Estrogen-induced gene expression and patient survival in high-grade serous carcinoma of the ovary

Background. Assessment of estrogen receptor (ER) expression has inconsistent utility as a prognostic marker in epithelial ovarian carcinoma. In breast and endometrial cancers, the use of estrogen-induced gene panels, rather than ER expression alone, has shown improved prognostic capability. Specifically, over-expression of estrogen-induced genes in these tumors is associated with a better prognosis and signifies estrogen sensitivity that can be exploited with hormone antagonizing agents. It was therefore hypothesized that estrogen-induced gene expression in ovarian carcinoma would successfully predict outcomes and differentiate between tumors of varying estrogen sensitivities. Methods. Two hundred nineteen (219) patients with ovarian cancer who underwent surgery at M. D. Anderson between 2004 and 2007 were identified. Of these, eighty-three (83) patients were selected for inclusion because they had advanced stage, high-grade serous carcinoma of the ovary or peritoneum, had not received neoadjuvant chemotherapy, and had readily available frozen tissue for study. All patients had also received adjuvant treatment with platinum and taxane agents. The expression of seven genes known to be induced by estrogen in the female reproductive tract (EIG121, sFRP1, sFRP4, RALDH2, PR, IGF-1, and ER) was measured using qRT-PCR. Unsupervised cluster analyses of multiple gene permutations were used to categorize patients as high or low estrogen-induced gene expressors. QPCR gene expression results were then compared to ER and PR immunohistochemical (IHC) expression. Cox proportional hazards models were used to evaluate the effects of both individual genes and selected gene clusters on patient survival. Results. Median follow-up time was 38.7 months (range 1-68 months). In a multivariate model, overall survival was predicted by sFRP1 expression (HR 1.10 [1.02-1.19], p=0.01) and EIG121 expression (HR 1.28 [1.10-1.49], p<0.01). A cluster defined by EIG121 and ER was further examined because that combination appeared to reasonably segregate tumors into distinct groups of high and low estrogen-induced gene expressors. Shorter overall survival was associated with high estrogen-induced gene expressors (HR 2.84 [1.11-7.30], p=0.03), even after adjustment for race, age, body mass index, and residual disease at debulking. No difference in IHC ER or PR expression was noted between gene clusters. Conclusion. In sharp contrast to breast and endometrial cancers, high estrogen-induced gene expression predicts shorter overall survival in patients with high-grade serous ovarian carcinoma. An estrogen-induced gene biomarker panel may have utility as prognostic indicator and may be useful to guide management with estrogen antagonists in this population.^

Predictors of Contralateral Breast Cancer in BRCA Negative Women

Breast cancer is the most common cancer diagnosis and second leading cause of death in women. Risk factors associated with breast cancer include: increased age, alcohol consumption, cigarette smoking, white race, physical inactivity, benign breast conditions, reproductive and hormonal factors, dietary factors, and family history. Hereditary breast and ovarian cancer syndrome (HBOC) is caused by mutations in the BRCA1 and BRCA2 genes. Women carrying a mutation in these genes are at an increased risk to develop a second breast cancer. Contralateral breast cancer is the most common second primary cancer in patients treated for a first breast cancer. Other risk factors for developing contralateral breast cancer include a strong family history of breast cancer, age of onset of first primary breast cancer, and if the first primary was a lobular carcinoma, which has an increased risk of being bilateral. A retrospective chart review was performed on a select cohort of women in an IRB approved database at MD Anderson Cancer Center. The final cohort contained 572 women who tested negative for a BRCA1 or BRCA2 mutation, had their primary invasive breast cancer diagnosed under the age of 50, and had a BRCAPro risk assessment number over 10%. Of the 572 women, 97 women developed contralateral breast cancer. A number of predictors of contralateral breast cancer were looked at between the two groups. Using univariable Cox Proportional Hazard model, thirteen statistically interesting risk factors were found, defined as having a p-value under 0.2. Multivariable stepwise Cox Proportional Hazard model found four statistically significant variables out of the thirteen found in the univariable analysis. In our study population, the incidence of contralateral breast cancer was 17%. Four statistically significant variables were identified. Undergoing a prophylactic mastectomy was found to reduce the risk of developing contralateral breast cancer, while not having a prophylactic mastecomy, a young age at primary diagnosis, having a positive estrogen receptor status of the primary tumor, and having a family history of breast cancer increased a woman’s risk to develop contralateral breast cancer.

