Searching for DNA methylation in patients triple-negative breast cancer: a liquid biopsy approach.

INTRODUCTION: Liquid biopsies are proving to have diagnostic and prognostic value in many different cancers, and in breast cancer they have the potential to improve outcomes by providing valuable information throughout a patient's cancer journey. However, patients with triple negative breast cancer (TNBC) have received little benefit from such liquid biopsies due to underlying limitations in the discovery and utility of robust biomarkers. Here, we examine the development of DNA methylation-based liquid biopsy assays for breast cancer and how they pertain to TNBC. AREAS COVERED: We conducted a systematic review of liquid biopsy assays for breast cancer and analyzed their relevance in TNBC. We show that the utility of DNA mutation-based assays is poor for TNBC due to the low mutational frequencies across the genome in this subtype. We offer a detailed review of mDETECT - a liquid biopsy specifically designed for assessing tumor burden in TNBC patients. EXPERT OPINION: DNA methylation are foundational and robust events that occur in cancer evolution and may differentiate almost all forms of cancer, including TNBC. Longitudinal patient monitoring using DNA methylation-based liquid biopsies offers great potential for improving the detection and management of TNBC.

A DNA methylation-based liquid biopsy for triple-negative breast cancer.

Here, we present a next-generation sequencing (NGS) methylation-based blood test called methylation DETEction of Circulating Tumour DNA (mDETECT) designed for the optimal detection and monitoring of metastatic triple-negative breast cancer (TNBC). Based on a highly multiplexed targeted sequencing approach, this assay incorporates features that offer superior performance and included 53 amplicons from 47 regions. Analysis of a previously characterised cohort of women with metastatic TNBC with limited quantities of plasma (<2 ml) produced an AUC of 0.92 for detection of a tumour with a sensitivity of 76% for a specificity of 100%. mDETECTTNBC was quantitative and showed superior performance to an NGS TP53 mutation-based test carried out on the same patients and to the conventional CA15-3 biomarker. mDETECT also functioned well in serum samples from metastatic TNBC patients where it produced an AUC of 0.97 for detection of a tumour with a sensitivity of 93% for a specificity of 100%. An assay for BRCA1 promoter methylation was also incorporated into the mDETECT assay and functioned well but its clinical significance is currently unclear. Clonal Hematopoiesis of Indeterminate Potential was investigated as a source of background in control subjects but was not seen to be significant, though a link to adiposity may be relevant. The mDETECTTNBC assay is a liquid biopsy able to quantitatively detect all TNBC cancers and has the potential to improve the management of patients with this disease.

Development and initial clinical correlation of a DNA methylation-based blood test for prostate cancer.

BACKGROUND: One of the principle limitations for more precise management of advanced prostate cancer is the lack of accurate biomarkers allowing estimation of tumor burden, ongoing assessment of progression, and response to treatment. Although prostate-specific antigen (PSA) performs modestly, nonsecreting cancers including those with early castrate-resistance warrant investigation of other predictive biomarkers. The objectives of these studies were to develop and perform initial validation of a circulating tumor DNA (ctDNA) methylation assay. METHODS: Methylation DETection of Circulating Tumor DNA (mDETECT) is a highly multiplexed targeted sequencing DNA methylation-based ctDNA blood test that captures the vast majority of prostate cancer phenotypes due to a careful development process that ensures that each probe region is methylated in at least 50% of all methylation-based subtypes and is not methylated in normal tissues. Next-generation sequencing of targeted polymerase chain reaction (PCR) products whose amplification is biased towards methylated DNA ensures the specificity of the assay by identifying multiple tumor-specific methylated CpG residues in each read. RESULTS: The final test is comprised of 46 PCR probes to 40 regions. It is relatively resistant to contaminating normal DNA and as a result functions in both serum and plasma samples. The assay was initially validated in a variety of prostate cancer cell lines to ensure specificity. Using a small number of longitudinal samples from prostate cancer patients initiating androgen deprivation therapy, the ability of mDETECT to track tumor burden was assessed compared with PSA. The mDETECT test signal generally paralleled that of PSA increasing and decreasing commensurate with tumor evolution in these patients. In two cases it appeared to anticipate clinical progression by a number of months compared to PSA and in a PSA nonproducing case, it was able to track tumor progression. CONCLUSIONS: mDETECT offers a promising tool for the assessment of prostate cancer burden based on the sensitive detection of prostate-specific ctDNA and requires further validation.

