L’objectif de ce travail a consisté à évaluer le rôle d’un microenvironnement avec une haplo-insuffisance hétérozygote du gène BRCA1 dans les événements précoces de la transformation tumorale du cancer du sein. Dans ce but, nous avons modélisé un stroma BRCA1-muté en utilisant des cellules souches / stromales mésenchymateuses (MSCs) obtenues par différenciation de cellules souches pluripotentes induites (iPSCs) issues d’une patiente porteuse de la mutation (MSCs BRCA1+/-). Ces cellules mutées pour BRCA1 ont été comparées à des MSCs sans la mutation (MSCs BRCA1+/+) générées à partir d’iPSCs BRCA1+/+. Ce travail de thèse a porté sur l’influence du stroma BRCA1-muté à travers deux axes : le caractère pro-angiogénique des MSCs BRCA1+/- et l’induction d’une transition épithélio-mésenchymateuse (TEM) sur des cellules mammaires normales (HME1).Nous montrons que les MSCs BRCA1-muté présentent des propriétés pro-angiogéniques significativement augmentées en surexprimant le facteur hypoxique HIF-1α et des facteurs de la famille du VEGF, PDGF et Angpt se traduisant par des capacités augmentées à former des structures vasculaires in vitro et in vivo. Les MSCs BRCA1-muté présentent également des capacités migratoires supérieures en produisant et sécrétant la périostine (POSTN), une protéine de la matrice extracellulaire impliquée dans l’adhésion, la motilité et la migration cellulaires. Ces capacités ont été validées par une approche de siRNA spécifique pour la POSTN. In vivo, nous montrons que la co-injection de MSCs BRCA1-muté et de cellules malignes mammaires murines (4T1-Luc-GFP) a permis d’augmenter significativement la croissance tumorale et la formation de métastases pulmonaires. Ces résultats sont corrélés avec la détection de la POSTN in situ et avec la formation d’un réseau vasculaire tumoral développé, quantifié par marquage du CD34. Par ailleurs nous avons démontré qu’un surnageant de MSCs BRCA1+/- peut induire une TEM des cellules HME1 en favorisant l’acquisition d’un phénotype souche cancéreux (CD24Low/CD44High) et en accélérant leur migration. Enfin nous avons initié la production in vitro d’organoïdes mammaires en utilisant des MSCs et des HME1 afin d’étudier plus précisément les mécanismes moléculaires de cette TEM après contact et des possibles événements précoces de la transformation maligne. Nos résultats indiquent que les MSCs peuvent participer à l’initiation tumorale et à la progression métastatique dans un contexte d’une mutation hétérozygote du gène BRCA1. La POSTN pourrait représenter à la fois un marqueur pronostique mais également une cible thérapeutique pour ces cancers du sein héréditaires. / The aim of this study was to evaluate the role of a BRCA1 heterozygous haplo-deficient microenvironment in the early events of tumour transformation of breast cancer. For this purpose we modeled a BRCA1-mutated stroma using mesenchymal stem / stromal cells (MSCs) obtained by differentiation of induced pluripotent stem cells (iPSCs) from a patient carrying the mutation (MSCs BRCA1+/-). These BRCA1-mutated cells were compared to MSCs without the mutation (MSCs BRCA1+/+) generated from iPSCs BRCA1+/+. This study focuses on two aspects of BRCA1-mutated stroma, namely the pro-angiogenic properties of BRCA1+/- MSCs and the induction of an epithelial-mesenchymal transition (EMT) on normal breast cells (HME1).We have shown that BRCA1-mutated MSCs exhibit enhanced pro-angiogenic properties by overexpressing the hypoxic factor HIF-1α and factors from VEGF, PDGF and Angpt families resulting in increased capacities to form vascular structures in vitro and in vivo. BRCA1-mutated MSCs exhibit also higher migratory capabilities by production and secretion of periostin (POSTN), an extracellular matrix protein, which is involved in cell adhesion, motility and migration. These capacities have been validated by a specific siRNA approach for POSTN. In vivo, the coinjection of BRCA1-mutated MSCs with murine breast cancer cell line (4T1-Luc-GFP) promotes tumour growth and the formation of lung metastases. These results are correlated with in situ POSTN detection and with the formation of a developed tumour vascular network, quantified by CD34 staining. We also demonstrated that supernatant of BRCA1+/- MSCs can induce an EMT on HME1 cells by promoting the acquisition of stemness properties (CD24Low/CD44High) and accelerating their migration. Finally we initiated the in vitro production of mammary organoids using MSCs and HME1 in order to study more precisely the molecular mechanisms of this EMT after contact and possible early events of the malignant transformation. These results indicate that MSCs can participate to tumour initiation and metastatic progression in heterozygous BRCA1-mutated background. POSTN could represent a prognostic marker and a therapeutic target for these hereditary breast cancers.

