JAK-STAT Signaling in Inflammatory Breast Cancer Enables Chemotherapy-Resistant Cell States.

Inflammatory breast cancer (IBC) is a difficult-to-treat disease with poor clinical outcomes due to high risk of metastasis and resistance to treatment. In breast cancer, CD44+CD24- cells possess stem cell-like features and contribute to disease progression, and we previously described a CD44+CD24-pSTAT3+ breast cancer cell subpopulation that is dependent on JAK2/STAT3 signaling. Here we report that CD44+CD24- cells are the most frequent cell type in IBC and are commonly pSTAT3+. Combination of JAK2/STAT3 inhibition with paclitaxel decreased IBC xenograft growth more than either agent alone. IBC cell lines resistant to paclitaxel and doxorubicin were developed and characterized to mimic therapeutic resistance in patients. Multi-omic profiling of parental and resistant cells revealed enrichment of genes associated with lineage identity and inflammation in chemotherapy-resistant derivatives. Integrated pSTAT3 chromatin immunoprecipitation sequencing and RNA sequencing (RNA-seq) analyses showed pSTAT3 regulates genes related to inflammation and epithelial-to-mesenchymal transition (EMT) in resistant cells, as well as PDE4A, a cAMP-specific phosphodiesterase. Metabolomic characterization identified elevated cAMP signaling and CREB as a candidate therapeutic target in IBC. Investigation of cellular dynamics and heterogeneity at the single cell level during chemotherapy and acquired resistance by CyTOF and single cell RNA-seq identified mechanisms of resistance including a shift from luminal to basal/mesenchymal cell states through selection for rare preexisting subpopulations or an acquired change. Finally, combination treatment with paclitaxel and JAK2/STAT3 inhibition prevented the emergence of the mesenchymal chemo-resistant subpopulation. These results provide mechanistic rational for combination of chemotherapy with inhibition of JAK2/STAT3 signaling as a more effective therapeutic strategy in IBC. SIGNIFICANCE: Chemotherapy resistance in inflammatory breast cancer is driven by the JAK2/STAT3 pathway, in part via cAMP/PKA signaling and a cell state switch, which can be overcome using paclitaxel combined with JAK2 inhibitors.

Intratumor heterogeneity defines treatment-resistant HER2+ breast tumors.

Targeted therapy for patients with HER2-positive (HER2+) breast cancer has improved overall survival, but many patients still suffer relapse and death from the disease. Intratumor heterogeneity of both estrogen receptor (ER) and HER2 expression has been proposed to play a key role in treatment failure, but little work has been done to comprehensively study this heterogeneity at the single-cell level. In this study, we explored the clinical impact of intratumor heterogeneity of ER protein expression, HER2 protein expression, and HER2 gene copy number alterations. Using combined immunofluorescence and in situ hybridization on tissue sections followed by a validated computational approach, we analyzed more than 13 000 single tumor cells across 37 HER2+ breast tumors. The samples were taken both before and after neoadjuvant chemotherapy plus HER2-targeted treatment, enabling us to study tumor evolution as well. We found that intratumor heterogeneity for HER2 copy number varied substantially between patient samples. Highly heterogeneous tumors were associated with significantly shorter disease-free survival and fewer long-term survivors. Patients for which HER2 characteristics did not change during treatment had a significantly worse outcome. This work shows the impact of intratumor heterogeneity in molecular diagnostics for treatment selection in HER2+ breast cancer patients and the power of computational scoring methods to evaluate in situ molecular markers in tissue biopsies.

Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis.

BACKGROUND: The microenvironment and stress factors like glucocorticoids have a strong influence on breast cancer progression but their role in the first stages of breast cancer and, particularly, in myoepithelial cell regulation remains unclear. Consequently, we investigated the role of glucocorticoids in ductal carcinoma in situ (DCIS) in breast cancer, focusing specially on myoepithelial cells. METHODS: To clarify the role of glucocorticoids at breast cancer onset, we evaluated the effects of cortisol and corticosterone on epithelial and myoepithelial cells using 2D and 3D in vitro and in vivo approaches and human samples. RESULTS: Glucocorticoids induce a reduction in laminin levels and favour the disruption of the basement membrane by promotion of myoepithelial cell apoptosis in vitro. In an in vivo stress murine model, increased corticosterone levels fostered the transition from DCIS to invasive ductal carcinoma (IDC) via myoepithelial cell apoptosis and disappearance of the basement membrane. RU486 is able to partially block the effects of cortisol in vitro and in vivo. We found that myoepithelial cell apoptosis is more frequent in patients with DCIS+IDC than in patients with DCIS. CONCLUSIONS: Our findings show that physiological stress, through increased glucocorticoid blood levels, promotes the transition from DCIS to IDC, particularly by inducing myoepithelial cell apoptosis. Since this would be a prerequisite for invasive features in patients with DCIS breast cancer, its clinical management could help to prevent breast cancer progression to IDC.

