SETD2表达缺失通过促进补体C5a分泌和中性粒细胞活化介导肾癌耐药的分子机制和临床意义研究

Metastatic renal cell carcinoma (mRCC) patients usually develop drug resistance during tyrosine kinase inhibitor (TKI) therapy. Chromatin remodeling plays a crucial role in mRCC development as well as TKI resistance. SET-domain containing 2 (SETD2) catalyzes H3K36 tri-methylation, while SETD2 mutation or down-regulation is quite common in mRCC samples. Our preliminary studies showed that SETD2 expression is often down-regulated in mRCC tissues compared with peri-tumor tissues. We also identified low SETD2 expression as a predictive marker for TKI resistance (J Urol, 2016). Furthermore, knockdown of SETD2 expression in RCC cell lines could result in augmented secretion of C5a, which would facilitate neutrophil chemotaxis and interleukin-8 secretion. Based on the preliminary data, the current project proposal is designed to illuminate the molecular mechanisms, functional significance and interventive strategies of aberrant chromatin remodeling including the activation of SETD2/C5a/neutrophil/interleukin-8 signaling in the TKI resistance mechanism. Human renal cell carcinoma cell lines, neutrophils, surgically resected renal cell carcinoma specimens and tumor bearing mouse models will be used in our attempts to establish the model of molecular regulation, TKI benefit prediction, survival assessment and interventive therapy based on the chromatin remodeling abnormalities and tumor microenvironment patterns in renal cell carcinoma. This project could help us lay the theoretical foundation for developing novel targeted agents against the interplay between renal cell carcinoma cells and neutrophils.

转移性肾癌患者死亡率较高，且大多数转移性肾癌患者经酪氨酸激酶抑制剂TKI靶向治疗后出现耐药。染色质重塑在肾癌发生发展和治疗抵抗过程中发挥关键作用，其中组蛋白H3第36位赖氨酸甲基转移酶SETD2表达改变在肾癌中较为常见，是促进肾癌发生发展的重要环节。项目申请人前期研究发现转移性肾癌肿瘤组织中SETD2表达水平显著低于癌旁组织，并且SETD2低表达提示较差的TKI治疗获益（J Urol, 2016）；而敲除肾癌细胞SETD2表达会显著促进补体C5a分泌，进而诱导中性粒细胞浸润活化并分泌IL-8。本申请项目拟在前期研究基础上，分析肾癌细胞SETD2表达缺失促进补体C5a分泌，并诱导中性粒细胞活化分泌IL-8和介导肾癌耐药的调控机制及其功能意义，建立染色质重塑调控肾癌对TKI靶向治疗耐药的分子机制、治疗反应性预测、生存评估和干预治疗模型，为开发阻断肾癌细胞与中性粒细胞相互作用的治疗药物奠定基础。

背景：免疫微环境的重构在肾癌的发生发展中发挥着重要的作用，随着高通量测序技术的成熟，对肾癌免疫微环境的刻画已经越发深入。靶向治疗已经在转移肾癌中取得了良好的疗效，而目前免疫治疗更是为诸多患者带来了生存获益，多项免疫联合靶向治疗已经成为一线治疗。然而目前仍然难以筛选出治疗敏感的患者，因此进一步明确肾癌的免疫微环境、挖掘潜在的治疗抵抗机制是极其关键的。CD8+ T细胞高浸润在肾癌中提示差预后 ，其可与巨噬细胞，调节性T细胞等构成抑制性的免疫微环境。CXCL13是一种趋化因子配体，对B淋巴细胞的归巢以及三级淋巴结构的形成都至关重要。既往研究多关注辅助T细胞分泌的CXCL13，CXCL13+CD8+ T细胞是否与肿瘤患者预后相关鲜有关注。.研究内容：我们通过制作肾癌患者组织芯片以及服用靶向药物的转移性肾癌患者的组织芯片，通过免疫组化研究各个患者的免疫浸润情况并通过聚类区分亚型，研究CXCL13+CD8+ T细胞的浸润情况。通过流式细胞技术研究新鲜肾癌组织样本的表达以及细胞的功能状态。.结果：我们通过无监督聚类鉴定出两种免疫亚型；其中以高浸润巨噬细胞、CD4+T细胞、CD8+T细胞、调节性T细胞为特征的肿瘤微环境分型TMEcluster-B，有着更短的OS(p<0.001;HR 2.629)，以及有着更差的靶向治疗反应性，表现为更短的OS(p<0.001;HR 2.223)。进一步通过大样本的ROC曲线分析发现该免疫分型联合IMDC预测模型能显著提高患者预后的预测效能。CXCL13+CD8+ T细胞的高浸润提示着更差的预后，OS缩短(p=0.013;HR 2.31)以及RFS缩短(P=0.043;HR 2.15)；高浸润CXCL13+CD8+ T细胞的患者免疫检查点表达上调(PD-1,Tim-3,TIGIT)以及效应分子下调(IFN-γ、TNF-α)。