CAFs旁分泌CXCL1活化Notch信号通路促进肝癌细胞“干性”的机制研究

Cancer stem cells (CSCs) drive hepatocellular carcinoma (HCC) progression and recurrence following therapy. Cancer-associated fibroblasts (CAFs) may contribute to stemness maintenance and cell plasticity in HCC. We’ve previously shown that CAFs feeders from clinical HCC patients demonstrated a powerful ability to maintain cancer stemness in CSC/CAF co-culture system. Most importantly, our findings revealed that CSC growth was regulated in a paracrine manner by CAFs through the CXCL1/CXCR2-Notch signaling pathway by using Human cytokine array and Human Cancer Stem Cells RT² Profiler™ PCR Array, which contain 80 human cytokines and 84 human cancer stem cell genes individually. In this study, we will confirm whether the inhibition of CXCL1 or CXCR2 signaling by a specific antibody or inhibitors could suppress Notch pathway or cancer stemness. In particular, siRNA ADAM17 or the inhibitor of ADAM17 (TAPI) will be utilized to knockdown or block ADAM17 in CSC/CAF co-culture system, and Notch pathway will be test. Subsequently, the rescue of ADAM17 knockdown by ectopic overexpression of NICD (Lenti- v NICD) downstream will be used to confirm that CAFs regulate the plasticity of HCC stemness via paracrine CXCL1/CXCR2-ADAM17–Notch pathway. Finally, we will explore the phosphorylation site of ADAM17 by PRM when it is activated by CXCL1/CXCR2 signaling. Together, the purpose of this study is to understand the role of CAFs in mediating the CSC phenotype and stemness of HCC. These findings may suggest promising functional biomarker and therapeutic target in anti-tumor treatment of patients with HCC.

肝癌干细胞是肝细胞癌转移复发的“源动力”，而肿瘤相关成纤维细胞（CAFs）在促进肿瘤细胞“干性”化中可能起重要作用。我们前期在CAFs与肝癌细胞的共培养体系中发现CAFs可促进肝癌细胞的“干性”，且经高通量蛋白芯片和基因芯片筛查初步确定CXCL1与Notch信号通路可能在CAFs促进肝癌细胞“干性”化中起关键作用。据此，本项目拟通过细胞因子的干预实验证实CAFs旁分泌CXCL1活化肝癌细胞Notch信号通路，再采用蛋白阻滞和慢病毒干扰技术序贯调控肝癌细胞的ADAM17与NICD，试图解析CAFs经CXCL1/CXCR2-ADAM17分子网络激活Notch信号通路的关键分子机制，并采用液相串联质谱分析筛查ADAM17磷酸化活化位点。旨在阐明CAFs旁分泌CXCL1激活Notch信号通路促进肝癌细胞“干性”的分子机制，可望从肿瘤微环境与肿瘤干细胞间对话这一角度为遏制肝癌复发转移提供新的靶标。

肝癌干细胞是肝细胞癌转移复发的“源动力”，而肿瘤相关成纤维细胞（CAFs）在促进肿瘤细胞“干性”化中可能起重要作用。我们前期在CAFs与肝癌细胞的共培养体系中发现CAFs可促进肝癌细胞的“干性”，且经高通量蛋白芯片和基因芯片筛查初步确定CXCL1与Notch信号通路可能在CAFs促进肝癌细胞“干性”化中起重要作用。实验过程中进一步探索发现STC1也可激活Notch信号通路，其与CXCL1在CAFs促进肝癌细胞“干性化”中起关键作用。本项目在向肝癌细胞中加入外源性CXCL1/STC1后，通过一系列“干性”鉴定实验证实CXCL1/STC1对肝癌细胞“干性”具有促进作用。CXCL1/STC1中和抗体可抑制其对肝癌“干性”的促进作用。在此基础上，检测Notch信号通路的变化，发现CXCL1/STC1可激活肝癌Notch信号通路。进一步的机制研究发现，CXCL1可以使ADAM17磷酸化从而激活Notch信号通路，而STC1直接结合在Notch1分子上激活Notch信号通路。总之，本课题阐明了CAFs旁分泌CXCL1/STC1促进肝癌“干性”的分子机制，可望从肿瘤微环境与肿瘤干细胞间对话这一角度为遏制肝癌复发转移提供新的靶标。