Brn-3b transcription factor in breast tumourigenesis: regulation of genes associated with growth and migration of cancer cells
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KeywordsBreast Cancer Estrogen Receptor HSP27 Protein Breast Epithelial Cell HSP27 Level
The Brn-3b transcription factor is elevated in many breast cancers compared with levels found in normal breast epithelial cells. High levels of Brn-3b increase growth and proliferation of cancer cells, both in vitro and in vivo, but also alter migration and confer resistance to growth inhibitory stimulus . Conversely, low levels of Brn-3b slow the growth of these cells. As a transcription factor, Brn3b changes the growth and behaviour of breast cancer cells by modifying the expression of target genes, either directly or indirectly upon association with the proliferation-associated estrogen receptor (ER) . A number of Brn-3b target genes have been identified that alter the growth and behaviour of these cancer cells. For instance, Brn-3b transactivates the promoter of the cyclin-dependent kinase, CDK4 , that is required for cell cycle progression and hence proliferation. Brn-3b also represses the promoter of the tumour suppressor gene, BRCA1, and inversely correlates with BRCA1 protein in tumour biopsies. We have recently demonstrated that the small heat shock protein, HSP27, is also regulated by Brn-3b . High expression of HSP27 in breast cancers is associated with increased anchorage-independent growth, increased invasiveness and resistance to chemotherapeutic drugs and poor prognosis. Thus, in cancers expressing high levels of Brn-3b the downstream target genes regulated by this transcription factor can alter the growth and behaviour of these cells.
Western blot analysis of tumour samples was used to correlate Brn-3b and HSP27 proteins. Transient co-transfection and reporter assays were used to look at the effects of Brn-3b and/or ER on the HSP27 promoter. Short hairpin RNA interference was used to target Brn-3b protein and to test its requirement for transactivation of the HSP27 promoter. EMSA was used to demonstrate direct binding of Brn-3b to a specific site in the HSP27 promoter. Chromatin immunoprecipitation (ChIP) was performed to show that Brn-3b was associated with the HSP27 promoter in intact cells.
Brn-3b protein levels correlated strongly with HSP27 levels in a significant number of breast cancer biopsies (R = 0.87) as well as in the breast cancer cell line, MCF7. Overexpression of Brn-3b in MCF7 cells resulted in increased HSP27 protein levels while reducing Brn-3b proteins using antisense correlated with decreased HSP27 compared with controls. Co-transfection analysis using a HSP27 reporter construct showed that Brn-3b could directly transactivate HSP27 promoter but cooperated with the ER for maximal expression. Decreasing Brn-3b using targeted RNA interference prevented activation of the HSP27 promoter by Brn-3b alone but also attenuated the response by ER. The Brn-3b site in the HSP27 promoter is flanked by two sequences that constitute half estrogen receptor elements. Site-directed mutagenesis demonstrated that this DNA sequence was required for maximal transactivation while the ChIP assay showed that Brn-3b protein binds to the HSP27 promoter in vivo.
The ability of Brn-3b to increase expression of proteins such as HSP27 in cancer cells may help to understand the altered growth and migration of tumour cells with elevated Brn-3b proteins. Therefore factors such as Brn-3b, which increase the expression of HSP27 in breast cancers, are likely to affect the progression of diseases, prognosis and outcome of treatment.
The authors thank Dr Daniel Ndisang, Dr Chandrakant Patel, Dr Jonothan Dennis and Dr Corrado D'Arrigo. This work was supported by the Breast Cancer Campaign (BCC) UK and the Association for International Cancer Research (AICR), UK.
- 4.Lee S, Ndisang D, Patel C, Dennis JH, D'Arrigo C, Farooqui-Kabir S, Heads RJ, Latchman DS, Budhram-Mahadeo V: Expression of the Brn3b transcription factor correlates with expression of HSP27 in breast cancer biopsies and is required for maximal activation of the HSP27 promoter. Cancer Res. 2005, 65: 3072-3080.PubMedGoogle Scholar