Cindy A. Wilson

Localization of human BRCA1 and its loss in high-grade, non-inherited breast carcinomas

Although the link between the BRCA1 tumour–suppressor gene and hereditary breast and ovarian cancer is established, the role, if any, of BRCA1 in non–familial cancers is unclear. BRCA1 mutations are rare in sporadic cancers, but loss of BRCA1 resulting from reduced expression or incorrect subcellular localization is postulated to be important in non–familial breast and ovarian cancers. Epigenetic loss, however, has not received general acceptance due to controversy regarding the subcellular localization of BRCA1 proteins, reports of which have ranged from exclusively nuclear, to conditionally nuclear, to the ER/golgi, to cytoplasmic invaginations into the nucleus. In an attempt to resolve this issue, we have comprehensively characterized 19 anti–BRCA1 antibodies. These reagents detect a 220–kD protein localized in discrete nuclear foci in all epithelial cell lines, including those derived from breast malignancies. Immunohistochemical staining of human breast specimens also revealed BRCA1 nuclear foci in benign breast, invasive lobular cancers and low–grade ductal carcinomas. Conversely, BRCA1 expression was reduced or undetectable in the majority of high–grade, ductal carcinomas, suggesting that absence of BRCA1 may contribute to the pathogenesis of a significant percentage of sporadic breast cancers.

HER-2 overexpression differentially alters transforming growth factor-β responses in luminal versus mesenchymal human breast cancer cells

IntroductionAmplification of the HER-2 receptor tyrosine kinase has been implicated in the pathogenesis and aggressive behavior of approximately 25% of invasive human breast cancers. Clinical and experimental evidence suggest that aberrant HER-2 signaling contributes to tumor initiation and disease progression. Transforming growth factor beta (TGF-β) is the dominant factor opposing growth stimulatory factors and early oncogene activation in many tissues, including the mammary gland. Thus, to better understand the mechanisms by which HER-2 overexpression promotes the early stages of breast cancer, we directly assayed the cellular and molecular effects of TGF-β1 on breast cancer cells in the presence or absence of overexpressed HER-2.MethodsCell proliferation assays were used to determine the effect of TGF-β on the growth of breast cancer cells with normal or high level expression of HER-2. Affymetrix microarrays combined with Northern and western blot analysis were used to monitor the transcriptional responses to exogenous TGF-β1 in luminal and mesenchymal-like breast cancer cells. The activity of the core TGF-β signaling pathway was assessed using TGF-β1 binding assays, phospho-specific Smad antibodies, immunofluorescent staining of Smad and Smad DNA binding assays.ResultsWe demonstrate that cells engineered to over-express HER-2 are resistant to the anti-proliferative effect of TGF-β1. HER-2 overexpression profoundly diminishes the transcriptional responses induced by TGF-β in the luminal MCF-7 breast cancer cell line and prevents target gene induction by a novel mechanism that does not involve the abrogation of Smad nuclear accumulation, DNA binding or changes in c-myc repression. Conversely, HER-2 overexpression in the context of the mesenchymal MDA-MB-231 breast cell line potentiated the TGF-β induced pro-invasive and pro-metastatic gene signature.ConclusionHER-2 overexpression promotes the growth and malignancy of mammary epithelial cells, in part, by conferring resistance to the growth inhibitory effects of TGF-β. In contrast, HER-2 and TGF-β signaling pathways can cooperate to promote especially aggressive disease behavior in the context of a highly invasive breast tumor model.

Novel consensus DNA-binding sequence for BRCA1 protein complexes.

Increasing evidence continues to emerge supporting the early hypothesis that BRCA1 might be involved in transcriptional processes. BRCA1 physically associates with more than 15 different proteins involved in transcription and is paradoxically involved in both transcriptional activation and repression. However, the underlying mechanism by which BRCA1 affects the gene expression of various genes remains speculative. In this study, we provide evidence that BRCA1 protein complexes interact with specific DNA sequences. We provide data showing that the upstream stimulatory factor 2 (USF2) physically associates with BRCA1 and is a component of this DNA‐binding complex. Interestingly, these DNA‐binding complexes are downregulated in breast cancer cell lines containing wild‐type BRCA1, providing a critical link between modulations of BRCA1 function in sporadic breast cancers that do not involve germline BRCA1 mutations. The functional specificity of BRCA1 tumor suppression for breast and ovarian tissues is supported by our experiments, which demonstrate that BRCA1 DNA‐binding complexes are modulated by serum and estrogen. Finally, functional analysis indicates that missense mutations in BRCA1 that lead to subsequent cancer susceptibility may result in improper gene activation. In summary, these findings establish a role for endogenous BRCA1 protein complexes in transcription via a defined DNA‐binding sequence and indicate that one function of BRCA1 is to co‐regulate the expression of genes involved in various cellular processes. Published 2003 Wiley‐Liss, Inc.†

Recent translational research: microarray expression profiling of breast cancer – beyond classification and prognostic markers?

Genomic expression profiling has greatly improved our ability to subclassify human breast cancers according to shared molecular characteristics and clinical behavior. The logical next question is whether this technology will be similarly useful for identifying the dominant signaling pathways that drive tumor initiation and progression within each breast cancer subtype. A major challenge will be to integrate data generated from the experimental manipulation of model systems with expression profiles obtained from primary tumors. We highlight some recent progress and discuss several obstacles in the use of expression profiling to identify pathway signatures in human breast cancer.

Evaluation of the arrayed primer extension resequencing assay for TP53 mutation detection.

Over the years we have screened for TP53 mutations in different patient materials using temporal temperature gel electrophoresis (TTGE) [1], followed by direct sequencing of samples with aberrant migrating bands to determine the nature of the sequence alteration. Mutations in the TP53 gene are associated with several different cancer types and have been shown to have both prognostic and predictive implications. In this project we are evaluating whether a commercial available array platform for sequencing the TP53 gene using a primer extension assay (APEX) is as sensitive, rapid and cost-effective as TTGE/sequencing. The array is designed by Asper Biotech [2]. Genomic DNA is amplified by PCR, and dUTP is incorporated. The amplification products are then concentrated and purified with spincolumns. Amplification products are fragmented by Uracil N-glycosylase, and unincorporated dNTPs are inactivated by shrimp alkaline phosphatase. The fragmented PCR products are mixed with thermosequenase and four fluorescence-labelled ddNTPs. The sample mixture is transferred to a chip that contains sequence-specific oligonucleotides. So far, exons 2–9 are included on the array. Genorama™ QuattroImager is used for scanning. The Genorama imaging system and genotyping software are used for imaging and semiautomatic sequence analysis. DNA samples from 48 primary breast carcinomas, 11 ovarian carcinomas and 34 cell lines were used for evaluation. Results from a titration experiment with different ratios of the Arg/Arg and Pro/Pro alleles on codon 72 in the TP53 gene showed that mutations could be detected even if the mutated cells were present in less than 5%. We have experienced that homozygous and hemizygous mutations occasionally are missed by the TTGE technique, but that they all were easily detected by APEX [3]. Detection of deletions and insertions, however, is not yet optimal using the APEX technology and they are frequently missed. For the tumour samples the resequencing efficiency using APEX was 92% for both DNA strands and 99.5% for sense and/or antisense strands. The strength of using the APEX technology is that both strands are simultaneously analyzed, and that no further sequencing is needed. It is rapid and sensitive. Cost-effectiveness is still under evaluation.