No description available.

Linear genomes end in characteristic structures consisting of repetitive DNA and proteins: the telomeres. These play two critical roles: on one hand they avoid the of loss of genetic information due to the incomplete replication of the chromosome ends and on the other, they provide capping structures for chromosome termini, differentiating them from double strand breaks. Telomeres contain specialized proteins (the shelterin complex), as well as proteins present elsewhere on the chromosomes (chromatin remodelling, DNA damage repair and response factors). Interestingly, several DNA damage factors are required for proper telomere maintenance, drawing a thin line between telomere protection and their recognition as broken DNA ends. Loss of telomere integrity has severe consequences for the cell, namely it can induce replicative senescence and cellular aging, or it can contribute to tumorigenesis. How telomeres are capped and how they are perceived by the cell when they become dysfunctional is essential for our understanding of the contribution of loss of telomere integrity to aging and disease. In order to unravel new factors involved in telomere maintenance, siRNA screens were performed. The optimization process has confirmed both telomeric foci and telomere dysfunction-induced foci (TIFs) as suitable readouts and the screens performed generated a list of potential candidate genes involved in telomere biology. Although some of the candidate genes tested in this work failed the validation process, other genes deserve further analysis. In addition this work also studied the role of several DNA damage factors at uncapped telomeres. Furthermore, BRCA1, CtIP and EXO1 were found to be critical for the formation of end-to-end fusions generated after TRF2 inactivation. The requirement of this proteins in this process, suggests that not only that not only the classical non-homologous end joining (C-NHEJ) pathway is active at TRF2-depelted telomeres, but emphasises the multiplicity of mechanisms that act to repair dysfunctional telomeres.

Breast cancer is a disease that afflicts a significant proportion of women globally. 5-10% of breast cancer cases are linked to inherited polymorphisms in critical genes such as BRCA1, a tumour suppressor essential for genomic stability. A dysfunctional BRCA1 gene can increase breast cancer risk by 60-80%. Previous microarray analysis established that differential gene expression in unperturbed Epstein-Barr virus transformed lymphocyte cell lines (EBV-LCL) was able to distinguish BRCA1 mutation carriers from controls with a high degree of accuracy. A 43-gene radiation-independent classifier for BRCA1 status was constructed. We hypothesize that this differential gene expression can be observed in a subset of these genes using quantitative PCR (qPCR) in both EBV-LCL and B-lymphocytes isolated from patients with known BRCA1 mutation carrier status. The 43-gene classifier was analyzed using gene ontology analysis and 4 target genes selected based on predictive value, expression intensity and gene ontology similarity. Genes selected were CXCR3, TBX21, MX2, and IFIT1, with GusB as an endogenous reference gene. EBV-LCL established from known BRCA1 mutation carriers and from BRCA1 wildtype individuals were obtained and RT-qPCR (reverse transcriptase qPCR) performed on isolated RNA. Our results showed significant downregulation of CXCR3 and TBX21 in BRCA1 mutation carriers (p=0.018 and p=0.003, respectively), as expected from previous microarray results. IFIT1, while showing a non-significant upregulation (p=0.183), agreed with the expected trend. MX2 did not show significant differential expression. These results indicate that differential gene expression has the potential to accurately distinguish pathogenic variants, even if it may require EBV immortalization of B-lymphocytes. To determine whether the assay could be extended to fresh blood samples, B-lymphocytes were isolated from patients with known BRCA1 mutation carrier status from North York General Hospital in Toronto, ON. An optimized protocol to enrich the B-lymphocyte population using magnetic separation was developed for this purpose. RT-qPCR using RNA isolated from these lymphocytes showed no significant differential gene expression in CXCR3 and TBX21. However, a low sample size, use of non-sequenced controls and a need for further qPCR optimization may call these results into question. In addition, problems with blood sample transportation from off-site sources resulted in an unacceptable drop in RNA integrity. While this gene expression assay may be limited to screening a small number of blood samples, results indicate that may still have clinical relevance that can be explored. This would necessitate further optimization of the qPCR methodology and resolution of the issues surrounding RNA integrity and sample transport. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-09-26 13:13:50.809

