Exosomes, or small extracellular vesicles (sEVs), serve as intercellular messengers with key roles in normal and pathological processes. Our previous work had demonstrated that Dsg2 expression in squamous cell carcinoma (SCC) cells enhanced both sEV secretion and loading of pro-mitogenic cargo. In this study, using wild-type Dsg2 and a mutant form that is unable to be palmitoylated (Dsg2cacs), we investigated the mechanism by which Dsg2 modulates SCC tumour development and progression through sEVs. We demonstrate that palmitoylation was required for Dsg2 to regulate sub-cellular localisation of lipid raft and endosomal proteins necessary for sEV biogenesis. Pharmacological inhibition of the endosomal pathway abrogated Dsg2-mediated sEV release. In murine xenograft models, Dsg2-expressing cells generated larger xenograft tumours as compared to cells expressing GFP or Dsg2cacs. Co-treatment with sEVs derived from Dsg2-over-expressing cells increased xenograft size. Cytokine profiling revealed, Dsg2 enhanced both soluble and sEV-associated IL-8 and miRNA profiling revealed, Dsg2 down-regulated both cellular and sEV-loaded miR-146a. miR-146a targets IRAK1, a serine-threonine kinase involved in IL-8 signalling. Treatment with a miR-146a inhibitor up-regulated both IRAK1 and IL-8 expression. RNAseq analysis of HNSCC tumours revealed a correlation between Dsg2 and IL-8. Finally, elevated IL-8 plasma levels were detected in a subset of HNSCC patients who did not respond to immune checkpoint therapy, suggesting that these patients may benefit from prior anti-IL-8 treatment. In summary, these results suggest that intercellular communication through cell-cell adhesion, cytokine release and secretion of EVs are coordinated, and critical for tumour growth and development, and may serve as potential prognostic markers to inform treatment options. ABBREVIATIONS Basal cell carcinomas, BCC; Betacellulin, BTC; 2-bromopalmitate, 2-Bromo; Cluster of differentiation, CD; Cytochrome c oxidase IV, COX IV; Desmoglein 2, Dsg2; Early endosome antigen 1, EEA1; Epidermal growth factor receptor substrate 15, EPS15; Extracellular vesicle, EV; Flotillin 1, Flot1; Glyceraldehyde-3-phosphate dehydrogenase, GAPH; Green fluorescent protein, GFP; Head and neck squamous cell carcinoma, HNSCC; Interleukin-1 receptor-associated kinase 1, IRAK1; Interleukin 8, IL-8; Large EV, lEV; MicroRNA, miR; Palmitoylacyltransferase, PAT; Ras-related protein 7 Rab7; Small EV, sEV; Squamous cell carcinoma, SCC; Tissue inhibitor of metalloproteinases, TIMP; Tumour microenvironment, TME.

中文翻译：

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miRNA 和细胞因子相关的细胞外囊泡介导鳞状细胞癌。

外泌体或小细胞外囊泡 (sEV) 作为细胞间信使在正常和病理过程中发挥关键作用。我们之前的工作已经证明，鳞状细胞癌 (SCC) 细胞中的 Dsg2 表达增强了 sEV 的分泌和促有丝分裂货物的负载。在这项研究中，我们使用野生型 Dsg2 和不能被棕榈酰化的突变形式 (Dsg2cacs)，研究了 Dsg2 通过 sEV 调节 SCC 肿瘤发展和进展的机制。我们证明 Dsg2 需要棕榈酰化来调节 sEV 生物发生所必需的脂筏和内体蛋白的亚细胞定位。内体途径的药理学抑制消除了 Dsg2 介导的 sEV 释放。在鼠异种移植模型中，与表达 GFP 或 Dsg2cacs 的细胞相比，表达 Dsg2 的细胞产生更大的异种移植肿瘤。与源自 Dsg2 过表达细胞的 sEV 共同处理会增加异种移植物的大小。细胞因子分析显示，Dsg2 增强了可溶性和 sEV 相关的 IL-8，并且 miRNA 分析显示，Dsg2 下调了细胞和 sEV 负载的 miR-146a。miR-146a 靶向 IRAK1，这是一种参与 IL-8 信号传导的丝氨酸-苏氨酸激酶。用 miR-146a 抑制剂治疗上调 IRAK1 和 IL-8 表达。HNSCC 肿瘤的 RNAseq 分析揭示了 Dsg2 和 IL-8 之间的相关性。最后，在对免疫检查点治疗没有反应的 HNSCC 患者子集中检测到 IL-8 血浆水平升高，这表明这些患者可能受益于先前的抗 IL-8 治疗。总之，这些结果表明，通过细胞间粘附、细胞因子释放和 EV 分泌的细胞间通讯是协调的，对肿瘤的生长和发展至关重要，并且可以作为潜在的预后标志物，为治疗方案提供信息。缩写 基底细胞癌，BCC；Betacellulin，BTC；2-溴棕榈酸酯，2-溴；分化簇，CD；细胞色素 c 氧化酶 IV、COX IV；Desmoglein 2, Dsg2; 早期内体抗原 1，EEA1；表皮生长因子受体底物 15，EPS15；细胞外囊泡，EV；Flotillin 1，Flot1；甘油醛-3-磷酸脱氢酶，GAPH；绿色荧光蛋白，GFP；头颈部鳞状细胞癌，HNSCC；白细胞介素 1 受体相关激酶 1，IRAK1；白细胞介素 8，IL-8；大型EV、lEV；微小RNA，和平号空间站；棕榈酰酰基转移酶，PAT；Ras相关蛋白7 Rab7；小型EV、sEV；鳞状细胞癌，鳞状细胞癌；金属蛋白酶组织抑制剂，TIMP；肿瘤微环境，TME。