Histamine receptor 1 inhibition enhances antitumor therapeutic responses through extracellular signal-regulated kinase (ERK) activation in breast cancer.

Histamine receptor 1 (HRH1) belongs to the rhodopsin-like G-protein-coupled receptor family. Its activation by histamine triggers cell proliferation, embryonic development, and tumor growth. We recently established that HRH1 is up-regulated in basal and human epidermal growth factor receptor 2 (HER2)-enriched human breast tumors and that its expression correlates with a worse prognosis. Nevertheless, the functional role of HRH1 in basal and HER2-targeted therapy-resistant breast cancer (BC) progression has not yet been addressed. Using terfenadine, a selective chemical inhibitor of HRH1, we showed that the inhibition of HRH1 activity in basal BC cells leads to sub-G0 cell accumulation, suppresses proliferation, promotes cell motility and triggers the activation of extracellular signal-regulated kinase (ERK) signaling, initiating the mitochondrial apoptotic pathway. Furthermore, HER2-targeted therapy-resistant cells express higher levels of HRH1 and are more sensitive to terfenadine treatment. Moreover, in vivo experiments showed that terfenadine therapy reduced the tumor growth of basal and trastuzumab-resistant BC cells. In conclusion, our results suggest that targeting HRH1 is a promising new clinical approach to consider that could enhance the effectiveness of current therapeutic treatment in patients with basal and BC tumors resistant to HER2-targeted therapies.

The Proliferative Activity of Mammary Epithelial Cells in Normal Tissue Predicts Breast Cancer Risk in Premenopausal Women.

The frequency and proliferative activity of tissue-specific stem and progenitor cells are suggested to correlate with cancer risk. In this study, we investigated the association between breast cancer risk and the frequency of mammary epithelial cells expressing p27, estrogen receptor (ER), and Ki67 in normal breast tissue. We performed a nested case-control study of 302 women (69 breast cancer cases, 233 controls) who had been initially diagnosed with benign breast disease according to the Nurses' Health Studies. Immunofluorescence for p27, ER, and Ki67 was performed on tissue microarrays constructed from benign biopsies containing normal mammary epithelium and scored by computational image analysis. We found that the frequency of Ki67(+) cells was positively associated with breast cancer risk among premenopausal women [OR = 10.1, 95% confidence interval (CI) = 2.12-48.0]. Conversely, the frequency of ER(+) or p27(+) cells was inversely, but not significantly, associated with subsequent breast cancer risk (ER(+): OR = 0.70, 95% CI, 0.33-1.50; p27(+): OR = 0.89, 95% CI, 0.45-1.75). Notably, high Ki67(+)/low p27(+) and high Ki67(+)/low ER(+) cell frequencies were significantly associated with a 5-fold higher risk of breast cancer compared with low Ki67(+)/low p27(+) and low Ki67(+)/low ER(+) cell frequencies, respectively, among premenopausal women (Ki67(hi)/p27(lo): OR = 5.08, 95% CI, 1.43-18.1; Ki67(hi)/ER(lo): OR = 4.68, 95% CI, 1.63-13.5). Taken together, our data suggest that the fraction of actively cycling cells in normal breast tissue may represent a marker for breast cancer risk assessment, which may therefore impact the frequency of screening procedures in at-risk women. Cancer Res; 76(7); 1926-34. ©2016 AACR.

Differential expression of neurogenes among breast cancer subtypes identifies high risk patients.

