PMC42, a novel model for the differentiated human breast

Cultured human breast carcinoma cell lines are important models for investigating the pathogenesis of breast cancer. Their use, however, is limited because of loss of expression of breast-specific markers and the development of a dedifferentiated phenotype after continuous culture. PMC42 is a unique human breast carcinoma line, previously shown to express secretory and myoepithelial markers. We have induced PMC42 cells to form hollow organoids in culture, similar to in vivo breast structures, using a combination of hormones including estrogen, progesterone, dexamethasone, insulin, and prolactin in combination with a permeable extracellular matrix. The organoids comprised polarized cells located around a central lumen. Expression of β-casein was demonstrated in cells within organoids using reverse transcriptase-polymerase chain reaction, Western blot analysis, and confocal immunofluorescence. In this in vitro system, milk-specific gene expression was induced through hormone and matrix interactions which may be similar to those operating in vivo. PMC42 is a novel model for investigations into the molecular mechanisms of carcinogenesis and differentiation in the human breast.

EpCAM aptamer-mediated survivin silencing sensitized cancer stem cells to doxorubicin in a breast cancer model

Understanding the molecular basis of drug resistance and utilising this information to overcome chemoresistance remains a key challenge in oncology. Here we report that survivin, a key protein implicated in drug resistance, is overexpressed in cancer stem cell pool of doxorubicin-resistant breast cancer cells. Moreover, by utilising an active targeting system consisting of an RNA aptamer targeted against the epithelial cell adhesion molecule and a Dicer substrate survivin siRNA, we could deliver a high dose of the siRNA to cancer stem cells in xenograft tumours. Importantly, silencing of survivin with this aptamer-siRNA chimera in cancer stem cell population led to the reversal of chemoresistance, such that combined treatment with low dose of doxorubicin inhibited stemness, eliminated cancer stem cells via apoptosis, suppressed tumour growth, and prolonged survival in mice bearing chemoresistant tumours. This strategy for in vivo cancer stem cell targeting has wide application for future effective silencing of anti-death genes and in fact any dysregulated genes involved in chemoresistance and tumour relapse.

Factors associated with breast and cervical cancer screening behaviours

To investigate the factors associated with the use of screening mammography for breast cancer and cervical smear tests for cervical cancer, a theoretical framework was used comprising elements from the Health Belief Model, the Theory of Reasoned Action, and illness representations from the self-regulatory model. Items reflecting older women’s illness representations about cancer and cancer screening were derived from an earlier qualitative study. Using a highly structured interview schedule, telephone interviews were conducted with 1,200 women aged 50-70 years. There were considerable similarities between the factors associated with both mammography and cervical smear test behaviours. The factors associated with screening mammography behaviour were: perceived barriers, perceived benefits, social influence, the illness representations, and marital status. The factors associated with cervical smear test behaviour were: perceived barriers, perceived benefits, emotions as a cause of cancer, feeling frightened of cancer, the illness representations, having a usual general practitioner, and being younger.

Extracellular matrix induces formation of organoids and changes in cell surface morphology in cultured human breast carcinoma cells PMC42-LA

In the lactating breast, the development of secretory alveoli consisting of differentiated cells arranged around a central lumen is dependent on signals from the extracellular environment of the cells. There are few cell lines that model this process. We previously showed that the human breast carcinoma line PMC42-LA can be induced to form organoids, reminiscent of secretory alveoli found in the lactating human breast. In this report, we used high-resolution scanning electron microscopy to show that the formation of organoids is accompanied by development of cell surface microvilli. Extracellular matrix-induced formation of microvilli occurred on the internal and external surfaces of cells in the organoids and not on surfaces in contact with the extracellular matrix. Organoid formation of PMC42-LA cells induced a rearrangement of the extracellular matrix, seen in the form of radiating fibers from the organoids. In summary, there is an interaction between PMC42-LA cells and the underlying extracellular matrix, which leads to the formation of polarized cells with well-developed microvilli. This is accompanied by organization of the extracellular matrix. PMC42-LA is a relevant model of the human breast for investigations into cell-cell and cell-matrix interactions.

Low-dose metronomic paclitaxel chemotherapy suppresses breast tumors and metastases in mice

This study investigated the use of low-dose metronomic (LDM) chemotherapy with paclitaxel in a highly metastatic mouse model of 4T1 breast cancers, and compared it with the maximum tolerable dose (MTD) therapy. LDM therapy displayed a stronger anti-tumor activity in suppressing primary and metastatic breast tumors with less degree of side effects, and stronger anti-angiogenic and anti-lymphangiogenic activities than MTD therapy. But MTD therapy showed stronger pro-apoptotic and anti-proliferative activities in situ. Paclitaxel therapy downregulated expression of vascular endothelial growth factor receptor-2 (VEGFR2) and up-regulated expression of thrombospondin-1. The results support the application of paclitaxel LDM therapy to treat advanced breast cancer.

