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The HIF1α-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element

Oncogene volume 28, pages 3671–3680 (2009)Cite this article

Abstract

The short isoform of single-minded 2 (SIM2s), a basic helix–loop–helix/PAS (bHLH/PAS) transcription factor, is upregulated in pancreatic and prostate tumours; however, a mechanistic role for SIM2s in these cancers is unknown. Microarray studies in prostate DU145 cells identified the pro-cell death gene, BNIP3 (Bcl-2/adenovirus E1B 19 kDa interacting protein 3), as a novel putative target of SIM2s repression. Further validation showed BNIP3 repression in several prostate and pancreatic carcinoma-derived cell lines with ectopic expression of human SIM2s. BNIP3 levels are enhanced in prostate carcinoma cells upon short interfering (si)RNA-mediated knockdown of endogenous SIM2s. Chromatin immunoprecipitation and promoter studies show that SIM2s represses BNIP3 through its activities at the proximal promoter hypoxia response element (HRE), the site through which the bHLH/PAS family member, hypoxia-inducible factor 1α (HIF1α), induces BNIP3. SIM2s attenuates BNIP3 hypoxic induction via the HRE, and increased hypoxic induction of BNIP3 occurs with siRNA knockdown of endogenous SIM2s in prostate PC3AR+ cells. BNIP3 is implicated in hypoxia-induced cell death processes. Prolonged treatment of PC3AR+ cells with hypoxia mimetics, DP and DMOG, confers hypoxia-induced autophagy, measured by enhanced LC3-II levels and SQSTM1/p62 turnover. We show that PC3AR+ cells expressing ectopic SIM2s have enhanced survival in these conditions. Induction of LC3-II and turnover of SQSTM1/p62 are attenuated in PC3AR+/SIM2s DMOG and hypoxia-treated cells, suggesting that SIM2s may attenuate autophagic cell death processes, perhaps through BNIP3 repression. These data show, for the first time, SIM2s cross-talk on an endogenous HRE. SIM2s' functional interference with HIF1α activities on BNIP3 may indicate a novel role for SIM2s in promoting tumourigenesis.

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Abbreviations

bHLH/PAS:

basic helix–loop–helix/PAS

BNIP3:

Bcl-2/adenovirus E1B 19 kDa interacting protein 3

HRE:

hypoxia response element

ARNT:

aryl hydrocarbon receptor nuclear translocator

RT–PCR:

reverse transcription–PCR

ChIP:

chromatin immunoprecipitation

DMOG:

dimethyloxallyl glycine

DP:

dipyridyl

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Acknowledgements

We thank M van Bekkum and C Bindloss for their kind technical assistance, and Dr S Woods for reagents and critical reading of the manuscript. PC3AR+ cells were a kind gift from Prof W Tilley, Hanson Research Institute, Adelaide, Australia. Tet-inducible lentiviral vectors were a kind gift from Dr S Barry, Children's Health Research Institute, SA, Australia (Drabsch et al., 2007). Anti-HIF1α-CAD antibody was obtained from the laboratory of Prof J Pouysségur, CNRS UMR6543, University of Nice, France (Richard et al., 1999). We acknowledge the contribution of the Australian Cancer Research Foundation for their support of the Adelaide Microarray Facility, Adelaide, Australia, which supplied, processed and analysed the microarrays. This work was supported by grants from the Australian Research Council and the Cancer Council South Australia.

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  1. Discipline of Biochemistry, School of Molecular & Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia

    A L Farrall & M L Whitelaw

  2. Centre for the Molecular Genetics of Development, Adelaide, South Australia, Australia

    A L Farrall & M L Whitelaw

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Correspondence to A L Farrall.

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Farrall, A., Whitelaw, M. The HIF1α-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element. Oncogene 28, 3671–3680 (2009). https://doi.org/10.1038/onc.2009.228

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