mPRα经PI3K/Akt/mTOR信号通路调控乳腺癌化疗敏感性机制的研究

Multidrug resistance (MDR) is a hurdle in the treatment of breast cancer, which is closely related to over-expression of breast cancer resistance protein (BCRP). We have reported that progesterone receptors (PRs) down-regulated the expression of BCRP via genomic pathway in PR posotive breast cancer. But we also found that progesterone could promote the BCRP expression in PR negative cancer which may be caused by out-nuclear PR via non-genomic pathway. The investigation about non-genomic PRs actions is a new field, which involved in three types of PR variants: membrane progesterone receptors (mPRs), progesterone receptor membrane components (PGRMCs) and nuclear progesterone receptors (nPRs). Among them, the mPRαis a major member in breast cancer.To these days, the mechanism of MDR regulated by mPRα is still poorly understood. In our pre-researching, we found that both mPRαand BCRP over expressed in breast cancer, which might be related to the activitation of PI3K signaling pathway. But it requires further investigation. From above, taking mPRα as a target according to the axis of mPRα，PI3K/Akt/mTOR pathway and BCRP, we try to clarify the relation between the expression of mPRα and BCRP from tissue level and verify whether the non-genomic PI3K/Akt/mTOR pathway involved in. Then we will explore if the rapamycin can strengthen the killing-cell effect of Taxol by reducing the expression of BCRP via PI3K/Akt/mTOR pathway and elucidate the possible mechanism. Taken together, the knowledge about the regulation of multidrug resistance mediated by BCRP via PI3K/Akt/mTOR signaling pathway will improved the new ideas for exploring the targets in increasing chemosensitivity in breast cancer.

多药耐药（MDR）是制约乳腺癌化疗有效性的主要原因之一。MDR发生与乳腺癌耐药蛋白（BCRP）过表达紧密相关。我们曾报道，孕激素受体（PR）可经基因组通路抑制BCRP表达；但PR能否经非基因组通路调控BCRP表达，仍未见研究。非基因组通路主要涉及三类孕激素受体：膜PR（mPRs）、PR膜元件（PGRMCs）和核外PRs；其中，mPRα在乳腺癌中高表达，但在调节BCRP表达中的作用机制仍不清楚。前期工作发现，mPRα与BCRP表达存在相关性，且可能与PI3K通路激活有关。基于此，本研究拟以mPRα为靶点，以mPRα-PI3K/Akt/mTOR通路-BCRP为轴线，从组织水平，明确mPRα与BCRP表达的相关性；从分子层次，研究mPRα经PI3K/Akt/mTOR通路调控BCRP表达的机制、探寻通路中的关键分子，阐明雷帕霉素增强乳腺癌化疗敏感性的可能机制，为临床提高保守治疗成功率提供理论基础。

化疗耐药是乳腺癌治疗常见的困境之一。约30%的浸润性乳腺癌因乳腺癌耐药蛋白（BCRP）的过表达而对多柔米星、多西紫杉醇等化疗药的反应性差，故BCRP是逆转乳腺癌耐药的重要靶点之一。雌、孕激素核受体在调控乳腺癌中BCRP表达的机制已被阐明，但孕激素膜受体α（mPRα）在调控耐药中的作用机制未见报道。本研究应用免疫组化的方法，检测了乳腺癌组织中BCRP、mPRα和磷酸化Akt1 （p-Akt1）的表达，并分析了mPRα和p-Akt1之间的相关性。为观察mPRα阳性乳腺癌中，其能否经PI3K/Akt/mTOR信号通路调节BCRP的表达，我们选择孕激素核受体阴性的乳腺癌细胞株、建立过表达BCRP蛋白的耐药细胞系MDA-MB-453/BCRP和 MDA-MB-231/BCRP；应用 co-IP法，检测mPRα与p-Akt1的相互结合；应用 western blotting 法，检测mPRα的特异性配体17β孕酮（17β-PG）调节BCRP表达的效应、PI3K/Akt/mTOR信号通路关键分子的表达和mTORC1的抑制剂雷帕霉素对BCRP表达的影响；应用CCK-8法，观察了雷帕霉素对乳腺癌耐药细胞株对化疗药多柔米星、多西紫杉醇敏感性的影响。结果显示，BCRP高表达与乳腺癌预后呈负相关； 17β-PG与mPRα结合后，可活化PI3K/Akt/mTOR信号通路继而调控BCRP的表达；雷帕霉素可下调BCRP的表达，并增强乳腺癌对化疗药的敏感性。综上所述，本研究阐述了BCRP是乳腺癌预后不良的危险因素，而mPRα可经PI3K/Akt/mTOR信号通路调控BCRP表达的机制。这为雷帕霉素联合化疗药，靶向增强mPRα阳性乳腺癌对化疗敏感性提供实验依据。此外，课题组还发现，mPRα确实能经非基因组通路调控MM-9的表达，以NF-κB为靶点可以抑制肿瘤细胞的迁移，这为靶向性抑制乳腺癌浸润转移的能力提供实验依据，并填补激素受体经非基因组通路调控MMP-9表达的研究空白。