金属硫蛋白MT1G在胰腺癌干性获得及其化疗耐药中的作用机制

Pancreatic cancer is one of the most aggressive cancers with lowest 5-year survival rate. Gemcitabine represents the first-line chemotherapeutic drug for pancreatic cancer; however drug resistance is frequently observed. Progression of pancreatic cancer after initial response to gemcitabine treatment is attributed to the expansion of a subpopulation with stemness properties within the tumor mass. Previously, we found that gemcitabine resistant cell line exhibited cancer stemness property. mRNA array analysis indicated that MT1G was listed among the highest downregulated genes in gemcitabine resistant cells. Our preliminary data showed that gemcitabine significantly induced MT1G expression in gemcitabine sensitive pancreatic cancer cells, but not resistant cells. Overexpression of MT1G reduced the sphere formation ability as well as drug resistance in gemcitabine resistant pancreatic cancer cells, suggesting that it might suppress stemness and drug resistance. In addition, NF-kB showed higher activity in gemcitabine resistant cells than that in sensitive cells, which was negatively correlated with MT1G expression. Furthermore, overexpression of MT1G inhibited NF-kB activation. Media from gemcitabine resistant cancer cells could induce drug resistance and promote stemness in gemcitabine sensitive pancreatic cancer cells. LC-MS identified that gemcitabine resistant pancreatic cells secreted MMP and IL, both of which are targets of NF-kB. Therefore, we hypothesize that MT1G might suppress pancreatic cancer stemness and drug resistance through NF-kB signaling pathway. In brief, this study aims to explore the role and mechanism of MT1G in drug resistance and stemness regulation, and demonstrate the molecular pathways of NF-kB regulated proteins in the development of drug resistance to provide solid foundation of inducing MT1G as a therapeutic strategy for improving the survival of gemcitabine resistant patients.

胰腺癌5年生存率极低，因肿瘤干细胞存在所致的耐药是导致一线化疗药物吉西他滨治疗失败的根本原因。本项目前期研究发现在具有肿瘤干细胞识别特征的胰腺癌吉西他滨耐药（GR）细胞中，金属硫蛋白MT1G表达下降。吉西他滨在敏感（GS）细胞显著诱导MT1G表达，而GR细胞则不明显。过表达MT1G导致GR细胞对吉西他滨的敏感性增强和肿瘤干性减弱，提示MT1G在胰腺癌干性形成与化疗耐药中的作用。GR细胞中NF-kB活性明显高于GS细胞，而MT1G过表达能显著抑制NF-kB的激活，质谱分析显示GR细胞大量分泌受NF-kB调控的MMP和IL等蛋白因子，可增强GS细胞的干性与耐药性。鉴于此，本项目旨在解析MT1G在胰腺癌干性形成与化疗耐药中的重要作用及其机制，阐明GR细胞分泌的蛋白因子促进GS细胞产生耐药的分子基础，并探讨利用MT1G诱导剂降低胰腺癌对吉西他滨耐药的潜在应用，为解决胰腺癌耐药提供实验依据。

化疗耐药是胰腺癌临床治疗中一个长期困扰的问题，胰腺肿瘤干细胞被认为是耐药的主要来源。本研究旨在探讨金属硫蛋白MT1G在胰腺癌细胞干性调控中的作用及其机制，以期为克服胰腺癌的化疗耐药提供新的策略。研究发现，MT1G基因启动子区高度甲基化导致其在胰腺癌耐药细胞中表达下调，MT1G低表达则促进了胰腺癌细胞的肿瘤干细胞特性和化疗耐药。在MT1G低表达的耐药细胞中，NF-kB活性增加，过表达MT1G可促进NF-kB的降解，进而负调控胰腺癌细胞的干性特征。在机制上，MT1G通过增强E3连接酶TRAF7的表达促进NF-kB p65亚基的降解。通过细胞分泌蛋白质谱分析，鉴定出在吉西他滨耐药细胞中差异表达、受NF-kB调控的关键细胞分泌因子Activin A。MT1G通过促进NF-kB的降解限制Activin A的表达与分泌，MT1G低表达的耐药细胞分泌更多的Activin A，可通过旁分泌作用以Smad4依赖或非依赖的方式促进诱导周围敏感细胞的干性增强并获得耐药能力。进一步利用Activin A抑制剂卵泡抑素联合吉西他滨，发现利用卵泡抑制素阻断Activin A信号传导可以克服吉西他滨耐药性。综上，本研究揭示了胰腺癌细胞干性调控中MT1G对NF-kB/Activin A通路的负调控作用及其机制，为深入理解胰腺癌化疗耐药的机理奠定了理论基础，获得的实验数据将为胰腺癌的临床治疗制订新的干预策略提供理论参考。