The nervous system is now recognized to be a relevant component of the tumor microenvironment. Receptors for neuropeptides and neurotransmitters have been identified in breast cancer. However, very little is known about the role of neurogenes in regulating breast cancer progression. Our purpose was to identify neurogenes associated with breast cancer tumorigenesis with a potential to be used as biomarker and/or targets for treatment. We used three databases of human genes: GeneGo, GeneCards and Eugenes to generate a list of 1266 relevant neurogenes. Then we used bioinformatics tools to interrogate two published breast cancer databases SAGE and MicMa (n=96) and generated a list of 7 neurogenes that are differentially express among breast cancer subtypes. The clinical potential was further investigated using the GOBO database (n=1881). We identified 6 neurogenes that are differentially expressed among breast cancer subtypes and whose expression correlates with prognosis. Histamine receptor1 (HRH1), neuropilin2 (NRP2), ephrin-B1 (EFNB1), neural growth factor receptor (NGFR) and amyloid precursor protein (APP) were differentially overexpressed in basal and HER2-enriched tumor samples and syntaxin 1A (STX1A) was overexpressed in HER2-enriched and luminal B tumors. Analysis of HRH1, NRP2, and STX1A expression using the GOBO database showed that their expression significantly correlated with a shorter overall survival (p < 0.0001) and distant metastasis-free survival (p < 0.0001). In contrast, elevated co-expression of NGFR, EFNB1 and APP was associated with longer overall (p < 0.0001) and metastasis-free survival (p < 0.0001). We propose that HRH1, NRP2, and STX1A can be used as prognostic biomarkers and therapeutic targets for basal and HER2-enriched breast cancer subtypes.

In situ single-cell analysis identifies heterogeneity for PIK3CA mutation and HER2 amplification in HER2-positive breast cancer.

Detection of minor, genetically distinct subpopulations within tumors is a key challenge in cancer genomics. Here we report STAR-FISH (specific-to-allele PCR-FISH), a novel method for the combined detection of single-nucleotide and copy number alterations in single cells in intact archived tissues. Using this method, we assessed the clinical impact of changes in the frequency and topology of PIK3CA mutation and HER2 (ERBB2) amplification within HER2-positive breast cancer during neoadjuvant therapy. We found that these two genetic events are not always present in the same cells. Chemotherapy selects for PIK3CA-mutant cells, a minor subpopulation in nearly all treatment-naive samples, and modulates genetic diversity within tumors. Treatment-associated changes in the spatial distribution of cellular genetic diversity correlated with poor long-term outcome following adjuvant therapy with trastuzumab. Our findings support the use of in situ single cell-based methods in cancer genomics and imply that chemotherapy before HER2-targeted therapy may promote treatment resistance.

Comparison of methods for the isolation of human breast epithelial and myoepithelial cells.

Two lineages, epithelial, and myoepithelial cells are the main cell populations in the normal mammary gland and in breast cancer. Traditionally, cancer research has been performed using commercial cell lines, but primary cell cultures obtained from fresh breast tissue are a powerful tool to study more reliably new aspects of mammary gland biology, including normal and pathological conditions. Nevertheless, the methods described to date have some technical problems in terms of cell viability and yield, which hamper work with primary mammary cells. Therefore, there is a need to optimize technology for the proper isolation of epithelial and myoepithelial cells. For this reason, we compared four methods in an effort to improve the isolation and primary cell culture of different cell populations of human mammary epithelium. The samples were obtained from healthy tissue of patients who had undergone mammoplasty or mastectomy surgery. We based our approaches on previously described methods, and incorporated additional steps to ameliorate technical efficiency and increase cell survival. We determined cell growth and viability by phase-contrast images, growth curve analysis and cell yield, and identified cell-lineage specific markers by flow cytometry and immunofluorescence in 3D cell cultures. These techniques allowed us to better evaluate the functional capabilities of these two main mammary lineages, using CD227/K19 (epithelial cells) and CD10/K14 (myoepithelial cells) antigens. Our results show that slow digestion at low enzymatic concentration combined with the differential centrifugation technique is the method that best fits the main goal of the present study: protocol efficiency and cell survival yield. In summary, we propose some guidelines to establish primary mammary epithelial cell lines more efficiently and to provide us with a strong research instrument to better understand the role of different epithelial cell types in the origin of breast cancer.

The Transmodulation of HER2 and EGFR by Substance P in Breast Cancer Cells Requires c-Src and Metalloproteinase Activation.

