Given the high risk of cancer development associated with BRCA1 mutation carriers, it is important that they be identified early and accurately. Identification is impeded however, by the large number of variants of unknown significance (VUS) and the pleiotropic nature of BRCA1 function. Our lab has developed a novel functional assay that identifies BRCA1 mutation carriers based on gene signatures in Epstein-Barr virus (EBV)-transformed lymphoblastoid cells (LCLs). Given that this assay was developed using blood-derived cells, we hypothesized that the same abnormally regulated genes would be detected in fresh blood samples and that this could be used to develop a novel blood-based functional assay for the determination of BRCA1 status in a patient population. The first part of the study used selected reaction monitoring-mass spectrometry (SRM-MS) as well as flow cytometry (FC) to determine protein expression in LCLs and identify potential targets for the development of a protein-based functional assay. Interestingly, many of the strong predictors of BRCA1 carriers were proteins involved in two signalling pathways: Interferon (IFN)-regulated signalling and B cell development. If validated in fresh blood samples, these observations represent potentially novel findings and suggest yet unexplored functions for BRCA1. Next, this study established the methodology to identify the desired cell population within a heterogeneous whole-blood sample by determining that LCLs express the pan B cell surface marker CD20. To determine which targets were detectable in CD20+ B cells isolated from fresh blood samples, mRNA and protein expression of the selected targets was examined using a sample cohort of BRCA1 mutation carrier and non-carrier control patients. The results showed that four mRNA targets, CXCR3, GLDC, IFIT1 and TBX21 were detectable in these fresh blood samples identifying them as appropriate targets for further development of a qRT-PCR-based assay. However, given poor RNA quality and changes in transcript expression levels as the age of the blood samples increased, the development of a protein-based assay is the best approach moving forward. Moreover, four proteins, IGHM, IGHD, CD24 and CXCR3, had high expression on CD20+ B cells, identifying them as the best targets for further development of a FC-based functional assay. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2014-01-31 20:49:06.393

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 2004. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Evidence indicates that the mammary epithelium is arranged in a hierarchy in which mature luminal and myoepithelial cells are derived from stem cells through a series of lineage-restricted intermediates. One of the more compelling hypotheses in breast cancer research is that transformation of a particular cell within the hierarchy will initiate a tumour with a specific molecular profile and clinical outcome. If this is true, valuable insight into tumourigenesis can be gained by investigating normal and malignant pathways of differentiation. A well-known tumour suppressor in breast cancer, BRCA1, plays a role in mammary epithelial differentiation. It has been proposed that haploinsufficiency or loss of BRCA1, either by germline mutation or sporadic downregulation, blocks differentiation producing a pool of genetically unstable mammary stem/progenitor cells that are prime targets for transformation. Thus, investigation of BRCA1 regulation and its role in differentiation are important to our understanding of breast cancer etiology. In this study, we determined that BRCA1 is at the end of a transcriptional network comprised of NRF-1 and GABP, a transcription factor comprised of two distinct subunits GABPalpha and GABPbeta. Decreased BRCA1 transcription in SK-BR-3 cells was found to be caused by aberrant activation of the GABPbeta promoter by an NRF-1 binding protein complex. We determined that the SWI/SNF family members BRG1, ARID1A and BAF155 may participate in the complex that activates GABPbeta transcription in conjunction with NRF-1. Examination of NRF-1, GABP and BRCA1 in 3D culture models suggests that mammary epithelial differentiation is biphasic with the transition between the phases being driven by changes in BRCA1 expression and localization. In the first phase, BRCA1 promotes differentiation in the nucleus, and in the second phase, BRCA1 is downregulated as a result of diminished GABP expression and relocated to an apical position, presumably to facilitate cell polarization. Following BRCA1 downregulation, NRF-1 and GABP levels increase indicating they are inducing oxidative phosphorylation in the second phase of differentiation. The involvement of NRF-1 and GABP in cellular respiration as well as differentiation through targets such as BRCA1 suggests that these proteins may integrate the cellular functions and mitochondrial metabolism required for mammary epithelial differentiation. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2012-09-11 14:22:26.407

Understanding the biological mechanisms underlying the initiation and progression of breast cancer is an important step for its prevention and treatment. We used an in vitro and in vivo model to demonstrate that p100/p52 and RelB are strongly activated in BRCA1-deficient mouse mammary progenitor cells and human BRCA1-mutation carriers. We found that NF-κB activation induces stem and progenitor cell expansion and inhibits differentiation. Knockdown and pharmacological inhibition showed that the progesterone-independent growth of BRCA1-deficient progenitor cells requires the alternative NF-κB activation mediated by ATM. Remarkably, treatment of mice with the NF-κB inhibitor dimethylaminoparthenolide (DMAPT) resulted in prolonged repression of BRCA1-deficient progenitor cell proliferation, revealing a possible approach to cyclic chemoprevention.