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

A growing body of literature suggests Breast Cancer-Associated Protein 1 (BRCA1) is important not only as a cause, but also as a target in the quest for cancer treatment. BRCA1 deficient cells treated with radiation as well as PARP inhibitors and other chemotherapeutics demonstrate a greater sensitivity than cells with wild type BRCA1. Inhibitors of BRCA1 would take advantage of this synthetic lethality and represent a significant advance in cancer treatment as well as an understanding of the biology of DNA repair. Despite significant study of BRCA1 protein and function, it is a large protein (220 KDa) that is still largely uncharacterized, but its N- and C-terminal domains have been described by significant structural data. The BRCT (BRCA1 C-Terminal) Domain is a phosphoprotein binding domain that is commonly mutated or lost in cancers and has a binding cleft seemingly very suitable for drug design. Small molecule screens have been conducted against this domain, but the resulting hits with moderate affinity have not been shown to induce BRCA1 deficient phenotypes. Phosphopeptides have also been studied as potential BRCA1 inhibitors, yet despite some having affinities in the mid-nanomolar range the presence of a phosphate is not without its pharmacologic challenges. We generated an mRNA display library with 1.3 x 10^13 cyclized peptides covalently attached to the mRNA that encoded them. Eight rounds of selection exposing the library to a GST-BRCT fusion resulted in selection of non-phosphorylated peptides that bind to a BRCT domain of BRCA1. The sequences resulting from the selection have common homologies and initial characterization has shown that these peptides may be the first viable non-phosphoserine containing inhibitors of BRCA1.

The Breast cancer gene 1 (BRCA1) codes for nuclear phosphoprotein with a key function in the regulation of DNA damage response. The BRCA1 protein contributes to the formation and regulation of protein supercomplexes that participates on the DNA double-strand break repair. These protein supercomplexes are formed by the protein-protein interactions between highly conservative protein motives in BRCA1 and its binding partners. Except to the wild type form of BRCA1 mRNA containing entire set of 22 exons coding for the 220 kD protein, numerous alternative splicing variants (ASVs) BRCA1 mRNA has been described. These ASVs code for BRCA1 isoforms lacking several critical functional domains. It has been proposed, that formation of BRCA1's ASVs represent a tool for regulation of BRCA1 function. Only poorly has been characterized a complex catalogue of in various human tissues and their expression. This study aims to address these questions. We optimized the identification of BRCA1's ASVs including those covering the entire transcripts of the wt BRCA1 mRNA with length exceeding 5.5 kb. In further analysis, we characterized 13 BRCA1's ASVs in RNA samples isolated from peripheral blood mononuclear cells (PBMNC) obtained from patients with breast cancer (BC) and control subjects. The majority of the identified...

A narrative qualitative research design was used to understand the stories of young women diagnosed with BRCA1 and BRCA 2 genetic mutation. Four participants were selected who met the following criteria: (a) the participant is diagnosed with BRCA1 or BRCA2 genetic mutation, b) is within the age range of 18 to 35, (c) is without a cancer diagnosis, and is (d) not currently pregnant and does not have children. The four participants were interviewed through open-ended inquiry. The participants’ narratives proved both similar and dissimilar. The themes were organized into within-case narratives and across-case narratives. The narratives revealed that young BRCA previvors face unique challenges and experiences, and many can be viewed from an underlying feminist lens. In response to the research questions, BRCA previvors revealed detailed narratives, explored issues of family planning, and explained the ways in which BRCA has changed their worldviews.