Platelets and Anti-Angiogenic Resistance in Ovarian Carcinoma

Background: Resistance to targeted anti-angiogenic therapy is a growing clinical concern given the disappointing clinical impact of anti-angiogenic. Platelets represent a component of the tumor microenvironment that are implicated in metastasis and represent a significant reservoir of angiogenic regulators. Thrombocytosis has been shown to be caused by malignancy and associated with adverse clinical outcomes, however the causal connections between these associations remain to be identified. Materials and Methods: Following IRB approval, patient data were collected on patients from four U.S. centers and platelet levels through and after therapy were considered as indicators of recurrence of disease. In vitro effects of platelets on cancer cell proliferation, apoptosis, and migration were examined. RNA interference was used to query signaling pathways mediating these effects. The necessity of platelet activation for in vitro effect was analyzed. In vivo orthotopic models were used to query the impact of thrombocytosis and thrombocytopenia on the efficacy of cytotoxic chemotherapy, the effect of aspirin on thrombocytosis and cancer, and platelet effect on anti-angiogenic therapy. Results: Platelets were found to increase at the time of diagnosis of ovarian cancer recurrence in a pattern comparable to CA-125. Platelet co-culture increased proliferation, increased migration, and decreased apoptosis in all cell lines tested. RNA interference implicated platelet derived growth factor alpha (PDGFRA) and transforming growth factor beta-receptor 1 (TGFBR1) signaling. Biodistribution studies suggested minimal platelet sequestration of taxanes. Blockade of platelet activation blocked in vitro effects. In vivo, thrombocytosis blocked chemotherapeutic efficacy, thrombocytopenia increased chemotherapeutic efficacy, and aspirin therapy partially blocked the effects of thrombocytosis. In vivo, withdrawal of anti-angiogenic therapy caused loss of therapeutic benefit with evidence of accelerated disease growth. This effect was blocked by use of a small-molecule inhibitor of Focal Adhesion Kinase. Anti-angiogenic therapy was also associated with increased platelet infiltration into tumor that was not seen to the same degree in the control or FAK-inhibitor-treated mice. Conclusions: Platelets are active participants in the growth and metastasis of tumor, both directly and via facilitation of angiogenesis. Blocking platelets, blocking platelet activation, and blocking platelet trafficking into tumor are novel therapeutic avenues supported by this data. Copyright © 2012 Justin Neal Bottsford-Miller, all rights reserved.

EVIDENCE OF HUMAN ENDOGENOUS RETROVIRUS K INVOLVEMENT IN HUMAN CANCER

Many lines of clinical and experimental evidence indicate a viral role in carcinogenesis (1-6). Our access to patient plasma, serum, and tissue samples from invasive breast cancer (N=19), ductal carcinoma in situ (N=13), malignant ovarian cancer (N=12), and benign ovarian tumors (N=9), via IRB-approved and informed consent protocols through M.D. Anderson Cancer Center, as well as normal donor plasmas purchased from Gulf Coast Regional Blood Center (N=6), has allowed us to survey primary patient blood and tissue samples, healthy donor blood from the general population, as well as commercially available human cell lines for the presence of human endogenous retrovirus K (HERV-K) Env viral RNA (vRNA), protein, and viral particles. We hypothesize that HERV-K proteins are tumor-associated antigens and as such can be profiled and targeted in patients for diagnostic and therapeutic purposes. To test this hypothesis, we employed isopycnic ultracentrifugation, a microplate-based reverse transcriptase enzyme activity assay, reverse transcription – polymerase chain reaction (RT-PCR), cDNA sequencing, SDS-PAGE and western blotting, immunofluorescent staining, confocal microscopy, and transmission electron microscopy to evaluate v HERV-K activation in cancer. Data from large numbers of patients tested by reverse transcriptase activity assay were analyzed statistically by t-test to determine the potential use of this assay as a diagnostic tool for cancer. Significant reverse transcriptase enzyme activity was detected in 75% of ovarian cancer patients, 53.8% of ductal carcinoma in situ patient, and 42.1% of invasive breast cancer patient samples. Only 11.1% of benign ovarian patient and 16.7% of normal donor samples tested positive. HERV-K Env vRNA, or Env SU were detected in the majority of cancer types screened, as demonstrated by the results shown herein, and were largely absent in normal controls. These findings support our hypothesis that the presence of HERV-K in patient blood circulation is an indicator of cancer or pre-malignancy in vivo, that the presence of HERV-K Env on tumor cell surfaces is indicative of malignant phenotype, and that HERV-K Env is a tumor-associated antigen useful not only as a diagnostic screening tool to predict patient disease status, but also as an exploitable therapeutic target for various novel antibody-based immunotherapies.

