McDyer, FA

Abstract Background: The BRCA1 tumour suppressor gene is mutated in the germline of women who are predisposed to developing breast cancer. Gene expression profiling has identified at least five different breast cancer subtypes with BRCA- mutated breast tumours clustering with triple negative breast cancers. The majority of BRCA1-mutated breast tumours are characterised as being negative for the estrogen receptor-α (ER-α), however, the underlying molecular biology of these tumours has not yet been fully determined. The aim of this study is to identify and characterise novel proliferation-associated BRCA1 target genes that are activated in BRCA1 mutated estrogen receptor negative (ER-ve) breast tumours. Methods: Gene expression profiling and data analysis was performed on a cohort of 46 FFPE (Formalin Fixed Paraffin Embedded) derived BRCA1 mutated (ER-ve) breast tumours and matched sporadic controls using the Almac Diagnostics Breast DSA research tool. Profiling was also performed on a panel of 15 breast cancer cell lines. Bioinformatics analysis was performed using Oncomine, DAVID and Metacore. High throughput siRNA screens using HiPerFect were performed on the Qiagen Flexiplate siRNA. Validation of gene targets was performed by qRT-PCR and ChIP assay. Clonogenic assays were performed to independently validate the effect of selected target genes on cell survival. Results: A list of differentially expressed transcripts was derived from the comparison of 23 (ER-ve) BRCA1 mutated breast tumours and 23 matched sporadic controls. A genome-wide ChIP-Chip promoter analysis was also performed in MCF7 breast cancer cells. By overlapping these two datasets, a list of tumour derived BRCA1 promoter bound target genes was identified. Functional analysis of this gene list has identified the main pathways and processes that are deregulated in BRCA1 mutated (ER-ve) breast cancer including: (1) immune response (2) induction of the epithelial to mesenchymal transition (EMT) (3) cell cycle regulation and (4) apoptosis and survival. Hierarchical clustering of these 46 breast tumours and 15 breast cancer cell lines was performed and the BRCA1 mutated (ER-ve) breast cancer cell lines (MDA436 and SUM149) were identified as those that best reflect the biology BRCA1 mutated (ER-ve) tumours. High throughput siRNA screening in these cell lines has identified a set of transcripts that when inhibited have a negative impact on cellular proliferation. Independent validation by qRT-PCR, ChIP assay, western blotting and clonogenic assays have confirmed HE4 (WFDC2) as a novel BRCA1 target gene that provides a growth advantage in BRCA1 mutated (ER-ve) breast cancer cells. Conclusions: Gene expression profiling of an extensive cohort of BRCA1 mutated (ER-ve) breast tumours and matched sporadic controls has identified a profile of BRCA1 mutated (ER-ve) breast cancer. This list has been further refined to generate a list of BRCA1 promoter bound transcriptional target genes. High throughout siRNA screening has revealed a panel of genes that are implicated in the proliferation of BRCA1 mutated (ER-ve) breast cancer. HE4 has been identified as a novel BRCA1 transcriptional target gene that promotes the survival of BRCA1 mutated (ER-ve) breast tumours. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD10-05.

Abstract Background: Approximately 5-10% of all breast cancers are hereditary and the majority of these arise due to germline mutations in the BRCA1 and BRCA2 tumour suppressor genes. BRCA1 is involved in multiple cellular pathways including DNA damage signalling, DNA repair, cell cycle regulation, protein ubiquitination, chromatin remodelling, transcriptional regulation and apoptosis. Several distinct pathological features can be used to estimate the likelihood of the presence of a BRCA1 mutation, however, it is not yet possible to fully categorise a BRCA1 mutated tumour. BRCA1-associated breast cancers are generally defined as being ER (Estrogen Receptor) negative and indeed triple negative for ER, PR and HER2. However, approximately 10-36% of BRCA1 mutated breast cancers are, in fact, ER positive. These tumours less frequently demonstrate the characteristics more commonly associated with BRCA 1 -associated breast cancers. Initial molecular evidence also suggests that there is heterogeneity within BRCA1-associated breast tumours and this is dependent on the presence or absence of the estrogen receptor. The aims of this study are to investigate the underlying biology of BRCA1-mutated (ER positive) and BRCA1-mutated (ER negative) breast tumours. Methods: Extensive gene expression profiling and data analysis was performed on a cohort of 70 FFPE (Formalin Fixed Paraffin Embedded) derived BRCA1 mutated breast tumours and matched sporadic controls using the ALMAC Breast Cancer DSA™ research tool. Within this dataset we have generated molecular profiles of: (1) BRCA 1 -mutated ER positive and (2) BRCA1-mutated ER negative breast cancer. Functional analysis was performed using DAVID and METACORE. Validation of gene targets was performed by qRT-PCR and Western blotting. Results: A list of differentially expressed transcripts was derived from the comparison of 35 BRCA1 mutant breast tumours and 35 matched sporadic controls. Further analysis based on the presence and absence of ER identified a set of transcripts defining BRCA1-mutated (ER positive) and BRCA1-mutated (ER negative) breast cancer. Functional analysis of these two datasets has identified the main pathways and processes that are deregulated. Specifically, BRCA1-deficiency in the absence of ER was associated with deregulation of pathways implicated in immune response whereas BRCA 1 deficiency in the presence of ER was associated with pathways implicated in cell adhesion and cytoskeletal remodelling. Validation of the key genes underlying these two BRCA1-deficient breast cancer subtypes has been performed. Discussion: This approach has revealed significant heterogeneity within BRCA1 mutated breast cancer based on the presence or absence of ER. Significant differences in the transcripts and molecular processes underlying BRCA1-mutated (ER positive) and BRCA1-mutated (ER negative) breast tumours have been identified. The ability to identify BRCA 1 -deficiency by gene expression profiling from FFPE derived breast tissue may also have significant clinical application. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-14.