Melissa A. Brown

Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers

Basal-like breast cancers arising in women carrying mutations in the BRCA1 gene, encoding the tumor suppressor protein BRCA1, are thought to develop from the mammary stem cell. To explore early cellular changes that occur in BRCA1 mutation carriers, we have prospectively isolated distinct epithelial subpopulations from normal mammary tissue and preneoplastic specimens from individuals heterozygous for a BRCA1 mutation. We describe three epithelial subsets including basal stem/progenitor, luminal progenitor and mature luminal cells. Unexpectedly, we found that breast tissue from BRCA1 mutation carriers harbors an expanded luminal progenitor population that shows factor-independent growth in vitro. Moreover, gene expression profiling revealed that breast tissue heterozygous for a BRCA1 mutation and basal breast tumors were more similar to normal luminal progenitor cells than any other subset, including the stem cell–enriched population. The c-KIT tyrosine kinase receptor (encoded by KIT) emerged as a key marker of luminal progenitor cells and was more highly expressed in BRCA1-associated preneoplastic tissue and tumors. Our findings suggest that an aberrant luminal progenitor population is a target for transformation in BRCA1-associated basal tumors .

B cell stimulatory factor-1/interleukin-4 mRNA is expressed by normal and transformed mast cells

BSF-1/interleukin-4, a product of activated T cells, has multiple biological activities that affect cells of most hematopoietic lineages. Among these is the ability of BSF-1 to costimulate the growth of mast cells and regulate the production of IgE. We demonstrate here that BSF-1 mRNA is expressed by a majority of transformed mast cell lines and by 5 IL-3-dependent non-transformed mast cell lines. BSF-1 activity, including the ability to enhance the growth of IL-3-dependent mast cells, was detected in the supernatants of transformed mast cells. The role of BSF-1 as a mast cell growth factor, its constitutive production by transformed mast cells, and the lack of IL-3 production by most of these cells raise the possibility that BSF-1 may act as an autocrine growth factor for some transformed mast cells. Furthermore, production of BSF-1 mRNA by nontransformed cells indicates that mast cells may be an important physiologic source of this factor.

Deletion mapping and expression in Escherichia coli of the large genomic segment of a birnavirus.

The large genomic segment of infectious bursal disease virus encodes a polyprotein in which the viral polypeptides are present in the following order: N-VP2-VP4-VP3-C. Expression in Escherichia coli of the large segment results in the processing of the polyprotein. The expression product reacts with a virus neutralizing and protective monoclonal antibody that recognizes a conformational epitope on the surface of the virus. Different regions of the large genomic segment were deleted at defined restriction sites and the truncated fragments were ligated to various expression vectors for high-level expression in E. coli. The expressed proteins were probed with three different monoclonal antibodies that recognize epitopes encoded by different regions of the large genomic segment. These deletion mapping studies suggest that VP4 is involved in the processing of the precursor polyprotein, and the conformational epitope recognized by the virus neutralizing monoclonal antibody is present within VP2.

Genetic and Histopathologic Evaluation of BRCA1 and BRCA2 DNA Sequence Variants of Unknown Clinical Significance

Classification of rare missense variants as neutral or disease causing is a challenge and has important implications for genetic counseling. A multifactorial likelihood model for classification of unclassified variants in BRCA1 and BRCA2 has previously been developed, which uses data on co-occurrence of the unclassified variant with pathogenic mutations in the same gene, cosegregation of the unclassified variant with affected status, and Grantham analysis of the fit between the missense substitution and the evolutionary range of variation observed at its position in the protein. We have further developed this model to take into account relevant features of BRCA1- and BRCA2-associated tumors, such as the characteristic histopathology and immunochemical profiles associated with pathogenic mutations in BRCA1, and the fact that approximately 80% of tumors from BRCA1 and BRCA2 carriers undergo inactivation of the wild-type allele by loss of heterozygosity. We examined 10 BRCA1 and 15 BRCA2 unclassified variants identified in Australian, multiple-case breast cancer families. By a combination of genetic, in silico, and histopathologic analyses, we were able to classify one BRCA1 variant as pathogenic and six BRCA1 and seven BRCA2 variants as neutral. Five of these neutral variants were also found in at least 1 of 180 healthy controls, suggesting that screening a large number of appropriate controls might be a useful adjunct to other methods for evaluation of unclassified variants.

