Expression data from non-malignant fallopian tube epithelium
ABSTRACT: Microarrays were used to examine gene expression changes that may be present in the fallopian tube epithelium of morphologically normal BRCA1 mutation positive and negative subjects. Fallopian tube epithelia has been implicated as an early point of origin for serous carcninoma. By examining the early events present in the microenvironment of this tissue between BRCA1 mutation carriers and non-carriers, we hoped to elucidate mechanisms that may lead to the development of epithelial ovarian cancer. Laser microcapture of samples from 12 BRCA1 mutation carriers and 12 non-mutation subjects was performed. Samples were further grouped according to menstrual cycle.
Project description:Mutations in BRCA1 and BRCA2 genes confer an increased lifetime risk for breast and ovarian cancer. Ovarian cancer risk can be decreased by risk-reducing salpingo-oophorectomy (RRSO). Studies on RRSO material have altered the paradigm of serous ovarian cancer pathogenesis. The purpose of this study was to identify candidate genes possibly involved in pathogenesis of serous ovarian cancer by carrying out a microarray analysis of differentially expressed genes in BRCA1/2- mutation positive ovarian and fallopian tube epithelium derived from RRSO surgery. Freshly frozen ovarian and fallopian tube samples from nine BRCA1/2 mutation carriers scheduled for RRSO were prospectively collected in comparison with five mutation-negative control patients undergoing salpingo-oophorectomy for benign indications. Microarray analysis of genome-wide gene expression was performed on ovarian and fallopian tube samples from BRCA1/2 and control patients. The validation of microarray data was performed by quantitative real-time polymerase chain reaction (qRT-PCR) in selected cases of RRSO samples, and also high grade serous carcinoma samples collected from patients with BRCA phenotype. From 22,733 genes, 454 transcripts were identified that were differentially expressed in BRCA1/2 mutation carriers when statistically compared to controls pooling all ovarian and fallopian tube samples together. Of these, 299 genes were statistically significantly downregulated and 155 genes were upregulated. Differentially expressed genes in BRCA1/2 samples reported here might be involved in serous ovarian carcinogenesis and provide interesting targets for further studies. Both fallopian tube and ovarian samples were collected from each BRCA1/2 mutation carrier resulting in eighteen mutation positive adnexal samples. Both fallopian tube and ovarian control samples were collected from one control patient while either ovarian or fallopian tube sample was available from four control patients, respectively, resulting in 6 adnexal control samples. High quality RNA was available from nine BRCA1/2-mutation positive ovarian and eight BRCA1/2-mutation positive fallopian tube samples and from three control ovarian and three control fallopian tube samples.
Project description:Approximately 5% of all breast cancers can be attributed to an inherited mutation in one of two cancer susceptibility genes, BRCA1 and BRCA2. We searched for genes that have the potential to distinguish healthy BRCA1 and BRCA2 mutation carriers from non-carriers based on differences in expression profiling. Using expression microarrays we compared gene expression of irradiated lymphocytes from BRCA1 and BRCA2 mutation carriers versus control non-carriers. We identified 137 probe sets in BRCA1 carriers and 1345 in BRCA2 carriers with differential gene expression. Gene Ontology analysis revealed that most of these genes relate to regulation pathways of DNA repair processes, cell cycle regulation and apoptosis. Real-time PCR was performed on the 36 genes which were most prominently differentially expressed in the microarray assay; 21 genes were shown to be significantly differentially expressed in BRCA1 or BRCA2 mutation carriers as compared to controls (p<0.05). Based on a validation study with 40 mutation carriers and 17 non-carriers, a multiplex model that included six or more coincidental genes of 18 selected genes was constructed in order to predict the risk of carrying a mutation. The results using this model showed sensitivity 95% and specificity 88%. In summary, our study provides insight into the biological effect of heterozygous mutations in BRCA1 and BRCA2 genes in response to ionizing irradiation induced DNA damage. We also suggest a set of 18 genes that can be used as a prediction and screening tool for BRCA1 or BRCA2 mutational carriers by using easily obtained lymphocytes. Using expression microarrays we compared gene expression of irradiated lymphocytes from BRCA1 and BRCA2 mutation carriers versus control non-carriers Fresh blood samples were obtained from 9 BRCA1 and 8 BRCA2 mutation carriers and 9 mutation-negative women. Lymphocytes were collected from fresh blood samples, and RNA was extracted one hour after γ-irradiation
Project description:Microarrays were used to examine gene expression changes that may be present in the fallopian tube epithelium of morphologically normal BRCA1 mutation positive and negative subjects. Fallopian tube epithelia has been implicated as an early point of origin for serous carcninoma. By examining the early events present in the microenvironment of this tissue between BRCA1 mutation carriers and non-carriers, we hoped to elucidate mechanisms that may lead to the development of epithelial ovarian cancer. Overall design: Laser microcapture of samples from 12 BRCA1 mutation carriers and 12 non-mutation subjects was performed. Samples were further grouped according to menstrual cycle.
