EGFR突变型肺腺癌经靶向治疗前后的甲基化改变及作用研究

Lung cancer is the leading cause of cancer death worldwide. It is well known that genetic alternation of epidermal growth factor receptor (EGFR) can predict outcome of non-small cell lung cancer (NSCLC) treatment, especially the EGFR tyrosine-kinase inhibitors (EGFR-TKIs) therapy. However EGFR mutation cannot explain all clinical outcomes of TKI therapy, around 10% of patients with mutated EGFR are resistant to the TKI therapy. Recent studies have demonstrated that cancer is as much an epigenetic disease as it is a genetic disease. Therefore, in addition to EGFR genotype, changes in epigenetic features such as CpG DNA methylation status of specific gene loci also contribute to the response to EGFR-TKI therapy. Firstly, our study has performed a genome-wide screening for the changes of DNA methylation before- and after-TKI treatment for EGFR mutated lung adenocarcinoma using methyl-sensitive cut counting sequencing (MSCC), a genome-wide methylation sequencing technology. Secondly, real DNA methylation changes will be identified and validated in the screeing samples and another independent cohort based on the other methylation methods, including bisulfite sequencing PCR and Sequenom MassArray. Thirdly, a subset of candidate genes will be validated by qRT-PCR on the RNA level and Western blot on the protein level. Furthermore, the biological function will be detected through over expression vector and siRNA cell lines of the candidate genes, for identifying the specific target genes. In the end, these target genes will be validated in the EGFR mutated lung adenocarcinoma further through correlating the gene methylation status with TKI response in the clinical practice. Our study might provide first epigenetic evidence for drug design and EGFR-TKI therapy in the future.

分子靶向治疗为肺癌的内科治疗提供了新的平台，针对EGFR-TKI的研究已成为肿瘤学界研究的热点。DNA甲基化作为一种较稳定的表观遗传修饰现象，为肺癌EGFR-TKI的治疗提供了新的研究方向。本研究首次运用MSCC全基因组甲基化测序技术，以接受TKI治疗的EGFR突变型肺腺癌为研究对象，首先从全基因组角度筛选经TKI治疗前后甲基化变化的位点和基因，并在筛查组和验证组中利用不同的甲基化分型方法进行验证，寻找与TKI疗效相关的甲基化差异基因。在此基础上通过对差异基因在转录水平和翻译水平的分析，明确在甲基化水平发生调控的目的基因。通过沉默/过表达目的基因，在体外探究其生物学功能，最终确定通过甲基化水平发生作用的靶标基因及其作用机制。进一步在临床接受TKI治疗的肺腺癌患者中再次验证靶标基因甲基化水平与TKI疗效的关系，以寻找真正的表观遗传学靶标基因，为TKI的治疗提供表观遗传学的理论依据。

肺癌是世界范围内癌症死亡的主要原因。近年来随着分子生物学技术的进展，抗肿瘤治疗已从传统的化疗发展到针对分子靶点的靶向治疗。然而，即使EGFR突变型肺癌患者接受TKI靶向治疗一段时间后均先后出现耐药，因此针对EGFR-TKI的深入研究已成为肿瘤学界研究的热点。作为近年来的研究热点，DNA甲基化是在不改变基因DNA序列的情况下通过对构象的修饰，影响相关基因表达最重要的表观遗传修饰方式，为肺癌EGFR-TKI的治疗提供了新的研究方向。.本研究首次运用MSCC全基因组甲基化测序技术，以接受TKI治疗的EGFR突变型肺腺癌为研究对象，首先从全基因组角度筛选经TKI治疗前后甲基化变化的位点和基因，并在筛查组中利用不同的甲基化分型方法对MSCC测序的结果进行验证，寻找并明确与TKI疗效相关的甲基化差异的目的基因。通过沉默/过表达目的基因，在体外探究其生物学功能，最终确定通过甲基化水平发生作用的靶标基因及其作用机制。.通过上述研究，在全基因组范围内以精确到单碱基的分辨率探究了DNA甲基化在TKI治疗前后的改变。后续对于MSCC全基因组范围内测序的筛选结果，经Sequenom EpiTYPER assay验证显示CBFA2T3和GABBR2基因的甲基化状态在TKI治疗前后差别明显。进一步功能学结果显示GABBR2基因可能通过参与厄洛替尼诱发的细胞凋亡起作用，是潜在的靶向治疗靶点。.本研究中最为关键的数据结果是GABBR2基因的过表达可以有效地逆转厄洛替尼诱导的EGFR突变型肺癌细胞系的凋亡，说明厄洛替尼的治疗效果可能与GABBR2的表达有非常密切的关系。对于EGFR突变型肺癌患者，厄洛替尼联合GABBR2抑制剂的治疗可能会起到良好的治疗效果，对肺癌靶向治疗的研究提供了有效的数据支持，具有非常重要的意义。