Retrotransposons constitute about a third of our genome. It is challenging to identify the causes and consequences of retrotransposon expression in human disease due to hundreds of active genomic copies and poor conservation across species. We profiled genomic insertions of retrotransposons in ovarian cancer. RNAs exhibiting Bayesian correlation with retrotransposon RNA were analyzed to identify potential causes and consequences of retrotransposon expression in ovarian and breast cancers. This strategy found divergent inflammatory responses associated with retrotransposon expression in ovarian and breast cancer. It identified new factors inducing the expression of endogenous retrotransposons, including anti-viral responses and the tumor suppressor BRCA1. In cell lines, mouse ovarian epithelial cells and patient-derived tumor spheroids, BRCA1 promoted the accumulation of retrotransposon RNA and facilitated transcription of active families of retrotransposons and their insertion into the genome. Intriguingly, elevated retrotransposon expression predicted survival in ovarian cancer patients. Retrotransposons are part of a complex regulatory network in ovarian cancer, including BRCA1 contributing to patient survival. The above-described analysis strategy could also be used to identify the regulators and impacts of retrotransposons in various contexts of biology and disease in humans.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 175-190).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 76-86).

Breast cancer is one of the most common malignancies in women, affecting one in twelve. Ovarian cancer, although not as frequent, is the leading cause of death from gynaecological cancer. Inherited predisposition to breast and ovarian cancer, which accounts for approximately 5 to 10% of these cancers, has been associated with mutations in the BRCA1 and BRCA2 genes. Mutations in both of these genes increase the lifetime risk of developing breast cancer by approximately 80%. BRCA1 confers a greater predisposition of ovarian cancer than BRCA2, however, BRCA2 has been associated with male breast cancer. Polymorphisms linked to BRCA1 and BRCA2 were studied to examine whether either of these genes were linked to breast and breast/ovarian cancer families. None of the five cancer families studied generated statistically significant lod scores although the segregation of a common haplotype with the disease in each family and positive lod scores did suggest that four of these families were linked to BRCA1 and the other to BRCA2. Subsequent mutation studies identified three germline mutations, thus confirming the initial linkage results in three families. A total of four deletions and six polymorphisms were identified in BRCA1 and BRCA2 from forty-eight breast and breast/ovarian cancer families, using SSCP analysis and PTT. The functional effect of these mutations is unclear although variable expression of the cancer phenotype suggests that other genes and environmental factors play an important role in the development of breast and ovarian cancer. Evidence of an abnormal protein was detected by the presence of clonal LOH of the normal allele, using BRCA1 antibodies in familial breast and ovarian tumours. In addition, BRCA1 immunostaining was negative in a greater proportion of benign tumours compared to malignant ovarian tumours. The loss of BRCA1 does not lead to malignancy, suggesting that BRCA1 may have another role in benign ovarian epithelial tumours.

DNA topoisomerase I (TopoI) is an essential enzyme involved in the relief of DNA supercoiling during replication. TopoI plays important role in various DNA events, however the recognition that it is the target of anticancer drug camptothecins (CPTs) led to the rapid growth in this field. CPTs inhibit TopoI during S phase and cause double stranded DNA lesions in rapidly dividing cells. This class of drug is used extensively in oncology clinical settings worldwide. However, resistance to this type of therapy has been found in approximately 70% of the patient population. Current evidence supports that degradation of TopoI by the Ubiquitin Proteasomal Pathway (UPP), and consequent compensation by Topoisomerase II expression may be involved in imparting drug resistance, but this mechanism requires much greater understanding. Protein-protein interaction studies have indicated that, BRCA1 is the E3 ligase for TopoI ubiquitination in response to CPT. BRCA1 impaired cells fail to ubiquitinate and degrade TopoI and are sensitive to CPTs. It is important to note that triple negative breast cancer patients have impaired BRCA1 function, higher mutation rate and/or a lower expression of BRCA1. The Bharti lab has shown that TopoI associates with BRCA1. Our work attempts to elucidate the nature of the interaction between BRCA1 and TopoI in the hope of better understanding the mechanism of resistance to camptothecin therapy in TNBC.

Background: A positive BRCA1/2 carrier status impacts an individual on various levels with implications to an entire family due to shared family genes. A gap exists in the research literature in the area of parental disclosure and non-disclosure of genetic test results to younger offspring. Additional studies in the area of parental disclosure and non-disclosure will help clinicians to better support parents and children during this process. Purpose: The purpose of this qualitative hermeneutic phenomenological research study was to attain an understanding of the lived experience of parents’ perceptions regarding the disclosure and non-disclosure of a positive BRCA1/2 test result to minors. Results: The essence of the lived experience of the 15 study participants was a parental desire for healthcare professionals to take the BRCA1/2 conversation a step further which was uncovered in the seven research themes. Discussion: For the study participants interviewed, stories reflected an identified need for axillary support that specifically pertained to the disclosure and non-disclosure decision-making process. Findings suggest ways in which parental support may be coordinated though intra and interdisciplinary team approaches to patient care. Implications: The findings from this study support the need for mixed methods studies of parental disclosure and non-disclosure of BRCA1/2 test results to minors. Specifically, studies assessing positive BRCA1/2 males and individuals from our gay community, members from our lower socioeconomic and diverse ethnic community, and fathers and children’s perceptions regarding the disclosure of parental BRCA1/2 test results to minors are warranted.