结直肠癌KRAS基因突变组成性活化核因子-κB的机制研究

Evidences showed that oncogenic KRAS-driven cancers required activation of nuclear factor-κB(NF-κB), however, the machanisms of constitutive activation of NF-κB induced by mutant KRAS were not fully understood. Our previous study showed that constitutive activation of NF-κB were observed in oncogenic KRAS-dependent colon cells and NF-κB activition required KRAS and MEK.And,the prevalence of constitutive NF-κB activation was significantly higher in colorectal cancer patients with KRAS G12 mutations than with KRAS G13 mutations. Based on the previous study, we will determine whether oncogenic KRAS activate NF-κB via MAPK signaling pathway with the methods of siRNA transfection, western blot and co-immunoprecipitation; investigate the effects of different expression of KRAS G12D or KRAS G13D on NF-κB activation, using a tetracycline inducible model; chart a comprehensive picture of the genes that interact with and participate to the NF-kB activation system by GeneChip analysis in colorectal cancers with KRAS mutations. This study would be helpful to better understand machanisms of constitutive NF-κB activation induced by mutant KRAS, which would provide a platform for development of NF-κB as a potential target for colorectal cancer with KRAS mutations.

研究表明KRAS突变导致的恶性生物学行为需依赖核因子-κB活化，但KRAS组成性活化核因子-κB的机制尚未充分阐明。我们前期在KRAS癌基因依赖的肠癌细胞株中观察到核因子-κB活化，而且核因子-κB活化依赖KRAS、MEK分子；另外，KRAS12位点突变的结直肠癌患者核因子-κB活化率显著高于13位点突变。在此基础上，本课题拟应用siRNA转染、蛋白印迹、免疫共沉淀等方法进一步明确KRAS通过MAPK途径旁路激活核因子-κB；通过构建四环素诱导表达KRAS G12D、KRAS G13D细胞模型以探明KRAS不同突变位点、不同表达状态对核因子-κB活化的影响；通过基因差异表达分析，勾画KRAS活化核因子-κB的背景图，探索不同基因背景对KRAS活化核因子-κB的影响。本课题将从不同层面和角度深化认识KRAS活化核因子-κB的机制，有望为核因子-κB作为KRAS突变结直肠癌的治疗靶点奠定基础。

研究表明KRAS突变肿瘤导致的恶性生物学行为需依赖核因子-κB活化，但KRAS组成性活化核因 子-κB的机制尚未充分阐明。我们研究发现SW620细胞株NF-κB活化依赖IKKα/β和ERK分子，RSK1、TBK1、IKKε蛋白并未发现磷酸化；KRAS突变肿瘤活化NF-κB很可能是通过TRAF6基因扩增这一桥梁，Spc-a-1细胞株NF-κB活化依赖于TRAF6，TRAF6参与调节细胞凋亡及侵袭活性，这一作用可能通过下调CD24、CXCR4实现；构建四环素诱导表达KRAS G12D、KRAS G13D的293T细胞模型以及不同浓度梯度的四环素诱导突变型KRAS基因的差异表达，但并没有在细胞模型中观察到NF-KB活化有显著差异；构建了NF-κB表达消减细胞模型，通过 Affymetrix表达谱芯片分析差异表达基因，发现MRPL3在NF-κB活化中起重要作用，参与调节细胞增殖、凋亡，该分子在肺腺癌中高表达；在组织标本中，发现单纯依靠P65或P50免疫组化染色并不能可靠判断NF-κB活化，两者同时表达反应患者预后不良；NF-κB活化和KRAS突变对肿瘤免疫微环境PD-L1可能具有潜在影响，我们在PD-L1表达进行了相关探索，发现PD-L1表达与TNM分期、病理类型、肿瘤淋巴细胞浸润有关、PD-L1瘤内表达具有较大的异质性。我们的实验结果为NF-κB作为肿瘤治疗靶点提供了可能的理论基础。本项目至今为止共发表SCI文章5篇，中文核心期刊1篇，目前尚有1-2篇SCI文章正在整理中，将于今后陆续发表，各项指标基本达到或超过预期目标。