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A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction
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AKT activity determines sensitivity to mammalian target of rapamycin (mTOR) inhibitors by regulating cyclin D1 and

Joseph F Gera1, Ingo K Mellinghoff, Yijiang Shi

  • 1Department of Medicine, West Los Angeles Veteran's Administration-UCLA Medical Center, University of California School of Medicine, Los Angeles, California 90073, USA. gera@ucla.edu

The Journal of Biological Chemistry
|October 25, 2003
PubMed

Insights

AKT activity influences cell sensitivity to mTOR inhibitors. By manipulating cyclin D1 and c-myc expression, researchers converted sensitive cells to a resistant phenotype, identifying key targets for drug development.

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Cancer Research

Background:

  • Mammalian target of rapamycin (mTOR) inhibitors induce G(1) arrest in cells.
  • AKT activity modulates cellular sensitivity to mTOR inhibitors.
  • Understanding the molecular mechanisms underlying this sensitivity is crucial for cancer therapy.

Purpose of the Study:

  • To identify genes translationally regulated by mTOR inhibitors in an AKT-dependent manner.
  • To investigate the role of specific genes in mediating cellular response to mTOR inhibition.
  • To explore therapeutic strategies for overcoming resistance to mTOR inhibitors.

Main Methods:

  • High-throughput microarray polysome analysis to assess mRNA translational efficiency and transcript levels.
  • Utilizing p27(kip1) 5'-untranslated region for ectopic expression of cyclin D1 and c-myc.
  • Transfection of cells with engineered constructs and treatment with rapamycin.
  • In vivo mouse xenograft model with CCI-779 treatment.

Main Results:

  • Identified cyclin D1 and c-myc as key transcripts with AKT-dependent differential expression following mTOR inhibition.
  • Demonstrated that bypassing mTOR inhibition-induced down-regulation of cyclin D1/c-myc confers resistance to G(1) arrest.
  • Confirmed AKT-dependent differential expression patterns in vivo using a mouse xenograft model.

Conclusions:

  • Cyclin D1 and c-myc are critical downstream targets regulated by AKT activity.
  • Down-regulation of cyclin D1 and c-myc expression is essential for sensitivity to rapamycin/CCI-779.
  • Targeting these pathways may offer novel strategies for enhancing mTOR inhibitor efficacy in cancer treatment.

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