BACKGROUND: Substance P (SP) is a pleiotropic cytokine/neuropeptide that enhances breast cancer (BC) aggressiveness by transactivating tyrosine kinase receptors like EGFR and HER2. We previously showed that SP and its cognate receptor NK-1 (SP/NK1-R) signaling modulates the basal phosphorylation of HER2 and EGFR in BC, increasing aggressiveness and drug resistance. In order to elucidate the mechanisms responsible for NK-1R-mediated HER2 and EGFR transactivation, we investigated the involvement of c-Src (a ligand-independent mediator) and of metalloproteinases (ligand-dependent mediators) in HER2/EGFR activation. RESULTS AND DISCUSSION: Overexpression of NK-1R in MDA-MB-231 and its chemical inhibition in SK-BR-3, BT-474 and MDA-MB-468 BC cells significantly modulated c-Src activation, suggesting that this protein is a mediator of NK-1R signaling. In addition, the c-Src inhibitor 4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline prevented SP-induced activation of HER2. On the other hand, SP-dependent phosphorylation of HER2 and EGFR decreased substantially in the presence of the MMP inhibitor 1-10, phenanthroline monohydrate, and the dual inhibition of both c-Src and MMP almost abolished the activation of HER2 and EGFR. Moreover, the use of these inhibitors demonstrated that this Src and MMP-dependent signaling is important to the cell viability and migration capacity of HER2+ and EGFR+ cell lines. CONCLUSION: Our results indicate that the transactivation of HER2 and EGFR by the pro-inflammatory cytokine/neuropeptide SP in BC cells is a c-Src and MMP-dependent process.

The neurokinin-1 receptor antagonist aprepitant is a promising candidate for the treatment of breast cancer.

The substance P (SP)/neurokinin (NK)-1 receptor system plays an important role in the development of cancer. No in-depth studies of the involvement of this system in breast cancer (BC) have been carried out, and the action exerted by the drug aprepitant on BC cells is currently unknown. We show the involvement of this system in human BC cell lines: i) these cells express mRNA for the NK-1 receptor; ii) they overexpress NK-1 receptors; iii) the NK-1 receptor is involved in their viability; iv) SP induces their proliferation; v) NK-1 receptor antagonists block SP-induced mitogen stimulation of these cells; vi) the specific antitumor action of such antagonists on these cells occurs through the NK-1 receptor; and vii) BC cell death is due to apoptosis. We also found NK-1 receptors and SP in all human BC samples studied. The NK-1 receptor may be a promising target in the treatment of BC and NK-1 receptor antagonists could be candidates as a new antitumor drug in the treatment of BC.

Non-cell-autonomous driving of tumour growth supports sub-clonal heterogeneity.

Cancers arise through a process of somatic evolution that can result in substantial sub-clonal heterogeneity within tumours. The mechanisms responsible for the coexistence of distinct sub-clones and the biological consequences of this coexistence remain poorly understood. Here we used a mouse xenograft model to investigate the impact of sub-clonal heterogeneity on tumour phenotypes and the competitive expansion of individual clones. We found that tumour growth can be driven by a minor cell subpopulation, which enhances the proliferation of all cells within a tumour by overcoming environmental constraints and yet can be outcompeted by faster proliferating competitors, resulting in tumour collapse. We developed a mathematical modelling framework to identify the rules underlying the generation of intra-tumour clonal heterogeneity. We found that non-cell-autonomous driving of tumour growth, together with clonal interference, stabilizes sub-clonal heterogeneity, thereby enabling inter-clonal interactions that can lead to new phenotypic traits.

GoIFISH: a system for the quantification of single cell heterogeneity from IFISH images.

Molecular analysis has revealed extensive intra-tumor heterogeneity in human cancer samples, but cannot identify cell-to-cell variations within the tissue microenvironment. In contrast, in situ analysis can identify genetic aberrations in phenotypically defined cell subpopulations while preserving tissue-context specificity. GoIFISHGoIFISH is a widely applicable, user-friendly system tailored for the objective and semi-automated visualization, detection and quantification of genomic alterations and protein expression obtained from fluorescence in situ analysis. In a sample set of HER2-positive breast cancers GoIFISHGoIFISH is highly robust in visual analysis and its accuracy compares favorably to other leading image analysis methods. GoIFISHGoIFISH is freely available at www.sourceforge.net/projects/goifish/.

JARID1B is a luminal lineage-driving oncogene in breast cancer.