Dynamic interactions between the promoter and terminator regions of the mammalian BRCA1 gene

The 85-kb breast cancer-associated gene BRCA1 is an established tumor suppressor gene, but its regulation is poorly understood. We demonstrate by gene conformation analysis in both human cell lines and mouse mammary tissue that gene loops are imposed on BRCA1 between the promoter, introns, and terminator region. Significantly, association between the BRCA1 promoter and terminator regions change upon estrogen stimulation and during lactational development. Loop formation is transcription-dependent, suggesting that transcriptional elongation plays an active role in BRCA1 loop formation. We show that the BRCA1 terminator region can suppress estrogen-induced transcription and so may regulate BRCA1 expression. Significantly, BRCA1 promoter and terminator interactions vary in different breast cancer cell lines, indicating that defects in BRCA1 chromatin structure may contribute to dysregulated expression of BRCA1 seen in breast tumors.

ORAI1-Mediated Calcium Influx in Lactation and in Breast Cancer

The entry of calcium into the mammary epithelial cell from the maternal plasma (i.e., calcium influx mechanisms) during lactation is poorly understood. As alterations in calcium channels and pumps are a key feature of some cancers, including breast cancer, understanding these calcium influx pathways may have significance beyond mammary biology. We show that the store-operated calcium influx protein, Orai1, is increased during lactation whereas the Orai1 activator Stim1, but not Stim2, is downregulated. Stim2 siRNA reduced basal calcium levels in a lactation model. Our results suggest that calcium influx is remodeled in mammary epithelial cells during lactation, with calcium influx increased through Orai1, activated by Stim2. Breast cancer cell lines had increased levels of ORAI1. ORAI1 siRNA in breast cancer cells reduced store-operated calcium entry and remodeled the calcium influx associated with invasive stimuli. Analysis of microarray data from 295 breast cancers showed that the transcriptional breast cancer subtype with the poorest prognosis (basal) was associated with an altered relationship between the ORAI1 regulators STIM1 and STIM2, and that women with breast cancers with STIM1high/STIM2low tumors had a significantly poorer prognosis. Our studies show that during lactation there is a remodeling in the nature of calcium influx and that alteration in the ORAI1 influx pathway may be a feature of some breast cancers, particularly those with the poorest prognosis. Our studies suggest that this pathway may be a novel therapeutic target for breast cancer treatment in these women. Mol Cancer Ther; 10(3); 448–60. ©2011 AACR.

Constitutional Methylation of the BRCA1 Promoter Is Specifically Associated with BRCA1 Mutation-Associated Pathology in Early-Onset Breast Cancer

Women carrying germline mutations in BRCA1 are at a substantially elevated risk of breast cancer and their tumors typically have distinctive morphologic features. We hypothesized that constitutional methylation of the BRCA1 promoter region could give rise to such breast cancers in women. We selected 255 women diagnosed with breast cancer before the age of 40 years for whom BRCA1 germline mutations had not been identified. Of them, 52 had five or more of nine BRCA1 mutation-associated morphologic features (group 1), 39 had four (group 2), and 164 had three or less (group 3). The prevalence of detectable BRCA1 promoter methylation in peripheral blood DNA decreased from 31% to 10% to 5% across groups 1–3, respectively (P = 0.000002), and was significantly greater than the 4% frequency in unaffected controls (P = 0.004). Peripheral blood methylation was associated with a 3.5-fold (95% CI, 1.4–10.5) increased risk of having early onset breast cancer. Methylation was consistently mosaic in the peripheral blood where the estimated allelic frequency of BRCA1 promoter methylation ranged from 0.1% to 17%. Group 1 women, but not group 3 women, with detectable methylation of peripheral blood DNA had high levels of BRCA1 promoter methylation of their tumor DNA, indicating that constitutional BRCA1 methylation strongly predisposes toward the development of BRCA1 methylated tumors that then have features resembling BRCA1 mutated tumors. Screening peripheral blood for BRCA1 promoter methylation might thus predict early-onset breast cancers. This raises the possibility of chemoprevention or other intervention to diminish the risk of developing breast cancer in these women. Cancer Prev Res; 4(1); 23–33. ©2010 AACR. Cancer Prev Res; 4(1); 23–33. ©2010 AACR.

