Prognostic indicators, measured at diagnosis, are important in breast cancer. They help clinicians select optimal treatment, provide rational bases for stratification of treatment trials and assist analysis of response to treatment. Univariate statistical survival curves have identified many such indicators. However, they do not explain why some patients, classified as favoured by one or other factor(s), experience early treatment failure, nor why a substantial number with unfavourable signs remain recurrence-free many years later. This study was undertaken to identify independent prognostic factors with the use of multivariate regression. A Cox proportional hazards model of disease-specific survival was based on 1184 primary breast cancer patients referred to the Cancer Control Agency of B.C. between 1975 and 1981 (median follow-up 60 months). Significant univariate associations with overall survival were found for estrogen receptor concentration ([ER]), axillary nodal status (NO, Nl-3, N4+), clinical stage (TNM I, II, III, IV), histologic differentiation and confluent tumor necrosis (minimal, marked). These factors were assessed at primary diagnosis. A subset of 859 patients with complete data on these variables and also histologic type, menopausal status, age, tumor size and treatment was used to fit the multivariate model. Nodal status was the most important independent factor but three others, TNM stage, [ER] and tumor necrosis, were needed to make adequate predictions. A derived Hazard Index defined risk groups with 8-fold variation in survival. Five-year predicted survival ranged from 36% (N4+, loge[ER]=0, marked necrosis) to 96% (NO, loge[ER]=6, no necrosis) with TNM I and 0% to 70% for the same categories in TNM IV. This wide variation occurred across all stages. Study of post-recurrence survival (369 patients) yielded a model with only three independent predictors: [ER], nodal status and tumor necrosis. Survival - overall, recurrence-free and post-recurrent - is predictable by modelling a few factors measureable at diagnosis. Use of ER concentration, rather than the more common ER status (+ or -), greatly strengthens the model. Presence of ER was also shown to be increasingly important as 'protective', attenuating the effect of other factors, as risk of mortality increases. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Much has been written on guiding children through trauma and life crises. Most sources focus on trauma such as violence, abuse, terminal illness or death, experienced by the children themselves. However, little has been published about a parent’s illness, especially the effects of a mother having breast cancer on her children. This research starts off by investigating the high prevalence of breast cancer in South Africa. Based on this evidence, it was argued that many families are affected, and it seems that little care, in terms of support, is given to the children of women with breast cancer. The aim of this research study was to analyse and describe the experiences and the meaning three children made whose mother was being treated with breast cancer. In the literature review, social constructionism, narrative therapy, and generally used approaches to supporting children during their parents’ illnesses were scrutinized. Thereafter, the research paradigm and design applicable to this study were examined. The emphasis was on the qualitative research paradigm, the exploratory case study design, the purposeful sample, as well as the methods used for data collection and data analysis. The data gathered during the research (therapeutic sessions with the three children), were analysed and documented. Each child’s data analysis started with a description of the child, after which a visual illustration of the analysis process and their experiences and meaning-making were discussed. According to the findings of this study, children explore their experiences and meaning-making freely in a narrative therapy approach. The experiences and the meanings they made, were unique and very different from each other. Developmental age and language usage played a role in their expression and sharing of meaning. Consequently, it would not be possible to use predetermined therapeutic ‘recipes’ or impose expert meaning onto children, since this would hinder the unique process of meaning-making and re-authoring. / Prof. J. Byrne

Transcription factor AP2C (TFAP2C) is believed to be involved in breast cancer carcinogenesis. However, the molecular mechanisms of regulating its trans-activation activity are not well understood. One of the potential mechanisms is through p53-mediated regulation. Using ChIP-seq analysis to map the TFAP2C occupancy across the genome, we found that the introduction of p53 to HCT 116 p53 -/- colon cancer cell line significantly augments TFAP2C occupancy on the promoter regions of a group of genes. Of these, six genes were further investigated. First, TFAP2C binding sites were identified in the center of ChIP-seq peaks on the promoters of the six genes and these were verified by gel shift assays. One of these genes, MUC1, was then determined to be activated by TFAP2C in MCF-7 breast cancer cell line. Subsequently, MUC1 was selected as the model target gene to elucidate the mechanism for p53-mediated enhancement of TFAP2C occupancy. We hypothesized that DNA methylation of the MUC1 promoter is altered by p53, leading to the increased TFAP2C occupancy to its TFBS on MUC1 promoter. To examine this, CpG methylation assay was performed. The result showed the DNA methylation of MUC1 promoter region remains identical with or without over-expression of p53 in HCT 116 p53 -/- cell line. From these studies, I conclude that 1) introduction of p53 augments TFAP2C binding on specific gene targets; 2) MUC1 gene is activated by TFAP2C and two TFAP2C binding sites were verified; 3) promoter DNA methylation does not explain the increased occupancy of TFAP2C on MUC1 promoter.