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Aberrant DNA methylation but not mutation of CITED4 is associated with alteration of HIF-regulated genes in breast cancer

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Abstract

CBP/p300-interacting transactivator with ED-rich carboxy-terminal domain 4 (CITED4) inhibits HIF-1α transactivation by binding to CBP/p300. We hypothesised that either somatic mutation or hypermethylation of the CITED4 gene underlies CITED4 down-regulation and thus enhanced HIF-1α expression in some breast tumours. DNA sequencing was used to screen for somatic mutations. Methylation-sensitive high resolution melting was performed to identify CITED4 methylation. RT-qPCR was carried out to measure the expression of CITED4 and selected HIF downstream targets. HIF-1α and downstream gene expression was assessed with immunohistochemistry. No somatic mutations of CITED4 were identified in 10 tumour cell lines and 100 breast carcinomas. However, CITED4 promoter methylation was identified in 5/168 breast carcinomas (four infiltrating ductal carcinomas and one infiltrating lobular carcinoma) and in 3/10 breast cancer cell lines (MDA-MB-453, MDA-MB-231 and Hs578T). CITED4 mRNA expression in cell lines was inversely correlated with DNA methylation. CITED4 mRNA expression was significantly increased in all three cell lines after 5-aza-2-deoxycytidine (DAC) treatment. Treatment of the MDA-MB-231 cell line with DAC followed by hypoxia (0.1% O2) resulted in down-regulation of expression of the HIF-1α downstream genes VEGFA and SLC2A1 (P = 0.0029). HIF-1α downstream SLC2A1 was decreased (P = 0.021) after CITED4 was re-expressed under hypoxia. Loss of expression of CITED4 in breast cancer may be due to DNA methylation but is unlikely to be due to mutation. Demethylation and histone modification can potentially reactivate CITED4 gene expression in some breast cancers and lead to changes in tumour behaviour. Strategies such as HDAC inhibitors may overcome this effect.

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Acknowledgments

We wish to thank the Molecular Pathology Research and Development group at the Peter MacCallum Cancer Centre for their help and support and Dr. Andreas Möller for instruction in using the hypoxic chamber. We would also like to thank Professor Adrian Harris for supplying us with the breast carcinoma sample DNAs and Australian Red Cross Blood Service for provision of donors for the blood collection. This study was funded by a grant from the Victorian Breast Cancer Research Consortium of Australia to SBF and a grant from the Cancer Council of Victoria to AD.

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Authors and Affiliations

  1. Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, VIC, 8006, Australia

    Katie T. Huang, Elena A. Takano, Thomas Mikeska, David J. Byrne, Alexander Dobrovic & Stephen B. Fox

  2. Department of Pathology, The University of Melbourne, Parkville, VIC, 3010, Australia

    Katie T. Huang, Thomas Mikeska, Alexander Dobrovic & Stephen B. Fox

Authors
  1. Katie T. Huang
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  2. Elena A. Takano
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  3. Thomas Mikeska
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  4. David J. Byrne
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  5. Alexander Dobrovic
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  6. Stephen B. Fox
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Corresponding author

Correspondence to Katie T. Huang.

Additional information

Katie T. Huang and Elena A. Takano contributed equally to this study.

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Huang, K.T., Takano, E.A., Mikeska, T. et al. Aberrant DNA methylation but not mutation of CITED4 is associated with alteration of HIF-regulated genes in breast cancer. Breast Cancer Res Treat 130, 319–329 (2011). https://doi.org/10.1007/s10549-011-1657-1

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  • DOI: https://doi.org/10.1007/s10549-011-1657-1

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