Reduced abundance of the E3 ubiquitin ligase E6AP contributes to decreased expression of the INK4/ARF locus in non-small cell lung cancer

Sci Signal. 2017 Jan 10;10(461):eaaf8223. doi: 10.1126/scisignal.aaf8223.

Abstract

The tumor suppressor p16INK4a, one protein encoded by the INK4/ARF locus, is frequently absent in multiple cancers, including non-small cell lung cancer (NSCLC). Whereas increased methylation of the encoding gene (CDKN2A) accounts for its loss in a third of patients, no molecular explanation exists for the remainder. We unraveled an alternative mechanism for the silencing of the INK4/ARF locus involving the E3 ubiquitin ligase and transcriptional cofactor E6AP (also known as UBE3A). We found that the expression of three tumor suppressor genes encoded in the INK4/ARF locus (p15INK4b, p16INK4a, and p19ARF) was decreased in E6AP-/- mouse embryo fibroblasts. E6AP induced the expression of the INK4/ARF locus at the transcriptional level by inhibiting CDC6 transcription, a gene encoding a key repressor of the locus. Luciferase assays revealed that E6AP inhibited CDC6 expression by reducing its E2F1-dependent transcription. Chromatin immunoprecipitation analysis indicated that E6AP reduced the amount of E2F1 at the CDC6 promoter. In a subset of NSCLC samples, an E6AP-low/CDC6-high/p16INK4a-low protein abundance profile correlated with low methylation of the gene encoding p16INK4a (CDKN2A) and poor patient prognosis. These findings define a previously unrecognized tumor-suppressive role for E6AP in NSCLC, reveal an alternative silencing mechanism of the INK4/ARF locus, and reveal E6AP as a potential prognostic marker in NSCLC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carcinoma, Non-Small-Cell Lung / genetics*
  • Carcinoma, Non-Small-Cell Lung / metabolism
  • Carcinoma, Non-Small-Cell Lung / pathology
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cell Line, Tumor
  • Cells, Cultured
  • Cyclin-Dependent Kinase Inhibitor p15 / genetics*
  • Cyclin-Dependent Kinase Inhibitor p15 / metabolism
  • Cyclin-Dependent Kinase Inhibitor p16 / genetics*
  • Cyclin-Dependent Kinase Inhibitor p16 / metabolism
  • Cyclin-Dependent Kinase Inhibitor p19 / genetics*
  • Cyclin-Dependent Kinase Inhibitor p19 / metabolism
  • DNA Methylation
  • E2F1 Transcription Factor / genetics
  • E2F1 Transcription Factor / metabolism
  • Embryo, Mammalian / cytology
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Kaplan-Meier Estimate
  • Lung Neoplasms / genetics*
  • Lung Neoplasms / metabolism
  • Lung Neoplasms / pathology
  • Mice, Knockout
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Promoter Regions, Genetic / genetics
  • Protein Binding
  • Ubiquitin-Protein Ligases / genetics*
  • Ubiquitin-Protein Ligases / metabolism

Substances

  • CDC6 protein, human
  • Cell Cycle Proteins
  • Cyclin-Dependent Kinase Inhibitor p15
  • Cyclin-Dependent Kinase Inhibitor p16
  • Cyclin-Dependent Kinase Inhibitor p19
  • E2F1 Transcription Factor
  • Nuclear Proteins
  • Ubiquitin-Protein Ligases