RARγ/Axin/β-catenin正反馈环路介导胆管癌抗药性的机制研究

Cholangiocarcinoma (CCA) is not only lack of susceptibility to present chemotherapy drugs, and is also prone to develop drug resistance in chemotherapy. In our previous project, we found that RARγ mediated the drug resistance of CCA through regulation of Wnt/β-catenin signaling pathway. However, the mechanisms are still unclear. Our preliminary experiments showed that RARγ could directly interact with Axin in cytoplasm, and the expression of RARγ and monocarboxylate transporter 1(MCT1) could be upregulated by Wnt/β-catenin signaling pathway. Furthermore, MCT1 was associated with the drug resistance of CCA. Therefore, our project was designed to study the followings: firstly, to investigate the mechanisms of RARγ on regulating Wnt/β-catenin and the positive feedback loop between RARγ and Wnt/β-catenin, which upregulate the MCT1; secondly, to determine the role of MCT1 in mediating drug resistance of CCA, and thirdly to evaluate the in vivo role of RARγ/β-catenin/MCT1 signaling chain in drug resistance of CCA in animal models and clinical samples, hence to clarify the mechanisms of RARγ/Axin/β-catenin positive feedback loop on the mediating drug resistance of CCA, and finally to provide the scientific basis for RARγ as a potential gene therapy target of CCA.

胆管癌对现有化疗药物不敏感，且极易产生抗药性。我们前期项目研究发现RARγ通过调控Wnt/β-catenin信号通路介导胆管癌产生抗药性，然而其具体机制尚不清楚。本项目预实验表明，RARγ可在胞浆与Axin直接相互作用；Wnt/β-catenin信号通路可上调靶基因单羧酸转运蛋白1（MCT1）及RARγ表达，且MCT1与胆管癌抗药性有关。因此，本项目拟先研究RARγ调控Wnt/β-catenin信号通路的具体机制，以及是否与Wnt/β-catenin信号通路形成正反馈环路进而调控MCT1表达升高；然后确定MCT1介导胆管癌抗药性的作用；最后在整体水平以动物模型和临床样本验证RARγ/β-catenin/MCT1信号链介导胆管癌抗药性的作用。本项目将最终阐明RARγ/Axin/β-catenin正反馈环路介导胆管癌抗药性的机制，以期为RARγ作为临床胆管癌潜在的基因治疗靶点提供科学实验基础。

胆管癌对现有化疗药物不敏感，且极易产生抗药性。前期我们研究发现核受体家族的视黄酸受体γ（RARγ）调控胆管癌细胞增殖、迁移侵袭、耐药产生等恶性表型。然而，RARγ调控胆管癌抗药性的分子机制尚不清楚，且其异常高表达的机制也尚未阐明。. 本研究发现RARγ通过在胞浆与Axin直接相互作用调控Wnt/β-catenin信号通路，从而上调靶基因单羧酸转运蛋白1（MCT1）介导胆管癌产生抗药性。为进一步全面分析RARγ调控胆管癌发生发展的作用机制，本研究首先运用蛋白组学联合生物信息学分析技术系统分析发现，RARγ敲低后介导胆管癌细胞诸多糖代谢相关酶类表达改变。进一步研究发现，RARγ敲低后显著抑制了胆管癌细胞葡萄糖摄取量、乳酸分泌量和胞内ATP产量等糖代谢相关指标。此外，海马仪检测发现，敲低RARγ可显著降低细胞外酸化率，耗氧率明显升高。为进一步研究RARγ调控胆管癌细胞糖代谢重编程的机制，我们从蛋白组学差异蛋白中筛选确定出RARγ调控靶蛋白醛酮还原酶家族1成员AKR1C1。抑制AKR1C1活性可抑制胆管癌细胞有氧糖酵解以及胆管癌细胞增殖。免疫组化检测发现AKR1C1在胆管癌组织中明显高表达，其高表达与胆管癌的肿瘤大小和病理分级的分化差密切相关，且与RARγ的高表达呈正相关。AKR1C1可作为胆管癌不良预后的预测指标。本课题还从肿瘤炎症、营养缺乏及缺氧三大微环境角度，研究胆管癌细胞微环境对RARγ表达的影响。结果表明，炎症因子、无糖培养、缺氧微环境可诱导胆管癌细胞RARγ表达升高，与三大微环境密切相关的NF-κB、STAT3、AMPK、HIF1α信号通路异常激活调控胆管癌细胞RARγ异常高表达。. 综上所述，RARγ通过调控Wnt/β-catenin／MCT1信号轴介导胆管癌耐药性的产生。同时RARγ可通过下游靶点AKR1C1调控胆管癌细胞有氧糖酵解，AKR1C1可能作为癌基因参与胆管癌的发生发展。胆管癌RARγ异常高表达与炎症微环境、营养缺乏微环境和缺氧微环境三大肿瘤微环境密切相关。本研究阐明了RARγ调控胆管癌耐药性及胆管癌糖代谢重编程的作用机制及其异常高表达的调控机制，为RARγ作为临床胆管癌治疗靶点提供科学实验依据。