Project description:The purpose of this study was to identify molecular alterations potentially involved in predisposition to adnexal serous carcinoma (SerCa) in the non-malignant fallopian tube epithelium (FTE) of BRCA1/2-mutation carriers, given recent evidence implicating the distal FTE as a common source for SerCa. Keywords: disease state analysis Overall design: We obtained and compared gene expression profiles of laser capture microdissected non-malignant distal FTE from 12 known BRCA1/2-mutation carriers (FTEb) and 12 control women (FTEn) during the luteal and follicular phase, as well as 13 high grade tubal and ovarian SerCa.
Project description:The purpose of this study was to identify molecular alterations potentially involved in predisposition to adnexal serous carcinoma (SerCa) in the non-malignant fallopian tube epithelium (FTE) of BRCA1/2-mutation carriers, given recent evidence implicating the distal FTE as a common source for SerCa. Experiment Overall Design: We obtained and compared gene expression profiles of laser capture microdissected non-malignant distal FTE from 12 known BRCA1/2-mutation carriers (FTEb) and 12 control women (FTEn) during the luteal and follicular phase, as well as 13 high grade tubal and ovarian SerCa.
Project description:The development of non-invasive primary cancer preventive measures in humans require a thorough understanding of the initial cancer-driving molecular mechanisms. High grade serous extra-uterine M llerian cancers (HGSEMC; formerly classified as ovarian/tubal/peritoneal cancer) present at a very late stage and less than 40% of women survive 5 years. Although the recent TCGA initiatives revealed key molecular changes in established cancers, very little is known about the initial molecular alterations in cancer development. Analysis of normal tissue at extensively high risk prior to the development of any microscopic alterations is critical. BRCA1/2 mutation carriers have an up to 30 40 fold increased risk to develop ovarian cancer, preferentially HGSEMC. Despite a plethora of evidence linking mutations in BRCA1 or BRCA2 to cancer development the core components such as organ specificity (i.e. to breast and Fallopian Tube) are still missing. The Fallopian Tube of BRCA mutation carriers offers a unique opportunity to study carcinogenesis because these cancers originate only from the distal (i.e. fimbrial) end of the Fallopian Tube (which is in close proximity to the ovary), and not from the proximal end (which is close to the uterus). The ovary which is in extreme close proximity to the fimbriae provides an excellent soil for cancer cells which are likely shed from the fimbriae and once the cancer has been discovered the big bulk of tumour is usually present on the ovary and hence the majority of HGSEMC are also referred to as ovarian cancer . To study the earliest steps of human carcinogenesis we performed epigenome-wide DNA methylation (DNAme) analyses (using the Illumina 450k DNA methylation bead-array assay assessing DNAme at ~480 000 CpG sites) in 215 microscopic normal Fallopian Tube samples of BRCA1/2 mutation carriers (n=56) and controls (n=59) who had their tubes/ovaries removed for prophylactic or other reasons, respectively (for 52 and 49 individuals respectively we analysed both fimbrial and proximal Fallopian Tubes). In order to adjust for any epigenetic effects which are not of immediate importance to the carcinogenic process, for each volunteer we analysed both the fimbrial (at risk) and the proximal (non at risk) portion of the tubes separately. Formalin fixed paraffin embedded (FFPE) tissue blocks were retrieved from UCL Biobank (NC09.13). Histopathological features of fimbrial and proximal section of Fallopian Tube from BRCA carriers and control cases were carefully examined. Cases negative for serous tubal intraepithelial carcinoma (STIC) lesions were chosen for DNA isolation to characterize pre-cancer epigenetic changes. For DNA isolation, a core of 3 x 0.6 mm was taken from each block representing fimbrial and proximal end of fallopian tubes from both BRCA carriers and matched control cases. The DNA was isolated using QIAamp(r) DNA FFPE Tissue Kit as per manufacturer's protocol with minor modifications (Dewaxing for 4 hours in xylene and proteinase digestion performed overnight, other procedures were as per the instructions). DNA was quantified using Nandrop and restored using the Infinium FFPE DNA Restore Kit and then 200 nanogram of DNA was bisulfite converted using the EZ DNA Methylation-Gold(tm) and subjected to methylation analysis on the Illumina Infinium Human Methylation450 BeadChip.
