c-Myc/ZNF652/Sonic Hedgehog调控大蒜活性成分二烯丙基三硫化物对肺癌干细胞干预作用的机制研究

Cancer stem cells (CSCs) play central role in the formation and development of cancer. To date, the involvement and function of ZNF652, a key transcription factor, in regulating Sonic Hedgehog signal pathway and lung CSCs has not been defined. The transcriptional regulation of c-Myc, a proto-oncogene, on ZNF652 has not been understood yet. Meanwhile, the interventional effects of diallyl trisulfide (DATS) on lung CSCs also remain to be explored. In our preliminary studies we found that lung CSCs upregulated c-Myc expression, downregulated ZNF652 expression, activated Sonic Hedgehog pathway as well as its target CSCs-related genes; ZNF652 transcriptionally repressed Gli expression, the key regulator of Sonic Hedgehog pathway; c-Myc transcriptionally repressed ZNF652 expression. We also showed that DATS reduced the sphere formation of lung cancer cells cultured in serum-free-medium, decreased CSCs markers’ expression, reduced c-Myc expression, increased ZNF652 expression, and suppressed Sonic Hedgehog activation. However, the role and regulation of c-Myc/ZNF652/Sonic Hedgehog axis in lung CSCs activity and DATS intervention is totally unknown. Using serum-free-medium cultured lung CSCs and nude mouse xenograft models, the proposed research will for the first time in the field to investigate the roles of c-Myc and ZNF652 in regulating Sonic Hedgehog pathway activation, target gene expression and lung CSCs activity. Regulation of Sonic Hedgehog signaling activity in lung CSCs will be examined. The transcriptional regulation of ZNF652 by c-Myc as well as Sonic Hedgehog by ZNF652 will be further explored. Moreover, this research will illustrate the role of c-Myc/ZNF652/Sonic Hedgehog in the interventional effects of DATS on lung CSCs. Findings from this research will provide important new insights into the molecular mechanisms of c-Myc/ZNF652/Sonic Hedgehog axis in regulating lung CSCs activity as well as its target intervention.

肿瘤干细胞对肿瘤发生、发展起决定性作用。目前关于人类基因组中最大转录因子家族锌指蛋白重要成员ZNF652调控Sonic Hedgehog（SHH）通路、大蒜主要活性成分二烯丙基三硫化物（DATS）对肺癌干细胞的干预作用及c-Myc对ZNF652的调控作用均未见报道。我们前期研究发现肺癌干细胞活性与上调c-Myc、下调ZNF652、激活SHH通路有关；ZNF652负向调控Gli转录；c-Myc抑制ZNF652转录；DATS降低肺癌干细胞活性。然c-Myc/ZNF652/Sonic Hedgehog是否真正介导肺癌干细胞活性及DATS的干预效应尚属未知。本课题拟采用体外细胞模型和动物模型相结合，创新性地探讨ZNF652对SHH活性、肺癌干细胞调控机制以及c-Myc对ZNF652的调控作用，并在此基础上研究DATS的干预效应。旨在为探索肺癌干细胞的调控机制并为其靶向干预提供新的重要科学依据。

锌指蛋白(ZNFs)是人类基因组中最大的转录因子家族，ZNFs可作为原癌基因或是肿瘤抑制因子参与肿瘤的调控。ZNF652是近年来新发现的转录抑制因子，然而，ZNF652在肺癌中的功能尚未见报道。本研究首次揭示了ZNF652在肺癌发生发展中的抑制作用。生物信息学研究表明，转录抑制因子ZNF652在非小细胞肺癌中低表达，并与肺癌患者的预后良好呈正相关。体外实验显示过表达ZNF652显著抑制细胞增殖、迁移和侵袭能力，并诱导细胞周期阻滞、促进ROS产生、导致DNA损伤和细胞凋亡。体内实验亦显示过表达ZNF652显著抑制肺癌细胞成瘤能力，表明ZNF652在肺癌中的抑癌功能。肿瘤干细胞在肿瘤包括肺癌的发生、发展过程中起着决定性的作用。ZNF652是否通过调控肺癌干细胞发挥抑癌作用及相关机制尚不清楚。研究结果显示，与贴壁细胞相比，肺癌干细胞低表达ZNF652。过表达ZNF652抑制悬浮细胞球的体积数量、降低CD133阳性细胞数量，下调肺癌干细胞标记分子的表达水平，并抑制肺癌干细胞Sonic Hedgehog信号通路的激活；而低表达ZNF652则显著促进肺癌干细胞干性并上调Sonic Hedgehog通路，表明ZNF652对肺癌干细胞的负向调控作用。本研究首次提出，Sonic Hedgehog信号通路中关键调控因子GLi是ZNF652的靶基因，ZNF652通过直接转录抑制Gli1和Gli2进而抑制该通路的激活。另外，研究结果显示，在肺癌干细胞中多能干细胞因子c-Myc直接负向转录调控ZNF652，表明c-Myc/ZNF652/Sonic Hedgehog信号轴在肺癌干细胞中起着的重要调控作用。近年来大量流行病学和实验研究表明，源于天然食物的多种植物化学物具有防癌和抗癌的作用。大蒜主要活性成分二烯丙基三硫化物 (DATS) 作为膳食来源丰富的植物化学物之一，具有抑制肿瘤细胞的作用。本研究进一步探讨DATS对肺癌细胞的干预作用以及c-Myc/ZNF652/Sonic Hedgehog轴在DATS抑制肺癌干细胞中的调控作用。细胞和裸鼠移植瘤实验均显示c-Myc/ZNF652/Sonic Hedgehog信号轴介导DATS对肺癌干细胞的抑制作用。综上所述，本项目为肺癌发生发展的研究提供新的调控机制和关键靶点，进而为以机制为导向的肺癌干细胞的干预研究提供新的科学依据和研究策略。