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第22839号 / 人健博第81号 / 新制||人健||6(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 恒藤 暁, 教授 田村 恵子, 教授 戸井 雅和 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Breast cancer is a highly heterogeneous disease with multiple drivers and a complex regulatory network. Periostin (Postn) is a matricellular protein involved in a plethora of cancer types and other diseases. More specifically, Postn has been shown to be involved in various processes of tumor progression such as angiogenesis, cell survival, invasion, and metastasis. A high Postn level in breast cancer has been corelated with a more aggressive phenotype. Despite extensive research, it remains unclear what Postn is doing to the cancer environment and how cancer cells regulate Postn. Here, we assessed the role and regulation mechanisms of Postn in ErbB2-mediated tumorigenesis. By crossing Postn deficient animals into the oncogenic NeuNDL model of ErbB2-positive breast cancer, we have shown that Postn deletion delays tumor onset and increases overall survival by affecting proliferation and apoptosis. These tumors also showed a decrease in collagen deposition which is the proposed mechanism for its effect in vivo. Using isolated cancer cells from the Postn deficient background we assessed re-expression of Postn which had no effect on in vitro tumorigenesis processes or in vivo subcutaneous growth in immunodeficient mice. Furthermore, we established an in vitro model to study the regulation of Postn using a bovine pituitary gland derived extract as a natural repressor of Postn. Using mass spectrometry and RNA sequencing, we identified potential regulators of Postn gene expression. We also showed a cross regulation between FGFR, TGFβ and PI3K/AKT pathways to regulate Postn expression. In ErbB2-mediated murine breast cancer cells, we found that TGFβ can induce Postn expression in a SMAD-independent manner while bFGF can repress Postn expression through a PKC-dependent pathway. Postn induction and repression by TGFβ and bFGF respectively, are both dependent on PI3K/AKT signaling. Overall, these results suggest a cancer-driving function for Postn and reveal a novel mechanism for regulating Postn expression.

Background: Cancer is a crippling disease affecting 32.6 million people globally. It is currently ranked as the leading cause of death worldwide, and is associated with significant morbidity and mortality. Despite advances made in the prevention, detection and treatment of malignancy, there remains a dearth of accurate and feasible prognostic and predictive factors for use in the management of patients suffering from cancer in developing countries. It is widely recognized that the immune system plays a fundamental role in regulating the development and progression of malignancy. T-lymphocytes in particular have been categorized into T-cell populations which either promote (T regulatory cells) or prevent (CD4+ and CD8+ T-lymphocyte) cancer development and progression. Aim: The aim of this thesis was to evaluate whether the levels of the respective pro- (T regulatory) and anti- (CD4+ and CD8+ T-lymphocytes) cancer T-lymphocyte subsets before treatment, and alterations of these levels following conventional anticancer treatment (surgery, chemotherapy and radiotherapy) could be used as markers of prognosis in conjunction with other well known prognostic factors, or as predictors of short term (5-7 weeks post-treatment) survival. Methods: Blood samples were collected from 25 breast cancer, and 10 oesophageal cancer patients before antitumor treatment, at day 1 post-treatment and at 5-7 weeks post-treatment. The circulating concentrations and percentages of CD4+, CD8+ and T regulatory cells were determined from whole blood by flow cytometry. These levels were used to determine the CD4: CD8+ T-cell ratio, the CD4+ Treg: total CD4+ Treg ratio and the CD8+ Treg: total CD8+ Treg ratio. Results: Early (stage 1 and 2) HER-2/neu negative breast cancer was associated with a higher CD4+ (558 versus 133 cells/μl; P=0.05) and CD8+ T-cell count (198 versus 58 cells/μl; P=0.05) as compared to early HER-2/neu positive breast cancer. Breast cancer patients showed a significant decline in CD8+CD25+CD127+ Treg cell subsets (P=0.012) following antitumor treatment. However, oesophageal cancer patients showed an increasing trend in CD8+CD127+FoxP3+ Treg cells at day 1 post-treatment (P=0.045) and at 5-7 weeks posttreatment (P=0.044). Patients who demised displayed a significantly lower