Flemming Joseph P, Hill Brianna L, Haque Mohammed W, Raad Jessica, Bonder Claudine S, Harshyne Larry A, Rodeck Ulrich, Luginbuhl Adam, Wahl James K, Tsai Kenneth Y, Wermuth Peter J, Overmiller Andrew M, Mahoney Mỹ G
Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA, USA.
Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, SA, Australia.
J Extracell Vesicles. 2020 Jul 13;9(1):1790159. doi: 10.1080/20013078.2020.1790159.
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.
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.
外泌体，即小细胞外囊泡（sEVs），作为细胞间信使在正常和病理过程中发挥关键作用。我们之前的研究表明，鳞状细胞癌（SCC）细胞中桥粒芯糖蛋白2（Dsg2）的表达增强了sEV的分泌以及促有丝分裂货物的装载。在本研究中，我们使用野生型Dsg2和一种无法进行棕榈酰化修饰的突变形式（Dsg2cacs），研究了Dsg2通过sEV调节SCC肿瘤发生和进展的机制。我们证明，棕榈酰化修饰是Dsg2调节脂质筏和sEV生物发生所需的内体蛋白亚细胞定位所必需的。内体途径的药理学抑制消除了Dsg2介导的sEV释放。在小鼠异种移植模型中，与表达绿色荧光蛋白（GFP）或Dsg2cacs的细胞相比，表达Dsg2的细胞产生了更大的异种移植肿瘤。与源自过表达Dsg2细胞的sEV共同处理增加了异种移植瘤的大小。细胞因子谱分析显示，Dsg2增强了可溶性和与sEV相关的白细胞介素8（IL-8），而微小RNA（miRNA）谱分析显示，Dsg2下调了细胞和sEV装载的miR-146a。miR-146a靶向IRAK1，一种参与IL-8信号传导的丝氨酸-苏氨酸激酶。用miR-146a抑制剂处理上调了IRAK1和IL-8的表达。头颈部鳞状细胞癌（HNSCC）肿瘤的RNA测序分析揭示了Dsg2与IL-8之间的相关性。最后，在一部分对免疫检查点治疗无反应的HNSCC患者中检测到血浆IL-8水平升高，这表明这些患者可能从先前的抗IL-8治疗中获益。总之，这些结果表明，通过细胞间粘附、细胞因子释放和EV分泌进行的细胞间通讯是协调的，对肿瘤生长和发展至关重要，并且可能作为潜在的预后标志物为治疗方案提供参考。
基底细胞癌，BCC；β细胞素，BTC；2-溴棕榈酸，2-Bromo；分化簇，CD；细胞色素c氧化酶IV，COX IV；桥粒芯糖蛋白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；小窝蛋白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EV，lEV；微小RNA，miR；棕榈酰转移酶，PAT；Ras相关蛋白7 Rab7；小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