Region-specific glucocorticoid receptor promoter methylation has both positive and negative prognostic value in patients with estrogen receptor-positive breast cancer.

BACKGROUND: The glucocorticoid receptor (NR3C1, GR) is frequently downregulated in breast tumors, and evidence suggests it acts as a tumor suppressor in estrogen receptor-positive (ER+) breast cancer. We previously found that methylation of the GR promoter CpG island represses gene expression and occurs in ER+ breast tumors. In this study, the prognostic and predictive value of GR methylation was examined in ER+ patients from the CCTG MA.12 clinical trial of tamoxifen versus placebo in women with early breast cancer. METHODS: We developed a targeted multiplex bisulfite next-generation sequencing assay to detect methylation at multiple GR promoter regions in DNA from formalin-fixed paraffin-embedded (FFPE) samples. Following validation in a small cohort of breast tumors, ER+ FFPE tumor samples from MA.12 (n = 208) were tested. Survival analyses evaluated the impact of GR promoter methylation on patient overall survival (OS) and disease-free survival (DFS). RESULTS: An analysis of TCGA data found that GR methylation is prevalent in ER+ tumors and is associated with decreased gene expression and analysis of public microarray data (KM Plotter) linked decreased GR expression to a poor outcome. In MA.12, two GR promoter regions (U and C) each had prognostic value, but with opposite effects on the outcome. U methylation was associated with poor OS (HR = 1.79, P = 0.041) whereas C methylation was associated with better OS (HR = 0.40, P = 0.040) and DFS (HR = 0.49, P = 0.037). The classification of patients based on the methylation status of the two regions was prognostic for OS (P = 0.006) and DFS (P = 0.041) and revealed a group of patients (U methylated, C unmethylated) with very poor outcomes. Placebo-treated patients in this high-risk group had worse OS (HR = 2.86, P = 0.002) and DFS (HR = 2.09, P = 0.014) compared to the rest of the cohort. CONCLUSION: Region-specific GR promoter methylation was an independent prognostic marker for patient survival and identified a subset of patients with poor prognosis, particularly without tamoxifen treatment. These findings provide a foundation for future studies into GR methylation as a promising prognostic biomarker in ER+ breast cancer.

Ovarian Cancers Harboring Inactivating Mutations in CDK12 Display a Distinct Genomic Instability Pattern Characterized by Large Tandem Duplications.

CDK12 is a recurrently mutated gene in serous ovarian carcinoma, whose downregulation is associated with impaired expression of DNA damage repair genes and subsequent hypersensitivity to DNA-damaging agents and PARP1/2 inhibitors. In this study, we investigated the genomic landscape associated with CDK12 inactivation in patients with serous ovarian carcinoma. We show that CDK12 loss was consistently associated with a particular genomic instability pattern characterized by hundreds of tandem duplications of up to 10 megabases (Mb) in size. Tandem duplications were characterized by a bimodal (∼0.3 and ∼3 Mb) size distribution and overlapping microhomology at the breakpoints. This genomic instability, denoted as the CDK12 TD-plus phenotype, is remarkably distinct from other alteration patterns described in breast and ovarian cancers. The CDK12 TD-plus phenotype was associated with a greater than 10% gain in genomic content and occurred at a 3% to 4% rate in The Cancer Genome Atlas-derived and in-house cohorts of patients with serous ovarian carcinoma. Moreover, CDK12-inactivating mutations together with the TD-plus phenotype were also observed in prostate cancers. Our finding provides new insight toward deciphering the function of CDK12 in genome maintenance and oncogenesis. Cancer Res; 76(7); 1882-91. ©2016 AACR.

Genomic hallmarks of homologous recombination deficiency in invasive breast carcinomas.