CD4+: CD8+ Tcell ratio (1.2 versus 2.2; P=0.044) before treatment, as well as a lower CD4+ (111 versus 390 cells/μl; P=0.0046) and CD8+ T-cell count (88 versus 160 cells/μl; P=0.058) at day 1 posttreatment, as compared to those who survived. While patients who survived displayed a higher CD8+ Treg: total CD8+ T-cell ratio (1.48 versus 0.53 P=0.0126) before treatment, and a higher CD4+CD25+CD127+ Treg cell level at day 1 post-treatment (9 versus 2 cells/μl; P=0.047), as compared to those who demised. Conclusion Taken together the findings of this thesis suggest that the CD4+ and CD8+ T-cell count and CD4:CD8 T-cell ratio may serve as predictors of short-term survival in the management of breast and oesophageal cancer. Furthermore, HER-2/neu receptor status may reflect the underlying immune status and antitumor effector activity of a patient. These findings also suggest that T regulatory cell subsets are a heterogeneous group of cells with variable frequencies at presentation and in responses to antitumor treatment depending on the cancer type, stage of disease and antitumor treatment employed. This further illustrates the need for more work aimed at better characterizing the different Treg cell subsets to better identify which subsets are associated with patient prognosis. In summary, this work presents evidence to suggest a role for the T-cell immune profile as a prognostic and predictive parameter in the management of cancer. It argues for early assessments of cancer patients’ immune status as part of the diagnostic work up and that the immune status is taken into consideration when decisions are made regarding treatment. / Dissertation (MSc)--University of Pretoria, 2014. / tm2015 / Immunology / MSc / Unrestricted

According to U.S. Breast Cancer Statistics, about 1 in 8 U.S. women will develop invasive breast cancer during their lifetime. Chemotherapeutics that are used on patients currently often lead to tumor resistance, bone marrow suppression and cachexia. This study evaluated a novel combination of three non-mutagenic compounds for their effectiveness against mammary tumor cells, toxicity towards immune cells, ability to provoke the expression of immunogenic cell death (ICD) markers, and killing in 3D tumor models. Methotrexate (MTX), 2-deoxyglucose (2DG), and wogonin (WGN) were combined at doses well below their EC50 values yet effectively killed human and mouse breast cancer cells. The combination inhibited cancer cell colony formation and induced a high degree of cell death in multiple malignant tumor cell lines. Importantly, the combination did not significantly inhibit the viability of peripheral-blood mononuclear cells (PBMCs), even when employed at 3X the concentration that killed cancer cells. In marked contrast, low-dose doxorubicin, a common therapeutic for breast cancers, significantly decreased PBMC viability and increased the percentage of cell death. Our novel combinatorial therapy (Trifecta) elicited the significant expression of three ICD hallmarks: calreticulin surface expression, ATP secretion, and HMGB-1 release. In all cases, Trifecta elicited an equal or greater degree of ICD-marker expression compared to doxorubicin, a known inducer of ICD. We show significant efficacy of Trifecta against human and mouse mammary 3D tumor models grown in Matrigel® ECM-complex containing culture medium, and reaffirm the marked resistance of tumorspheres towards the conventional chemotherapeutic doxorubicin. The effectiveness of Trifecta in an acceptable surrogate model for mouse studies bodes well for translation of our findings to the clinic. In conclusion, Trifecta has proven highly effective against tumor cells grown either as monolayers or tumorspheres, without significant cytotoxic effects towards proliferating immune cells. Furthermore, treatment with this combination elicits ICD, which has the potential to prime an adaptive immune response against tumor cells and prevent future relapse. The drugs chosen for our combination target metabolic pathways that cancer cells are heavily dependent upon and do not interact with or induce mutations in DNA. These properties place Trifecta at the forefront of developing anticancer therapies. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

Based on the findings from breast cancer cells and nude mouse assays, we noticed that fatty acid metabolism was influenced by BDE-47 exposure. To have a comprehensive understanding of the impact, we performed targeted metabolomics analysis of fatty acids. Short-chain fatty acids (SCFAs) and hydroxylated short-chain fatty acids (OH-SCFAs) are crucial intermediates related to a variety of diseases, such as bowel disease, cardiovascular disease, renal disease and cancer. We developed a global profiling method to screen SCFAs and OH-SCFAs by tagging these analytes with d0/d6-N, N-dimethyl-6,7-dihydro-5H-pyrrolo[3,4-d] pyrimidine-2-amine (d0/d6-DHPP) and UHPLC-MS/MS in parallel reaction monitoring (PRM) mode. The derivatization procedure was simple and rapid. The targeted compounds could be derivatized within three minutes under mild condition and analyzed without the need of further purification. The derivatization significantly improved the chromatographic performance and mass