In cancer cells, elevated Brn-3b transcription factor enhances proliferation in-vitro and increases tumour growth in-vivo, whilst conferring drug resistance and migratory potential, whereas reducing Brn-3b slows growth, both in-vitro and in-vivo. Brn-3b regulates distinct groups of key target genes that control cell growth and behaviour. Brn-3b is elevated in >65% of breast cancer biopsies, but mechanisms controlling its expression in these cells are not known.

Bioinformatics analysis was used to identify the regulatory promoter region and map transcription start site and transcription factors binding sites. Polymerase chain reaction (PCR) cloning was used to generate promoter constructs for reporter assays. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis were used to confirm transcription start site and auto-regulation. Breast cancer cell, MCF7 and Cos7 cells were used. Cells grown in culture were transfected with Brn-3b promoter and treated with growth factors or estradiol to test for effects on promoter activity. Quantitative reverse transcriptase PCR (q-RT-PCR) and immunoblotting were used to confirm changes in gene /protein expression.

We cloned the Brn-3b promoter, mapped the transcription start site and showed stimulation by estradiol and growth factors, nerve growth factor (NGF) and epidermal growth factor (EGF), which are implicated in breast cancer initiation/progression. The effects of GFs are mediated through mitogen activated protein kinase (MAPK) pathway, whereas hormone effects act via the estrogen receptor, ERalpha. Brn-3b also auto-regulate its expression and co-operates with ERalpha to further enhance levels.

Key regulators of growth in cancer cells e.g. estrogens and growth factors can stimulate Brn-3b expression and auto-regulation also contributes to increasing Brn-3b in breast cancers. Since increasing Brn-3b profoundly enhances growth in these cells, then understanding how Brn-3b is increased in breast cancers will help to identify strategies for reducing its expression and thus effects on target genes, thereby reversing its effects in breast cancer cells.

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