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Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
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The HIF1alpha-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the

A L Farrall1, M L Whitelaw

  • 1Discipline of Biochemistry, School of Molecular & Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia. alexandra.farrall@adelaide.edu.au

Oncogene
|August 12, 2009
PubMed

Insights

The short isoform of single-minded 2 (SIM2s) transcription factor represses the pro-cell death gene BNIP3 in pancreatic and prostate cancers. SIM2s may promote tumor growth by interfering with hypoxia-induced cell death pathways.

Area of Science:

  • Molecular Biology
  • Cancer Biology
  • Cellular Signaling

Background:

  • The short isoform of single-minded 2 (SIM2s), a bHLH/PAS transcription factor, is upregulated in pancreatic and prostate tumors.
  • The mechanistic role of SIM2s in these cancers remains largely unknown.

Purpose of the Study:

  • To investigate the role of SIM2s in pancreatic and prostate cancer.
  • To identify novel targets of SIM2s repression and elucidate its mechanism of action.

Main Methods:

  • Microarray analysis in prostate DU145 cells to identify SIM2s targets.
  • Validation of BNIP3 repression in various cancer cell lines.
  • Short interfering (si)RNA-mediated knockdown of SIM2s.
  • Chromatin immunoprecipitation and promoter assays.
  • Hypoxia treatment with mimetics (DP and DMOG).
  • Analysis of autophagy markers (LC3-II and SQSTM1/p62).

Main Results:

  • BNIP3 was identified as a novel putative target gene repressed by SIM2s.
  • SIM2s represses BNIP3 expression via the hypoxia response element (HRE) on its promoter.
  • SIM2s attenuates hypoxia-induced BNIP3 expression and interferes with hypoxia-inducible factor 1alpha (HIF1alpha) activity.
  • SIM2s expression enhances cancer cell survival under hypoxia.
  • SIM2s attenuates hypoxia-induced autophagy, suggesting a role in inhibiting autophagic cell death.

Conclusions:

  • SIM2s directly represses BNIP3 expression at the HRE, interfering with HIF1alpha-mediated induction.
  • SIM2s may promote tumorigenesis by repressing BNIP3 and attenuating hypoxia-induced cell death and autophagy.

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