Roles of heterogeneous nuclear ribonucleoproteins A and B in cell proliferation

Overexpression of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2 and B1 has been observed in a variety of tumour types, however, it is unknown whether this dysregulation is a consequence of, or a driving force for, unregulated cell proliferation. We have shown that the levels of hnRNPs A1, A2 and B1, but not A3, are modulated during the cell cycle of Colo16 squamous carcinoma cells and HaCaT immortalized keratinocytes, suggesting that A1, A2 and B1 are needed at particular cell cycle stages. However, the levels of hnRNP A1, A2 and B1 mRNAs were constant, indicating that regulation of protein levels was controlled at the level of translation. RNAi suppression of hnRNP A1 or A3 alone did not affect the proliferation of Colo16 cells but the proliferation rate was significantly reduced when both were suppressed simultaneously, or when either was suppressed together with hnRNP A2. Reducing hnRNP A2 expression in Colo16 and HaCaT cells by RNAi led to a non-apoptotic-related decrease in cell proliferation, reinforcing the view that this protein is required for cell proliferation. Suppression of hnRNP A2 in Colo16 cells was associated with increased p21 levels but p53 levels remained unchanged. In addition, expression of BRCA1 was downregulated, at both mRNA and protein levels. The observed effects of hnRNP A2 and its isoforms on cell proliferation and their correlation with BRCA1 and p21 expression suggest that these hnRNP proteins play a role in cell proliferation.

Virus-Neutralizing and Passively Protective Monoclonal-Antibodies to Infectious Bursal Disease Virus of Chickens

Murine monoclonal antibodies (MAbs) were produced to assist in the identification and characterization of the virus-neutralizing epitopes of infectious bursal disease virus (IBDV). Only MAbs that reacted in Western blotting with viral protein 2 (VP2) or immunoprecipitated VP2 neutralized the infectivity of the virus in cell culture and passively protected young chickens from infection. Three of the neutralizing MAbs did not react with denatured viral proteins. Additivity enzyme-linked immunosorbent assays indicated that the six virus-neutralizing MAbs recognized two spatially independent epitopes. The ability of two of the virus-neutralizing MAbs to neutralize a variant of IBDV that had escaped neutralization by all the other MAbs confirmed the existence of two distinct neutralizing epitopes. The results support the hypothesis that there are at least two non-overlapping epitopes recognized by the virus-neutralizing MAbs reported in this study, although these may still be within one conformational site on VP2 of IBDV.

A guide for functional analysis of BRCA1 variants of uncertain significance

Germline mutations in the tumor suppressor gene BRCA1 confer an estimated lifetime risk of 56–80% for breast cancer and 15–60% for ovarian cancer. Since the mid 1990s when BRCA1 was identified, genetic testing has revealed over 1,500 unique germline variants. However, for a significant number of these variants, the effect on protein function is unknown making it difficult to infer the consequences on risks of breast and ovarian cancers. Thus, many individuals undergoing genetic testing for BRCA1 mutations receive test results reporting a variant of uncertain clinical significance (VUS), leading to issues in risk assessment, counseling, and preventive care. Here, we describe functional assays for BRCA1 to directly or indirectly assess the impact of a variant on protein conformation or function and how these results can be used to complement genetic data to classify a VUS as to its clinical significance. Importantly, these methods may provide a framework for genome‐wide pathogenicity assignment. Hum Mutat 33:1526–1537, 2012.