Therapeutic strategies targeting Homologous Recombination Deficiency (HRD) in breast cancer requires patient stratification. The LST (Large-scale State Transitions) genomic signature previously validated for triple-negative breast carcinomas (TNBC) was evaluated as biomarker of HRD in luminal (hormone receptor positive) and HER2-overexpressing (HER2+) tumors. The LST genomic signature related to the number of large-scale chromosomal breakpoints in SNP-array tumor profile was applied to identify HRD in in-house and TCGA sets of breast tumors, in which the status of BRCA1/2 and other genes was also investigated. In the in-house dataset, HRD was predicted in 5% (20/385) of sporadic tumors luminal or HER2+ by the LST genomic signature and the inactivation of BRCA1, BRCA2 or RAD51C confirmed this prediction in 75% (12/16) of the tested cases. In 14% (6/43) of tumors occurring in BRCA1/2 mutant carriers, the corresponding wild-type allele was retained emphasizing the importance of determining the tumor status. In the TCGA luminal and HER2+ subtypes HRD incidence was estimated at 5% (18/329, 95%CI: 5-8%) and 2% (1/59, 95%CI: 2-9%), respectively. In TNBC cisplatin-based neo-adjuvant clinical trials, HRD is shown to be a necessary condition for cisplatin sensitivity. This analysis demonstrates the high performance of the LST genomic signature for HRD detection in breast cancers, which suggests its potential as a biomarker for genetic testing and patient stratification for clinical trials evaluating platinum salts and PARP inhibitors.

Expression microarray identifies the unliganded glucocorticoid receptor as a regulator of gene expression in mammary epithelial cells.

BACKGROUND: While glucocorticoids and the liganded glucocorticoid receptor (GR) have a well-established role in the maintenance of differentiation and suppression of apoptosis in breast tissue, the involvement of unliganded GR in cellular processes is less clear. Our previous studies implicated unliganded GR as a positive regulator of the BRCA1 tumour suppressor gene in the absence of glucocorticoid hormone, which suggested it could play a similar role in the regulation of other genes. METHODS: An shRNA vector directed against GR was used to create mouse mammary cell lines with depleted endogenous levels of this receptor in order to further characterize the role of GR in breast cells. An expression microarray screen for targets of unliganded GR was performed using our GR-depleted cell lines maintained in the absence of glucocorticoids. Candidate genes positively regulated by unliganded GR were identified, classified by Gene Ontology and Ingenuity Pathway Analysis, and validated using quantitative real-time reverse transcriptase PCR. Chromatin immunoprecipitation and dual luciferase expression assays were conducted to further investigate the mechanism through which unliganded GR regulates these genes. RESULTS: Expression microarray analysis revealed 260 targets negatively regulated and 343 targets positively regulated by unliganded GR. A number of the positively regulated targets were involved in pro-apoptotic networks, possibly opposing the activity of liganded GR targets. Validation and further analysis of five candidates from the microarray indicated that two of these, Hsd11b1 and Ch25h, were regulated by unliganded GR in a manner similar to Brca1 during glucocorticoid treatment. Furthermore, GR was shown to interact directly with and upregulate the Ch25h promoter in the absence, but not the presence, of hydrocortisone (HC), confirming our previously described model of gene regulation by unliganded GR. CONCLUSION: This work presents the first identification of targets of unliganded GR. We propose that the balance between targets of liganded and unliganded GR signaling is responsible for controlling differentiation and apoptosis, respectively, and suggest that gene regulation by unliganded GR may represent a mechanism for reducing the risk of breast tumourigenesis by the elimination of abnormal cells.

Frequent promoter hypermethylation and expression reduction of the glucocorticoid receptor gene in breast tumors.

Previous studies have found that expression of the Glucocorticoid Receptor (GR) is altered or reduced in various cancers, while the GR promoter has been shown to be methylated in gastric, lung, and colorectal cancers. Examining a small cohort of matched normal and breast cancer samples we found that GR levels were dramatically reduced in almost all tumors in relation to their normal tissue. The methylation status of the GR promoter was assessed to determine if this observed decrease of expression in breast tumors could be due to epigenetic regulation. While it was not methylated in normal tissue, the GR proximal promoter was methylated in 15% of tumor samples, particularly, but not exclusively, in Estrogen Receptor positive tumors. GR expression in these tumors was particularly low and loss of GR expression was specifically correlated with methylation of the proximal promoter GR B region. Overall, these results show that hypermethylation of the promoter in tumors is a frequent event and suggests that GR may act as a tumor suppressor in breast tissue.