Regressive logistic and proportional hazards disease models for within-family analyses of measured genotypes, with application to a CYP17 polymorphism and breast cancer

Various statistical methods have been proposed to evaluate associations between measured genetic variants and disease, including some using family designs. For breast cancer and rare variants, we applied a modified segregation analysis method that uses the population cancer incidence and population-based case families in which a mutation is known to be segregating. Here we extend the method to a common polymorphism, and use a regressive logistic approach to model familial aggregation by conditioning each individual on their mother's breast cancer history. We considered three models: 1) class A regressive logistic model; 2) age-of-onset regressive logistic model; and 3) proportional hazards familial model. Maximum likelihood estimates were calculated using the software MENDEL. We applied these methods to data from the Australian Breast Cancer Family Study on the CYP17 5UTR TC MspA1 polymorphism measured for 1,447 case probands, 787 controls, and 213 relatives of case probands found to have the CC genotype. Breast cancer data for first- and second-degree relatives of case probands were used. The three methods gave consistent estimates. The best-fitting model involved a recessive inheritance, with homozygotes being at an increased risk of 47% (95% CI, 28-68%). The cumulative risk of the disease up to age 70 years was estimated to be 10% or 22% for a CYP17 homozygote whose mother was unaffected or affected, respectively. This analytical approach is well-suited to the data that arise from population-based case-control-family studies, in which cases, controls and relatives are studied, and genotype is measured for some but not all subjects.

Therapeutic effects of matrine on primary and metastatic breast cancer

Matrine, one of the main components extracted from a traditional Chinese herb, Sophora flavescens Ait, has displayed anti-cancer activity in several types of cancer cells. This study aims to evaluate the therapeutic benefits of matrine on primary and metastatic breast cancer. Matrine inhibited the viability of and induced apoptosis in human MCF-7 and mouse 4T1 breast cancer cells in a dose-dependent manner in vitro as shown by MTT assay, flow cytometry and laser scanning confocal microscopy. Administration of matrine inhibited the growth of primary tumors and their metastases to lungs and livers, in a dose-dependent manner, in a highly metastatic model of 4T1 breast cancer established in syngeneic Balb/c mice. Tumors from matrine-treated mice had a smaller proliferation index, shown by immunostaining with an anti-Ki-67 antibody, a greater apoptosis index, shown by TUNEL-staining, and a less microvessel density, shown by immunostaining with an anti-CD31 A antibody, compared to the controls. Western blot analysis of tumoral homogenates indicated that matrine therapy reduced the ratio of Bcl-2/Bax, downregulated the expressions of VEGF and VEGFR-2, and increased the activation of caspase-3 and caspase-9. This study suggests matrine may be a potent agent, from a natural resource, for treating metastatic breast cancer because of its anti-apoptotic, anti-proliferative and anti-angiogenic activities.

Does Candida and/or Staphylococcus play a role in nipple and breast pain in lactation? A cohort study in Melbourne, Australia

Objective: To investigate Candida species and Staphylococcus aureus and the development of 'nipple and breast thrush' among breastfeeding women. Design: Prospective longitudinal cohort study. Setting: Two hospitals in Melbourne, Australia (one public, one private) with follow-up in the community. Participants: 360 nulliparous women recruited at 36 weeks' gestation from November 2009 to June 2011. Participants were followed up six times: in hospital, at home weekly until 4 weeks postpartum and by telephone at 8 weeks. Main outcome measures: Case definition 'nipple and breast thrush': burning nipple pain and breast pain (not related to mastitis); detection of Candida spp (using culture and PCR) in the mother's vagina, nipple or breast milk or in the baby's mouth; detection of S aureus in the mother' nipple or breast milk. Results: Women with the case definition of nipple/ breast thrush were more likely to have Candida spp in nipple/breast milk/baby oral samples (54%) compared to other women (36%, p=0.014). S aureus was common in nipple/breast milk/baby samples of women with these symptoms as well as women without these symptoms (82% vs 79%) (p=0.597). Time-to-event analysis examined predictors of nipple/breast thrush up to and including the time of data collection. Candida in nipple/breast milk/baby predicted incidence of the case definition (rate ratio (RR) 1.87 (95% CI 1.10 to 3.16, p=0.018). We do not have evidence that S aureus colonisation was a predictor of these symptoms (RR 1.53, 95% CI 0.88 to 2.64, p=0.13). Nipple damage was also a predictor of these symptoms, RR 2.30 (95% CI 1.19 to 4.43, p=0.012). In the multivariate model, with all three predictors, the RRs were very similar to the univariate RRs. This indicates that Candida and nipple damage are independent predictors of our case definition.

