Suppression of the NF-κB cofactor Bcl3 inhibits mammary epithelial cell apoptosis and, in breast tumours, correlates with poor prognosis

Abstract

Background Several transcription factors have been shown to play important roles in the regulation of apoptosis at the onset of murine mammary involution. These include LIF-activated STAT3, c/ebpdelta, Ap-1 and IKK/NF-κB-mediated regulation of death receptor ligands. A study of STAT3 and STAT5 transcriptional targets in mammary epithelial cells in vitro showed that both c/ebpdelta and c-fos (a component of Ap-1) were upregulated by STAT3, suggesting a degree of interdependence between these transcription factor pathways in mediating their apoptotic effects. Interestingly, while no NF-κB or IKK genes were significantly regulated by STATs, the NF-κB cofactor gene, Bcl3, was found to be a principal transcriptional target of STAT3. This factor plays a role in altering the transcriptional capacity of specific NF-κB subunits and has previously been described as an oncogene in B-cell lymphomas. In this study we set out to establish whether Bcl3 had a role in regulating the cell fate of mammary epithelial cells either in the normal mammary gland or in mammary/breast cancer. Methods Archived material representing a range of tumour grades and types was collected from breast cancer patients immediately after surgery (tumour tissues = 122, normal tissues = 32). The median follow-up of the patients was 120 months (range 12 to 156 months). QRT-PCR for Bcl3 was performed and this information was used to determine statistically significant correlations with the clinical data on breast pathology. MCF7, T47D and MDA-MB231 human breast cancer cell lines were subjected to Bcl3-specific siRNA knockdown and subsequently assessed for cell motility characteristics using ECIS technology. Bcl3-knockout mice were assessed histologically for alterations in apoptosis rate during the adult pregnancy cycle. Western blots, quantitative PCR and DNA binding assays were used to determine the activity of molecular markers of apoptosis in these animals. Bcl3-deficient animals were crossed with mmtv-neu (c-erbB2) mice to establish the role of Bcl3 in primary (neu-dependent) mammary tumour growth, and magnetic resonance imaging was performed on tumour-bearing animals, to establish metastasis rates in the presence/absence of Bcl3. Results An analysis of 122 human breast cancer tissues showed that Bcl3 gene expression was suppressed in a significant proportion of invasive tumours, which correlated with poor prognosis. This also correlated with a significant decrease in Bcl3 gene expression in human breast cancer cell lines exhibiting increased motility characteristics. The effects of siRNA-mediated knockdown of Bcl3 are ongoing. In the mouse mammary gland, Bcl3 expression was restricted to epithelial cells during the first 24 hours of involution. Bcl3 deficiency resulted in a transient delay in the appearance of apoptotic bodies in the early involuting mammary gland in Bcl3-/- mice, while pSTAT3 levels were unchanged compared with equivalent timepoints in control animals. The activities of initiator/executor caspases of both intrinsic and extrinsic pathways were significantly decreased in Bcl3-/- tissues at this time, which correlated with decreases in the expression of key regulators of intrinsic/extrinsic apoptosis. Results from the ongoing magnetic resonance imaging study of tumour incidence/progression in mmtv-neu/Bcl3-/- mice will be presented. Conclusion These observations suggest that Bcl3 promotes apoptosis in the mammary gland and provides preliminary evidence of cross-talk between STAT3 and NF-κB pathways, both of which have been implicated in breast cancer. Our current data on Bcl3 in primary breast tumours and breast cancer cell lines contrasts with other studies, to suggest that Bcl3 suppresses the metastatic progression of primary breast cancer and has a neutral role in breast cancer incidence or primary tumour growth.