DataSheet_2_Hypoxia-Induced Intracellular and Extracellular Heat Shock Protein gp96 Increases Paclitaxel-Resistance and Facilitates Immune Evasion in Breast Cancer.docx

BackgroundsHypoxia contributes to cancer progression, drug resistance and immune evasion in various cancers, including breast cancer (BC), but the molecular mechanisms have not been fully studied. Thus, the present study aimed to investigate this issue.
MethodsThe paclitaxel-sensitive BC (PS-BC) cells were administered with continuous low-dose paclitaxel treatment to establish paclitaxel-resistant BC (PR-BC) cells. Exosomes were isolated/purified by using the commercial kit, which were observed by Transmission electron microscopy (TEM). Cell viability was measured by MTT assay, cell apoptosis was determined by flow cytometer (FCM). Gene expressions were respectively measured by Real-Time qPCR, Western Blot and immunofluorescence staining assay. The peripheral mononuclear cells (PBMCs) derived CD8+ T cells were obtained and co-cultured with gp96-containing exosomes, and cell proliferation was evaluated by EdU assay. ELISA was employed to measure cytokine secretion in CD8+ T cells’ supernatants.
ResultsHSP gp96 was significantly upregulated in the cancer tissues and plasma exosomes collected from BC patients with paclitaxel-resistant properties. Also, continuous low-dose paclitaxel treatment increased gp96 levels in the descendent PR-BC cells and their exosomes, in contrast with the parental PS-BC cells. Upregulation of gp96 increased paclitaxel-resistance in PS-BC cells via degrading p53, while gp96 silence sensitized PR-BC cells to paclitaxel treatments. Moreover, PR-BC derived gp96 exosomes promoted paclitaxel-resistance in PS-BC cells and induced pyroptotic cell death in the CD8+ T cells isolated from human peripheral blood mononuclear cells (pPBMCs). Furthermore, we noticed that hypoxia promoted gp96 generation and secretion through upregulating hypoxia-inducible factor 1 (HIF-1), and hypoxia increased paclitaxel-resistance and accelerated epithelial-mesenchymal transition (EMT) in PS-BC cells.
ConclusionsHypoxia induced upregulation of intracellular and extracellular gp96, which further degraded p53 to increase paclitaxel-sensitivity in BC cells and activated cell pyroptosis in CD8+ T cells to impair immune surveillance.

背景：低氧（Hypoxia）可在包括乳腺癌（Breast Cancer, BC）在内的多种恶性肿瘤中促进肿瘤进展、诱导化疗耐药及免疫逃逸，但其相关分子机制尚未完全明确。为此，本研究拟针对该问题展开探究。
方法：本研究通过持续低剂量紫杉醇处理紫杉醇敏感型乳腺癌（Paclitaxel-Sensitive Breast Cancer, PS-BC）细胞，构建紫杉醇耐药型乳腺癌（Paclitaxel-Resistant Breast Cancer, PR-BC）细胞模型。采用商业化试剂盒分离纯化外泌体，通过透射电子显微镜（Transmission Electron Microscopy, TEM）对外泌体进行形态观察。采用MTT法检测细胞活力，流式细胞仪（Flow Cytometer, FCM）检测细胞凋亡情况。分别通过实时定量聚合酶链反应（Real-Time Quantitative PCR, Real-Time qPCR）、蛋白质免疫印迹（Western Blot）及免疫荧光染色实验检测基因表达水平。分离获取外周血单个核细胞（Peripheral Mononuclear Cells, PBMCs）来源的CD8+ T细胞，与携带gp96的外泌体共培养后，采用EdU法检测细胞增殖情况。采用酶联免疫吸附实验（Enzyme-Linked Immunosorbent Assay, ELISA）检测CD8+ T细胞上清液中的细胞因子分泌水平。
结果：在携带紫杉醇耐药表型的乳腺癌患者的肿瘤组织及血浆外泌体中，热休克蛋白gp96（HSP gp96）的表达水平显著上调。与亲本PS-BC细胞相比，经持续低剂量紫杉醇处理获得的子代PR-BC细胞及其分泌的外泌体中gp96的表达水平亦显著升高。gp96过表达可通过降解p53增强PS-BC细胞的紫杉醇耐药性，而敲低gp96则可使PR-BC细胞对紫杉醇治疗重新敏感。此外，PR-BC细胞来源的携带gp96的外泌体可增强PS-BC细胞的紫杉醇耐药性，并诱导人外周血单个核细胞（Peripheral Blood Mononuclear Cells, pPBMCs）分离得到的CD8+ T细胞发生细胞焦亡（Pyroptosis）。进一步研究发现，低氧可通过上调缺氧诱导因子1（Hypoxia-Inducible Factor 1, HIF-1）促进gp96的产生与分泌，同时可增强PS-BC细胞的紫杉醇耐药性并加速上皮间质转化（Epithelial-Mesenchymal Transition, EMT）进程。
结论：低氧可诱导细胞内及细胞外gp96的表达上调，后者进一步通过降解p53增强乳腺癌细胞的紫杉醇耐药性，并通过激活CD8+ T细胞的细胞焦亡过程损害机体免疫监视功能。