spectrometry response. The d6-DHPP tagged standards were used as internal standards, which remarkably reduced the matrix effects. The use of high resolution PRM mode made it possible to identify unknown SCFA and OH-SCFA species. The developed method was successfully applied to the analysis of mouse feces, serum, and liver tissue samples harvested from the breast cancer nude mice that had been exposed to BDE-47. By using the developed method, 40 analytes (10 SCFAs and 30 OH-SCFAs) were characterized. Semi-quantitative analysis indicated that the exposure of BDE-47 to the mice altered the SCFA and OH-SCFA metabolism, especially in the high dose group. In addition, medium- and long-chain fatty acids (MLFAs) are essential energy sources in cells and possess vital biological functions. Characteristics of MLFAs in biosamples can contribute to the understanding of biological process and the discovery of potential biomarkers for relevant diseases. However, there are obstacles of the MLFAs determination because of the poor ionization efficiency in mass spectrometry and structural similarity. Herein, a derivatization strategy was developed by labeling with d0-DHPP and detecting with UHPLC-MS/MS in multiple reaction monitoring (MRM) mode. The parallel isotope labeled internal standards were generated by tagging d6-DHPP to MLFAs. The simple and rapid derivatization procedure and mild reaction conditions greatly reduced the potential of MLFA degradation. With the methodology, the chromatography performance was greatly improved, and the mass spectrum response was enhanced up to 1, 600 folds. Finally, the developed derivatization method was applied to serum samples to analyze the alteration of MLFAs induced by BDE-47 exposure in breast cancer nude mice. The semi-quantitative results demonstrated that the BDE-47 exposure significantly influenced the MLFA metabolism. Together, mass spectrometry-based targeted and nontargeted metabolomics of in vitro and in vivo studies suggested that BDE-47 impacted multiple metabolic pathways and was positively associated with breast tumor growth in mice. This study might further our understanding of the health risks of BDE-47 to breast cancer.;Polybrominated diphenyl ethers (PBDEs) are commonly used to prevent the development of fire in various factory products. Due to the adverse effects on human health and bio-accumulation capacity, PBDEs are considered as one kind of persistent organic pollutants. 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is one of the most frequently detected PBDE congeners in humans. Although numerous studies have shown the close connection between BDE-47 and human health, few reports were related to breast carcinoma. In vivo study of the association between BDE-47 and breast cancer was also scarce. In this study, both in vitro and in vivo experiments were conducted to explore the influence of BDE-47 to breast cancer. Firstly, we performed the in vitro study by exposing different concentrations of BDE-47 (5, 10 µM) to MCF-7 breast cancer cells. Nontargeted metabolomics analysis was conducted by using ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS). Results showed that the toxicity to MCF-7 cells gradually increased when the concentration of BDE-47 exceeded 1 µM in the medium. Pyrimidine metabolism, purine metabolism and pentose phosphate pathway (PPP) were the most influenced metabolic pathways, and the metabolites in the three metabolic pathways were significantly downregulated. Moreover, the increase of reactive oxygen species was detected by using the 2',7'-dichlorodihydrofluorescein diacetate staining assay. Results suggested that the BDE-47 induced oxidative stress by downregulating the NADPH generation in PPP. The pyrimidine metabolism and purine metabolism might be downregulated by the downregulation of mRNA transcripts. Therefore, BDE-47 could induce oxidative stress in breast cancer cells by inhibiting PPP and disordering the metabolism of the entire cell subsequently. Secondly, we constructed a breast cancer nude mouse model, performed in vivo exposure of BDE-47 to the mice, and conducted mass spectrometry-based metabolomics and lipidomics analysis to investigate the metabolic changes in mice. Results showed that the tumor sizes were positively associated with the dosage of BDE-47. Metabolomics and lipidomics profiling analysis indicated that BDE-47 induced significant alterations of metabolic pathways in livers, including glutathione metabolism, ascorbate and aldarate metabolism, and lipids metabolism, etc. The upregulations of phosphatidylcholines and phosphatidylethanolamines suggested the membrane remodeling, and the downregulations of Lyso-phosphatidylcholines and Lyso-phosphatidylethanolamines might be associated with the tumor growth. Targeted metabolomics analysis revealed that BDE-47 inhibited fatty acid β-oxidation (FAO) and induced incomplete FAO. The inhibition of FAO and downregulation of PPARγ would contribute to inflammation, which could promote tumor growth. In addition, BDE-47 elevated the expression of the cytokines TNFRSF12A, TNF-α, IL-1β and IL-6, and lowered the cytokines SOCS3 and the nuclear receptor PPARα. The changes of cytokines and receptor may contribute to the tumor growth of mice.

