ABSTRACT

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小泡介导鳞状细胞癌。

摘要

外泌体或小的细胞外囊泡（sEVs）充当细胞间信使，在正常和病理过程中起关键作用。我们以前的工作已经证明，Dsg2在鳞状细胞癌（SCC）细胞中的表达增强了sEV的分泌和促有丝分裂原货物的负载。在这项研究中，使用野生型Dsg2和无法被棕榈酰化的突变形式（Dsg2cacs），我们研究了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脂筏和sEV生物发生所必需的内体蛋白的亚细胞定位。内体途径的药理抑制作用废除了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。与源自过表达Dsg2的细胞的sEV的共处理增加了异种移植物的大小。细胞因子分析显示，Dsg2增强了可溶性和sEV相关的IL-8，而miRNA分析显示，Dsg2下调了细胞和sEV加载的miR-146a。miR-146a靶向IRAK1，IRAK1是一种参与IL-8信号传导的丝氨酸-苏氨酸激酶。用miR-146a抑制剂处理可上调IRAK1和IL-8的表达。HNSCC肿瘤的RNAseq分析显示Dsg2与IL-8之间存在相关性。最后，在对免疫检查点疗法无反应的一部分HNSCC患者中检测到IL-8血浆水平升高，表明这些患者可能受益于先前的抗IL-8治疗。综上所述，

缩略语

基底细胞癌，BCC；Betacellulin，BTC；2-溴棕榈酸酯，2-溴; 分化集群，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；胞外囊泡，EV；Flotillin 1，Flot1; 3-磷酸​​甘油醛脱氢酶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；IL-8，白介素8；大型电动汽车 微小RNA，miR; 棕榈糖基转移酶 Ras相关蛋白7 Rab7; 小型EV，sEV；鳞状细胞癌； SCC；金属蛋白酶组织抑制剂TIMP；肿瘤微环境，TME