基于necroptosis的肺癌化疗药物耐受和化疗增敏分子机理研究

Necroptosis, a programmed cell necrosis, is the latest advance in the cell death research field, which may establish a novel mechanism for tumor chemoresistance and open a new avenue for improving chemotherapy efficacy. Our previous study have found that RIP3 (receptor interacting protein kinase-3), the key molecule of necroptosis signaling pathway, was significantly downregulated in human lung cancer tissues and lung cancer cell lines. Importantly, it was found that restoration of RIP3 expression obviously promoted sensitivity of lung cancer cells to chemotherapeutic drugs. In this project, with in vitro cell culture system and in vivo animal models, we will systematically determine if RIP3 molecule and necroptosis contribute substantially to anticancer activity of chemotherapy and sensitizing necroptosis overcomes chemoresistance in lung cancer cells, particularly focusing on epigenetic regulatory mechanisms involving RIP3 expression and intervention of these mechanisms for the restoration of RIP3 expression and necroptosis and overcoming chemoresistance in lung cancer. The expected outcomes of this project will provide experimental and theoretical basis for the development of new anticancer strategy targeting necroptosis, and also may be applied to other diseases, thus have a more broad application prospects.

necroptosis（程序性细胞坏死）是细胞死亡研究领域的最新进展，可能为揭示肿瘤化疗耐药机制、实现化疗增敏开拓新的方向。我们前期研究发现，该通路关键信号分子RIP3在人肺癌组织和多种肺癌细胞株中表达显著降低，而重建RIP3分子表达明显促进肺癌细胞对化疗药物敏感性。基于这些发现，本课题拟通过体内外实验，阐明RIP3分子和necroptosis在化疗药物抗癌活性中的重要性，探明活化necroptosis能否逆转肺癌细胞耐药性，并立足于RIP3分子的表观遗传学调控，阐明在肺癌细胞中RIP3分子表达抑制进而导致necroptosis阻断的分子机理，探寻有效解除RIP3分子表达抑制从而重建necroptosis、有效杀灭癌细胞新途径，为开拓以necroptosis为靶向的肿瘤治疗新策略奠定实验和理论基础。研究结果对necroptosis新认识也可能应用于其它疾病的防治，因而具有更广阔的应用前景。

化学治疗是肺癌的主要治疗手段之一，虽然普遍认为诱导癌细胞发生凋亡是化疗药物杀灭癌细胞的主要机理，但尽管已进行了大量的调控细胞凋亡、逆转耐药的研究，这些努力并未显著提高肺癌化疗效果。面临这一困境，我们跳出“细胞凋亡”的范畴，从细胞死亡新途径---necroptosis（程序性细胞坏死）入手，研究necroptosis在化疗药物抗癌活性中的作用。本研究首先以体外培养的多种肺癌细胞株为研究对象，采用多种方法对necroptosis通路进行调控，包括用针对necroptosis通路关键信号分子的化学抑制剂来抑制necroptosis通路，通过基因转染过表达RIP3分子来上调necroptosis通路，或RNA干扰技术抑制MLKL分子表达以下调necroptosis通路，发现在肺癌细胞株中调控necroptosis通路可有效影响细胞对化疗药物敏感性；然后分别以稳定过表达RIP3的肺癌细胞株和稳定表达MLKL-shRNA、MLKL分子表达被抑制的肺癌细胞株建立体内裸鼠成瘤模型，从上调necroptosis通路和抑制necroptosis通路两个不同的方向研究，发现活化necroptosis通路可有效增加肺癌细胞对化疗药物的体内敏感性；在此基础上，进一步从表观遗传学入手，发现肺癌组织中RIP3的表达较正常肺组织显著下降，而且肺癌组织中RIP3启动子区域存在甲基化，采用DAC处理、siRNA抑制甲基化关键酶DNMT1或低剂量长时间木犀草素处理来抑制RIP3 启动子甲基化，上调RIP3在肺癌细胞株中的表达，均可促进肺癌细胞株对化疗药物的敏感性；最后以数据库中的相关肺癌资料为对象，进一步分析Kaplan-Meier Plotter数据库中资料显示RIP3分子高表达组的总体预后显著好于RIP3分子低表达组。以上结果揭示了necroptosis在化疗药物发挥抗癌活性中的重要性，necroptosis通路失活在肺癌细胞对化疗药物耐药中具有一定的作用，而活化necroptosis能够增加肺癌细胞对化疗药物的敏感性，在肺癌细胞中RIP3分子及MLKL分子是necroptosis通路的关键调控分子，RIP3启动子的甲基化是参与necroptosis通路调控的重要表观遗传学调控机制，本研究为开拓以necroptosis为靶向的肿瘤治疗新策略奠定了一定的实验和理论基础。