NICD/CBF1/SIX1信号途径调控非小细胞肺癌放射敏感性的机制研究

The retinal determination gene network (RDGN) are recently found closely implicating in lung development and lung cancer initiation, and operating mainly through DACH1/EYA1/SIX1 pathway. Notch signaling pathway mediated by transcriptional factor C protein binding factor 1(CBF1) represents as a pivotal mechanism involved in the modulation of radiosensitivity. Preliminary findings suggested that JAG1 upregulated Notch signals as well as modulating transcription of DACH1 and SIX1. In lung tumor tissues underwent neo-adjuvant radiotherapy, abundance of Notch positively correlated with SIX1. Screening promoter region of SIX1 revealed several CBF1 binding sites and JAG1 transcriptionally upregulated SIX1 expression. Based on the above findings, this project hypothesized that Notch signaling activated SIX1 through CBF1 route and synergically enhanced resistance of non-small cell lung cancer (NSCLC) to radiation together with RDGN. The study approach includes molecular and cell biological methods and relevant functional experiments. Consisting of aspects of molecular, cell line, animal model and specimen tissue sample, the study is to elucidate the underlying mechanisms by which Notch signaling modulat RDGN and relevant biological functions. Confirmation of interactions between the emerging RDGN and Notch signaling may provide rationale for identifying them as novel targets in radiation-sensitizing of NSCLC.

新近发现的RDGN网络与肺癌发生密切相关，通过DACH1/EYA1/SIX1通路发挥调控作用。转录因子CBF1介导的Notch信号通路是调控放射敏感性的关键机制之一。课题组前期发现：配体JAG1上调Notch通路的同时，调节DACH1和SIX1转录；新辅助放疗后的肺癌组织中Notch与SIX1表达正相关；SIX1基因启动子区含有CBF1位点，且JAG1上调SIX1转录活性。据此，本研究假设Notch信号通路通过Notch受体胞内域（NICD）和转录因子CBF1调控SIX1，Notch信号与RDGN网络协同调控非小细胞肺癌放疗抵抗。本项目拟在分子、细胞、动物模型及临床组织标本四个层面，利用细胞生物学、分子生物学、组织细胞功能试验等方法深入研究Notch信号调控RDGN网络的机制及生物学意义，明确RDGN这一新的调控网络与Notch信号通路之间的相互作用，为增强非小细胞肺癌放疗敏感性提供新靶点

肺癌是我国乃至全世界癌症死亡率居首位的肿瘤，对于早中期不能手术和局部晚期非小细胞肺癌患者而言，放射治疗是主要的治疗方法之一，然而原发或继发性放疗抵抗极大地限制了其临床有效性。研究表明，视网膜决定基因网络（Retinal Determination Gene Network, RDGN）中关键信号分子同源框蛋白SIX1在多种人类癌症中均呈现过表达，而其在非小细胞肺癌中的表达模式及生物学作用仍有待探索。在本研究中，对一项包含226例非小细胞肺癌患者的数据库进行分析发现，SIX1表达水平与肺癌患者预后呈负相关。肺癌组织中SIX1的mRNA水平高于正常组织，且与肿瘤大小成正比，且SIX1 mRNA丰度高的肺癌患者较SIX1低表达患者总生存时间与无复发时间明显缩短。在SIX1低表达的A549及H1299细胞中进行SIX1过表达慢病毒转染，结果发现，SIX1过表达可促进非小细胞肺癌细胞的迁移、侵袭与小鼠体内成瘤等生物学行为。此外，非小细胞肺癌细胞中SIX1的过表达可显著增强细胞放射抗性，并增强DNA损伤修复。数据库与Western blot及RT-PCR分析显示，SIX1可诱导非小细胞肺癌细胞中Vimentin等间质相关分子表达上调及E-cadherin等上皮相关分子表达下调。同时，SIX1可诱导Notch信号通路激活。我们进一步发现，SIX1诱导的上皮-间质转化和辐射抗性可以被Notch抑制剂逆转。总之，这些结果显示SIX1是通过调控Notch信号通路从而增强非小细胞肺癌上皮-间质转化和抗辐射能力。结果表明，SIX1诱导的上皮-间质转化可能对增强放射抗性有显著的贡献。因此，SIX1可以作为非小细胞肺癌放射治疗中潜在的生物标志物或治疗靶点。