AGR2, a novel metastasis inducing protein with an effect on breast cancer patient survival
Breast Cancer Research
Epidemiological studies have shown that only about 20% of the familial clustering of breast cancer is explained by the known highly penetrant mutations in BRCA1 and BCRA2. We have set out to search for the genes for the remaining 80%. Twin studies indicate a predominant role of shared genes rather than a shared environment; the patterns of occurrence of breast cancer in families are consistent with a major polygenic component. Methods We have assembled a population based set of 5,000 breast
... of 5,000 breast cancer cases and 5,000 controls from the East Anglian population. We have simple clinical and epidemiological information, including family history, and samples of blood and paraffin embedded tumour. We have used association studies based on single nucleotide polymorphisms, first with candidate genes and then in a genome-wide scan of 266,000 single nucleotide polymorphisms, to search for the putative predisposing genes. We have as yet searched only for common variants (frequency >5%). Results We have modelled the effects of polygenic predisposition in the East Anglian population, and have shown that the model predicts a wide distribution of individual risk in the population, such that half of all breast cancers may occur in the 12% of women at greatest risk. Both the candidate gene-based and genome-wide scans have provided provisional identification of a number of novel susceptibility genes, and these are currently being confirmed by a world-wide consortium of independent laboratories totalling 20,000-plus cases and controls. No single gene so far identified contributes more than 2% of the total inherited component, consistent with a model in which susceptibility is the result of a large number of individually small genetic effects. S2 Translating breast cancer research into clinical practice -new approaches and better outcomes SRD Johnston Breast Cancer Research 2006, 8(Suppl 2):S2 Background Ten to twenty per cent of breast tumours exhibit a basallike genetic profile and these tumours carry a poor prognosis. Breast tumours which contain germline mutations for BRCA1 commonly exhibit a molecular profile similar to basal breast tumours. BRCA1 is a tumour suppressor gene which is mutated in up to 5-10% of breast cancer cases and is involved in multiple cellular processes including DNA damage control, cell cycle checkpoint control, apoptosis, ubiquitination and transcriptional regulation. Methods Microarray-based profiling was carried out using the HCC1937EV and HCC1937BR breast cancer cell lines. Basal gene and protein expression levels were analysed by qRT-PCR and western blotting. ChIP analyses were performed and demonstrated that BRCA1 regulates basal gene expression through a transcriptional mechanism involving c-myc. Results We have previously carried out microarray-based expression profiling to examine differences in gene expression when BRCA1 is reconstituted in BRCA1 mutated HCC1937 breast cancer cells. We observed that p-cadherin and the cytokeratin 5 and cytokeratin 17 genes, which are strongly correlated with the basal phenotype, are differentially expressed when BRCA1 is reconstituted. In addition, qRT-PCR and ChIP analysis of BRCA1 reconstituted cells show that BRCA1 represses the expression of these basal genes by a transcriptional mechanism. Furthermore, abrogation of endogenous BRCA1 protein in the T47D cell line using siRNA results in reexpression of these basal genes, suggesting that BRCA1 expression levels may be important in basal gene expression. We have also demonstrated that BRCA1 is physically associated with the promoter regions of basal genes through an association with c-myc. Consequently, we have confirmed that siRNA inhibition of c-myc in T47D cells results in re-expression of these genes. Conclusions Our results suggest that BRCA1 is involved in the transcriptional regulation of genes associated with the basal phenotype and that BRCA1 controls basal gene expression through a transcriptional mechanism involving c-myc. Further work is now concentrating on defining the relationship between BRCA1 and basal gene expression and how this may affect clinical responses to breast cancer chemotherapy. Acknowledgement This work is funded by Breast Cancer Campaign. Background Inherited mutations in BRCA2 are associated with a predisposition to early-onset breast cancers. The underlying basis of tumourigenesis is thought to be linked to defects in DNA double-strand break repair by homologous recombination (HR), as indicated by the spontaneous chromosomal instability phenotype of BRCA2-defective cell lines. The BRCA2 protein interacts with ssDNA and the RAD51 recombination protein, and is proposed to recruit RAD51 to the damage site for the HR repair. Methods Recombinant BRCA2 fragments that cover the entire length of BRCA2 were tested for interaction with RAD51 and for their phosphorylation using cell free extracts. An antibody that specifically recognises BRCA2 phosphorylated