METHODS: Both our pre-established cellular necroptotic model and newly established Dox-induce intratumoral necroptosis model were used to investigate how necroptosis affect HNSCC progression. Transcriptomic alterations in peri-necroptotic tumor cells were analyzed by RNA-seq and validated in the NME in mice and patients\' samples. The differential DAMPs compositon among apopotosis. Necrosis, and necroptosis were analyzed by label-free proteomic technique, and the necroptosis-specific DAMPs were then identified and validated. The potential receptor for ISG15 were simulated using molecular docking and further validated by in vitro assays. Then the ISG15-RAGE axis was blocked by either knockdown of necroptotic-ISG15 release and RAGE inhibitor FPS-ZM1, and the impact on tumor progression were tested. Last, we further tested our findings in a HNSCC-patients cohort.
RESULTS: Necroptosis played a crucial role in driving tumor-cell invasiveness and lymphatic metastasis via tumor-type dependent DAMPs-releasing. Mechanistically, necroptotic DAMPs induced peri-necroptotic EMT via NF-κB and STAT3 signaling. Furthermore, intrinsic orchestration between necroptotic and cGAS-STING signaling resulted in producing a group of interferon stimulated genes (ISGs) as HNSCC-dependent necroptotic DAMPs. Among them, ISG15 played an essential role in reprogramming the NME. We then identified RAGE as a novel receptor for extracellular ISG15. Either blockage of ISG15 release or ISG15-RAGE interaction dramatically impeded necroptosis-driven EMT and lymphatic metastasis in HNSCC. Lastly, clinicopathological analysis showed high ISG15 expression in NME. Extensive necroptosis and high tumor-cell RAGE expression correlated with tumor progression and poor survival of HNSCC patients.
CONCLUSIONS: Our data revealed a previously unknown cGAS-ISG15-RAGE dependent reprogramming of the necroptotic microenvironment which converts the necroptotic stress into invasive force to foster HNSCC-cell dissemination. By demonstrating the programmatic production of ISG15 via necroptosis-cGAS orchestration and its downstream signaling through RAGE, we shed light on the unique role of ISG15 in HNSCC progression. Targeting such machineries may hold therapeutic potential for restoring intratumoral survival stress and preventing lymphatic metastasis in HNSCC.
方法:我们预先建立的细胞坏死模型和新建立的Dox诱导的瘤内坏死模型均用于研究坏死如何影响HNSCC进展。通过RNA-seq分析周围坏死肿瘤细胞的转录组学改变,并在小鼠和患者样品中的NME中进行验证。凋亡之间的差异DAMPs复合物。坏死,和坏死通过无标记蛋白质组学技术进行分析,然后鉴定和验证坏死凋亡特异性DAMP。使用分子对接模拟ISG15的潜在受体,并通过体外测定进一步验证。然后通过敲除坏死的ISG15释放和RAGE抑制剂FPS-ZM1来阻断ISG15-RAGE轴,并测试对肿瘤进展的影响。最后,我们在HNSCC患者队列中进一步检验了我们的发现.
结果:坏死性凋亡在通过肿瘤类型依赖性DAMPs释放驱动肿瘤细胞侵袭和淋巴转移中起关键作用。机械上,坏死性DAMPs通过NF-κB和STAT3信号诱导坏死性EMT。此外,坏死和cGAS-STING信号之间的内在协调导致产生一组干扰素刺激基因(ISG),作为HNSCC依赖性坏死DAMPs。其中,ISG15在重新编程NME中发挥了重要作用。然后,我们将RAGE鉴定为细胞外ISG15的新型受体。ISG15释放的阻断或ISG15-RAGE相互作用极大地阻碍了HNSCC中的坏死性凋亡驱动的EMT和淋巴转移。最后,临床病理分析显示ISG15在NME中高表达。广泛的坏死性凋亡和高肿瘤细胞RAGE表达与HNSCC患者的肿瘤进展和低生存率相关。
结论:我们的数据揭示了一种以前未知的cGAS-ISG15-RAGE依赖的坏死微环境重编程,将坏死应力转化为侵袭力以促进HNSCC细胞播散。通过证明通过坏死-cGAS编排及其通过RAGE的下游信号传导来编程生产ISG15,我们阐明了ISG15在HNSCC进展中的独特作用。靶向此类机器可能具有恢复肿瘤内生存压力和预防HNSCC淋巴转移的治疗潜力。