AHR/NRF1信号通路调控线粒体生物合成在黑素瘤生长转移中的关键作用研究

Mitochondrial biogenesis plays a vital role in the maintenance of mitochondrial homeostasis and cell survival in melanoma, involved in the tumorigenesis, progression and drug resistance of melanoma, but the specific mechanism remains unclear. Our previous study found that the transcription factor AHR was expressed at a high level in melanoma cells and at a low level in melanocytes. AHR signal pathway could promote mitochondrial biogenesis, stabilize mitochondrial homeostasis and maintain survival of both melanoma cells and melanocytes. We further performed mechanism research and found that AHR signal pathway could upregulate mtDNA level and ATP production and promote mitochondrial biogenesis through directly inducing the expression and function of NRF1, which might be a novel mechanism for regulating mitochondrial biogenesis in melanoma. Then, we put forward the innovative hypothesis: AHR signal pathway could upregulate the expression of NRF1 and its downstream targets, which could further promote mitochondrial biogenesis, maintain mitochondrial homeostasis and cell survival involved in the pathogenesis of melanoma. In the present research project, we plan to study the regulatory mechanism of AHR involved in mitochondrial biogenesis in melanoma, which might provide novel strategy for treatment of melanoma.

线粒体生物合成对于黑素瘤线粒体稳态和细胞存活至关重要，参与黑素瘤生长、侵袭、转移、代谢及耐药过程，但其调控机制不明。我们前期研究首次发现，转录因子AHR在黑素瘤细胞中高表达、在黑素细胞中低表达，AHR信号通路可促进线粒体生物合成、维持线粒体稳态，促进黑素瘤细胞及黑素细胞存活；生物信息学预测及机制研究首次发现，AHR可能通过直接调控NRF1转录表达，促进线粒体DNA及ATP合成，成为调节黑素瘤线粒体生物合成的重要新机制。据此我们提出创新性假说：AHR信号通路通过调控NRF1表达及功能，促进线粒体生物合成，进而维持线粒体数量、结构和功能，促进黑素瘤生长、存活、转移和能量代谢，在黑素瘤发生发展过程中发挥关键作用。本项目拟在细胞、动物和黑素瘤临床样本中，运用细胞和分子生物学技术，系统研究AHR/NRF1信号通路介导线粒体生物合成调控黑素瘤生长转移的关键机制，为黑素瘤治疗提供新的理论依据及策略方向。

线粒体生物合成是黑素瘤发生进展的关键，可维持黑素瘤线粒体稳态、肿瘤细胞存活及高能量代谢需要，在黑素瘤生长、侵袭、转移和耐药过程中发挥核心作用，但其调控机制不明。我们课题组研究发现AHR在黑素瘤细胞中高表达、在黑素细胞中低表达，且二者存在功能性AHR信号通路。我们检测AHR对黑素瘤生长转移的影响，结果显示：AHR表达下调导致黑素瘤细胞出现生长停滞、黑素瘤细胞侵袭迁移能力减弱，激活AHR信号通路后能够部分逆转并促进黑素瘤侵袭迁移；黑素瘤组织AHR表达水平显著高于正常对照，肢端、四肢、外阴及肛周的AHR表达量与正常对照有差异；TCGA数据库分析提示AHR表达水平与黑素瘤患者生存相关。我们检测AHR对线粒体生物合成的影响，结果显示：抑制AHR信号通路后，线粒体内膜ROS水平明显升高，线粒体膜电位显著降低，细胞内ATP水平显著降低，线粒体DNA拷贝数显著减少，提示AHR参与调控线粒体生物合成。我们进一步进行机制研究，结果显示：激活AHR信号通路，能够促进线粒体生物合成相关调控分子NRF1及下游TFAM的表达，抑制AHR信号通路后二者的mRNA及蛋白表达水平均明显下调；CLC Main Workbench预测分析发现NRF1基因启动子区含有2个AHR结合位点，应用染色质免疫共沉淀实验证实，应用AHR激动剂能够促进AHR与NRF1基因启动区-34~-30区域结合，应用AHR抑制剂能够抑制AHR与NRF1的结合，提示AHR/NRF1是线粒体生物合成的作用机制。上述结果高度提示：AHR信号通路可能通过调控线粒体生物合成影响黑素瘤发病过程，AHR/NRF1信号通路可能是调控黑素瘤线粒体生物合成的关键机制所在。靶向调控线粒体生物合成有望有效改善黑素瘤患者的疗效和预后，成为黑素瘤治疗新策略。