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

Oncotarget. 2016 Dec 6;7(49):80190-80207. doi: 10.18632/oncotarget.13376.

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.

Keywords: DNA licensing; GANT61; GLI; transcription.

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Binding Sites
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cell Death / drug effects
  • Colonic Neoplasms / drug therapy*
  • Colonic Neoplasms / genetics
  • Colonic Neoplasms / metabolism
  • Colonic Neoplasms / pathology
  • DNA Replication / drug effects*
  • Dose-Response Relationship, Drug
  • Forkhead Box Protein M1 / genetics
  • Forkhead Box Protein M1 / metabolism
  • Gene Expression Regulation, Neoplastic / drug effects*
  • HT29 Cells
  • Histones / metabolism
  • Humans
  • Minichromosome Maintenance Complex Component 2 / genetics
  • Minichromosome Maintenance Complex Component 2 / metabolism
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Origin Recognition Complex / genetics
  • Origin Recognition Complex / metabolism
  • Promoter Regions, Genetic
  • Pyridines / pharmacology*
  • Pyrimidines / pharmacology*
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transcription, Genetic / drug effects*
  • Transcriptional Elongation Factors / genetics
  • Transcriptional Elongation Factors / metabolism
  • Transfection
  • Zinc Finger Protein GLI1 / antagonists & inhibitors*
  • Zinc Finger Protein GLI1 / genetics
  • Zinc Finger Protein GLI1 / metabolism

Substances

  • Antineoplastic Agents
  • CDT1 protein, human
  • Cell Cycle Proteins
  • FOXM1 protein, human
  • Forkhead Box Protein M1
  • GANT 61
  • GLI1 protein, human
  • H2AX protein, human
  • Histones
  • NSMF protein, human
  • Nuclear Proteins
  • ORC4 protein, human
  • Origin Recognition Complex
  • Pyridines
  • Pyrimidines
  • SUPT5H protein, human
  • Transcription Factors
  • Transcriptional Elongation Factors
  • Zinc Finger Protein GLI1
  • RNA Polymerase II
  • MCM2 protein, human
  • Minichromosome Maintenance Complex Component 2