mPRα介导孕激素下调Notch-1改善肺腺癌细胞对EGFR-TKIs耐药机制研究

Primary and acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) emerges as the bottleneck of targeted therapies. Our previous study shows that progesterone through the mediation of membrane progesterone receptor alpha (mPRα), modulates the sensitivity of lung adenocarcinoma cells to EGFR-TKIs, while progesterone down-regulates the expression of Notch-1 as well. As we all know, Notch-1 facilitates cells to undergo EMT and upregulate EGFR, and ultimately leads to EGFR-TKIs resistance. Here, we raise our hypothesis: P4’s action through the mediation of mPRα, down-regulate Notch-1, by reversing EMT or down-regulating EGFR, ultimately sensitizes lung adenocarcinoma cells to EGFR-TKIs. On this premise, by utilizing co-immunoprecipitation, we aim to explore the interaction between mPRα and Notch-1, Notch-1 and EGFR; by immunofluorescence confocal technique and isolation of caveolar fraction, we try to identify the co-location of the above three proteins. By CRISPR/Cas9 gene knockout system and gene transfection plasmid system to modulate the expression of mPRα and Notch-1, we plan to investigate the expression of Notch-1, EGFR, EMT associated proteins and cell sensitivity to EGFR-TKIs, prior to vs. postior to P4 treatment. At last, we establish a nude mouse model of human lung adenocarcinoma to verify the above hypothesis in vivo. The objective of this study is to clarify the underlying mechanisms of P4’s action mediated by mPRα, through down-regulating Notch-1 to sensitize lung adenocarcinoma cells to EGFR-TKIs.

EGFR酪氨酸激酶抑制剂（EGFR-TKIs）耐药逐渐成为肺腺癌靶向治疗的瓶颈，我们前期研究发现孕激素（P4）通过孕激素膜受体mPRα介导调节肺腺癌对EGFR-TKIs敏感性，P4可下调Notch-1表达，而Notch-1促进EMT发生和上调EGFR，且与耐药相关。我们课题假设：mPRα介导P4下调Notch-1，逆转EMT或下调EGFR，可改善EGFR-TKIs耐药。本项目拟采用免疫共沉淀验证mPRα与Notch-1、Notch-1与EGFR的相互作用，免疫荧光及小窝片段分离明确三者的共定位情况；利用基因过表达、CRISPR/Cas9基因敲除调节mPRα及Notch-1表达，研究P4处理前后EGFR、EMT相关蛋白表达及细胞对EGFR-TKIs敏感性变化；构建肺腺癌动物模型验证以上假说。以期阐明mPRα介导P4下调Notch-1改善肺腺癌细胞对EGFR-TKIs耐药的分子机制。

EGFR酪氨酸激酶抑制剂（EGFR-TKIs）的发现是晚期非小细胞肺癌治疗领域的里程碑，但EGFR-TKIs耐药或缺乏EGFR有义突变的患者逐渐成为肺腺癌靶向治疗的瓶颈。本项目采用激光共聚焦和免疫共沉淀验证mPRα与EGFR的细胞定位和相互作用关系。mPRα特异性激动剂Org OD 02-0（Org）和/或EGFR-TKIs吉非替尼（Gef）作用于不同肺腺癌细胞系A549（EGFR野生型）、PC-9GR（耐药型）或PC-9（有义突变型） 24h、48h或72h，CCK-8法检测细胞的增殖活性。稳定转染慢病毒LV-shmPRα敲减肺腺癌细胞系的mPRα表达，Org和/或Gef分别作用于肺腺癌细胞系A549、PC-9GR或PC-9 48h，CCK-8法检测细胞的增殖活性。A549细胞稳定转染慢病毒LV-shmPRα或空载体，构建裸鼠皮下成瘤模型，1周后给予Org和/或Gef腹腔注射3周，第4周处死小鼠，组织免疫荧光法检测各组小鼠瘤体标本的TUNEL表达。结果发现激光共聚焦结果显示mPRα定位于肺腺癌的细胞膜和细胞质中，且mPRα和EGFR共定位于肺腺癌细胞膜和细胞质中。免疫共沉淀结果表明：Org处理肺腺癌细胞48h后，利用mPRα抗体可以沉淀肺腺癌细胞的EGFR蛋白；采用EGFR抗体亦可以沉淀mPRα蛋白，二者存在直接相互作用。Org或Gef可抑制A549、PC-9GR或PC-9细胞的增殖活性，Org联合Gef抑制作用显著增强。mPRα表达下调后，Org对肺腺癌细胞增殖的抑制作用显著减弱，且Org促进Gef抑制肺腺癌细胞增殖的作用减弱。联合Org和Gef注射小鼠对比单独使用Gef显著抑制了瘤体生长，而当mPRα下调时，Org对Gef抗肿瘤的促进作用消失。联合Org和Gef处理小鼠比单独使用Gef显著提高了肿瘤细胞的凋亡水平，而当mPRα下调时，Org对增强Gef促肿瘤细胞凋亡的作用基本消失。联合Org和Gef处理小鼠比单独使用Gef显著降低了肿瘤细胞的增殖水平，而当mPRα下调时，Org对增强Gef抑制肿瘤细胞增殖的作用基本消失。本研究从分子、细胞和动物三个水平进一步研究mPRα在肺腺癌中的非基因组作用，并对mPRα特异性活化调控肺腺癌对EGFR-TKIs敏感性的分子机制进行探索，期望寻找到新的EGFR-TKIs增敏靶点。