Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8525, Japan.
Advanced Research Center for Oral and Craniofacial Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8525, Japan.
Cells. 2020 Mar 19;9(3):755. doi: 10.3390/cells9030755.
Tumor cells exhibit therapeutic stress resistance-associated secretory phenotype involving extracellular vesicles (EVs) such as oncosomes and heat shock proteins (HSPs). Such a secretory phenotype occurs in response to cell stress and cancer therapeutics. HSPs are stress-responsive molecular chaperones promoting proper protein folding, while also being released from cells with EVs as well as a soluble form known as alarmins. We have here investigated the secretory phenotype of castration-resistant prostate cancer (CRPC) cells using proteome analysis. We have also examined the roles of the key co-chaperone CDC37 in the release of EV proteins including CD9 and epithelial-to-mesenchymal transition (EMT), a key event in tumor progression. EVs derived from CRPC cells promoted EMT in normal prostate epithelial cells. Some HSP family members and their potential receptor CD91/LRP1 were enriched at high levels in CRPC cell-derived EVs among over 700 other protein types found by mass spectrometry. The small EVs (30-200 nm in size) were released even in a non-heated condition from the prostate cancer cells, whereas the EMT-coupled release of EVs (200-500 nm) and damaged membrane vesicles with associated HSP90α was increased after heat shock stress (HSS). GAPDH and lactate dehydrogenase, a marker of membrane leakage/damage, were also found in conditioned media upon HSS. During this stress response, the intracellular chaperone CDC37 was transcriptionally induced by heat shock factor 1 (HSF1), which activated the CDC37 core promoter, containing an interspecies conserved heat shock element. In contrast, knockdown of CDC37 decreased EMT-coupled release of CD9-containing vesicles. Triple siRNA targeting CDC37, HSP90α, and HSP90β was required for efficient reduction of this chaperone trio and to reduce tumorigenicity of the CRPC cells in vivo. Taken together, we define "stressome" as cellular stress-induced all secretion products, including EVs (200-500 nm), membrane-damaged vesicles and remnants, and extracellular HSP90 and GAPDH. Our data also indicated that CDC37 is crucial for the release of vesicular proteins and tumor progression in prostate cancer.
肿瘤细胞表现出与治疗应激相关的分泌表型，涉及细胞外囊泡（EVs），如肿瘤小体和热休克蛋白（HSPs）。这种分泌表型发生在细胞应激和癌症治疗过程中。HSPs 是应激反应性分子伴侣，促进蛋白质的正确折叠，同时也通过 EV 以及一种称为警报素的可溶性形式从细胞中释放。我们在这里使用蛋白质组分析研究了去势抵抗性前列腺癌（CRPC）细胞的分泌表型。我们还研究了关键共伴侣 CDC37 在 EV 蛋白（包括 CD9 和上皮-间充质转化（EMT））释放中的作用，这是肿瘤进展中的关键事件。来自 CRPC 细胞的 EV 促进了正常前列腺上皮细胞的 EMT。在通过质谱法发现的超过 700 种其他蛋白质类型中，HSP 家族成员及其潜在受体 CD91/LRP1 在 CRPC 细胞衍生的 EV 中高度富集。即使在没有加热的情况下，前列腺癌细胞也会释放小 EV（30-200nm 大小），而在热休克应激（HSS）后，与 EMT 相关的 EV 释放（200-500nm）和带有 HSP90α 的受损膜囊泡增加。在 HSS 后，在条件培养基中还发现了 GAPDH 和乳酸脱氢酶，这是膜渗漏/损伤的标志物。在这种应激反应中，热休克因子 1（HSF1）转录诱导了细胞内伴侣 CDC37，激活了包含种间保守热休克元件的 CDC37 核心启动子。相比之下，CDC37 的敲低减少了含有 CD9 的囊泡的 EMT 相关释放。靶向 CDC37、HSP90α 和 HSP90β 的三重 siRNA 是减少这种伴侣三联体并降低体内 CRPC 细胞致瘤性所必需的。总之，我们将“应激组”定义为细胞应激诱导的所有分泌产物，包括 EV（200-500nm）、膜损伤囊泡和残余物以及细胞外 HSP90 和 GAPDH。我们的数据还表明，CDC37 对于前列腺癌中囊泡蛋白的释放和肿瘤进展至关重要。
