ISG15在鼻咽癌微环境中的调控作用及机制

Cytokines in tumor microenvironment is a key to regulate cancer stem cell (CSC) phenotype, and contributes to tumor relapse and metastasis. By RNA-seq, we identified that Interferon-Stimulated Gene 15 (ISG15) was overexpressed in nasopharyngeal carcinoma (NPC) tissues, promoted a CSC phenotype, and were correlated with tumor recurrence and shorter survival. Further studies revealed that secretory ISG15 was elevated in the culture media of ISG15 overexpression NPC cells and up-regulated M2-type cytokines in monocytes. Thus, we hypothesize autocrine and paracrine of ISG15 in tumor microenvironment modify the phenotypes of tumor cells and macrophages, and contribute to cancer cell stemness. To clarify the role and mechanism of ISG15 autocrine in NPC, variation of tumor phenotypes will be observed by using recombinated ISG15 and its nertralizing antibody, and membrane protein receptor of ISG15 will be identify by pull-down and mass spectrometry. In order to reveal the effect of ISG15 paracrine in NPC, the change of M2 phenotype of macrophages and the subsequently influence on tumor cells will be studied in vitro and in vivo, as well as the molecular mechanism will be investigated. Our studies focus on the microenvironment regulations of ISG15 for malignant transformation and provide a new molecular target for NPC therapy.

肿瘤微环境中的细胞因子是调控肿瘤细胞干性的关键，与肿瘤复发、转移相关。通过转录组测序，我们发现ISG15在鼻咽癌中高表达，ISG15可增强鼻咽癌细胞干性，与肿瘤复发及不良预后相关，进一步发现过表达ISG15的鼻咽癌细胞培养上清中分泌型ISG15增多，后者可上调单核细胞M2表型细胞因子。因此我们推测鼻咽癌细胞可通过自分泌和旁分泌ISG15，参与对肿瘤细胞和巨噬细胞的调控。本项目拟采用重组ISG15及其中和抗体，明确鼻咽癌自分泌ISG15的促癌作用；通过免疫沉降-质谱分析，阐明分泌型ISG15的膜蛋白受体及促癌的机制；通过体内外实验明确ISG15对巨噬细胞表型及功能的作用及机制，继而观察ISG15处理后巨噬细胞对鼻咽癌细胞表型的影响，从微环境调控的途径探讨ISG15对鼻咽癌细胞恶性转化的作用及机制，为鼻咽癌临床治疗提供新的靶标及依据。

干扰素刺激基因15（interferon-stimulated gene 15, ISG15）是最早被发现的一种类泛素化蛋白，不仅具有抗病毒作用，还与多种癌症密切相关。局部复发和远处转移是鼻咽癌患者死亡的主要原因，课题组前期研究发现ISG15在鼻咽癌组织中高表达与预后不良相关。有研究发现，肿瘤细胞分泌的ISG15可减弱树突细胞的抗原提呈能力。然而，目前尚未发现ISG15在鼻咽癌微环境中的研究。本课题将从肿瘤微环境角度，探讨ISG15能否作为鼻咽癌微环境因子影响巨噬细胞表型，进而促进鼻咽癌恶性进展。.在鼻咽癌组织中检测到IFN-α和IFN-βmRNA水平均上调；而IFN-β处理后的鼻咽癌细胞内和分泌的ISG15表达明显增加。免疫荧光实验发现ISG15主要在鼻咽癌组织的T细胞和巨噬细胞中表达，且在后者表达更强。这表明鼻咽癌细胞能分泌ISG15，且ISG15可能在鼻咽癌微环境中起细胞因子作用。 .通过免疫组化实验及临床病例资料分析发现，ISG15+ CD163+ TAMs高表达组总生存期及无病生存期更短，且ISG15+ CD163+ TAMs数目和临床晚期显著相关。这提示表达ISG15+ CD163+ TAMs与鼻咽癌患者不良预后正相关。.用重组人ISG15蛋白（rISG15）诱导人外周血来源巨噬细胞，通过流式发现其M2表型表面标记物CD163的表达增加；Real-time PCR发现M2类细胞因子（IL-10、TGF-β）mRNA的表达上调。动物实验发现rISG15诱导的人巨噬细胞可促进肿瘤生长。这表明ISG15 可诱导巨噬细胞 M2 表型，进而促进鼻咽癌的恶性进展。.细胞因子芯片实验发现rISG15诱导的巨噬细胞上清中CCL18的增加最明显。Transwell 实验发现rISG15诱导的巨噬细胞上清可促进鼻咽癌细胞迁移，而使用CCL18多克隆抗体或ISG15受体抑制剂可阻断该过程。rISG15处理的巨噬细胞包膜上检测其受体LFA-1的表达，而LFA-1抑制剂A286982可抑制rISG15诱导的CCL18的分泌。此外，在rISG1诱导的巨噬细胞中，SRC信号被激活。而LFA-1和SRC抑制剂均可阻碍rISG15诱导的SRC信号的激活。这表明ISG15诱导的M2型巨噬细胞通过LFA-1-SFKs信号通路增强CCL18的分泌，从而促进鼻咽癌恶性进展。