头颈部鳞癌中STAT3介导CAF转录调控PD-L2及糖基化修饰与西妥昔单抗敏感性的机制研究

Carcinoma-associated fibroblasts (CAF) activated the critical signaling pathway thereby inducing the immune escape is the leading cause for the failure of anti-EGFR therapy in head and neck squamous cell carcinoma (HNSCC). We previously found that CAF significantly activated signal transducers and activators of transcription 3 (STAT3) signaling pathway and PD-L2 expression. Furthermore, the deregulated of STAT3/PD-L2, aberrant glycosylation of PD-L2 and strong co-locolization of EGFR/PD-L2 were detected in the cetuximab resistant HNSCC patients, compared to the sensitive patients. Based on these data, we will test our hypothesis “CAF promote the STAT3 signaling pathway thereby both trans-activating PD-L2 and modulating its glycosylation modification, and then consequently initiating the immune evasion and cetuximab resistance”. Chromatin immunoprecipitation (CHIP), and luciferase reporter assay will be performed to explore the transactivation of PD-L2 by STAT3. Lectin microarrays and TCGA data analysis will be utilitized to explore the mechanism of STAT3 mediated the aberrant glycosylation of PD-L2. Finally, the combination therapy of small molecule inhibitor of STAT3 and cetuximab will be used to study the effect of the inhibition tumor immune evasion and enhanced the drug sensitivity of cetuximab in both HNSCC cell experiment and animal study. In summary, this research proposal will reveal a novel mechanism of STAT3-mediated drug resistance and immune escape in the tumor microenvironment and may present STAT3 as the core of the combination therapy in HNSCC.

肿瘤相关成纤维细胞(CAF)激活炎症通路介导的免疫逃逸是头颈部鳞癌(HNSCC)抗EGFR治疗失败的重要原因。我们前期发现，CAF激活STAT3通路并调控PD-L2；西妥昔单抗不敏感患者STAT3/PD-L2高表达、PD-L2异常糖基化修饰且与EGFR共定位。据此，我们提出“STAT3介导CAF调控PD-L2转录及糖基化修饰影响HNSCC西妥昔单抗敏感性”的科学假说。拟通过: CHIP和荧光素酶报告实验证明STAT3转录PD-L2；凝集素微阵列结合TCGA大数据分析探究STAT3调控PD-L2糖基化修饰的机制；体内外实验研究STAT3小分子抑制剂联合西妥昔单抗抑制免疫逃逸进而提高HNSCC治疗效果。预期结果将从CAF介导免疫逃逸的新角度揭示HNSCC中STAT3影响西妥昔单抗敏感性的作用机制，并为开发新一代以STAT3为核心的联合治疗策略提供依据。

肿瘤微环境介导的免疫逃逸是导致头颈部鳞癌(HNSCC)抗EGFR治疗效果不佳的重要原因。翻译后修饰，尤其是糖基化修饰，决定免疫检查点的稳定性和免疫调节功能。我们发现西妥昔单抗不敏感患者中，PD-L2高表达且存在糖基化修饰，据此，本项目拟对PD-L2糖基化修饰调控西妥昔单抗疗效的分子机制及其靶向治疗开展研究。.本课题发现，PD-L2在超过70%头颈部鳞癌患者中高表达，其表达水平与病理分级和淋巴结转移密切相关，构建随机森林生存模型发现肿瘤浸润的CD3+和CD8+T细胞与肿瘤细胞表达的PD- L2的组合，即基于PD-L2的免疫分子模型可实现患者的预后分层，该模型优于AJCC分期和肿瘤细胞分化程度构建的临床因素模型。本课题阐明了PD-L2在HNSCC中的表达水平及临床意义，证实PD-L2是HNSCC中关键的免疫检查点分子，可以作为潜在的治疗靶点。相关研究成果以通讯作者发表在Oncoimmunology杂志。此外，我们总结了PD-L2在不同肿瘤的表达和功能，PD-L2的调节机制和靶向PD-L2的治疗策略。相关研究成果以通讯作者发表在British Journal of Cancer杂志。.我们进一步发现，PD- L2存在N-连接的糖基化修饰且明确了糖基化修饰的4个具体位点N64，N57， N163 和N189；揭示岩藻糖基转移酶FUT8是介导PD-L2糖基化修饰的关键糖基转移酶。阐明糖基化修饰是维持PD-L2蛋白稳定性及与PD-1相结合发挥免疫逃逸功能的关键因素。此外，基于单细胞转录组测序分析及免疫组化染色结果，我们证实PD- L2的表达尤其是糖基化修饰程度与抗EGFR治疗西妥昔单抗的治疗效果密切相关。糖基化PD-L2与EGFR形成复合物，不依赖于表皮生长因子EGF持续激活EGFR/STAT3信号通路，介导西妥昔单抗的耐药。该课题从免疫分子的异常糖基化修饰介导免疫逃逸的角度阐明西妥昔单抗的耐药机制，为HNSCC的综合治疗开辟了新的思路，具有重要的转化医学意义。相关研究成果以通讯作者发表在Journal for Immunotherapy of Cancer杂志。.在国家自然科学基金面上项目的资助支持下，本课题组发表SCI文章共计6篇，累计累计影响因子超过40，其中单篇最高影响因子13.429。本项目顺利完成了预定的全部研究任务，达到了预期的效果。