Microenvironmental regulation of BRCA1 gene expression by c-Jun and Fra2 in premalignant human ovarian surface epithelial cells.

Reduced BRCA1 gene expression is common in the sporadic form of ovarian carcinoma. The spread of this highly lethal cancer often begins when tumor cell clusters are shed into the fluid of the abdominopelvic cavity such that they can float freely before seeding distant sites on the peritoneal walls and organs. Thus, the microenvironment that tumor cells find themselves in changes dramatically during these early shedding and floating stages of transperitoneal metastasis. To mimic this microenvironmental change in vitro, we released premalignant human ovarian surface epithelial cells from the substratum and forced them to cluster in suspension. Under these conditions, steady state levels of BRCA1 mRNA and protein fell significantly and the transcriptional activation state of the BRCA1 promoter was suppressed. Analysis of the promoter indicated that the previously identified "CRE" element located within the "positive regulatory region" (PRR) contributed to this suppression. More specifically, we show that the suppression was mediated, at least in part, by a suspension culture-driven decrease in the levels of two members of the AP1 transcription factor complex, c-Jun and Fra2, that bind to the CRE element. Therefore, a microenvironmental change that is manifested during the initial stages of ovarian carcinoma dissemination may, potentially, help suppress BRCA1 expression in sporadic tumors and thus promote their progression.

The unliganded glucocorticoid receptor positively regulates the tumor suppressor gene BRCA1 through GABP beta.

Loss of BRCA1 tumor suppressor function is a critical event in breast tumorigenesis. We have previously identified the stress hormone hydrocortisone as a negative regulator of BRCA1 expression in nonmalignant mammary cells. Here, we have identified a direct role for the unliganded glucocorticoid receptor (GR) in BRCA1 upregulation in the absence of hydrocortisone. The positive regulatory effect of GR is lost upon the addition of hydrocortisone. We have shown that GR interacts with the BRCA1 promoter only in the absence of hydrocortisone, and that this interaction is mediated through the ß-subunit of the ets transcription factor GA-binding protein (GABP) at the RIBS promoter element. GR and GABPß interact in both coimmunoprecipitation and mammalian two-hybrid assays, and this interaction involves the N-terminal to central regions of both proteins. This work presents the first evidence of a ligand-independent role for GR as a positive regulator of gene expression, and loss of GR from the BRCA1 promoter in response to stress hormones leads to decreased BRCA1 expression. Because low levels of BRCA1 have been implicated in the development of sporadic breast cancer, this may represent a novel mechanism through which prolonged stress signaling increases breast cancer risk.

Stimulation of the hypothalamic ventromedial nuclei by pituitary adenylate cyclase-activating polypeptide induces hypophagia and thermogenesis.

Numerous studies have demonstrated that the hypothalamic ventromedial nuclei (VMN) regulate energy homeostasis by integrating and utilizing behavioral and metabolic mechanisms. The VMN heavily express pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptors (PAC1R). Despite the receptor distribution, most PACAP experiments investigating affects on feeding have focused on intracerebroventricular administration or global knockout mice. To identify the specific contribution of PACAP signaling in the VMN, we injected PACAP directly into the VMN and measured feeding behavior and indices of energy expenditure. Following an acute injection of PACAP, nocturnal food intake was significantly reduced for 6 h after injections without evidence of malaise. In addition, PACAP-induced suppression of feeding also occurred following an overnight fast and could be blocked by a specific PAC1R antagonist. Metabolically, VMN-specific injections of PACAP significantly increased both core body temperature and spontaneous locomotor activity with a concurrent increase in brown adipose uncoupling protein 1 mRNA expression. To determine which signaling pathways were responsive to PACAP administration into the VMN, we measured mRNA expression of well-characterized hypothalamic neuropeptide regulators of feeding. One hour after PACAP administration, expression of pro-opiomelanocortin mRNA was significantly increased in the arcuate nuclei (ARC), with no changes in neuropeptide Y and agouti-related polypeptide mRNA levels. This suggests that PAC1R expressing VMN neurons projecting to pro-opiomelanocortin neurons contribute to hypophagia by involving melanocortin signaling. While the VMN also abundantly express PACAP protein, the present study demonstrates that PACAP input to the VMN can influence the control of energy homeostasis.

