌细胞从原发部位向周围播散，在癌转移过程中发挥重要作用。为研究Goosecoid 在乳腺癌EMT调节中的分子机制，我们用Dual luciferase assay (Promega)证明Goosecoid抑制上皮细胞标记基因CDH1启动子活性。用TGF-beta 处理MCF-7细胞后诱导 Goosecoid表达，抑制E-cadherin 表达。构建了pcDNA3.1-GSC-Flag 表达载体，用anti-Flag antibody亲和纯化Goosecoid蛋白复合物，用SDS-PAGE分离和质谱鉴定了与Goosecoid相互作用蛋白，用Co-IP 和FRET 对Goosecoid和ENO1蛋白的相互作用进行了验证，包装了带有eGFP标签的慢病毒， 建立稳定表达Goosecoid 和ENO1 的MCF-7 细胞系，进一步分析Goosecoid 和ENO1 在乳腺癌细胞上皮细胞标记和间质细胞标记的表达、增殖、细胞周期，侵袭、迁移及在裸鼠体内成瘤及转移的影响。我们的实验结果证明Goosecoid参与TGF-beta 信号转导通路诱导乳腺癌细胞EMT，但其上下游调节机制有待进一步研究。部分实验结果在2013年SCBA国际会议上交流，部分结果发表在2013年的ABBS上，并对基金资助进行了标注。