EPB41L3基因超甲基化作为胶质母细胞瘤预后及抗血管生成治疗反应的分子标记物研究

Glioblastomas (GBMs) are the most frequent and vicious subtype of all primary central nervous systems (CNS) tumors. Molecular and clinical heterogeneity critically hindered better treatment outcomes for this deadly disease. DNA methylation marks have long been the leading candidates for cancer biomarker in predicting prognosis and the response to chemotherapeutic regimens such as temozolomide. A four-CpGs panel including EPB41L3 was identified by incorporating genome-wide DNA methylation data and clinical information, and was combined using a risk-score model. The risk score signature robustly predicted overall survival (OS) in all discovery and validation cohorts, and in a treatment-independent manner. It also predicted progression-free survival (PFS) in available patients. The defined risk subgroups were molecularly distinct; high-risk tumors were biologically more aggressive with concordant activation of pro-angiogeneic signaling at multi-molecular levels. Accordingly we observed better OS benefits of bevacizumab-contained therapy to high-risk patients in independent sets, supporting its implication in guiding usage of anti-angiogeneic therapy. Further gene set enrichment analysis showed that high-risk group marked by EPB41L3 hypermethylation were enriched with cancer-promoting signatures relating to angiogenesis. Therefore we hypothesize that EPB41L3 gene silencing induced by promoter hypermethylation is crucial to the regulation of angiogenesis of GBM. A combination of large-scale prospective clinical study, analysis of histopathological samples, cell culture, chemical biology and protein biochemistry will be incorporated to investigate the function and underlying mechanisms of EPB41L3-mediated angiogenesis of GBM and thus shed light on targeting EPB41L3 as a potential therapeutic option for this devastating disease, as well as using this CpGs panel as a predictive indicator to anti-angiogeneic therapy response and prognosis of GBM patients to improve personalized treatment.

胶质母细胞瘤的分子和临床异质性是导致疗效不佳的重要原因。我们研究发现EPB41L3基因的异常甲基化与该类患者的预后相关。预后较差的高危组肿瘤表现为EPB41L3超甲基化，出现特异性血管生成信号通路的基因集富集，并对抗血管生成靶向药物贝伐单抗治疗具有显著的生存获益。临床标本验证发现EPB41L3基因表达与其启动子甲基化负相关，且EPB41L3表达分别与微血管密度、VEGF表达负相关，因此我们设想EPB41L3超甲基化通过下调EPB41L3基因表达，上调VEGF表达而参与调控肿瘤的血管生成。我们拟通过大样本前瞻性研究分析EPB41L3超甲基化与胶质母细胞瘤预后和贝伐单抗治疗反应的相关性，并借助组织病理学、细胞培养及动物实验方法，明确EPB41L3超甲基化在肿瘤血管生成中的作用，探索其对贝伐单抗疗效的影响，并验证去甲基化药物对其表达的逆转，为抗血管治疗提供疗效预测的分子标记物和分子治疗的新靶点。

胶质母细胞瘤(glioblastoma, GBM)是最常见且恶性程度最高的中枢神经系统原发性肿瘤，不同GBM患者肿瘤的分子和临床异质性是标准化治疗对预后改善不明显的重要原因。近年来随着高通量检测手段的广泛应用，对基因组水平的肿瘤表观遗传学谱的绘制，提供了以基于多个位点的甲基化谱作为更强有力的分子标记物的手段。本课题组利用全球最大的癌症基因组计划(The Cancer Genome Atlas, TCGA)数据库中GBM样本的全基因组甲基化芯片数据、全基因组表达谱数据及对应的临床信息，采用R软件平台大数据算法的多分子平台的整合分析方案，从数万个甲基化位点中，筛选出一组基于4个位点CpG岛（CpGs island, CGI）：骨髓基质细胞抗原2(bone marrow stromal cell antigen, BST2)、壳多糖酶3样蛋白1 (chitinase-3-like protein 1, CHI3L1)、红细胞膜带4.1类3蛋白(Erythrocyte Membrane Protein Band 4.1 Like 3, EPB41L3)和纤调蛋白(fibromodulin, FMOD)的异常甲基化谱的预后风险评估模型，能稳定地预测患者的生存期。基于此模型对生存期的预测可将GBM患者分为低危组与高危组，两组肿瘤的生物学特性和恶性程度具有显著差异。EPB41L3超甲基化为此预后风险评估模型中的关键分子，高危组肿瘤表现为EPB41L3超甲基化，伴有特异性血管生成相关信号通路的基因集富集。我们进一步在组织学水平证实EPB41L3基因表达与其启动子甲基化负相关，且EPB41L3表达分别与微血管密度、VEGF表达负相关。在体内、体外实验分别通过过表达和干涉上调、下调EPB41L3基因表达，证实EPB41L3作为抑癌基因的潜能，具有抑制GBM细胞增殖、侵袭、转移，以及血管生成的功能，且其通过作用于VEGF通路调控GBM的血管生成。本研究建立的预后风险预测模型能稳定预测GBM患者的生存期及对抗血管药物贝伐单抗的获益可能，并验证了EPB41L3超甲基化作为新分子靶点的潜能，为表观遗传学药物应用于GBM的治疗提供科学依据。