生长在低氧龛环境中的BCSCs是乳腺癌耐药的根源。低氧可激活GRP78介导的ERS，但是否与BCSCs耐药相关？未见报道。针对低氧龛发现参与GRP78介导的ERS的调控因素是研究BCSCs耐药的切入点。课题组发现BCSCs药物敏感性与GRP78表达相关；HIF-2α在BCSCs中特异高表达；HIF-2α阻断剂增加BCSCs药物敏感性、并与GRP78表达变化相关；但HIF-2α对GRP78介导的BCSCs耐药调控机制尚不明确。本课题拟通过检测BCSCs-MCF7/S细胞及荷瘤组织中干预GRP78表达对BCSCs药物敏感性及IREl-XBPl／PERK-eIF2-ATF4／ATF6等ERS-UPR通路功能变化，比较HIF-2α抑制剂作用前后异同，明确HIF-2α通过GRP78介导的ERS调控BCSCs药物敏感性作用与机制。本课题将为深入探讨BCSCs介导的乳腺癌耐药性研究提供新思路和新靶点。

Breast cancer stem cells (BCSCs) which survive in hypoxia niche may be the root of drug resistance. It has been reported that hypoxia microenvironment can activate glucose-regulated protein 78 (GRP78)-mediated endoplasmic reticulum stress (ERS), but it is unknown whether this is related to drug resistance in BCSCs. So, perhaps it will be a worth exploring issue that hypoxia niche may be involved in GRP78-mediated ERS to regulate drug resistance of BCSCs. In our study, we have found that the expression of GRP78 was related to BCSCs drug sensitivity and HIF-2α was high expressed in BCSCs. We also found when BCSCs were treated with HIF-2α inhibitor, the drug sensitivity increased and GRP78 expression changed. However, how HIF-2α regulates GRP78-mediated BCSCs drug resistance is still unclear. Hence, to elucidate the potential mechanism, we plan to interfere the expression of GRP78 in BCSCs-MCF7/S cells and xdenograft, detect its impact on BCSCs drug sensitivity and the changed functions of IREl-XBPl/PERK-eIF2-ATF4/ATF6 ERS signal pathways, then compare the different effects with or without HIF-2α inhibitor treatment. Our study will propose a new target or a new method for deep researching the mechanism of BCSCs drug resistance.

生长在低氧龛微环境中的乳腺癌干细胞（Breast cancer stem cells，BCSCs）是乳腺癌耐药的根源，低氧诱导因子参与肿瘤的发生发展及耐药复发，但其在BCSCs中介导化疗抵抗的机制尚未完全阐明。本项目通过TCGA数据库分析发现乳腺癌患者HIF-2α（而非HIF-1α）与干细胞标志物ALDH1A1、CD44、BCRP及P-gp表达显著正相关。发现经无血清非粘附悬浮培养具有对紫杉醇耐药等BCSCs样特性的MCF7 MS和T47D MS细胞高表达HIF-2，且紫杉醇可诱导MCF7 MS和T47D MS细胞进一步高表达HIF-2α，提示HIF-2α可能与乳腺癌细胞干性形成及耐药密切相关。经慢病毒转染沉默HIF-2α抑制了BCSCs干性，逆转对PTX的耐药；反之，过表达HIF-2α增强了乳腺癌细胞干性转化，诱导对PTX的耐药。在机制上，我们首次证实了抑制HIF-2α可通过下调SOD2表达，增强mtROS水平及细胞内氧化态环境，增强PDI的表达与酶活性，抑制GRP78表达及UPR通路活性，降低BCSCs干性，逆转化疗耐药的新机制。本项目进一步靶向HIF-2α的PAS活性区域对接、筛选、合成小分子化合物ComA，证实了ComA与HIF-2α的PAS区的高度亲和力，特异抑制HIF-2α表达，抑制BCSCs增殖，协同增敏PTX作用，也通过调控SOD2/mtROS/PDI抑制UPR通路发挥作用机制，且沉默HIF-2α与ComA的作用与机制均在荷瘤鼠水平得到了证实。本项目以肿瘤低氧微环境为切入点，发现了HIF-2α是介导BCSCs化疗药物抵抗的关键靶点，阐明了BCSCs中特异高表达的HIF-2α通过SOD2/mtROS/PDI调控UPR通路介导BCSCs耐药的新机制。本项目进行过程中研发的靶向抑制HIF-2α的小分子先导化合物ComA，可特异拮抗乳腺癌药物抵抗、特别是拮抗肿瘤干性转化介导的药物抵抗。特异拮抗HIF-2α的先导化合物将有望成为新型的乳腺癌治疗药物。