Recurrent mutations in histone-modifying enzymes imply key roles in tumorigenesis, yet their functional relevance is largely unknown. Here, we show that JARID1B, encoding a histone H3 lysine 4 (H3K4) demethylase, is frequently amplified and overexpressed in luminal breast tumors and a somatic mutation in a basal-like breast cancer results in the gain of unique chromatin binding and luminal expression and splicing patterns. Downregulation of JARID1B in luminal cells induces basal genes expression and growth arrest, which is rescued by TGFß pathway inhibitors. Integrated JARID1B chromatin binding, H3K4 methylation, and expression profiles suggest a key function for JARID1B in luminal cell-specific expression programs. High luminal JARID1B activity is associated with poor outcome in patients with hormone receptor-positive breast tumors.

Genetic and phenotypic diversity in breast tumor metastases.

Metastatic disease is the main cause of cancer-related mortality due to almost universal therapeutic resistance. Despite its high clinical relevance, our knowledge of how cancer cell populations change during metastatic progression is limited. Here, we investigated intratumor genetic and phenotypic heterogeneity during metastatic progression of breast cancer. We analyzed cellular genotypes and phenotypes at the single cell level by performing immunoFISH in intact tissue sections of distant metastatic tumors from rapid autopsy cases and from primary tumors and matched lymph node metastases collected before systemic therapy. We calculated the Shannon index of intratumor diversity in all cancer cells and within phenotypically distinct cell populations. We found that the extent of intratumor genetic diversity was similar regardless of the chromosomal region analyzed, implying that it may reflect an inherent property of the tumors. We observed that genetic diversity was highest in distant metastases and was generally concordant across lesions within the same patient, whereas treatment-naïve primary tumors and matched lymph node metastases were frequently genetically more divergent. In contrast, cellular phenotypes were more discordant between distant metastases than primary tumors and matched lymph node metastases. Diversity for 8q24 was consistently higher in HER2(+) tumors compared with other subtypes and in metastases of triple-negative tumors relative to primary sites. We conclude that our integrative method that couples ecologic models with experimental data in human tissue samples could be used for the improved prognostication of patients with cancer and for the design of more effective therapies for progressive disease.

Inference of tumor evolution during chemotherapy by computational modeling and in situ analysis of genetic and phenotypic cellular diversity.

Cancer therapy exerts a strong selection pressure that shapes tumor evolution, yet our knowledge of how tumors change during treatment is limited. Here, we report the analysis of cellular heterogeneity for genetic and phenotypic features and their spatial distribution in breast tumors pre- and post-neoadjuvant chemotherapy. We found that intratumor genetic diversity was tumor-subtype specific, and it did not change during treatment in tumors with partial or no response. However, lower pretreatment genetic diversity was significantly associated with pathologic complete response. In contrast, phenotypic diversity was different between pre- and posttreatment samples. We also observed significant changes in the spatial distribution of cells with distinct genetic and phenotypic features. We used these experimental data to develop a stochastic computational model to infer tumor growth patterns and evolutionary dynamics. Our results highlight the importance of integrated analysis of genotypes and phenotypes of single cells in intact tissues to predict tumor evolution.

Substance P autocrine signaling contributes to persistent HER2 activation that drives malignant progression and drug resistance in breast cancer.

ERBB receptor transmodulation by heterologous G-protein-coupled receptors (GPCR) generates functional diversity in signal transduction. Tachykinins are neuropeptides and proinflammatory cytokines that promote cell survival and cancer progression by activating several GPCRs. In this work, we found that the pain-associated tachykinin Substance P (SP) contributes to persistent transmodulation of the ERBB receptors, EGFR and HER2, in breast cancer, acting to enhance malignancy and therapeutic resistance. SP and its high-affinity receptor NK-1R were highly expressed in HER2(+) primary breast tumors (relative to the luminal and triple-negative subtypes) and were overall correlated with poor prognosis factors. In breast cancer cell lines and primary cultures derived from breast cancer samples, we found that SP could activate HER2. Conversely, RNA interference-mediated attenuation of NK-1R, or its chemical inhibition, or suppression of overall GPCR-mediated signaling, all strongly decreased steady-state expression of EGFR and HER2, establishing that their basal activity relied upon transdirectional activation by GPCR. Thus, SP exposure affected cellular responses to anti-ERBB therapies. Our work reveals an important oncogenic cooperation between NK-1R and HER2, thereby adding a novel link between inflammation and cancer progression that may be targetable by SP antagonists that have been clinically explored.