at serine 3291 was generated and used to analyse the phosphorylation status of endogenous BRCA2 during the cell cycle and after DNA damaging treatment. A cell line that stably expresses a C-terminal BRCA2 fragment was generated, to allow the analysis of RAD51 interactions and ability to promote homologous recombinational repair (HRR). Results We found that the C-terminal region of BRCA2, which directly interacts with RAD51, contains a site (S3291) that is phosphorylated by cyclin-dependent kinases. Phosphorylation of S3291 increases as cells progress towards mitosis, and was shown to block C-terminal interactions between BRCA2 and RAD51. However, DNA damage overcomes cell cycle regulation by reducing S3291 phosphorylation and stimulating interactions with RAD51. HRR is defective in cells overexpressing the C-terminal fragment of BRCA2, indicating that interactions between RAD51 and the C-terminal region of endogenous BRCA2 are important for repair. Conclusion We suggest that S3291 phosphorylation provides a molecular switch that can regulate RAD51-mediated HRR. Loss of phosphorylation in response to DNA damage allows interactions between RAD51 and the C-terminal region of BRCA2 and may facilitate the loading of RAD51 on damaged DNA . Importantly, a S3291 nonphosphorylatable mutation (P3292L) has been found in familial breast cancer patients, implicating a role of S3291 phosphorylation in the maintenance of genome integrity. Chromosome translocations that form fusion transcripts and/or activate expression of genes by promoter insertion are key events in leukaemias and lymphomas, and mesenchymal tumours, but it has been fashionable to think they are irrelevant to the common epithelial cancers such as breast cancer. However, that view is now being challenged [1-4]; in particular, we have shown that NRG1 is translocated in breast cancers . It seems likely that some translocations in breast cancers target specific genes at their breakpoints, and this is particularly likely for reciprocal translocations. We are cataloguing translocation breakpoints in breast cancer cell lines and tumours. We use array painting, in which individual chromosomes are purified in a cell sorter and their DNA hybridized to microarrays. We have analysed all the chromosomes of three breast cancer lines to 1 Mb resolution or better. A striking finding was that reciprocal and more complex balanced translocations are far more frequent than expected. Together the three lines had at least 14 balanced translocations, almost three times more than identified by cytogenetics -the cryptic ones involved small fragments, or were obscured by subsequent rearrangement. Furthermore, several translocation breaks were in genes, including known cancer-critical genes such as EP300/p300 and CTCF. This supports the emerging idea that chromosome rearrangement plays a major role in the gene changes that cause breast cancer. ) Breast epithelial stem cells are thought to be the primary targets in the etiology of breast cancer. Since breast cancers mostly express estrogen receptor-alpha (ERα), we examined the biology of these cells and their relationship to stem cells in normal human breast epithelium. We employed several complementary approaches to identify putative stem cell markers, to characterise an isolated stem cell population and to relate these to cells expressing ERα. ERα-positive cells were found to coexpress the putative stem cell markers p21 CIP1 and Msi-1. Human breast epithelial cells with Hoechst dye-effluxing 'side population' (SP) properties characteristic of mammary stem cells in mice were demonstrated to be undifferentiated cells by lack of expression of myoepithelial and luminal epithelial membrane markers. These SP cells were sixfold enriched for ERα-positive cells and expressed several-fold higher levels of the ERα, p21 CIP1 and Msi1 genes than non-SP cells. In contrast to non-SP cells, SP cells formed branching structures in matrigel which included cells of both luminal and myoepithelial lineages. The data suggest a model where scattered ERα-positive cells are stem cells that self-renew through asymmetric cell division and generate patches of transit amplifying and differentiated cells. In recent studies we have been investigating breast cancers for the presence of a stem cell population. Using a nonadherent culture method analogous to neurosphere culture that enriches for neural stem cells, we have demonstrated that breast cancer cell lines and primary tumours contain a self-renewing population that is highly regulated by the Notch receptor signaling pathway. Inhibitors of this pathway could represent a new therapeutic modality in breast cancer, perhaps through combination with current treatments. Breast Cancer Research Vol 8 Suppl 2 Breast cancer research: the past and the future 1. Jhappan C, Gallahan D, Stahle C, Chu E, Smith GH, Merlino G, Callahan R: Expression of an activated Notch-related int3 transgene interfers with cell differentiation and induces neoplastic transformation in mammary and salivary glands. Genes Dev 1992, 6:345-355.