Daniel Ndisang

The Brn-3b POU family transcription factor represses expression of the BRCA-1 anti-oncogene in breast cancer cells

The BRCA-1 tumour supressor gene was identified on the basis of mutations which occur in familial breast cancer indicating that its inactivation can cause this disease. Although BRCA-1 does not appear to be mutated in sporadic breast cancer, its expression has been shown to be reduced in tumour material from such cases. We show here that mammary tumours which have reduced levels of BRCA-1 expression show enhanced expression of the Brn-3b POU family transcription factor at both the mRNA and protein levels. This elevated expression of Brn-3b is not found in normal mammary cells, benign tumours or in malignant tumour samples which do not exhibit reduced levels of BRCA-1. In contrast, no correlation was noted between BRCA-1 and expression of the related factor Brn-3a. Moreover, Brn-3b but not Brn-3a can strongly repress the BRCA-1 promoter approximately 20-fold in mammary tumour cells. To our knowledge, this is the first report of a transcription factor which regulates BRCA-1 expression. Thus, Brn-3b may play an important role in regulating expression of BRCA-1 in mammary tumours with enhanced expression of Brn-3b resulting in reduced BRCA-1 expression and thereby being potentially important in tumour development.

Expression of the Brn-3b transcription factor correlates with expression of HSP-27 in breast cancer biopsies and is required for maximal activation of the HSP-27 promoter

In breast cancer, overexpression of the small heat shock protein, HSP-27, is associated with increased anchorage-independent growth, increased invasiveness, and resistance to chemotherapeutic drugs and is associated with poor prognosis and reduced disease-free survival. Therefore, factors that increase the expression of HSP-27 in breast cancer are likely to affect the prognosis and outcome of treatment. In this study, we show a strong correlation between elevated levels of the Brn-3b POU transcription factor and high levels of HSP-27 protein in manipulated MCF-7 breast cancer cells as well as in human breast biopsies. Conversely, HSP-27 is decreased on loss of Brn-3b. In cotransfection assays, Brn-3b can strongly transactivate the HSP-27 promoter, supporting a role for direct regulation of HSP-27 expression. Brn-3b also cooperates with the estrogen receptor (ER) to facilitate maximal stimulation of the HSP-27 promoter, with significantly enhanced activity of this promoter observed on coexpression of Brn-3b and ER compared with either alone. RNA interference and site-directed mutagenesis support the requirement for the Brn-3b binding site on the HSP-27 promoter, which facilitates maximal transactivation either alone or on interaction with the ER. Chromatin immunoprecipitation provides evidence for association of Brn-3b with the HSP-27 promoter in the intact cell. Thus, Brn-3b can, directly and indirectly (via interaction with the ER), activate HSP-27 expression, and this may represent one mechanism by which Brn-3b mediates its effects in breast cancer cells.

The Brn-3a transcription factor plays a critical role in regulating human papilloma virus gene expression and determining the growth characteristics of cervical cancer cells.

The Brn-3a POU family transcription factor has previously been shown to activate the human papilloma virus type 16 (HPV-16) promoter driving the expression of the E6- and E7-transforming proteins. Moreover, Brn-3a is overexpressed approximately 300-fold in cervical biopsies from women with cervical intra-epithelial neoplasia type 3 (CIN3) compared with normal cervical material. To test the role of Brn-3a in cervical neoplasia we have manipulated its expression in cervical carcinoma-derived cell lines with or without endogenous HPV genes. In HPV-expressing cells, reduction in Brn-3a expression specifically reduces HPV gene expression, growth rate, saturation density and anchorage-independent growth, whereas these effects are not observed when Brn-3a expression is reduced in cervical cells lacking HPV genomes. Together with our previous observations, these findings indicate a critical role for Brn-3a in regulating HPV gene expression and thereby in controlling the growth/transformation of cervical cells.

The Brn-3a transcription factor plays a key role in regulating the growth of cervical cancer cells In vivo

The cellular Brn-3a transcription factor is known to activate transcription of the genes encoding the human papilloma virus E6 and E7 proteins and is over-expressed in women with cervical neoplasia. We show that cervical cell lines with reduced Brn-3a expression show a greatly reduced ability to form tumours in nude mice compared to control cells and also show reduced expression of the HPV E6 and cellular Bcl-2 oncogenes. These effects are also observed in cervical cells over-expressing the related Brn-3b factor, which is known to antagonize activation of HPV gene expression by Brn-3a. These results demonstrate, for the first time, that inhibition of Brn-3a expression or enhanced Brn-3b expression can inhibit cervical cell-derived tumour growth in vivo as well as in vitro. Hence they establish Brn-3a as a key factor in cervical tumorigenesis and as a potential therapeutic target in human cervical neoplasia.