Breast cancer is the most common cancer in women among nearly every racial and ethnic group. About one in eight women in the US will get breast cancer in her lifetime. Sigma 2 receptor has long been implicated in breast carcinogenesis and compounds binding to this receptor have been developed as imaging agents for breast cancer, but its molecular identity had been elusive until 2017 when TMEM97 was identified as sigma 2 receptor. It is highly important to determine whether the biological functions of TMEM97 protein defined in previous studies are overlapping with those linked to sigma 2 receptor. In this study, we found that TMEM97 is highly expressed in ER positive breast tumors and its expression levels are associated with poor overall survival rate of breast cancer patients, and its expression patten is strongly correlated with ER and PR but not with HER2 status. Breast cancer cells with TMEM97 overexpression showed growth advantage over the control cells both in nutrition sufficient and starvation conditions. The differences were more pronounced under estrogen depleted conditions. In breast cancer MCF7 and T47D cells, TMEM97/sigma 2 receptor could regulate ERα transcriptional activities, and also regulate mTOR/S6K1 signaling pathways. The increased level of active, phosphorylated ERα, and the increased resistance to Tamoxifen treatment could be blocked by a mTOR inhibitor. TMEM97 in ER positive breast cancer cells could retard and delay the estradiol caused ERα turnover because of the accelerated the ERα protein synthesis offsetting the degradation caused by estradiol. TMEM97 increases the transactivated ERα, specifically for the nuclear soluble and chromatin bounded ERα. These observations suggest that TMEM97/sigma 2 receptor participates in breast tumor cell growth driven by estrogen signaling and further regulates the transcriptional activities of ERα through modulating ERα binding to estradiol and subsequent nuclear localization. Further, increased TMEM97 expression renders breast cancer cells with increased resistance toward endocrine therapeutics such as tamoxifen, which make TMEM97 a valid target of intervention to modulate ER activities to reduce resistance toward endocrine therapy.

Breast cancer (BC) is the second leading cause of death in Canadian women. Detection of the disease at an early stage greatly increases the average 5-year survival rate, however non-invasive early detection methods are not available to-date. Cells release various types of extracellular vesicles (EVs) to mediate intercellular communication by transferring signals in the form of bioactive molecules such as proteins, metabolites, and nucleic acids. Understanding the composition of these biomolecules may shed light on the physiological state of the cell of origin. Therefore, EVs are a promising source of biomarkers for non-invasive detection of BC. However, the surface proteome of EVs is not yet understood well enough to propose BC biomarkers that could be detected directly from biofluids. In this study, small EVs (sEVs) and medium EVs (mEVs) were isolated by differential ultracentrifugation from breast cancer MDA-MB-231 and MCF7, and non-cancerous breast epithelial MCF10A cell lines and analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. EV proteins were analyzed by two approaches: (1) global proteomic analysis and (2) enrichment of EV surface proteins by labelling surface-accessible proteins with a Sulfo-NHS-SS-Biotin reagent. Potential BC biomarkers were obtained from the first approach (1) by identifying the presence of cell line specific sEV proteins, filtering for membrane/surface proteins using UniProt annotations, and predicting the co-localization of proteins on sEVs with known EV marker proteins (CD63, CD9, CD81) by correlation analysis. This resulted in 11 potential BC sEV biomarkers (C8A, AXL, ST14, FAM20B, PROM2, CLDN3, ITGA7, MEGF10, SHISA2, GJC1, IFNGR1); the presence of ST14, CLDN3 and ITGA7 was validated by Western blot analysis. The surface labelling approach (2) enriched proteins previously not identified using the first approach (1). Potential general BC biomarkers were selected from surface proteins commonly identified from MDA-MB-231 and MCF7, but not identified in MCF10A EVs. Annotation with known BC disease associations from DisGeNET yielded 9 and 2 potential surface proteins on sEVs and mEVs, respectively. This study demonstrates the emerging role of EVs as a rich source of known and novel biomarkers which may be used for non-invasive detection of BC.