Lifetime prevalence and risk factors for cancer in a Mexican American population with severe health disparities: The Cameron County Hispanic Cohort

Retrospective data from the Cameron Country Hispanic Cohort (1) were analyzed to assess the burden of cancer in the Mexican American population living in Brownsville TX. Data provided by the study participants for themselves and their parents and other extended relatives on cancer and related risk factors were used to determine both the prevalence of cancer and these risk factors as well as any associations between them. Lifetime incidence of cancer among the study participants was of 2.8%. Lifetime incidence of cancer among the parents of the study population was calculated for cancer in general and for specific cancer sites to determine the ranking of occurrence of each type of cancer. Some cancer types in this population were ranked higher than what would be expected when compared with national data from Hispanics in the U.S, these were: Liver cancer (3rd vs. 7th nationally in males and 6th vs. 13th nationally in females), stomach cancer (4th vs. 8th nationally in males and 5th vs. 11th nationally in females) and ovarian cancer (3rd vs. 8th nationally in females). A significant association with cancer was found for being born in the United States compared to being born elsewhere (O.R. 1.62, 95% C.I. 1.01–2.60) among study participants and the same association was also found between birth of parents in the United States regardless of gender for cancers in general (O.R. 1.38 95% C.I. 1.12–1.70), stomach cancer (O.R. 1.92 95% C.I. 1.01–3.67) and colorectal cancer (O.R. 2.93 95% C.I. 1.28–6.72). Having been born in the United States and having a family history of cancer was also found to be significantly associated with other risk factors for cancer such as obesity, diabetes and insulin resistance, both among the parents and the participant population, suggesting these interactions are complex. These high rates of cancer and particular prominence of less usual cancer such as liver and ovary in health disparities warrant evaluation of early detection strategies.^

Biological effects of PEA3 expression in {\it HER-2\/}/{\it neu\/}-overexpressing human cancer cells and the molecular mechanisms of PEA3-mediated transcriptional repression on {\it HER-2\/}/{\it neu\/}

HER-2/neu is a receptor tyrosine kinase highly homologous with epidermal growth factor receptor. Overexpression and/or amplification of HER-2/neu has been implicated in the genesis of a number of human cancers, especially breast and ovarian cancers. Transcriptional upregulation has been shown to contribute significantly to the overexpression of this gene. Studies on the transcriptional regulation of HER-2/neu gene are important for understanding the mechanism of cell transformation and developing the therapeutic strategies to block HER-2/neu-mediated cancers. PEA3 is a DNA binding transcriptional factor and its consensus sequence exists on the HER-2/neu promoter. To examine the role of PEA3 in HER-2/neu expression and cell transformation, we transfected PEA3 into the human breast and ovarian cancer cells that overexpress HER-2/neu and showed that PEA3 dramatically represses HER-2/neu transcription. PEA3 suppresses the oncogenic neu-mediated transformation in mouse fibroblast NIH 3T3 cells. Expression of PEA3 selectively blocks the growth of human cancer cells that overexpress HER-2/neu and inhibits their colony formation. It does not occur in the cancer cells expressing basal level of HER-2/neu. Further studies in the orthotopic ovarian cancer model demonstrated that expression of PEA3 preferentially inhibits growth and tumor development of human cancer cells that overexpress HER-2/neu, the tumor-bearing mice survived significantly longer if treated by injection of the PEA3-liposome complex intraperitoneally. Immunoblotting and immunohistochemical analysis of the tumor tissues indicated that PEA3 mediates the tumor suppression activity through targeting HER-2/neu-p185. Thus, PEA3 is a negative regulator of HER-2/neu gene expression and functions as a tumor suppressor gene in the HER-2/neu-overexpressing human cancer cells.^ The molecular mechanisms of PEA3 mediated transcriptional repression were investigated. PEA3 binds specifically at the PEA3 site on HER-2/neu promoter and this promoter-binding is required for the PEA3 mediated transcriptional repression. Mutation of the PEA3 binding site on HER-2/neu promoter causes decreased transcriptional activity, indicating that the PEA3 binding site is an enhancer-like element in the HER-2/neu-overexpressing cells. We therefore hypothesized that in the HER-2/neu-overexpressing cells, PEA3 competes with a transactivator for binding to the PEA3 site, preventing the putative factor from activating the transcription of HER-2/neu. This hypothesis was supported by the data which demonstrate that PEA3 competes with another nuclear protein for binding to the HER-2/neu promoter in vitro, and expression of a truncated protein which encodes the DNA binding domain of PEA3 is sufficient to repress HER-2/neu transcription in the HER-2/neu-overexpressing human cancer cells. ^