While the etiology of ovarian cancer (OC) is not completely understood, evidence suggests that chronic inflammation may promote malignant transformation. However, familial history remains the strongest risk factor for developing OC and is associated with germline BRCA1 mutations, such as the 185delAG mutation. Normal human ovarian surface epithelial cells expressing the 185delAG mutant, BRAT, exhibit molecular and pathological changes that may contribute to OC oncogenesis. In the current study, I sought to determine whether BRAT could promote an inflammatory phenotype by investigating BRAT's impact on the expression of the proinflammatory cytokine, Interleukin-1β (IL-1β). Using a culture model system of normal human ovarian surface epithelial (OSE) cells with and without the BRCA1 185delAG frameshift mutation, BRAT, I investigated BRAT's role in IL-1β expression. OSE cells stably expressing the 185delAG mutation and ovarian surface epithelial cells with endogenous 185delAG were analyzed for differential target gene expression by real time PCR, western blot, ELISA, luciferase reporter and siRNA assays. Normal and malignant breast epithelial cell lines transiently expressing BRAT were also evaluated by real time PCR to determine whether BRAT-induced IL-1β expression is tissue specific. BRAT-expressing OSE cells exhibited enhanced IL-1β mRNA and protein expression. However, expression of BRAT in all breast cell lines failed to significantly alter IL-1β expression levels so that BRAT-mediated IL-1β expression promoting a chronic inflammatory phenotype conducive to malignant transformation may be limited to the ovary. Secondly, since OSE cells expressing the BRCA1 185delAG mutation have increased levels of IL-1β that may contribute to malignant transformation, in a pilot study, I sought to assess whether elevated urinary levels of IL-1β are associated with OC as well as compare urinary IL-1β levels with clinical parameters. Urinary and serum levels of IL-1β were analyzed by ELISA and biostatistical analysis from a patient cohort consisting of healthy women (N=10), women with ovarian benign disease (N=23), women with OC (N=32), women with other benign gynecological conditions (N=22), and women with other gynecological cancers (N=6). Urinary IL-1β levels were elevated in patients with ovarian benign disease and a first degree family history of ovarian and/or breast cancer. Urinary IL-1β levels were also correlated with increased body mass index. Urinary and serum IL-1β levels were increased in ovarian benign and OC patient samples supporting the theory of elevated urinary IL-1β being associated with cancer progression. Lastly, I sought to begin early molecular characterization of BRCA1 185delAG to better understand its role in ovarian transformation. I isolated 185delAG protein expressed in E. coli and utilized web tools to analyze the amino acid sequence to determine the molecular and structural characteristics. The study results showed the predicted BRCA1 185delAG protein product is an ordered, self-aggregating, alpha helical protein structurally and molecularly distinct from wild-type BRCA1. The BRCA1 185delAG amino acid sequence contained domains with resemblance to the Peptidase M20 family. Isolation of the BRCA1 185delAG protein product will allow for further protein analysis to better understand its' oncogeneic functions; as well as, elucidate the mechanism of tissue-specific BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC.

Prophylactic salpingo-oophorectomy, the preventive removal of the ovaries and fallopian tubes, is recommended to women who carry a BRCA1 or BRCA2 mutation in order to reduce the risk of breast, ovarian and fallopian tube cancer. The short and long term health and quality of life effects of this procedure are not well understood. We examined the actual and perceived reduction in cancer risk associated with this surgery. The impact of prophylactic salpingo-oophorectomy on health-related quality of life, psychological distress, cancer worry, menopausal symptoms, and sexual function during the year following surgery was also evaluated. In our prospective study, prophylactic salpingo-oophorectomy was associated with an 80% reduction in ovarian and fallopian tube cancer risk. The residual risk for primary peritoneal cancer was 0.2% per year or 4.3% at 20 years after salpingo-oophorectomy. Most women accurately perceived their risk of breast cancer. However, the risk for ovarian cancer was overestimated, particularly by women who carry a BRCA2 mutation. Physical and mental health-related quality of life did not decrease in the year following surgery; and psychological distress was similar to levels experienced by the general population. Most women were significantly less worried about cancer after the surgery, however, a subset of women continued to experience significant cancer specific distress after prophylactic salpingo-oophorectomy. Women who underwent prophylactic salpingo-oophorectomy when premenopausal experienced a significant worsening of vasomotor symptoms and a decline in sexual functioning. Hormone replacement therapy mitigated these symptoms, but not to pre-surgical levels. Dyspareunia was somewhat alleviated by hormone replacement therapy, however, the decrease in sexual pleasure was not. Satisfaction with the decision to undergo prophylactic salpingo-oophorectomy was high regardless of these symptoms. These studies will provide women who are considering prophylactic salpingo-oophorectomy with information about the reduction in cancer risk associated with the surgery and the possible effects experienced during the year following surgery.