EGFR-TKI持续暴露诱导肺癌细胞TYK2/STAT3过度激活的分子机制及生物学影响研究

EGFR-TKI has been used as first-line treatment for non-small cell lung cancer patients with EGFR activation mutations.However, there are still a part of patients with wild type EGFR were treated by EGFR-TKI. Our previous experiments confirmed that continuous EGFR - TKI exposure lead to multi chemotherapy drug resistance in EGFR wild type lung cancer cells. The underlying mechanism is the excessive activation of STAT3. The STAT3-mediated drug resistance phenomena are also observed in EGFR mutation cell line with EGFR-TKI acquired resistance, which reveal common molecular signal compensation follow by EGFR pathway continuous inhibition. Previous investigations have shown that STAT3 is a oncogene and its activation is closely related to tumor metastasis, treatment resistance and immune escape. Therefore, it is very important to clarify the molecular mechanism of excessive activation of STAT3 induced by EGFR-TKI. Our previous study suggested that TYK2 probably is a upstream molecule of STAT3 over-activation, and HSP90 may be involved. In current study, we will investigate the molecular mechanism of over-activation of TYK2/STAT3 and the influence on lung cancer biological behavior, what is more. Furthermore, molecular intervention strategy will also be investigated based on the results of molecular events. The implementation of this project will help understand the molecular compensatory mechanism response to EGFR-TKI continuous exposure in lung cancer, promote the rational use of EGFR-TKI, and provide more scientific treatment strategies followed by EGFR-TKI resistance and new insights for clinical translation of new molecular targeting drugs.

EGFR-TKI已用于EGFR激活突变的非小细胞肺癌一线治疗，但仍有部分EGFR野生型患者接受EGFR-TKI治疗。我们前期证实，野生型肺癌细胞在EGFR-TKI持续暴露后导致多化疗药物抵抗，其机制为STAT3过度激活，这一耐药机制也存在于EGFR-TKI获得性耐药的EGFR突变细胞系，提示EGFR通路持续抑制后分子信号代偿具有共性。研究证实，STAT3激活与肿瘤转移、治疗抵抗及免疫逃逸密切相关，因此，STAT3过度激活的分子机制亟待阐明。我们前期证实TYK2可能为STAT3过度激活的上游分子，并有HSP90参与，本项目拟进一步研究TYK2/STAT3过度激活的分子机制及对肺癌细胞生物学行为的影响，并探讨分子干预策略。项目实施将有助深入理解EGFR-TKI持续暴露下肺癌的分子代偿机制，推动EGFR-TKI合理用药，并为EGFR-TKI耐药后后续治疗策略和新型分子靶向药物临床转化提供新思路。

本项目首先确定对于EGFR野生型的非小细胞肺癌细胞，持续EGFR-TKIs（吉非替尼，厄洛替尼，埃克替尼等）暴露，会导致序贯使用的化疗疗效产生影响。研究按照计划，探究EGFR-TKIs暴露处理导致细胞对化疗药物敏感性降低的机制，我们发现STAT3和Akt的磷酸化水平增加可能是导致细胞对化疗药物敏感性的主要机制，并且证明在EGFR-TKIs暴露处理后STAT3从与Akt相互独立又互补的信号通路中转变为调控Akt磷酸化的主要蛋白分子，占信号传导主导地位。在体内，我们也证实了经连续三周吉非替尼和厄洛替尼灌胃处理的裸鼠异种移植瘤模型中，存在STAT3磷酸化修饰水平增加。对于STAT3激活的机制研究，由于EGFR激酶结构域功能复杂，并且下游分子庞大，我们通过多种技术发现TYK2是STAT3激活的主要调控蛋白。尽管采用多种方法，还是未能明确TYK2激活的清晰机制，我们发现HSP90，FAK，IL-23等多种分子参与TYK2的激活。但我们使用TYK-2抑制剂处理细胞，发现STAT3磷酸化修饰可以被明显抑制，并且部分逆转化疗抵抗，据此我们认为TYK2抑制剂具有潜在应用价值。. 此外，在此项目支持下，我拓展延伸相关研究内容。我们发现EGFR-TKI持续暴露会导致正常成年小鼠免疫细胞亚群变化。厄洛替尼持续用药后小鼠外周血、脾脏及骨髓中MDSC数量增加，其中以多形核细胞亚型为主（CD11b+Ly-6G+Ly-6Clow，PMN-MDSC）。通过转录组测序我们还发现EGFR-TKIs诱导的MDSC具有高表达免疫抑制分子的表型。并且证实清除MDSC有助于增强EGFR-TKI耐药后PD-1单抗疗效。针对一部分服用EGFR-TKIs肺癌患者主诉服药期间出现明显记忆力减退、思维迟缓、注意力集中困难等认知改变的情况，我们也通过体外研究，问卷调查等方式开展了相关研究。. 在本项目支持下共发表 SCI 论文 2 篇，目前正在撰写论文2篇，培养博士后2名，培养博士研究生4名，硕士研究生3名。