ABSTRACT

CAV1 (caveolin 1) expression and secretion is associated with prostate cancer (PCa) disease progression, but the mechanisms underpinning CAV1 release remain poorly understood. Numerous studies have shown CAV1 can be secreted within exosome-like vesicles, but antibody-mediated neutralization can mitigate PCa progression; this is suggestive of an inverted (non-exosomal) CAV1 topology. Here we show that CAV1 can be secreted from specific PCa types in an inverted vesicle-associated form consistent with the features of bioactive CAV1 secretion. Characterization of the isolated vesicles by electron microscopy, single-molecule fluorescence microscopy and proteomics reveals they represent a novel class of exosomes ~40 nm in diameter containing ~50-60 copies of CAV1 and, strikingly, are released via a non-canonical secretory macroautophagy/autophagy pathway. This study provides novel insights into a mechanism whereby CAV1 translocates from a normal plasma membrane distribution to an inverted secreted form implicated in PCa disease progression.

Abbreviations: 3-MA: 3-methyladenine; APEX: a modified soybean ascorbate peroxidase; ATG5: autophagy related 5; ATG9A: autophagy related 9A; ATG12: autophagy related 12; BHK: baby hamster kidney; C-exosomes: caveolin-exosomes; CAMKK2/CAMKKβ: calckum/calmodulin dependent protein kinase kinase 2; CAV1: caveolin 1; DAB: 3,3′-diaminobenzidine; DAPK: death associated protein kinase; EEA1: early endosome antigen 1; EM: electron microscopy; FCS: fluorescence correlation spectroscopy; GBP: GFP/YFP-binding peptide; GFP: green fluorescent protein; GOLGA2: golgin A2; ILVs: intralumenal vesicles; LC3: microtubule-associated protein 1 light chain 3; MBP: maltose binding protein; MTORC1: mechanistic target of rapamycin kinase complex 1; MVBs: multivesicular bodies; PBS: phosphate-buffered saline; PCa: prostate cancer; PI3K: phosphoinositide 3-kinase; PM: plasma membrane; SFM: serum-free medium; TSG101: tumor susceptibility 101; WCL: whole cell lysates; WT: wild type; YFP: yellow fluorescent protein; βoG: β-octylglucoside

摘要

CAV1 (caveolin 1) 表达和分泌与前列腺癌 (PCa) 疾病进展有关，但支持 CAV1 释放的机制仍然知之甚少。大量研究表明 CAV1 可以在外泌体样囊泡中分泌，但抗体介导的中和可以减缓 PCa 进展；这暗示了倒置的（非外泌体）CAV1 拓扑。在这里，我们显示 CAV1 可以从特定的 PCa 类型以倒置的囊泡相关形式分泌，这与生物活性 CAV1 分泌的特征一致。通过电子显微镜、单分子荧光显微镜和蛋白质组学对分离的囊泡进行表征表明，它们代表了一类新型外泌体，直径约 40 nm，含有约 50-60 个 CAV1 拷贝，并且引人注目的是，通过非经典的分泌性巨自噬/自噬途径释放。这项研究为 CAV1 从正常质膜分布转位到与 PCa 疾病进展有关的倒置分泌形式的机制提供了新的见解。

缩写：3-MA：3-甲基腺嘌呤；APEX：改性大豆抗坏血酸过氧化物酶；ATG5：自噬相关5；ATG9A：自噬相关的9A；ATG12：自噬相关12；BHK：小仓鼠肾；C-外泌体：caveolin-外泌体；CAMKK2/CAMKKβ：钙/钙调蛋白依赖性蛋白激酶激酶 2；CAV1：小窝蛋白 1；DAB：3,3'-二氨基联苯胺；DAPK：死亡相关蛋白激酶；EEA1：早期内体抗原 1；EM：电子显微镜；FCS：荧光相关光谱；GBP：GFP/YFP 结合肽；GFP：绿色荧光蛋白；GOLGA2：高尔金A2；ILVs：腔内囊泡；LC3：微管相关蛋白 1 轻链 3；MBP：麦芽糖结合蛋白；MTORC1：雷帕霉素激酶复合物 1 的机械靶点；MVBs：多泡体；PBS：磷酸盐缓冲液；PCa：前列腺癌；PI3K：磷酸肌醇 3-激酶；PM：质膜；可持续森林管理：无血清培养基；TSG101：肿瘤易感性101；WCL：全细胞裂解物；WT：野生型；YFP：黄色荧光蛋白；βoG：β-辛基葡萄糖苷