脂质蓄积产生的雌激素激活c-MET转录促进非小细胞肺癌EGFR-TKI耐药

EGFR-TKI是非小细胞肺癌重要一线治疗，但耐药限制了疗效。我们率先发现雌激素（E2）通过受体β（ERβ）促进TKI耐药，但具体机制及E2来源不明。我们另一项研究发现TKI治疗后脂质蓄积也促进耐药。预实验提示E2可能源于脂质蓄积，E2激活的ERβ可能通过结合多个转录调控序列上调c-MET表达（MET扩增促耐药关键环节）促进耐药。本项目拟扩大耐药前后样本，分析脂质蓄积与E2合成、ERβ和c-MET表达与耐药相关性；在细胞中探讨脂质蓄积促进E2合成及机制；构建c-MET过表达耐药细胞，探讨ERβ激活c-MET转录及耐药效应，明确MET调控序列上ERβ结合位点及调控作用；构建脂质蓄积、TKI耐药和PDX动物模型，验证脂质蓄积对E2合成的促进作用、阻断E2/ERβ对耐药的逆转作用。本项目从“溯源”（E2来源）和“追根”（E2促耐药）两方面探讨TKI耐药新理论，为抗雌激素逆转TKI耐药提供依据。

Epidermal Growth Factor Receptor- Tyrosine Kinase Inhibitor (EGFR-TKI) is an important first-line defense for non-small lung cancer (NSCLC), unfortunately, its effect eventually has been highly limited by drug resistance. Complex mechanisms impede the pace of research into reversing resistance, therefore it is urgent to seek new therapeutic targets and explore novel combined therapeutic strategies on the basis of systematic exploration of the mechanism.. Our previous study firstly put forward that estrogen (E2)/estrogen receptor β (ERβ) promotes TKI resistance, but still both the source and mechanism of E2 promoting TKI resistance are not clear. Our another research found that lipid accumulation, which results from TKI treatment in NSCLC cell also promotes the drug resistance. Based on the evidences of preliminary experiments, we speculate the source of E2 promoting resistance may be due to lipid accumulation, which results from TKI therapy, and the mechanism may be related to transcription of c-MET, activated by E2/ERβ. The results of ChIP-PCR show that E2-activated ERβ could bind to multiple transcriptional regulators of MET gene to activate transcription of MET, which is the key of MET amplification promoting resistance.. This project intends to expand paired NSCLC samples（n=100 ）before and after TKI resistance to analyze the correlation between lipid accumulation and E2 synthesis, and ERβ/c-MET expression and TKI resistance by performing IHC assay on paraffin sections. The investigation for the role and pathway of lipid accumulation on promoting E2 synthesis will be performed by selecting NSCLC cell lines with third-generation EGFR-TKI sensitive mutation (PC-9 with 19-Del; H1975 with both L858R and T790M mutation). Constructing TKI-resistant cell lines mediated by c-MET over-expression through single-cell cloning culture and the single-cell sequencing, investigation for the effect of ERβ on up-regulating c-MET expression and promoting TKI resistance will be carried out. Furthermore, the ChIP-PCR assay and the dual-luciferase reporter gene assay will be performed for identification of the binding sites and regulatory role of ERβ on MET regulatory sequence. Finally, three animal models including lipid accumulation, TKI resistance and PDX（Patient-Derived tumor Xenograft）will be constructed to verify the promoting effect of lipid accumulation on E2 synthesis and the reversing effect of blocking E2/ERβ on TKI resistance mediated by c-MET over-expression.. This project will systematically explore the mechanism of TKI resistance promoted by E2/ERβ on the basis of "tracing source" (E2 source) and "tracing mechanism" (E2-mediated TKI resistance mechanism), providing a theoretical basis for the combination of anti-estrogen to reverse TKI resistance.