Measurement of Brn-3a levels in Pap smears provides a novel diagnostic marker for the detection of cervical neoplasia

OBJECTIVES We have previously demonstrated that Brn-3a cellular transcription factor activates transcription of the human papillomavirus (HPV) E6 and E7 oncogenes in human cervical cancer cells and that Brn-3a levels are dramatically elevated in biopsies from women with high-grade cervical neoplasia. The aim of this study was to establish the relationship between Brn-3a levels in Pap smears and the histological diagnoses. We also analysed whether Brn-3a levels can be used in combination with Pap smear to predict the presence of cervical intraepithelial lesion. METHODS Two hundred thirty-eight women who were referred with abnormal Pap smear underwent a diagnostic colposcopy, repeat in-study Pap smear, colposcopically directed biopsy, and assessment of Brn-3a and HPV-16 E6 m-RNA levels. Data were analysed to assess the association between Brn-3a levels and the histological diagnosis. RESULTS Brn-3a was readily measured in smears and showed a statistically significant correlation with the grade of cervical abnormality. Positive Brn-3a is associated with increased relative risk of higher-grade lesion. Moreover, measurement of Brn-3a levels in smears can be used to detect a significant proportion of cervical lesions that were missed by Pap smear. CONCLUSION Measurement of Brn-3a levels in routinely taken Pap smears is a feasible technique that correlates with the severity of the epithelial abnormality and is a useful adjunct to cytology. Brn-3a appears to have great promise since it detects activation of oncogenic HPVs rather than simply detecting their presence, as is currently being done.

The Brn-3b POU family transcription factor represses plakoglobin gene expression in human breast cancer cells

The Brn‐3b transcription factor has been shown to be overexpressed in human breast cancer cells and contributes toward cell growth regulation. Using micro‐arrays, more than 50 cancer‐related genes regulated by Brn‐3b in human breast cancer cells have been identified. For example, Brn‐3b activates the cell cycle regulator CDK4 that provides a mechanism by which Brn‐3b controls the growth of breast cancer cells. Here, we show that Brn‐3b regulates plakoglobin (γ‐catenin), a member of the catenin family involved in cell‐cell adhesion and signal transduction. Brn‐3b expression inversely correlates with plakoglobin expression at both mRNA and protein levels in breast cancer cell lines and human breast biopsies. In contrast, no significant correlation was observed between Brn‐3b expression and β‐catenin, or between Brn‐3b expression and E‐cadherin expression. Brn‐3b represses the plakoglobin promoter via a Brn‐3 consensus binding site contained within the region −965 to −593 relative to the transcriptional start site. Both repression of the promoter and binding of Brn‐3b are lost when this site is mutated. To our knowledge, this is the first time that a Brn‐3b POU family transcription factor has been shown to regulate a member of the catenin family, which provides insight into the molecular mechanisms by which Brn‐3b expression may favour breast cancer progression and tumor invasion.

The Brn-3a POU family transcription factor stimulates p53 gene expression in human and mouse tumour cells.

The Brn-3a POU family transcription factor is able to induce the expression of genes encoding anti-apoptotic proteins such as Bcl-2 and Bcl-x and protects neuronal cells from apoptosis. This effect is opposed by the pro-apoptotic p53 protein which completely inhibits the ability of Brn-3a to activate the Bcl-2 and Bcl-x promoters. Here we demonstrate that Brn-3a is able to stimulate p53 expression. Thus, in co-transfection experiments, Brn-3a activates the p53 promoter acting via a region from +22 to +67, located between the most proximal (+1) and the most distal (+105) transcriptional start sites. Similarly, reduction of Brn-3a expression using anti-sense constructs reduces endogenous p53 expression in human neuroblastoma or cervical carcinoma cell lines growing in vitro and as tumours in nude mice whilst increasing Brn-3a levels enhances p53 expression. These results suggest the existence of a negative feedback loop in which elevated Brn-3a expression induces the expression of p53 which, in turn, antagonises the anti-apoptotic activity of Brn-3a.

The HPV Cellular Transactivator Brn-3a Can Be Used to Predict Cervical Adenocarcinoma and Squamous Carcinoma Precancer Lesions in the Developed and Developing Worlds

The cellular transactivator Brn-3a has previously been shown to be expressed at elevated levels in the cervix of women with squamous cell carcinoma of the cervix (SCC) and to activate the expression of HPV E6 mRNA. In this study, we show that common and rare cervical precancer lesions, including those of adenocarcinoma (AC), which are usually difficult to diagnose using classical procedures, also expressed high levels of Brn-3a and can be diagnosed by measuring the levels of Brn-3a and E6 mRNAs.