Smad3: 非小细胞肺癌EGFR-TKI治疗耐药的新靶点？

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) are widely used for advanced non-small-cell lung cancer patients (NSCLC). In clinical, dramatic responses have been observed soon after initiation of treatment, particularly in EGFR mutations. However, drug resistance eventually develops in most patients despite the initial good response. Intense research has identified two major mechanisms of resistance to TKI: primary and acquired resistance. We recently observed the transforming growth factor-β (TGF-β) signaling pathways is strongly antiproliferative, which plays an important role in tumorigenesis, cancer progression and metastasis via crosstalk with Smad and EGFR signaling pathways. To our knowledge, TGF-β signaling pathway has two major mechanisms to affect the outcome of EGFR-TKI. First, epithelial-mesenchymal transition (EMT) is one of the important mechanisms of TKI resistance, and changes in EMT status can explain acquired resistance to EGFR-TKI. Secondly, Bcl-2 (BIM) associated with TKI primary resistance, and one target of Smad3 in inducing apoptosis is BIM, a key antiapoptotic inhibitor. We determined the genotypes of 33 single-nucleotide polymorphisns (SNPs) from 13 genes involved in the TGF-β signaling pathway, and their association with survival time after treatment of TKI was analyzed. Just Smad3 rs11632964 was associated with a longer survival time, and the association was significant in the patients with EGFR mutant tumors. ..The purpose of our study is to determine the role of Smad3 in resistance of EGFR-TKI, and the relationship of Smad3 with EMT and BIM. Our hypothesis is that Smad3 involves in resistance though EMT and BIM, and it is possible to partly reverse resistance of TKI by regulating Smad3. We focus on NSCLC cells, in which expression of proteins about EMT and BIM can be mediated by Smad3, to investigate the underlying mechanisms of proteins expression regulation. Western blot analysis, real-time quantitative reverse transcriptase PCR, luciferase reporter gene assays, RNA interference and chromatin immunoprecipitation assay were performed on human NSCLC cell lines A549, PC9, PC9/G2, H3255 and H1975. The tumor xenograft of A549, PC9 and PC9/G2 cells in nude mice will be established. Our study can investigate the mechanism the role of Smad3 in resistance to EGFR-TKI, and suggest new targets for resistance.

表皮生长因子受体酪氨酸激酶抑制剂 (EGFR-TKI)为非小细胞肺癌的治疗打开了一片新的天地，然而耐药是制约EGFR-TKI进一步发展的瓶颈，目前临床上能逆转耐药的方法还很少。我们的前期研究发现转化生长因子β（TGF-β）信号传导通路中Smad3的遗传变异与EGFR敏感突变患者使用TKI后的耐药相关，提示Smad3可能参与了EGFR-TKI治疗耐药的发生，且在原发性和获得性耐药方面都发挥重要作用。本项目拟从体内和体外实验探索Smad3通过上皮-间质转化（EMT）和促凋亡蛋白（BIM）相关途径参与TKI耐药的机制；通过裸鼠移植瘤模型及Smad3 siRNA沉默后移植瘤模型的对比，探讨逆转EGFR-TKI耐药的方法。本项目的研究结果不仅可以揭示Smad3参与EGFR-TKI耐药的机制，更可能使Smad3成为对抗耐药的新靶点，为临床治疗TKI耐药提供新的理论依据。

本项目发现转化生长因子（TGF-β）信号传导通路中Smad3的遗传变异与EGFR敏感突变患者使用TKI后的耐药相关，提示Smad3可能参与EGFR-TKI治疗耐药，且在原发性和获得性耐药方面都发挥重要作用。本项目从体内和体外试验及临床研究，进一步明确Smad3基因沉默后，肺腺癌细胞对TKI处理后的凋亡能够减少。本项目同时纳入111例晚期NSCLC患者采用吉非替尼治疗后进入研究，确立了与Smad3密切相关的BIM基因缺失与患者使用EGFR-TKI后的OS, PFS相关，而在EGFR突变阳性的患者中，这种相关性表现的更为明显。其他基因位点的遗传变异均没有显著性差异。为了探寻逆转TKI耐药的方法，本项目组通过细胞和动物水平，研究放疗如何改变Smad3、BIM表达水平的分子机制，以及依赖MKP2的抑制BIM的磷酸化和降解，在逆转靶向治疗耐药中的分子机制，并为放疗介入TKI治疗寻找最佳时间点。TKI联合放疗作用于小鼠移植瘤后，RT-PCT 、Western Blot检测到Smad3、Bcl2家族促凋亡蛋白mRNA和蛋白水平均有所升高，而TKI联合早放疗这些指标变化更为明显。本项目的主要研究成果是通过动物实验探讨Smad3通过BIM对TKI耐药的作用机制，并且找到逆转TKI耐药的方式，证实TKI联合早放疗从机制上能最有效的逆转TKI耐药，为临床医生选择适当的放射介入时机提供依据，这必将是今后在临床上得到推广的有利条件。