Serotonin mediated changes in corticotropin releasing factor mRNA expression and feeding behavior isolated to the hypothalamic paraventricular nuclei.

Fenfluramine reduces hunger and promotes body weight loss by increasing central serotonin (5-HT) signaling. More recently, neuropeptides have been linked to the regulation of feeding behavior, metabolism and body weight. To examine possible interactions between 5-HT and neuropeptides in appetite control, fenfluramine (200 nmol/0.5 µl/side) was administered directly into the hypothalamic paraventricular nuclei (PVN) of male rats. Bilateral fenfluramine produced significant hypophagia and increased expression of PVN corticotropin releasing factor (CRF) mRNA and neuropeptide Y (NPY) mRNA in the arcuate nucleus within the first hour after drug administration. Fenfluramine's effects on feeding behavior and mRNA expression were blocked by PVN injections of a 5-HT(1-2) receptor antagonist, metergoline (15 nmol/0.5 µl/side). These data suggest that 5-HT neurons targeting hypothalamic paraventricular CRF neurons may participate in an appetite control circuit for reducing food intake.

Decreased expression of BRCA1 in SK-BR-3 cells is the result of aberrant activation of the GABP Beta promoter by an NRF-1-containing complex.

BACKGROUND: BRCA1 has recently been identified as a potential regulator of mammary stem/progenitor cell differentiation, and this function may explain the high prevalence of breast cancer in BRCA1 mutation carriers, as well as the downregulation of BRCA1 in a large proportion of sporadic breast cancers. That is, loss of BRCA1 function results in blocked differentiation with expansion of the mammary stem/progenitor cells. Because BRCA1 also maintains genomic integrity, its loss could produce a pool of genetically unstable stem/progenitor cells that are prime targets for further transforming events. Thus, elucidating the regulatory mechanisms of BRCA1 expression is important to our understanding of normal and malignant breast differentiation. RESULTS: Loss of BRCA1 expression in the ErbB2-amplified SK-BR-3 cell line was found to be the result of loss of activity of the ets transcription factor GABP, a previously characterized regulator of BRCA1 transcription. The expression of the non-DNA binding GABPß subunit was shown to be deficient, while the DNA binding subunit, GABPα was rendered unstable by the absence of GABPß. Deletion analysis of the GABPß proximal promoter identified a potential NRF-1 binding site as being critical for expression. Supershift analysis, the binding of recombinant protein and chromatin immunoprecipitation confirmed the role of NRF-1 in regulating the expression of GABPß. The siRNA knockdown of NRF-1 resulted in decreased GABPß and BRCA1 expression in MCF-7 cells indicating that they form a transcriptional network. NRF-1 levels and activity did not differ between SK-BR-3 and MCF-7 cells, however the NRF-1 containing complex on the GABPß promoter differed between the two lines and appears to be the result of altered coactivator binding. CONCLUSIONS: Both NRF-1 and GABP have been linked to the regulation of nuclear-encoded mitochondrial proteins, and the results of this study suggest their expression is coordinated by NRF-1's activation of the GABPß promoter. Their linkage to BRCA1, a potential breast stem cell regulator, implies a connection between the induction of mitochondrial metabolism and breast differentiation.

Stress and breast cancer: from epidemiology to molecular biology.

Stress exposure has been proposed to contribute to the etiology of breast cancer. However, the validity of this assertion and the possible mechanisms involved are not well established. Epidemiologic studies differ in their assessment of the relative contribution of stress to breast cancer risk, while physiological studies propose a clear connection but lack the knowledge of intracellular pathways involved. The present review aims to consolidate the findings from different fields of research (including epidemiology, physiology, and molecular biology) in order to present a comprehensive picture of what we know to date about the role of stress in breast cancer development.