Medical diagnosis by fuzzy standard additive model with wavelets

This paper proposes a combination of fuzzy standard additive model (SAM) with wavelet features for medical diagnosis. Wavelet transformation is used to reduce the dimension of high-dimensional datasets. This helps to improve the convergence speed of supervised learning process of the fuzzy SAM, which has a heavy computational burden in high-dimensional data. Fuzzy SAM becomes highly capable when deployed with wavelet features. This combination remarkably reduces its computational training burden. The performance of the proposed methodology is examined for two frequently used medical datasets: the lump breast cancer and heart disease. Experiments are deployed with a five-fold cross validation. Results demonstrate the superiority of the proposed method compared to other machine learning methods including probabilistic neural network, support vector machine, fuzzy ARTMAP, and adaptive neuro-fuzzy inference system. Faster convergence but higher accuracy shows a win-win solution of the proposed approach.

Tumour but not stromal expression of β3 integrin is essential, and is required early, for spontaneous dissemination of bone-metastatic breast cancer.

Although many preclinical studies have implicated β3 integrin receptors (αvβ3 and αIIbβ3) in cancer progression, β3 inhibitors have shown only modest efficacy in patients with advanced solid tumours. The limited efficacy of β3 inhibitors in patients could arise from our incomplete understanding of the precise function of β3 integrin and, consequently, inappropriate clinical application. Data from animal studies are conflicting and indicate heterogeneity with respect to the relative contributions of β3-expressing tumour and stromal cell populations in different cancers. Here we aimed to clarify the function and relative contributions to metastasis of tumour versus stromal β3 integrin in clinically relevant models of spontaneous breast cancer metastasis, with particular emphasis on bone metastasis. We show that stable down-regulation of tumour β3 integrin dramatically impairs spontaneous (but not experimental) metastasis to bone and lung without affecting primary tumour growth in the mammary gland. Unexpectedly, and in contrast to subcutaneous tumours, orthotopic tumour vascularity, growth and spontaneous metastasis were not altered in mice null for β3 integrin. Tumour β3 integrin promoted migration, protease expression and trans-endothelial migration in vitro and increased vascular dissemination in vivo, but was not necessary for bone colonization in experimental metastasis assays. We conclude that tumour, rather than stromal, β3 expression is essential and is required early for efficient spontaneous breast cancer metastasis to bone and soft tissues. Accordingly, differential gene expression analysis in cohorts of breast cancer patients showed a strong association between high β3 expression, early metastasis and shorter disease-free survival in patients with oestrogen receptor-negative tumours. We propose that β3 inhibitors may be more efficacious if used in a neoadjuvant setting, rather than after metastases are established. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Bone-derived soluble factors and laminin-511 cooperate to promote migration, invasion and survival of bone-metastatic breast tumor cells

Tumor intrinsic and extrinsic factors are thought to contribute to bone metastasis but little is known about how they cooperate to promote breast cancer spread to bone. We used the bone-metastatic 4T1BM2 mammary carcinoma model to investigate the cooperative interactions between tumor LM-511 and bone-derived soluble factors in vitro. We show that bone conditioned medium cooperates with LM-511 to enhance 4T1BM2 cell migration and invasion and is sufficient alone to promote survival in the absence of serum. These responses were associated with increased secretion of MMP-9 and activation of ERK and AKT signaling pathways and were partially blocked by pharmacological inhibitors of MMP-9, AKT-1/2 or MEK. Importantly, pre-treatment of 4T1BM2 cells with an AKT-1/2 inhibitor significantly reduced experimental metastasis to bone in vivo. Promotion of survival and invasive responses by bone-derived soluble factors and tumor-derived LM-511 are likely to contribute to the metastatic spread of breast tumors to bone.

Transitioning to routine breast cancer risk assessment and management in primary care: what can we learn from cardiovascular disease?

To capitalise on advances in breast cancer prevention, all women would need to have their breast cancer risk formally assessed. With ~85% of Australians attending primary care clinics at least once a year, primary care is an opportune location for formal breast cancer risk assessment and management. This study assessed the current practice and needs of primary care clinicians regarding assessment and management of breast cancer risk. Two facilitated focus group discussions were held with 17 primary care clinicians (12 GPs and 5 practice nurses (PNs)) as part of a larger needs assessment. Primary care clinicians viewed assessment and management of cardiovascular risk as an intrinsic, expected part of their role, often triggered by practice software prompts and facilitated by use of an online tool. Conversely, assessment of breast cancer risk was not routine and was generally patient- (not clinician-) initiated, and risk management (apart from routine screening) was considered outside the primary care domain. Clinicians suggested that routine assessment and management of breast cancer risk might be achieved if it were widely endorsed as within the remit of primary care and supported by an online risk-assessment and decision aid tool that was integrated into primary care software. This study identified several key issues that would need to be addressed to facilitate the transition to routine assessment and management of breast cancer risk in primary care, based largely on the model used for cardiovascular disease.