Intrinsic oxidative stress in cancer cells: A biochemical basis for therapeutic selectivity using ROS -generating agents

A major goal of chemotherapy is to selectively kill cancer cells while minimizing toxicity to normal cells. Identifying biological differences between cancer and normal cells is essential in designing new strategies to improve therapeutic selectivity. Superoxide dismutases (SOD) are crucial antioxidant enzymes required for the elimination of superoxide (O2·− ), a free radical produced during normal cellular metabolism. Previous studies in our laboratory demonstrated that 2-methoxyestradiol (2-ME), an estradiol derivative, inhibits the function of SOD and selectively kills human leukemia cells without exhibiting significant cytotoxicity in normal lymphocytes. The present work was initiated to examine the biochemical basis for the selective anticancer activity of 2-ME. Investigations using two-parameter flow cytometric analyses and ROS scavengers established that O2·− is a primary and essential mediator of 2-ME-induced apoptosis in cancer cells. In addition, experiments using SOD overexpression vectors and SOD knockout cells found that SOD is a critical target of 2-ME. Importantly, the administration of 2-ME resulted in the selective accumulation of O 2·− and apoptosis in leukemia and ovarian cancer cells. The preferential activity of 2-ME was found to be due to increased intrinsic oxidative stress in these cancer cells versus their normal counterparts. This intrinsic oxidative stress was associated with the upregulation of the antioxidant enzymes SOD and catalase as a mechanism to cope with the increase in ROS. Furthermore, oxygen consumption experiments revealed that normal lymphocytes decrease their respiration rate in response to 2-ME-induced oxidative stress, while human leukemia cells seem to lack this regulatory mechanism. This leads to an uncontrolled production of O2·−, severe accumulation of ROS, and ultimately ROS-mediated apoptosis in leukemia cells treated with 2-ME. The biochemical differences between cancer and normal cells identified here provide a basis for the development of drug combination strategies using 2-ME with other ROS-generating agents to enhance anticancer activity. The effectiveness of such a combination strategy in killing cancer cells was demonstrated by the use of 2-ME with agents/modalities such as ionizing radiation and doxorubicin. Collectively, the data presented here strongly suggests that 2-ME may have important clinical implications for the selective killing of cancer cells. ^

A single nucleotide polymorphism in the RAD51 gene modifies cancer risk in BRCA2 but not BRCA1 carriers

BRCA1 and BRCA2 carriers are at increased risk for both breast and ovarian cancer, but estimates of lifetime risk vary widely, suggesting their penetrance is modified by other genetic and/or environmental factors. The BRCA1 and BRCA2 proteins function in DNA repair in conjunction with RAD51. A preliminary report suggested that a single nucleotide polymorphism in the 5′ untranslated region of RAD51 (135C/G) increases breast cancer risk in BRCA1 and BRCA2 carriers. To investigate this effect we studied 257 female Ashkenazi Jewish carriers of one of the common BRCA1 (185delAG, 5382insC) or BRCA2 (6174delT) mutations. Of this group, 164 were affected with breast and/or ovarian cancer and 93 were unaffected. RAD51 genotyping was performed on all subjects. Among BRCA1 carriers, RAD51-135C frequency was similar in healthy and affected women [6.1% (3 of 49) and 9.9% (12 of 121), respectively], and RAD-135C did not influence age of cancer diagnosis [Hazard ratio (HR) = 1.18 for disease in RAD51-135C heterozygotes, not significant]. However, in BRCA2 carriers, RAD51-135C heterozygote frequency in affected women was 17.4% (8 of 46) compared with 4.9% (2 of 41) in unaffected women (P = 0.07). Survival analysis in BRCA2 carriers showed RAD51-135C increased risk of breast and/or ovarian cancer with an HR of 4.0 [95% confidence interval 1.6–9.8, P = 0.003]. This effect was largely due to increased breast cancer risk with an HR of 3.46 (95% confidence interval 1.3–9.2, P = 0.01) for breast cancer in BRCA2 carriers who were RAD51-135C heterozygotes. RAD51 status did not affect ovarian cancer risk. These results show RAD51-135C is a clinically significant modifier of BRCA2 penetrance, specifically in raising breast cancer risk at younger ages.