Region-specific alterations in glutamate receptor expression and subcellular distribution following extinction of cocaine self-administration.

A role for glutamatergic signaling in the nucleus accumbens (NA) in both the expression and extinction of cocaine seeking has been suggested. The effects of extinction following cocaine self-administration on the expression and synaptosomal distribution of GluR1 and NMDAR1 glutamate receptor subunits in the NA shell and core and the dorsolateral striatum were examined. Rats self-administered cocaine or had access to saline for 14 days followed by a period of extinction training, home-cage exposure, or placement in the self-administration chambers with levers retracted in the absence of discrete cues. Self-administration followed by home-cage exposure reduced GluR1 expression in the NA shell and NMDAR1 expression in the dorsolateral striatum without affecting expression in the NA core. These effects were not observed following extinction. Extinction training increased synaptosomal GluR1 in the NA shell and core and NMDAR1 in the dorsolateral striatum while decreasing synaptosomal NMDAR1 in the shell. Extinction but not home-cage exposure was associated with altered expression and synaptosomal content of the scaffolding proteins PICK1 and PSD95.Following extinction, synaptosomal PICK1 decreased in the dorsolateral striatum while total PICK1 expression was increased in the shell. The synaptosomal PSD95 was decreased in the NA shell, while total PSD95 expression was increased in the core. These data suggest that extinguished cocaine seeking is associated with changes in GluR1 and NMDAR1 expression and subcellular distribution that are region-specific and consist of both a reversal of cocaine-induced adaptations and emergent extinction-related alterations that include receptor subunit redistribution and may involve alterations in scaffolding proteins.

Evaluation of the function of a type I peritrophic matrix as a physical barrier for midgut epithelium invasion by mosquito-borne pathogens in Aedes aegypti.

In addition to modulating blood meal digestion and protecting the midgut epithelial cells from mechanical and chemical damage, a biological function attributed to the mosquito type I peritrophic matrix (PM) is preventing or reducing pathogen invasion, especially from Plasmodium spp. Previously, we demonstrated that chitin is an essential component of the PM and is synthesized de novo in response to blood feeding in Aedes aegypti. Therefore, knocking down chitin synthase expression by RNA interference severely disrupts formation of the PM. Utilizing this artificial manipulation, we determined that the absence of the PM has no effect on the development of Brugia pahangi or on the dissemination of dengue virus. However, infectivity of Plasmodium gallinaceum is lower, as measured by oocyst intensity, when the PM is absent. Our findings also suggest that the PM seems to localize proteolytic enzymes along the periphery of the blood bolus during the first 24 hours after blood feeding. Finally, the absence of the PM does not affect reproductive fitness, as measured by the number and viability of eggs oviposited.

Hydrocortisone down-regulates the tumor suppressor gene BRCA1 in mammary cells: a possible molecular link between stress and breast cancer.

Psychological stress has been correlated with breast cancer development in numerous epidemiological studies. However, physiological and molecular models which may account for this association are not readily available. We have found that the stress hormone hydrocortisone (cortisol) down-regulates the expression of the breast cancer susceptibility gene BRCA1 in the nonmalignant mouse mammary cell line EPH4. This effect is concentration-dependent, is reliant on the continuous presence of hydrocortisone, and is not affected by the addition of lactogenic hormones, or growth conditions. Hydrocortisone was also found to negate a known positive effect of estrogen on BRCA1 expression and, therefore, may interfere with estrogen-related signaling in mammary epithelial cells. The repressive effect of hydrocortisone is diminished or lost in the mouse mammary lines HC-11 and SP1, respectively, suggesting regulation of the BRCA1 may differ between lines. We have uncovered two promoter regulatory sites, which are involved in BRCA1 regulation by hydrocortisone, namely the RIBS and UP regulatory elements. Binding of the transcription factor GABP to both sites is lost upon hydrocortisone addition, though the levels of these factors are not altered by hydrocortisone treatment. Because BRCA1 activity is important for a number of intracellular pathways involved in prevention of tumorigenesis, its observed down-regulation may represent a novel molecular mechanism for cortisol's involvement in breast cancer development.