Project description:Germline BRCA1 or BRCA2 mutations (mtBRCA1 and mtBRCA2) dramatically increase risk for high-grade serous ovarian cancer (HGSOC), the most commonly diagnosed histotype. Other risk factors for this cancer, which originates primarily in the distal fallopian tube epithelium (FTE), implicate ovulation. To test whether mtBRCA1 or mtBRCA2 FTE cells respond differently to peri-ovulatory follicular fluid (FF) exposure than control patient FTE, gene expression profiles from primary FTE cultures were compared at baseline, 24h after FF exposure, and 24h after FF replacement with culture medium. Hierarchical clustering revealed both FF exposure and BRCA mutation status affect gene expression, with BRCA1 mutation having the greatest impact. Analysis revealed increased NFκB and EGFR signaling at baseline, with increased interferon signaling after recovery from FF exposure in mtBRCA1 samples. Inhibition of EGFR signaling and ISGylation by increased BRCA1 expression was verified in an immortalized FTE cell line, OE-E6/E7, stably transfected with BRCA1. Suppression of ISG15 and ISGylated protein levels by BRCA1 expression was found to be mediated by decreased NFκB signaling and was transiently suppressed by FF exposure. This study demonstrates increased NFκB signaling associated with decreased BRCA1 expression resulting in increased ISG15 and ISGylation following FF exposure, which could represent potential targets for chemoprevention. Overall design: Fallopian tube epithelium from BRCA1 and BRCA2 mutation carriers and non-mutated women were seeded in collagen-IV coated transwells (n=8 for mtBRCA1, mtBRCA2 and control). The lower chamber contained cuture medium for the duration of the experiment. Upper chamber first contained culture medium, this was replaced with follicular fluid for 24h, and this was then replaced with fresh culture medium for 24h. RNA was extracted from cells after 24h FF exposure (F24), and after recovery from FF exposure (FC). Cells maintained only in culture medium were used as a control (C24).
Project description:The fallopian tube transports the gametes to the fertilization site and delivers the embryo to the uterus at the optimal time for implantation. Progesterone and the classical progesterone receptor (PGR) are known to be involved in regulating both tubal ciliary beating and muscular contractions, possibly involving both genomic and non-genomic actions. To provide more clues on the mechanisms involved, we investigated the effect of progesterone on gene expression in mice fallopian tubes in vitro at early (20 min) and later (2 h, 8 h) time-points using microarray and/or quantitative PCR. In parallel, oocyte cumulus complex transport was investigated in ovulating mice injected with one of the PGR antagonists, Org 31710 or CDB2194. Microarray analyses did not reveal any apparently regulated genes 20 min after progesterone treatment, in agreement with a proposed non-genomic action of progesterone controlling ciliary beating. After 2 h, 11 genes were significantly up-regulated. Analyses by quantitative PCR at 2 h and 8 h showed a consistent up-regulation of endothelin 1 (Edn1) and a down-regulation of its receptor Ednra by progesterone. We also show that treatment with progesterone receptor antagonist before ovulation accelerates the transport of the oocyte cumulus complex. This is the first study showing that progesterone regulates Edn1 and Ednra in the fallopian tube. Together with previous studies on endothelin-mediated effects on muscular contractions in the fallopian tube, the results from this study suggest that endothelin is a mediator of the progesterone-controlled effects on muscular contraction, and eventually gamete transport, in the fallopian tube. 16 pooled samples from mice fallopian tubes were exposed in vitro to progesterone for up to 2 hours.
Project description:We report mapping of the PAX8 cistrome in three high grade serous ovarian cancer cell lines (KURAMOCHI, OVSAHO, and JHOS4) compared to three benign immortalized fallopian tube cell lines (FT33, FT194, and FT246). We identified a highly conserved PAX8 binding pattern common across benign fallopian tube cell lines that was distinct from the unique PAX8 binding patterns seen in each cancer cell line. Comparison of benign and malignant Mullerian cell lines.
Project description:Female BRCA1 mutation carriers have a nearly 80% probability of developing breast cancer during their life-time. We hypothesized that the breast epithelium at risk in BRCA1 mutation carriers harbors mammary epithelial cells (MECs) with altered proliferation and differentiation properties. Microarray studies revealed that PMEC colonies from BRCA1 mutation carriers anticipate expression profiles found in BRCA1-related tumors, and that the EGFR pathway is upregulated in BRCA1 mutation carriers compared ton non BRCA1 mutation carriers. Keywords: Class comparison and pathway analysis 10 colonies were collected and RNA was isolated using the Absolutely RNA Nanoprep kit, Stratagene. The arrays included duplicates from four normal controls and from two BRCA1 mutation carriers and single arrays from another two BRCA1 mutation carriers.