Cancer-predisposing mutations within the RING domain of BRCA1: Loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity

BRCA1 is a breast and ovarian cancer-specific tumor suppressor that seems to be involved in transcription and DNA repair. Here we report that BRCA1 exhibits a bona fide ubiquitin (Ub) protein ligase (E3) activity, and that cancer-predisposing mutations within the BRCA1 RING domain abolish its Ub ligase activity. Furthermore, these mutants are unable to reverse γ-radiation hypersensitivity of BRCA1-null human breast cancer cells, HCC1937. Additionally, these mutations within the BRCA1 RING domain are not capable of restoring a G2 + M checkpoint in HCC1937 cells. These results establish a link between Ub protein ligase activity and γ-radiation protection function of BRCA1, and provide an explanation for why mutations within the BRCA1 RING domain predispose to cancer. Furthermore, we propose that the analysis of the Ub ligase activity of RING-domain mutations identified in patients may constitute an assay to predict predisposition to cancer.

Lost in Translation? Russian Media Portrayals and Laypersons’ Interpretations of Angelina Jolie’s Breast Cancer Discourse

In May 2013, Angelina Jolie revealed that because she had a family history of breast and ovarian cancer and carried a rare BRCA gene mutation, she had undergone a preventive double mastectomy. Media coverage has been extensive around the world, including in Russia, not an English-language country, where all global news is inevitably filtered by translation. After examining the reactions of Russian mass media and members of the public to Jolie’s disclosure, I consider what transformations have occurred with Jolie’s message in the process of cross-cultural transfer. I explore the mass media portrayal of Jolie’s announcement, laypersons’ immediate and prolonged reactions, and the reflections of patients involved directly in the field of hereditary breast cancer. To my knowledge, this multifaceted and bilingual project is the first conceptualization of Jolie’s story as it has been translated in a different sociocultural environment. I start with examination of offline and online publications that appeared in Russia within two months after Jolie’s announcement. In this part of my analysis, I conceptualize the representation of Jolie’s case in Russian mass media and grasp what sociocultural waves were generated by this case among general lay audiences. Another part of my study contains the results of qualitative in-depth interviews. Eight women with a family history of hereditary breast cancer were recruited to participate in the research. The findings represent Jolie’s case through the eyes of Russian women with the same gene mutation as Jolie. Consolidating my findings, I argue that Jolie’s announcement was misinterpreted and misrepresented by Russian mass media, as well as misunderstood by a considerable part of the media audience. Jolie’s perspective on hereditary breast cancer mostly remained unheard among members of the Russian public. I make suggestions about the reasons for such a phenomenon, and demonstrate how Jolie’s case is implicated in politics, economics, and the culture of contemporary Russia.

Dissection moléculaire de la sénescence cellulaire induite par le stress et la thérapie dans le cancer de l’ovaire et son impact sur la réponse des patientes

Le cancer de l’ovaire (COv) est le cancer gynécologique le plus létal chez la femme et les traitements existants, chirurgie et chimiothérapie, ont peu évolué au cours des dernières décennies. Nous proposons que la compréhension des différents destins cellulaires tels que la sénescence que peuvent choisir les cellules du cancer de l’ovaire en réponse à la chimiothérapie pourrait conduire à de nouvelles opportunités thérapeutiques. La sénescence cellulaire a été largement associée à l’activité de la protéine TP53, qui est mutée dans plus de 90% des cas de cancer de l’ovaire séreux de haut grade (COv-SHG), la forme la plus commune de la maladie. Dans nos travaux, à partir d’échantillons dérivés de patientes, nous montrons que les cultures primaires du cancer de l’ovaire séreux de haut grade exposées au stress ou à des drogues utilisées en chimiothérapie entrent en senescence grâce à l’activité d’un isoforme du gène CDKN2A (p16INK4A). Dans ces cellules, nous avons évalué les caractéristiques fondamentales de la sénescence cellulaire tels que les altérations morphologiques, l’activité béta galactosidase associée à la sénescence, les dommages à l’ADN, l’arrêt du cycle cellulaire et le phénotype sécrétoire associé à la sénescence. En utilisant des micromatrices tissulaires construites à partir d’échantillons humains de COv-SHG pré- et post-chimiothérapie, accompagnées de leurs données cliniques, nous avons quantifié des marqueurs de sénescence incluant une diminution de la prolifération cellulaire quelques semaines après chimiothérapie. De façon intéressante, l’expression de p16INK4A dans les échantillons de COv-SHG prétraitement corrèle avec une survie prolongée des patientes suite au traitement. Ceci suggère ainsi pour la première fois un impact biologique bénéfique pour la présence de cellules cancéreuses qui sont capable d’activer la sénescence, particulièrement pour le traitement du cancer de l’ovaire. Dans le but de complémenter les thérapies actuelles avec des approches de manipulation pharmacologique de la sénescence, nos résultats suggèrent qu’il serait important de déterminer l’impact positif ou négatif de la sénescence induite par la thérapie sur la progression de la maladie et la survie, pour chaque type de cancer de façon indépendante.

