mPRα介导孕激素抑制肺腺癌细胞生长的快速非基因组作用机制

The biological characteristics of lung adenocarcinoma are closely related to female hormones. Our previous studies have found that progesterone inhibits the growth of lung adenocarcinoma cells through membrane progesterone receptor α (mPRα) mediated pathway. However, the detailed molecular mechanisms are still unknown. We hypothesize that progesterone mediated by mPRα may activate the rapid non-genomic action with decreasing intracellular cAMP/PKA levels and inhibiting cell metabolism. By utilizing the cutting-age technologies such as yeast two-hybrid, laser co-focus microscopy, CRISPR/Cas9 gene knockout system, protein phosphorylation chip and lung cancer animal models, we plan to screen and identify the key proteins of mPRα associated signal transducing network. We will investigate the changes in the PKA-related metabolic enzymes, phosphorylation levels of various functional proteins and/or functional alternations of ion channels in plasma membrane, prior to vs. postior to mPRα gene knockout. We establish nude mouse model of human lung adenocarcinoma, and then study the inhibitory effect of P4/Org alone or with gefitinib or PP1on the growth of lung adenocarcinoma xenografts . The objectives of this study are to identify or clarify the non-genomic mechanisms of inhibitory effect of progesterone/mPRα in lung adenocarcinoma cancer cells, to perfect and discover a new mechanism of non-genomic tumor-inhibition by progesterone which is mediated by mPRα, in the meanwhile to provide clues for new treatment targets for lung adenocarcinoma and retard EGFR-TKIs resistance.

肺腺癌生物学特性与女性激素密切相关。我们前期研究发现孕激素膜受体mPRα介导孕激素（P4）能抑制肺腺癌细胞生长，但分子机制不清。我们提出mPRα介导P4通过影响细胞膜离子通道功能、下调cAMP/PKA活性，抑制PKA调控的新陈代谢途径呈现快速非基因组抑癌效应。拟采用核外酵母双杂交、活细胞成像术等筛选及鉴定mPRα的相互作用蛋白质和调控网络；用CRISPR/Cas9基因敲除术、蛋白质磷酸化芯片、激光共聚焦及流式细胞术等研究孕激素处理前后、mPRα基因敲除前后肺腺癌细胞内依赖PKA的新陈代谢酶及功能蛋白的磷酸化水平差异及质膜上离子通道功能变化；肺腺癌动物模型观察P4/Org单独或联合吉非替尼、PP1等对肺腺癌移植瘤生长的抑制情况。以期阐明或发现新的由mPRα介导孕激素启动的抑制肺腺癌细胞生长的快速非基因组作用机制，同时为寻找肺腺癌治疗新靶点和减缓EGFR-TKIs耐药提供新思路。

肺腺癌生物学特性与女性激素密切相关。我们前期研究发现孕激素膜受体mPRα介导孕激素（P4）能快速抑制肺腺癌细胞生长，但具体的分子机制不清。本项目在前期研究基础上，利用细胞、动物和临床标本阐明了mPRα通过EGFR-SRC-ERK1/2途径介导P4抑制肺腺癌细胞生长，改善肺腺癌对EGFR-TKI的敏感性的机制；并通过膜酵母双杂交技术以及免疫共沉淀技术筛选和鉴定了与mPRα具有相互作用的靶蛋白。同时利用细胞实验证明了PTEN通过下调PI3K/AKT/hTERT通路诱导肺腺癌A549细胞细胞周期阻滞；Twist1、RhoC在肺腺癌的EMT过程中起着重要作用。此外在临床水平阐明了mPRα、孕激素水平与患者临床病理特征的关系。我们的研究阐明了mPRα介导孕激素启动的抑制肺腺癌细胞生长的潜在的快速非基因组作用机制，为寻找肺腺癌治疗新靶点和减缓EGFR-TKIs耐药提供新思路。