Targeting GLI by GANT61 involves mechanisms dependent on inhibition of both transcription and DNA licensing

Ruowen Zhang,Jiahui Wu,Janet A Houghton,Sylvain Ferrandon,Katie J Glowacki

doi:10.18632/oncotarget.13376

Ruowen Zhang, Jiahui Wu + Show 3 more

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https://doi.org/10.18632/oncotarget.13376

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Abstract

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.

Highlights

The GLI genes, GLI1 and GLI2, are transcription factors that regulate target genes at the distal end of the canonical Hedgehog (HH) signaling pathway (SHH> PTCH- > SMO- > GLI)
In this study we demonstrate that FOXM1 is a transcriptional target of GLI1
GLI is a nodal point of activation for oncogenic signaling pathways, including oncogenic KRAS/BRAF, which is channeled through GLI, driving GLI to a higher activating state [22]

Summary

Introduction

The GLI genes, GLI1 and GLI2, are transcription factors that regulate target genes at the distal end of the canonical Hedgehog (HH) signaling pathway (SHH> PTCH- > SMO- > GLI) Their expression in these processes is tightly regulated, with GLI1 and GLI2 being activated and deactivated at critical times during regulation of the normal cellular processes of embryogenesis, tissue patterning, and differentiation [1,2,3]. GANT61, an experimental agent in preclinical studies, was originally identified in a cell-based screen for small molecule inhibitors of GLI1-mediated transcription [20] This agent has induced extensive cytotoxicity in human www.impactjournals.com/oncotarget models of colon cancer [21,22,23], suggesting that GLI is a critical target in colon cancer cell survival, and in other cancers where GLI is constitutively activated and/ or an oncogenic KRAS-GLI axis drives proliferation. We have previously demonstrated that GANT61 inhibits GLI-dependent transcription in these models, binding to GLI proteins and not to DNA or other transcription factors [24]

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