Polymerizable Peptide Monomers for the Targeted and Intracellular Delivery of Cancer Therapeutics

Thesis (Ph.D.)--University of Washington, 2016-06

Brca1 inactivation induces p27Kip1-dependent cell cycle arrest and delayed development in the mouse mammary gland

One common characteristic of breast cancers arising in carriers of the predisposition gene BRCA1 is a loss of expression of the CDK inhibitor p27(Kip1) (p27), suggesting that p27 interacts epistatically with BRCA1. To investigate this relationship, we examined expression of p27 in mice expressing a dominant negative allele of Brca1 (MMTV-trBr) in the mammary gland. While these mice rarely develop tumors, they showed a 50% increase in p27 protein and a delay in mammary gland development associated with reduced proliferation. In contrast, on a p27 heterozygote background, MMTV-trBrca1 mice showed an increase in S phase cells, and normal mammary development. p27 was the only protein in the cyclin cyclin-dependent kinase network to show altered expression, suggesting that it may be a central mediator of cell cycle arrest in response to loss of function of BRCA1. Furthermore, in human mammary epithelial MCF7 cells expressing BRCA1-specific RNAi and in the BRCA1-deficient human tumor cell line HCC1937, p27 is elevated at the mRNA level compared to cells expressing wild-type BRCA1. We hypothesize that disruption of BRCA1 induces an increase in p27 that inhibits proliferation. Accordingly, reduction in p27 expression leads to enhancement of cellular proliferation in the absence of BRCA1.

Reduced expression of chemokine (C-C motif) ligand-2 (CCL2) in ovarian adenocarcinoma

Chemokine (C-C motif) ligand-2 (CCL2) is a chemoattractant and activator of macrophages and is a key determinant of the macrophage infiltrate into tumours. We demonstrate here that CCL2 is expressed in normal human ovarian surface epithelium ( HOSE) cells and is silenced in most ovarian cancer cell lines, and silenced or downregulated in the majority of primary ovarian adenocarcinomas. Analysis of the CCL2 locus at 17q11.2-q12 showed loss of heterozygosity (LOH) in 70% of primary tumours, and this was significantly more common in tumours of advanced stage or grade. However, we did not detect any mutations in the CCL2 coding sequence in 94 primary ovarian adenocarcinomas. These data support the hypothesis that CCL2 may play a role in the pathobiology of ovarian cancers, but additional studies will be required to evaluate this possibility.

Identification of two evolutionarily conserved and functional regulatory elements in intron 2 of the human BRCA1 gene

Cross-species comparative genomics is a powerful strategy for identifying functional regulatory elements within noncoding DNA. In this paper, comparative analysis of human and mouse intronic sequences in the breast cancer susceptibility gene (BRCA1) revealed two evolutionarily conserved noncoding sequences (CNS) in intron 2, 5 kb downstream of the core BRCA1 promoter. The functionality of these elements was examined using homologous-recombination-based mutagenesis of reporter gene-tagged cosmids incorporating these regions and flanking sequences from the BRCA1 locus. This showed that CNS-1 and CNS-2 have differential transcriptional regulatory activity in epithelial cell lines. Mutation of CNS-1 significantly reduced reporter gene expression to 30% of control levels. Conversely mutation of CNS-2 increased expression to 200% of control levels. Regulation is at the level of transcription and shows promoter specificity. Both elements also specifically bind nuclear proteins in vitro. These studies demonstrate that the combination of comparative genomics and functional analysis is a successful strategy to identify novel regulatory elements and provide the first direct evidence that conserved noncoding sequences in BRCA1 regulate gene expression. (c) 2005 Elsevier Inc. All rights reserved.