University of Macau / Faculty of Health Sciences

background The extracellular matrix receptor integrin ανβ6 is known to potentiate breast cancer (BrCa) cell invasion, metastasis and tumour-trophic growth factor receptor crosstalk during tumourigenesis. Monoclonal antibody blockade of ανβ6 diminishes invasion in vitro and arrests BrCa tumour growth and metastasis in vivo. Aberrant integrin activation status has been implicated in progression to metastatic disease in BrCa; with differential internalisation and endocytic trafficking kinetics reported for active versus inactive integrin species in malignant disease. Despite its emerging potential for targeted therapy, little is known regarding regulation of integrin ανβ6-mediated activation and signalling during progression to an invasive, metastatic state. It is hypothesised that the aetiopathological significance of integrin ανβ6 during neoplastic transformation and malignant progression in BrCa is dependent specifically upon its activation status and associated conformation, since this active state will permit establishment of known integrin-mediated oncogenic signalling underpinning acquisition of a malignant phenotype, including activation of invasion and metastasis. results Canonical integrin activation studies using divalent cations and cognate ligand stimulation indicated antibodies 6.2E5 and 6.2G2 recognise activation-associated epitopes, which are also ligand-induced binding sites (LIBS) in live-labelled cells by FCM and IMF. However, their utility to discriminate the active fraction distinct from the total or inactive fractions of ανβ6 by IHC in primary BrCa samples could not be robustly established. Evaluation of the 6.2E5 and 6.2G2 epitopes in the MCF10 isogenic model revealed that relative surface abundance of these active epitopes determined by FCM was not significantly altered; but their subcellular redistribution upon neoplastic transformation and malignant progression was observed by IMF, implicating derailed internalisation and trafficking of active ανβ6 during breast tumourigenesis and metastatic disease progression. Proteomic interrogation and network analysis of the 2D-enriched adhesion assays identified 7 novel putative molecular regulators of a ligand-engaged, activated ανβ6-mediated adhesion environment (DMBT-1, MARCKS, MXRA5, SEPT6, SEPT9, MYH9, MYH10) in the BT-20 TNBC cell line. Functional validation of these candidate mediators of the "β6 adhesome" by siRNA strategies was not achieved due to inconsistent stable knockdown. Phosphoproteomic definition of LAP ligand-engaged, active ανβ6-mediated signalling ("β6 kinome") during receptor-ligand internalisation revealed EGFR-dependency for downstream ERK1/2 signal activation in BT-20 and SUM159, but not MDA-MB-468 TNBC cells. Kinase substrate enrichment analysis (KSEA) identified 5 novel putative mediators of downstream ανβ6 signalling (COT, MAPKAPK2, PDPK1, Nuak1, TBK1) and implicated Akt1 isoform-specific activation downstream of ανβ6-LAP internalisation. Following LAP-induced ανβ6 activation and internalisation, EGFR underwent phosphorylation at multiple known activation sites, including a residue (Thr693) critical for EGFR receptor internalisation; suggesting integrin ανβ6-EGFR reciprocity during respective receptor activation and internalisation. conclusion The active conformer of integrin ανβ6 may be studied using antibodies 6.2E5 and 6.2G2 in live-labelled cells by FCM and IMF. Subcellular redistribution of activation-associated epitopes during BrCa progression implicates derailed internalisation and intracellular trafficking kinetics of active ανβ6 during tumourigenesis, while protein expression studies identified 7 putative molecular regulators of ligand-engaged, active ανβ6-mediated adhesion. Integrin ανβ6-mediated signalling during internalisation revealed an ανβ6-EGFRAkt1 signalling axis during breast tumourigenesis and disease progression, while further understanding of integrin biology and growth factor receptor crosstalk may provide additional rationale for potential combination therapies in breast cancer.

No description available.

