涎腺恶性多形性腺瘤中E-cadherin表达沉默的组蛋白甲基化调控

Malignant pleomorphic adenoma(carcinoma ex pleomorphic adenoma, Ca-ex-PA) is an aggressive salivary gland malignancy that usually develops in a primary or recurrent pleomorphic adenoma(PA). It is also a frequent salivary malignant tumor in China. The characteristic that the malignant components usually coexist with benign elements in an individual neoplasm makes the tumor to be as a good model for tumor research. Previously, we demonstrated that reduceded E-cadherin expression correlated with the tumorgenesis and invasion of tumor,and DNA methylation, an epigenetic event, was found to contribute to the inactivation of E-cadherin in Ca-ex-PA. But in some cases neither DNA methylation nor loss of heterozygosity were found and the causes leading to gene silencing in these tumors were still unknown. Histone methylation and DNA methylation usually act together and the former is now recognized as another epigenetic mechanism for gene silencing in human cancers. Some experts even think that histone methylation would be an "initiator" for gene silencing. We therefore raised the question weather histone methylation at the E-cadherin gene promoter was responsible for E-cadherin downregulation in Ca-ex-PA. .Analysis of patients samples,however,is hampered by technical limitations in the study of chromatin structure from pathology archives that usually consist of heavily fixed,paraffin-embedded material. In this study, the detection for histone methylation, including H3K9,H3K4 and H3K27, etc, will be processed on paraffin embedded pathology tissues by means of PAT-ChIP-qPCR (pathology tissue-chromatin immunoprecipitation). We will divide Ca-ex-PA samples into several distinct groups based on E-cadherin expression level and E-cadherin promoter methylation status.The results from PAT-ChIP-qPCR will be compared and statistically analyzed among these distinct groups. Then the histone methylation status in association with DNA methylation, gene silencing, clinicopathological features of tumors and prognosis of patients will be discussed,and therefore,the question whether histone methylation is an independent mechanism in gene silencing will be determined. Some experiments on Ca-ex-PA cell lines will be done to further comfirm the results from above examinations. Owing to the reversible nature of histone methylation, study on histone methylation at E-cadherin gene promoter in Ca-ex-PA will aid us in searching for potential therapeutic targets to block the downregulation of E-cadherin.

恶性多形性腺瘤(Ca-ex-PA)是常见恶性涎腺肿瘤，肿瘤中良、恶性成份共存，是很好的肿瘤研究天然模型。我们前期研究发现其恶变、侵袭与E-cadherin下调有关，DNA甲基化是致该基因下调的重要原因，但仍有部分病例其基因失活机理不明。H3K9等位点组蛋白甲基化修饰是与DNA甲基化、基因沉默密切相关的调控机制，并可能是基因沉默的"始动者"，我们设想组蛋白甲基化可能是上述部分病例E-cadherin失活的机制。与以往研究多基于细胞系或组蛋白总体修饰水平不同，本研究拟针对病理标本，通过对良、恶性成份E-cadherin基因启动子区组蛋白甲基化修饰的差异性分析，包括H3K9、H3K4、H3K27等位点，结合体外动态抑制实验结果，揭示E-cadherin特异性组蛋白甲基化修饰方式，探讨其是否为独立于DNA甲基化的另一表观调控机制以及与肿瘤恶变、侵袭、预后相关性，为肿瘤治疗提供靶点分析。

恶性多形性腺瘤（MPA）是常见唾液腺肿瘤，寻找有效预后评价指标及靶向治疗靶点是其重要研究课题。361例MPA分析发现侵袭亚型是影响患者生存的主要亚型，T分期、N 分期是疾病特异性生存时间独立预测因子。MPA中以肌上皮成分恶变为主（肌上皮癌亚型）者，总生存时间、疾病特异性生存时间、无病生存时间、无转移生存时间均优于腺上皮成分恶变（腺癌亚型）为主病例，肿瘤细胞癌变亚型是侵袭性MPA重要预后评估指标，具有较强可操作性和临床指导意义。侵袭性MPA腺癌亚型中E-cadherin表达明显高于肌上皮癌亚型，E-cadherin与细胞分化类型相关但与预后无关。腺癌亚型CDH1启动子区平均甲基化率低于肌上皮癌亚型，经去甲基化药物处理后，肌上皮癌细胞CDH1甲基化率显著降低，E-cadherin蛋白和mRNA表达升高，细胞迁移能力降低，相反，体外加入TGF-β1，使腺癌细胞CDH1甲基化程度提高，蛋白和mRNA表达降低，细胞迁移能力提高，提示DNA甲基化是导致E-cadherin表达下调的机制之一，而H3K9me3与DNA甲基化发挥协同作用。在细胞构成与MPA类似的腺样囊性癌(ACC)中同样发现，H3K9me3表达在腺上皮和肌上皮细胞之间无差异，表达程度是患者总生存时间、无病生存时间的预测因子。ChIP on chip检测发现与H3K9me3结合的基因有551个，其中EDNRB和CHL1与ACC中H3K9me3关系密切，H3K9me3可能通过调控EDNRB、CHL1在部分ACC中促进肿瘤生长、淋巴结转移。MPA腺癌亚型不仅E-cadherin高表达，且Her-2癌基因高表达，阳性率为40.5%，Her-2表达与性别、组织学分级和N分期相关，高表达患者总生存时间和疾病特异性生存时间显著降低，提示Her-2 扩增是MPA的重要分子事件，与细胞分化相关，是患者预后差的重要指标。因此对侵袭性MPA肌上皮癌亚型患者（大多数呈现E-cadherin低表达）和腺癌亚型的部分病例（呈Her-2扩增）分别采用去甲基化或/和组蛋白甲基化抑制剂、Her-2靶向治疗是具有潜在应用价值的治疗策略。除与肿瘤治疗相关之外，某些分子标志物在疑难病例诊断中亦具有很高价值，如MYB蛋白及基因扩增检测有助于鉴别ACC和筛状型基底细胞肿瘤。总之本研究结果将有助于MPA预后评估、为晚期患者提供新治疗途径，提高MPA患者生存率。