HDAC-miR212/132-SOX4轴活化促进肺腺癌EGFR-TKI继发性耐药的机制研究

TKI acquired resistance is a difficult problem in the treatment of EGFR mutant lung cancer. Our previous research based on microarray data provided different gene expression profiles (DEG) of TKI resistant lung cancer cells. It was predicted and verified that the histone deacetylase (HDAC) inhibitor VPA can reversal of TKI resistance through MAPK and AKT signaling pathway inhibition. Besides, It was found that the transcription factor SOX4, an important "hub genes" in the network composed by DEG, was closely related to MAPK, AKT and other signaling pathways. Moreover, It was also found that the expression of SOX4 increased in TKI resistant lung cancer cells, but decreased after HDAC inhibition. Because HDAC can inhibit the expression of miR-212/132 and SOX4 is a common target gene of miR-212/132, It is speculated that HDAC inhibits miR-212/132 transcription, upregulates SOX4, therefore activates MAPK and AKT signaling pathway, which may be a new mechanism for TKI resistance, but also provides a new therapeutic target for lung cancer. In this project, we plan to use the methods including positive and reverse experiment, structure and function verification, based on clinical specimens, animal cells and luciferase reporter gene model, to investigate the mechanism of lung cancer TKI resistance resulted from HDAC-miR212/132-SOX4 axis activation，and provide the basic research for the treatment of lung cancer.

TKI获得性耐药是EGFR突变型肺癌治疗难题。我们前期据基因芯片数据分析得到TKI耐药肺癌细胞差异基因表达谱（DEG）。预测并验证了组蛋白去乙酰化酶（HDAC）抑制剂VPA可阻滞MAPK、AKT等信号通路而逆转TKI耐药。还发现此DEG组成的网络中，转录因子SOX4属重要“节点”基因，它和MAPK、AKT等信号通路关系密切，在耐药株中表达上升，但在HDAC受抑后表达减少。由于HDAC可抑制miR212/132表达，SOX4是miR212/132共同靶基因。故推测HDAC抑制miR212/132转录，上调SOX4，活化MAPK、AKT等信号通路，可能是TKI耐药的新机制和治疗的靶点。我们拟用临床标本、细胞和动物模型、荧光素酶报告基因模型等，采用临床随访、正向和反向实验、结构功能验证等方法，证明上述HDAC-miR212/132-SOX4轴活化导致肺癌TKI耐药的机制。为肺癌治疗提供基础研究。

肺癌发生发展，以及TKI获得性耐药是EGFR突变型肺癌的治疗难题,其机制繁多，涉及大量基因变化。所以，通过生物信息学分析肺癌发生发展和TKI耐药后海量的基因变化谱，找到其中居于核心地位的靶分子，可能开发出有效的治疗策略。.我们应用生物信息学分析表明：肺癌细胞对比正常细胞，TKI耐药的肺癌细胞对比TKI敏感的肺癌细胞，均存在若干差异表达基因（DEGs）。它们的生物学功能富集类型均与细胞周期、增殖、调亡和生存能力有关，涉及多种代谢途径，包括能量代谢途径。提示这些机制可能参与了肿瘤发生、进展和TKI耐药。进一步分析表明，其中某些基因和肺癌的不良预后密切相关，包括SOX4、SIRT6、MTIF2、ACOX1、MRPL17、CAV1、CAV1等。这些分子可能为为肺癌治疗的靶目标。.以对TKI敏感的肺腺癌细胞株HCC827细胞为基础，构建了TKI耐药的HCC827－ER细胞，对比HCC827细胞，发现HCC827－ER与HCC827存在不同的表型：HDAC、SOX4、AKT、MAPK、JAK等分子表达上升、miRNA212/132表达下降，以及更高的HIF-1a表达和糖酵解水平等。.分别抑制TKI耐药肺癌细胞的HDAC、SOX4或HK2表达后，均发现TKI耐药程度减弱，survivin表达下降，Caspase3表达上升，AKT、MAPK活性减弱，糖酵解水平减少和细胞ATP生成减少，提示这些基因参与了TKI耐药过程，其中miR212/132处于HDAC和SOX4的中介地位。由于SOX4是HDAC和miRNA212/132影响TKI耐药表型的下游分子，提示HDAC-miR212/132-SOX4轴活化促进肺腺癌EGFR-TKI继发性耐药的机制存在。.进一步发现SIRT6是SOX4的下游分子，是SOX4导致上述TKI耐药的中介机制。SIRT6也能增强细胞糖酵解，活化HK2，产生更多的乳酸和能量，提高细胞生存能力。而且，HK2活化（导致糖酵解增强）能直接诱导TKI耐药。这提示，HDAC-miR212/132-SOX4轴活化后促进肺腺癌TKI耐药的机制和上调SIRT6表达和增强细胞糖酵解水平，让细胞生存能力上升有关。.由于种种因素导致细胞能量生成增加，可能是驱动细胞恶性表型维持及耐药等生存能力增加的重要因素，3代TKI耐药或其它靶向药物耐药，也可能存在类似的机制，值得进一步研究。