Abstract Breast cancer is highly prevalent in the United States with an estimated 260,000 women diagnosed with invasive breast cancer in 2018 alone. There is a growing need to identify the molecular drivers of metastatic breast cancer as the molecular mechanisms responsible for the transition from normal mammary epithelial cells to aggressive cancer cells remain poorly understood. Understanding this transition may reveal a therapeutic target for aggressive breast cancer. Small, noncoding RNAs (ncRNA), such as microRNAs (miRNAs), have recently been discovered to promote initiation, progression, and metastasis of breast cancer. Similar in size to miRNAs, tRNA-derived small RNAs (tsRNAs) are a novel class of small ncRNA whose expression may differentiate between cancer types and cancer cell lines. TsRNAs are created during the maturation process of primary tRNA transcripts, where the 3-prime end of the tRNA is cleaved by RNaseZ, resulting in a 16-48 nucleotide long strand of RNA. Although similar in size to miRNA, the functions of tsRNA are largely unknown. Previously identified two tsRNA, ts-2 and ts-112, that are expressed at 10-fold higher levels in the MCF10CA1a aggressive breast cancer cell line than the normal-like MCF10A mammary epithelial cell line. Further, ts-2 and ts-112 are detected at similarly high levels in female human embryonic cells, displaying oncofetal expression. For these reasons we hypothesize that ts-2 and ts-112 promote breast cancer characteristics. Custom inhibitors of ts-2 and ts-112 were transfected into the aggressive breast cancer cell line MCF10CA1a in vitro. The following phenotypic assays were conducted to determine the function of ts-2 and ts-112 in the MCF10CA1a cell line: proliferation, cell cycle, and wound healing. Following transfection of ts-2 inhibitors, in the proliferation assay showed a 15-20% reduction in growth of aggressive cancer cells. Ts-2 inhibition also saw an increase in population doubling time of 7% from 12 to 14 hours. These results suggest that ts-2 may play a role in cell cycle progression. Following ts-112 inhibition in the aggressive MCF10CA1a breast cancer cell line, cell cycle analysis revealed a statistically significant decrease in the number of cells in G1 phase and an increase in S phase. Using a candidate approach we analyzed the effect of ts-2 and ts-112 inhibition on G1/S phase and S/G2 phase checkpoint markers by qPCR. The inhibition of these tsRNA showed no effect on the chosen genes. Our data are in support for ts-2 and ts-112 having a role in aggressive breast cancer and may be tumor promoting In on-going studies, the capacity of tsRNAs to act as predictive biomarkers of long-term breast cancer risk is being evaluated in serum collected from women at elevated risk. Analyzing ts-2 and ts-112 biological consequences following inhibition furthers our understanding of the molecular mechanisms that are responsible for aggressive breast cancer. Continued study of tsRNA function could produce a model of their role in aggressive breast cancer and thus metastasis. Understanding tsRNA function in metastatic breast cancer in turn could lead to their use as a possible biomarker or therapeutic target.

Thesis (M Med. (Family Medicine))-- University of Limpopo, 2009. / Introduction Inspired by the late presentation for care and consequently the diagnosis of breast cancer done at an advanced stage of the disease in majority of cases, this study aimed to explore the knowledge and practices related to breast cancer from patients presenting at Princess Marina Hospital (PMH) for care. Methodology The descriptive qualitative method using interviews (free attitude) was chosen to understand the trend of late presentation among participants, with following opening questions: 1. Can you please tell me all you know about the cancer of the breast? 2. How have you been treating your breast condition (growth/wound/pain) before you decided to come to PMH? Sampling was purposeful with a sample of twelve. Out of eleven interviews done with breast cancer patients fulfilling the criteria of inclusion, ten were used in the final analysis. Interviews were recorded (audiotape), transcribed verbatim and translated. Emerging themes were identified and coded into different categories Results This study noted a poor knowledge and understanding of patients about cancer of the breast. The knowledge and practice of the common well established screening methods like self breast examination (SBE) was equally poor. In majority, participants delayed going to the hospital as a result of the preceding( poor knowledge and understanding about Ca breast ), as well as the influence of lays beliefs and advices received from the surrounding. In some cases however advices from the surrounding resulted in timely medical consultation. Unexpectedly, Poor clinical practice of health worker in some cases and decision maker‟s inadequate involvement on issue of cancer awareness were other important themes which emerged during analysis of the results. Conclusions Cancer awareness together with consistent use of early detection measures by adhering to screening methods should be taken seriously and done throughout the country for the benefit of all potential victims, to address the poor knowledge, misconceptions and inappropriate health seeking behavior encountered in case of breast cancer.

