Zhang Ruowen, Wu Jiahui, Ferrandon Sylvain, Glowacki Katie J, Houghton Janet A
Department of Oncology, Division of Drug Discovery, Southern Research, Birmingham, AL, USA.
Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.
Oncotarget. 2016 Dec 6;7(49):80190-80207. doi: 10.18632/oncotarget.13376.
The GLI genes are transcription factors and in cancers are oncogenes, aberrantly and constitutively activated. GANT61, a specific GLI inhibitor, has induced extensive cytotoxicity in human models of colon cancer. The FOXM1 promoter was determined to be a transcriptional target of GLI1. In HT29 cells, inhibition of GLI1 binding at the GLI consensus sequence by GANT61 led to inhibited binding of Pol II, the pause-release factors DSIF, NELF and p-TEFb. The formation of R-loops (RNA:DNA hybrids, ssDNA), were reduced by GANT61 at the FOXM1 promoter. Pretreatment of HT29 cells with α-amanitin reduced GANT61-induced γH2AX foci. Co-localization of GLI1 and BrdU foci, inhibited by GANT61, indicated GLI1 and DNA replication to be linked. By co-immunoprecipitation and confocal microscopy, GLI1 co-localized with the DNA licensing factors ORC4, CDT1, and MCM2. Significant co-localization of GLI1 and ORC4 was inhibited by GANT61, and enrichment of ORC4 occurred at the GLI binding site in the FOXM1 promoter. CDT1 was found to be a transcription target of GLI1. Overexpression of CDT1 in HT29 and SW480 cells reduced GANT61-induced cell death, gH2AX foci, and cleavage of caspase-3. Data demonstrate involvement of transcription and of DNA replication licensing factors by non-transcriptional and transcriptional mechanisms in the GLI-dependent mechanism of action of GANT61.
GLI基因是转录因子，在癌症中为癌基因，异常且持续激活。GANT61是一种特异性GLI抑制剂，在结肠癌的人类模型中诱导了广泛的细胞毒性。FOXM1启动子被确定为GLI1的转录靶点。在HT29细胞中，GANT61抑制GLI1在GLI共有序列处的结合，导致RNA聚合酶II、暂停释放因子DSIF、NELF和p-TEFb的结合受到抑制。在FOXM1启动子处，GANT61减少了R环（RNA:DNA杂交体，单链DNA）的形成。用α-鹅膏蕈碱预处理HT29细胞可减少GANT61诱导的γH2AX病灶。GANT61抑制了GLI1和BrdU病灶的共定位，表明GLI1与DNA复制有关。通过免疫共沉淀和共聚焦显微镜观察，GLI1与DNA许可因子ORC4、CDT1和MCM2共定位。GANT61抑制了GLI1和ORC4的显著共定位，并且在FOXM1启动子的GLI结合位点出现了ORC4的富集。发现CDT1是GLI1的转录靶点。在HT29和SW480细胞中过表达CDT1可减少GANT61诱导的细胞死亡、gH2AX病灶和半胱天冬酶-3的切割。数据表明，在GANT61的GLI依赖性作用机制中，转录和DNA复制许可因子通过非转录和转录机制发挥作用。
