癌症相关成纤维细胞通过SDF1/FEZF1-AS1/PP2A/GLUT1信号轴调控胰腺癌有氧糖酵解的研究

Aerobic glycolysis is a metabolic pathway which is indispensible for pancreatic cancer cells to survive and proliferate, and the tumor microenvironment plays a vital role in regulating the heterogeneity of tumor metabolism. Cancer-associated fibroblasts (CAFs) are the major stromal cells in pancreatic cancer. In our previous research, we found that CAFs could promote the aerobic glycolysis of pancreatic cancer cells by regulating the expression level of GLUT1 in a SDF1-related paracrine manner, but the concrete mechanism remains unclear. Further preliminary experiments prompted that CAFs might upregulated the expression level of lncRNA FEZF1-AS1 via the SDF1/CXCR4 axis. Our study next indicated that lncRNA FEZF1-AS1 possibly mediated the ubiquitylation and degradation of PP2A protein and subsequently modulated the expression level of AKT kinase dependent target gene GLUT1. As mentioned above, we put forward the scientific hypothesis that cancer-associated fibroblasts regulate the aerobic glycolysis of pancreatic cancer through the SDF1/FEZF1-AS1/PP2A/GLUT1 axis. Several important experimental methods, including RNA pull-down assay, dual-luciferase reporter gene assay, protein ubiquitination detection and extracellular acidifcation rate assay, will be used to illuminate the exact molecular mechanism that how CAFs modulate the aerobic glycolysis of pancreatic cancer cells in terms of differential expression of lncRNAs. We aim to lay the foundation for the development of specific drugs targeting on both energy metabolism and fibroinflammatory microenvironment of pancreatic cancer.

有氧糖酵解是胰腺癌细胞赖以生存的代谢途径，而肿瘤微环境是影响肿瘤代谢异质性的关键因素。癌症相关成纤维细胞（CAFs）是胰腺癌最主要的间质细胞，我们前期发现CAFs能旁分泌SDF1上调GLUT1表达而促进胰腺癌细胞有氧糖酵解，但具体调控机制远未明确。进一步预实验提示：CAFs可能通过SDF1/CXCR4轴上调lncRNA FEZF1-AS1表达，而后者可能增强PP2A的泛素化降解、进而介导AKT激酶依赖性的靶基因GLUT1表达。据此，本项目提出“癌症相关成纤维细胞通过SDF1/FEZF1-AS1/PP2A/GLUT1信号轴调控胰腺癌有氧糖酵解”的科学假说，拟通过RNA pull-down、双荧光素酶报告基因检测、蛋白泛素化检测、细胞外酸化率检测等方法，首次从lncRNA角度阐明CAFs调控胰腺癌细胞有氧糖酵解的具体机制，为开发针对胰腺癌“能量代谢+纤维炎性微环境”双靶向药物奠定基础。

胰腺癌是常见的消化系统恶性肿瘤，起病隐匿，发展迅速，预后极差。近年对肿瘤细胞能量代谢的研究为胰腺癌提供新的诊治方向。有氧糖酵解是胰腺癌细胞赖以生存的代谢途径，而肿瘤微环境是影响肿瘤代谢异质性的关键因素。癌症相关成纤维细胞（CAFs）是胰腺癌最主要的间质细胞，在肿瘤代谢中具有重要作用。本项目研究发现（1）高通量筛选及共培养实验提示CAFs大量旁分泌的趋化因子SDF1促进胰腺癌细胞有氧糖酵解；（2）CAFs通SDF1/CXCR4轴调控胰腺癌细胞GLUT1表达；（3）高通量测序及qRT-PCR验证发现SDF1/CXCR4信号轴通过上调lncRNA FEZF1-AS1的转录，上调GLUT1的表达；（4）RNA pull down、CO-IP及截断实验等验证lncRNA FEZF1-AS1直接结合胞浆PP2A蛋白，作为分子支架募集CRL4 E3泛素连接酶、促进PP2A的泛素化降解，以蛋白质翻译后修饰途径促进基线水平AKT蛋白的持续磷酸化激活、继而上调GLUT1表达；（5）构建了小鼠胰腺癌原位成瘤模型，在体内验证靶向抑制SDF1/FEZF1-AS1/PP2A/GLUT1信号轴对肿瘤生长的影响，结果提示共注射过表达SDF1的CAFs显著促进胰腺癌原位肿瘤的生长，而敲降lncRNA FEZF1-AS1的表达逆转录了过表达SDF1的CAFs促进胰腺癌肿瘤生长的作用。此外，敲降lncRNA FEZF1-AS1的表达显著延长过表达SDF1的CAFs诱导后的荷瘤小鼠的生存时间；（6）临床资料提示lncRNA FEZF1-AS1在胰腺癌肿瘤组织中的表达相比癌旁组织中的表达明显上调，且lncRNA FEZF1-AS1高表达与胰腺癌患者肿瘤分化程度及病理分期呈正相关。本研究首次从lncRNA角度阐明CAFs调控胰腺癌细胞有氧糖酵解的具体机制，为开发针对胰腺癌“能量代谢+纤维炎性微环境”双靶向药物奠定基础。

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