A novel activating role of SRC and STAT3 on HGF transcription in human breast cancer cells.

We have previously determined that the HGF promoter can be transactivated by a combination of activated Src and wild-type Stat3 in the mouse breast cell lines HC11 and SP1. To determine if this pathway is of relevance for the human disease, a series of human breast and other human cells lines were examined, and the status of key proteins in these cells determined. All of the human breast cell lines exhibited strong transactivation by a combination of activated Src and Stat3. This activation was dependent on a Stat3 recognition element present at nt-95. The exception was the ErbB2 over-expressing cell line SK-BR-3 where Stat3 alone could transactivate HGF though Src augmented this effect. Increased phosphorylation of Stat3 tyrosine 705 was also observed in this line. Analysis of three ovarian cell lines revealed that Src/Stat3 expression was not able to activate the HGF promoter in two of these lines (SKOV3 and IOSE-80PC). Src/Stat3 expression did activate HGF transcription in OVCAR3 cells, but this effect was not mediated by the Stat3 site at nt-95. Stat3 phosphorylation at tyrosine 705 was observed in IOSE-80PC cells, but was insufficient to allow for activation of the HGF promoter. Human kidney (HEK293) and cervical carcinoma (HeLa) cells were also not Src/Stat3 permissive, despite high levels of Stat3 phospho-Y705. These results suggest that human breast cells are a uniquely permissive environment for HGF transactivation by Src/Stat3 which may allow for the inappropriate activation of HGF transcription during the early stages of breast transformation. This could lead to paracrine or autocrine activation of the Met receptor in breast carcinoma cells.

Characterization of a negative transcriptional element in the BRCA1 promoter.

INTRODUCTION: Decreased transcription of the BRCA1 gene has previously been observed to occur in sporadic breast tumours, making elucidation of the mechanisms regulating the expression of this gene important for our understanding of the etiology of the disease. METHODS: Transcriptional elements involved in the regulation of the BRCA1 promoter were analysed by co-transfection experiments into the human MCF-7 and T-47D breast cancer cell lines. RESULTS: We have identified a repressor element, referred to as the UP site, within the proximal BRCA1 promoter whose inactivation results in increased promoter activity. An E2F recognition element, previously suggested to mediate repression via E2F-6, is adjacent to the UP site and its inactivation also leads to increased BRCA1 expression. These two elements appear to form a composite repressor element whose combined effect is additive. The UP element is composed of two sequences, one of which binds the ubiquitously expressed ets family transcription factor GABP alpha/beta. This site is distinct from a previously identified GABP alpha/beta site, the RIBS element, though the RIBS site appears to be necessary for derepression of the promoter via mutations in the UP site. Knockdown of GABP alpha using an shRNA vector confirms that this protein is important for the function of both the RIBS and UP sites. CONCLUSION: The identification of a repressor element in the BRCA1 promoter brings a new level of complexity to the regulation of BRCA1 expression. The elements characterized here may play a normal role in the integration of a variety of signals, including two different growth related pathways, and it is possible that loss of the ability to derepress the BRCA1 promoter during critical periods may contribute to breast transformation.

Regulation of the BRCA1 promoter in ovarian surface epithelial cells and ovarian carcinoma cells.

As BRCA1 expression is often suppressed in sporadic ovarian carcinoma we characterized the regulation of the 231nt proximal 'L6' fragment of the BRCA1 promoter in two human ovarian surface epithelial cell and two sporadic ovarian carcinoma cell lines. Two individual regulatory elements within L6, the 'RIBS' element and the potential 'CRE' element were each necessary, but alone not sufficient for L6 activation in all four cell lines. The latter element showed some affinity for the CREB transcription factor, but cAMP pathway stimulation failed to promote its activation. This element did, however, interact with, and was activated by, c-Jun and Fra2 which suggests that it can interact with AP1-like transcription factors and that it may act co-operatively with RIBS-binding factors to regulate BRCA1 transcription in ovarian cells.