雌激素受体β剪切变异体介导EGFR突变型非小细胞肺癌EGFR TKIs耐药的分子机制

Cytoplasmic estrogen receptorβ (ERβ) can activate non-genomic signal pathways, which might lead to resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs). Our previous study have showed that ERβ1（wild type) was distributed in cell nuleus while ERβ2 and ERβ5 was expressed mainly in cytoplasm. We proposed that interactions between ERβ1 and ERβ2 or ERβ5 activated non-genomic signaling that mediated EGFR TKIs resistance. To deeply clarify the molecular mechanism of EGFR TKIs resistance mediated by ERβ in lung cancer patients, Stable lung cancer cell lines expressing both high level of ERβ1 and ERβ2 or ERβ5 was established to validate the interactions between different variant isoforms and reveal the molecular mechanism of EGFR TKIs resistance mediated by estrogen receptor β. We also prove that the resistance mediated by ERβ could be reversed by ER antagonist fulvestrant in vivo and in vitro experiments. At last, We will detect the expression of ERβ1 ,ERβ2 and ERβ5 in non small cell lung cancers using ERβ variant isoforms specific antibodies and investigate the correlation between the expression of ERβ isoforms and outcome of EGFR TKIs treatment. The results will provide theoretical evidence for well- defined cohorts from EGFR mutant lung cancer patients who can benefit from anti-estrogen treatment.

胞浆中雌激素受体β能够激活非基因组信号通路，可能与EGFR TKIs耐药相关。我们前期研究发现ERβ1（野生型）主要表达于细胞核，ERβ2和ERβ5主要定位于细胞浆，我们推测的机制是ERβ2或ERβ5与ERβ1结合后将ERβ1留在细胞浆中进而激活非基因组传导通路介导EGFR TKIs耐药。为了进一步阐明ERβ剪切变异体介导肺癌EGFR TKIs耐药的分子机制，我们拟建立稳定共表达ERβ1和ERβ2或ERβ5的肺癌细胞系，验证不同剪切变异体之间的相互作用以及介导EGFR TKI耐药的分子机制，体内外实验验证ER抑制剂氟维司群可以逆转ERβ介导的EGFR TKIs耐药，利用ERβ剪切变异体特异性抗体检测非小细胞肺癌中ERβ1、ERβ2和ERβ5的表达，分析与EGFR TKIs疗效的关系。该研究为选择优势人群给予抗雌激素治疗提供理论依据。

本研究在前期研究结果基础上进一步对雌激素受体介导EGFR突变型肺腺癌EGFR TKI原发耐药的分子机制进一步探索，首先建立了单独表达ERβ1和共表达ERβ1/5的EGFR突变型肺腺癌稳定细胞系，免疫荧光法检测发现单独表达ERβ1主要定位于细胞核中，而双转之后一部分留在细胞浆中；western blot发现双转染细胞系EGFR下游信号通路在EGFR TKI作用下仍处于活化状态，但更下游的mTOR信号通路被抑制，这一分子机制与吉非替尼耐药有关，细胞存活实验（CCK8）也提示双转染的细胞对于吉非替尼敏感性有所下降。结果显示雌激素（E2）和ERβ1起到核心作用。我们利用临床组织标本进行免疫组化染色，发现胞浆中ERβ1表达与EGFR突变型肺腺癌患者预后差关系密切，这提示将来可以通过ERβ1在细胞中表达不同来筛选合适的患者给予抗雌激素治疗来延长患者的PFS。