Oral administration of iron-saturated bovine lactoferrin-loaded ceramic nanocapsules for breast cancer therapy and influence on iron and calcium metabolism

We determined the anticancer efficacy and internalization mechanism of our polymeric-ceramic nanoparticle system (calcium phosphate nanocores, enclosed in biodegradable polymers chitosan and alginate nanocapsules/nanocarriers [ACSC NCs]) loaded with iron-saturated bovine lactoferrin (Fe-bLf) in a breast cancer xenograft model. ACSC-Fe-bLf NCs with an overall size of 322±27.2 nm were synthesized. In vitro internalization and anticancer efficacy were evaluated in the MDA-MB-231 cells using multicellular tumor spheroids, CyQUANT and MTT assays. These NCs were orally delivered in a breast cancer xenograft mice model, and their internalization, cytotoxicity, biodistribution, and anticancer efficacy were evaluated. Chitosan-coated calcium phosphate Fe-bLf NCs effectively (59%, P≤0.005) internalized in a 1-hour period using clathrin-mediated endocytosis (P≤0.05) and energy-mediated pathways (P≤0.05) for internalization; 3.3 mg/mL of ACSC-Fe-bLf NCs completely disintegrated (~130-fold reduction, P≤0.0005) the tumor spheroids in 72 hours and 96 hours. The IC50 values determined for ACSC-Fe-bLf NCs were 1.69 mg/mL at 10 hours and 1.62 mg/mL after 20 hours. We found that Fe-bLf-NCs effectively (P≤0.05) decreased the tumor size (4.8-fold) compared to the void NCs diet and prevented tumor recurrence when compared to intraperitoneal injection of Taxol and Doxorubicin. Receptor gene expression and micro-RNA analysis confirmed upregulation of low-density lipoprotein receptor and transferrin receptor (liver, intestine, and brain). Several micro-RNAs responsible for iron metabolism upregulated with NCs were identified. Taken together, orally delivered Fe-bLf NCs offer enhanced antitumor activity in breast cancer by internalizing via low-density lipoprotein receptor and transferrin receptor and regulating the micro-RNA expression. These NCs also restored the body iron and calcium levels and increased the hematologic counts.

Targeting VEGF with LNA-stabilized G-rich oligonucleotide for efficient breast cancer inhibition

In this study, we investigated the efficacy of an LNA (locked nucleic acid)-modified DNA aptamer named RNV66 targeting VEGF against various breast cancer cell lines. Our results demonstrate that RNV66 efficiently inhibits breast cancer cell proliferation both in vitro and in vivo. Introduction of LNA nucleotides were crucial for higher efficacy. Furthermore, the binding interaction of RNV66 with VEGF was investigated using molecular dynamic simulations leading to the first computational model of the LNA aptamer-VEGF complex blocking its interaction with VEGF-receptor.

Multimodal iron oxide (Fe₃O₄)-saturated lactoferrin nanocapsules as nanotheranostics for real-time imaging and breast cancer therapy of claudin-low, triple-negative (ER-/PR-/HER2-)

AIM: To unravel the multimodal nanotheranostic ability of Fe3O4-saturated bovine lactoferrin nanocapsules (FebLf NCs) in claudin-low, triple-negative breast cancer model. MATERIALS & METHODS: Xenograft study was performed to examine biocompatibility, antitumor efficacy and multimodal nanotheranostic action in combination with near-infrared live mice imaging. RESULTS: FebLf NCs exhibited a size range of 80 nm ± 5 nm with observed superparamagnetism. FebLf NCs successfully internalized into breast cancer cells through receptor-mediated endocytosis and induced apoptosis through the downregulation of inhibitor of apoptosis survivin and livin proteins. Investigations revealed a remarkable biocompatibility, anticancer efficacy of the FebLf NCs. Near-infrared imaging observations confirmed selective localization of multimodal FebLf NCs at the tumor site and lead to time-dependent reduction of tumor growth. CONCLUSION: FebLf NCs can be safe, biocompatible nanotheranostic approach for real-time imaging and monitoring the effect of drugs in real time and have potentials in future clinical trials.