Investigation into the molecular mechanism of the antiapoptotic functions of CTCF in breast cancer cells using a proteomics approach
Breast Cancer Researchvolume 10, Article number: P19 (2008)
CCCTC binding protein (CTCF) is a highly conserved and ubiquitous transcription factor with versatile functions. It is involved in transcriptional regulation, chromatin insulation and epigenetic control . Although CTCF has features of a tumour suppressor gene, it is overexpressed in breast cancer cells; this phenomenon is associated with the resistance of these cells to apoptosis . The aim of the present study is to investigate the molecular mechanisms of the CTCF-dependent resistance of breast cancer cells to apoptosis.
A proteomics approach was used to generate protein profiles of breast cancer cells, ZR75.1, with normal and reduced levels of CTCF. In the latter cells CTCF was knocked-down using siRNA and iRNA. Cell extracts were analysed using two-dimensional PAGE, and differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight or liquid chromatography/mass spectrometry/mass spectrometry.
More than 20 putative candidates have so far been obtained; they belong to various protein families involved in the control of signalling, metabolic, apoptotic, stress response and mammary gland specific regulatory pathways. One of the candidates, the proapoptotic protein Bax, was further validated as a target for negative regulation by CTCF. We demonstrated that expression of Bax correlated inversely with CTCF levels. Furthermore, Bax promoter was negatively regulated by CTCF in reporter assays. Two putative CTCF binding sites were identified within the promoter of Bax gene; contact nucleotides were determined by footprinting and methylation interference assays.
Our data suggest that high levels of CTCF may cause repression of Bax and inhibition of apoptosis. Lower levels of CTCF lead to activation of Bax, resulting in apoptosis. Selective reduction of CTCF can therefore be an attractive option in the development of antibreast cancer therapies.
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Docquier F, Farrar D, D'Arcy V, Chernukhin I, Robinson AF, Loukinov D, Vatolin S, Pack S, Mackay A, Harris RA, Dorricott H, O'Hare MJ, Lobanenkov V, Klenova E: Heightened expression of CTCF in breast cancer cells is associated with resistance to apoptosis. Cancer Res. 2005, 65: 5112-5122. 10.1158/0008-5472.CAN-03-3498.
Supported by Breast Cancer Campaign and CONACyT (National Council of Science and Technology, Mexico).