Molecular profiling of human mammary gland links breast cancer risk to a p27(+) cell population with progenitor characteristics.

Early full-term pregnancy is one of the most effective natural protections against breast cancer. To investigate this effect, we have characterized the global gene expression and epigenetic profiles of multiple cell types from normal breast tissue of nulliparous and parous women and carriers of BRCA1 or BRCA2 mutations. We found significant differences in CD44(+) progenitor cells, where the levels of many stem cell-related genes and pathways, including the cell-cycle regulator p27, are lower in parous women without BRCA1/BRCA2 mutations. We also noted a significant reduction in the frequency of CD44(+)p27(+) cells in parous women and showed, using explant cultures, that parity-related signaling pathways play a role in regulating the number of p27(+) cells and their proliferation. Our results suggest that pathways controlling p27(+) mammary epithelial cells and the numbers of these cells relate to breast cancer risk and can be explored for cancer risk assessment and prevention.

Cellular heterogeneity and molecular evolution in cancer.

Intratumor heterogeneity represents a major obstacle to effective cancer treatment and personalized medicine. However, investigators are now elucidating intratumor heterogeneity at the single-cell level due to improvements in technologies. Better understanding of the composition of tumors, and monitoring changes in cell populations during disease progression and treatment, will improve cancer diagnosis and therapeutic design. Measurements of intratumor heterogeneity may also be used as biomarkers to predict the risk of progression and therapeutic resistance. We summarize important considerations related to intratumor heterogeneity during tumor evolution. We also discuss experimental approaches that are commonly used to infer intratumor heterogeneity and describe how these methodologies can be translated into clinical practice.

Amplified loci on chromosomes 8 and 17 predict early relapse in ER-positive breast cancers.

Adjuvant hormonal therapy is administered to all early stage ER+ breast cancers, and has led to significantly improved survival. Unfortunately, a subset of ER+ breast cancers suffer early relapse despite hormonal therapy. To identify molecular markers associated with early relapse in ER+ breast cancer, an outlier analysis method was applied to a published gene expression dataset of 268 ER+ early-stage breast cancers treated with tamoxifen alone. Increased expression of sets of genes that clustered in chromosomal locations consistent with the presence of amplicons at 8q24.3, 8p11.2, 17q12 (HER2 locus) and 17q21.33-q25.1 were each found to be independent markers for early disease recurrence. Distant metastasis free survival (DMFS) after 10 years for cases with any amplicon (DMFS = 56.1%, 95% CI = 48.3-63.9%) was significantly lower (P = 0.0016) than cases without any of the amplicons (DMFS = 87%, 95% CI = 76.3% -97.7%). The association between presence of chromosomal amplifications in these regions and poor outcome in ER+ breast cancers was independent of histologic grade and was confirmed in independent clinical datasets. A separate validation using a FISH-based assay to detect the amplicons at 8q24.3, 8p11.2, and 17q21.33-q25.1 in a set of 36 early stage ER+/HER2- breast cancers treated with tamoxifen suggests that the presence of these amplicons are indeed predictive of early recurrence. We conclude that these amplicons may serve as prognostic markers of early relapse in ER+ breast cancer, and may identify novel therapeutic targets for poor prognosis ER+ breast cancers.

Evolutionary pathways in BRCA1-associated breast tumors.

BRCA1-associated breast tumors display loss of BRCA1 and frequent somatic mutations of PTEN and TP53. Here we describe the analysis of BRCA1, PTEN, and p53 at the single cell level in 55 BRCA1-associated breast tumors and computational methods to predict the relative temporal order of somatic events, on the basis of the frequency of cells with single or combined alterations. Although there is no obligatory order of events, we found that loss of PTEN is the most common first event and is associated with basal-like subtype, whereas in the majority of luminal tumors, mutation of TP53 occurs first and mutant PIK3CA is rarely detected. We also observed intratumor heterogeneity for the loss of wild-type BRCA1 and increased cell proliferation and centrosome amplification in the normal breast epithelium of BRCA1 mutation carriers. Our results have important implications for the design of chemopreventive and therapeutic interventions in this high-risk patient population.