平滑肌肉瘤间叶-上皮转化(MET)的临床意义及相关分子机制

Soft tissue leiomyosarcoma is typical type of mesenchymal tumor with poor prognosis because of lack of effective therapeutic approaches. We recently reported for the first time that this type of mesenchymal tumor exhibits an epithelial transition (mesenchymal to epithelial transition, MET) which is manifested by the expression of E-cadherin and other epithelial cell markers. Importantly, this transition is significantly associated with better survival. We intend to further validate and investigate the clinical significance and its associated molecular mechanism using technologies including next generation sequencing of whole genome and transcriptome, tissue microarray, cell signaling expression array, Real-Time PCR, Western Blotting, gene transfection, cell culture, and tumor xenograft mouse model. Our goals are to identify the drivers at genetic and epigenetic level, miRNA regulations, TGFβ and other signaling pathway regulator that are critical for the MET and to identify potentially novel therapeutic targets for leiomyosarcoma patients. Using large human clinical leiomyosarcoma samples, we will confirm the MET and its clinical significance. Using our established leiomyosarcoma cell line system, we will characterize the critical transcription factors such as SNAI and TWIST and important signaling pathways associated with the transition. We will also investigate how the modification of these pathways in the transition impact on the sensitivity of chemotherapy and radiotherapy. Using xenograft mouse model, we will determine how the transition affect tumorigenesis, invasion, metastasis, and response to chemotherapy, radiotherapy, and selected targeted therapy. With these in vitro and vivo experiments, we seek to clarify the mechanism of this clinical relevant transition with the aim of developing novel biomarkers and therapeutic targets for soft tissue leiomyosarcoma patients.

软组织平滑肌肉瘤是典型间叶源性肿瘤，但临床工作中我们发现部分肿瘤细胞呈上皮样改变。我们前期对此进行研究发现部分肿瘤细胞表达上皮性标记，出现间叶-上皮转化（MET）且与患者预后相关。基于此，拟用组织样本分析、肿瘤细胞培养、移植瘤裸鼠模型等方法，探讨平滑肌肉瘤中MET的临床意义及相关分子机制。通过大样本临床病例及组织标本分析，验证平滑肌肉瘤中MET现象并分析其与肿瘤复发、转移、放化疗敏感性、预后间关系，探讨MET的临床意义。通过诱导或阻断平滑肌肉瘤细胞中MET的实验研究，探讨MET影响肿瘤复发、转移、放化疗敏感性的分子机制包括分子遗传学异常、miRNA调节、 SNAI等关键转录因子、肿瘤细胞增殖凋亡及DNA损伤修复等信号通路改变。通过移植平滑肌肉瘤裸鼠模型研究，体内验证组织及细胞系实验结果，筛选针对MET的潜在分子治疗靶点。通过三个层面的研究，明确平滑肌肉瘤中MET的临床意义及其相关分子机制。

我们前期研究发现部分软组织平滑肌肉瘤显示间叶-上皮样表型转化（Mesenchymal to epithelial transition, MET）且可能和预后相关。基于此，拟：1.验证MET及其临床意义。2.探讨miR-506与MET的关系。 3.探讨miR-506与DNA损伤同源重组修复信号通路的关系。4.探讨miR-506的生物学功能及其机制。研究结果发现：1. 61例平滑肌肉瘤中E-cadherin阳性 27例，阳性率 39.7%。E-cadherin的表达与beta-catenin呈正相关关系，与 Vimentin及N-cadherin呈负相关关系。平滑肌肉瘤中E-cadherin表达阳性的患者预后较好，其无病生存期及总生存均优于表达阴性患者。平滑肌肉瘤中Vimentin及N-cadherin呈高表达状态，高表达的患者总生存、无病生存及无疾病进展生存均差。2.LMS中DNA损伤同源重组修复相关蛋白RAD51、RAD52、ATM及ATR的阳性表达率分别为45.9%（28/61）、62.3%（38/61）、16.4%（10/61）和60.7%（37/61）。Kaplan-Meier分析显示RAD51, RAD52, ATM和ATR是影响LMS患者OS的重要因素，COX回归分析显示RAD52, ATM和ATR是影响LMS患者OS的独立预后因子。ATM是LMS患者DFS的独立影响因子，而RAD52是PFS的独立影响因子。3. qRT-PCR定量平滑肌肉瘤37例组织样本中miR-506的表达情况，结果显示平滑肌肉瘤组织中miR-506低于正常组织，miR-506的表达与总生存、无病生存及无疾病进展生存无显著相关性。4. miR-506的高表达可显著降低平滑肌肉瘤细胞的增殖、侵袭、迁移及克隆形成能力。5.平滑肌肉瘤中miR-506的表达可调节MET及DNA损伤同源重组修复信号通路，从而显著促进肿瘤细胞对顺铂等药物的敏感性。这些研究结果显示平滑肌肉瘤中存在MET及DNA损伤同源重组修复信号通路异常，且miR-506对其有调节作用，并影响平滑肌肉瘤对化疗药物的敏感性，从而为改善平滑肌肉瘤化疗疗效提供新的策略。