Thesis (MPH)--University of Limpopo, 2010. / Background: Screening for early detection and diagnosis of diseases and health conditions is an important public health principle. Breast cancer examination is whereby a woman will examine the breast by Breast Self Examination (BSE), Clinical Breast Examination (CBE), and Mammogram. Objective: The main aim of the study was to assess the knowledge, attitude and practice of breast cancer examination among women attending Extension 2 clinic in Gaborone, Botswana. Method: The cross-sectional quantitative study design to examine knowledge, attitude and practice of women attending Extension 2 clinic in Gaborone was carried out between August and September 2009 using an interviewer administered questionnaire designed by the researcher. Results: The study was conducted among 375 women attended at extension 2 clinic. Study participants had low knowledge of breast cancer examination. The overall mean knowledge score was 49.7%. The commonest presentation of breast cancer which is a painless breast lump only a third 128(34.1%) of the respondents knew about it. The participants had a positive attitude towards breast cancer examination. Practice of breast cancer examination was unacceptable. Out of 238 Of those who practiced breast self examination (63.5% ) (BSE), only 88(23.5%) of the respondents practiced monthly as required. Similarly only 85(22.7%) of the respondents had visited a doctor for clinical breast examination (CBE) in the past year. Mammogram practice was also unacceptable only 6 (1.6%) of the respondents had done mammogram in the past 2 years. There was no association between socio-demographic characteristics with the knowledge attitude and practice of breast cancer examination. Conclusion: The results of this study suggested that women attending at extension 2 clinic had low knowledge of breast cancer examination. Despite having positive attitude towards breast cancer examination, minority practiced breast self examination, clinical breast examination and mammogram. There was no association between socio-demographic characteristics with the knowledge of breast cancer. Therefore the Government should develop a policy on breast cancer screening. Awareness and advocacy campaign on breast cancer screening should be increased in the country.

acase@tulane.edu / 1 / Emily Carron

Breast cancer is the most common cause of cancer death in Australian women. Current pathological analysis examines a small section of tissue for cellular and plasma abnormalities using a light microscope. However, this method of diagnosis, despite being the current gold standard, has its limitations, where human error and professional experience can influence a patient’s diagnosis. A potential alternative or adjunct to conventional histopathology for classifying tissue disease status is offered by Small Angle X-ray Scattering (SAXS). At the time of commencement of this work, there had been several small scale studies which examined the potential of SAXS to classify the disease status of breast tissue. These tended to focus on the supramolecular structure of collagen fibrils found in the breast, where it is known that the degradation of these fibres is related to the spread of disease. Most previous studies also used a synchrotron as an X-ray source, due to the intense and highly collimated flux available. This study used a synchrotron source, but also evaluated the use of a laboratory X-ray source, as a more convenient and relatively inexpensive alternative that could one day find application in the clinic. The work presented in this thesis analyses the largest cohort of patients and breast tissue samples studied to date using SAXS: 130 patients with 543 tissue samples. Tissues were sourced from surgical waste and classified into four groups: invasive carcinoma, benign, normal, and mammoplasty. Mammoplasty tissue samples were harvested from patients undergoing breast reduction and/or reconstruction, where no history or presence of disease was indicated. Normal tissue was sampled from patients with known disease, but pathological analysis of the tissue core diagnosed it as normal. A comprehensive analysis of the scattering patterns was carried out, analysing features arising from the collagen structure and orientation, the total scattered intensity, and adipose tissue in the breast. Features related to the axial D-spacing of the collagen fibrils within the breast tissue as well as the integrated scattering intensity (called amorphous scatter) demonstrated the highest ability to discriminate tissue types, in SAXS images acquired from both the synchrotron source and the laboratory X-ray source. The amorphous scatter intensities obtained using a synchrotron source showed highly significant differences (p < 0.01) for almost all of the tissue pair comparisons: invasive carcinoma vs. benign, invasive carcinoma vs. normal, invasive carcinoma vs. mammoplasty, benign vs. mammoplasty, and normal vs. mammoplasty. However, no significant difference was seen in the amorphous scatter between benign versus normal tissues (p = 0.30). The amorphous scatter values increased with severity of disease, i.e. it was the highest for invaded tissues and decreased progressively from benign to normal to mammoplasty. There was a significant difference between normal and mammoplasty tissue types using the amorphous scatter as a discriminator (p = 0.0025). Pathological assessment cannot differentiate between these two tissue types, which suggests that there may be changes occurring in these tissue structures at the supramolecular level that can be characterised using SAXS. The ability of SAXS to reveal structural differences between normal and mammoplasty tissue types is highly significant, for both disease diagnosis and treatment, as well as for understanding disease progression. For example, these differences might aid in determining surgical margin clearance of excised breast lesions as well as potentially provide a means of pre-screening or perhaps improve false-negative rates of diagnosis. The potential of SAXS to reveal macroscopic extent and directional spread of disease was explored using two-dimensional mapping of the amorphous scatter. These maps showed broad agreement with histopathological diagnosis, but further investigation regarding their reliability and interpretation for clinical utility is still needed. Changes in both the amorphous scatter and the axial D- spacing were seen in tissue samples up to 6 cm away from the primary site of disease. In particular, a significant decrease in both parameters was seen between the centre of the tumour (at 0 cm) and 2 cm away, suggesting that closer examination of the tissue structures over the disease/healthy tissue border may provide information regarding the mechanisms of metastasis and growth of cancerous tumours. The combination of the amorphous scattering results from the two X-ray sources indicates that the size of the scatterers may be the key in classifying tissue types. The synchrotron source was able to access a lower q-range (q = 0.1-0.6 nm-1) and the laboratory source covered a larger q-range (q = 0.25-2.3 nm-1). Mammoplasty tissues appear to be characterised by large scattering components (d > 25.13 nm), whereas normal tissues are characterised by slightly smaller scattering components (10.47 nm < d < 25.13 nm) and benign tissues by even smaller scattering components (4.83 < d < 10.47 nm). It appears that the size of the scatterers contributing to the total scattering intensity decreases with severity of disease, which was seen independently with both X-ray sources. Further investigation is warranted to determine the biological origin of these differences. These results also suggest that the optimum SAXS instrument may need to cover a scattering vector range of q < 0.25 nm-1 to identify differences in healthy tissue types, and q > 2.3 nm-1 to possibly investigate invasive carcinoma tissue types. A SAXS apparatus that can examine a large q-range may provide all of the necessary information from the amorphous scatter to differentiate between tissue groups. The periodic structure of collagen fibrils along their longitudinal axis can be characterised by the axial D-spacing, where this spacing was found to change with the presence of disease. The axial D-spacing for healthy breast tissues was found to be significantly lower in normal and mammoplasty tissues compared to invaded tissues (p = 0.0050 and p = 0.0093, respectively). However, no significant differences between the other tissue group pairs were seen (p > 0.05). These differences were evident in classification modelling of the four tissue groups, where the amorphous scatter and the amplitude of a collagen axial peak were used to build a probability model for disease status. The model showed high sensitivities (> 70%) and widely variable specificities (ranged from 18-97%) for the data examined with the synchrotron source. This means that the model was a good indicator of disease, but poor at indentifying healthy tissue types. The work presented in this thesis shows that SAXS is capable of distinguishing breast tissue types with high sensitivity and has the potential to become a significant tool for the investigation of cancer progression or even diagnosis. Further investigation into the amorphous scatter and axial D-spacing in particular may provide insight into the biological mechanisms related to tissue degradation associated with invasive disease.

Loss of proliferative control is a cornerstone of cancer development, induced by deregulation of mitogenic signalling, insensitivity to anti-proliferative signals and direct changes in cell cycle proteins. In breast cancer these alterations are frequently targeted through cyclins D1 and E, leading to defects in G1/S transition. I have investigated the role of two potential pro-proliferative oncogenes in breast cancer, id1 andwt1. Each protein promotes proliferation in distinct contexts, with unique consquences for breast cancer cells. Using a 3D culture model of non-transformed mammary epithelial cells, I identified that id1 undergoes downregulation via rapid proteosomal degradation and cytoplasmic relocalisation during mammary epithelial morphogenesis. Overexpression of Id1 led to an increase in acinar size via an increase in S phase, and wa dependent on the presence of an intact HLH domain in Id1. Co-expression with the proto-oncogene Bcl2 led to a more disorganised acinar structure, indicating that Id1 overexpression primed the cells for further oncogenic insult. Further, Id1 overexpression was unable to increase acinar size in cyclin D1-/- acini, indicating that Id1 is dependent on cyclin D1 for its proliferative effects. Overall these data identified Id1 as capable of altering the proliferation of normal mammary epithelial cells, a crucial step in early breast carcinogenesis. Wt1 was originally identified as a tumour suppressor, but our data lends support to Wt1 acting as an oncogene in breast cancer. Wt1 is expressed highly in a range of breast cancer cell lines, and is strongly regulated by progestins. Using siRNA, we identified that Wt1 is likely to be a molecular intermediary of progestin as the downregulation of Wt1 mimics a subset of progestin effects on cell proliferation and lipid synthesis. Conversely, the overexpression of the major Wt1 isoform, Wt1 (+/+), led to attenuation of progestin-induced differentiation and growth arrest via maintenance of cyclin D1 levels. The effects of Wt1 overexpression were specific to progestins, and did not affect the actions of anti-estrogens or androgens. Consequently the overexpression of Wt1 (+/+) may disrupt the endocrine response in mammary epithelial cells, and contribute to excess proliferation and failure to differentiate during breast oncogenesis.

Introduction - This study re - examined the epidemiology of bilateral breast cancer with regard to the age at diagnosis and histology of the first breast cancer, and examined the effect of bilateral breast cancer on breast cancer survival. Methods - A cohort of US women with breast cancer was identified using cancer registry data for the period 1973 to 2000 obtained from the Surveillance, Epidemiology, and End Results ( SEER ) Program. In this cohort, incidence cases of bilateral breast cancer were identified and rates calculated per 1,000 person - years and the effect on survival of a diagnosis of a bilateral breast cancer was determined using time - dependent proportional hazard regression. Results - The overall incidence of bilateral breast cancer was 5.5 per 1,000 person - years and, apart from an elevation in incidence in the first year, was constant over time. In age - cohorts of young women, age - specific rates of bilateral breast cancer were found to decline as these women aged, approaching the incidence observed in older age cohorts. In older age - cohorts, age - specific rates were comparatively constant until age 75 - 79 years, after which age - specific rates began to decline regardless of age at first diagnosis.Differences in the crude incidence of bilateral breast cancer in sub - cohorts of women with lobular carcinoma ( 6.56 per 1,000 person - years ) and infiltrating ductal carcinoma ( 5.31 per 1,000 person - years ) were largely explained by differential incidence in the first year following diagnosis of the first breast cancer. Diagnosis of bilateral breast cancer increased the risk of breast cancer mortality, independent of the interval between the first and second breast cancer. Stage of both the first and second breast cancers was found to be the most important determinant of risk. Conclusions - This study found that the pattern of age - specific incidence of bilateral breast cancer was consistent with effects already well established in the literature describing the incidence of first primary breast cancer - pre - menopausal effects in young women and underascertainment in older women. Estimates of the incidence of bilateral breast cancer were subject to bias caused by an elevation in the incidence in the first year following diagnosis of the first breast cancer. This was most likely an effect of increased surveillance. This explained to a large extent, associations between the histology of the first breast cancer and the incidence of bilateral breast cancer observed in earlier studies. This study challenged the currently accepted view that bilateral breast cancer was a sign of increased susceptibility to breast cancer. Instead it is argued that the constant annual incidence of bilateral breast cancer suggests a final, discrete stage in a multistage carcinogenesis process. It is further argued that the observed incidence of bilateral breast cancer allows us to estimate the incidence of breast cancer in the population among women reaching this final stage within their lifetime. It is conservatively estimated that by age 75 to 79 years only half the women in the population have reached this final stage. This implies that in half the population of women, breast cancer either never initiates or progresses so slowly that the final stage of carcinogenesis is not reached within their lifetime. / Thesis (Ph.D.)--School of Population Health and Clinical Practice, 2006.