TGF-β Biology in Mammary Development and Breast Cancer

Transforming growth factor-β1 (TGF-β) was first implicated in mammary epithelial development by Daniel and Silberstein in 1987 and in breast cancer cells and hormone resistance by Lippman and colleagues in 1988. TGF-β is critically important for mammary morphogenesis and secretory function through specific regulation of epithelial proliferation, apoptosis, and extracellular matrix. Differential TGF-β effects on distinct cell types are compounded by regulation at multiple levels and the influence of context on cellular responses. Studies using controlled expression and conditional-deletion mouse models underscore the complexity of TGF-β biology across the cycle of mammary development and differentiation. Early loss of TGF-β growth regulation in breast cancer evolves into fundamental deregulation that mediates cell interactions and phenotypes driving invasive disease. Two outstanding issues are to understand the mechanisms of biological control in situ and the circumstances by which TGF-β regulation is subverted in neoplastic progression.The discovery of a “transforming growth factor” in normal tissue and serum in the early 1980s rapidly led to the identification of a large family of polypeptides whose action is involved in all aspects of development, homeostasis, and cancer (Moses and Roberts 2008). The activity of transforming growth factor-β1 (TGF-β) was first implicated in mammary epithelial development in 1987 by a canonical experiment by Daniel and Silberstein. Pellets containing TGF-β implanted into mouse mammary gland during ductal morphogenesis were shown to induce rapid regression of advancing endbuds, which was among the first demonstration of its potent inhibitory, rather than transforming, activity (Silberstein and Daniel 1987). However, soon after, Lippman and colleagues showed that TGF-β was produced by breast cancer cells, which in turn contributed to their hormone resistance (Knabbe et al. 1987). These two diametrically opposed actions have continued to fascinate those studying its sundry roles in mammary biology and breast cancer. After nearly a quarter century, this brief article underscores the major two themes in mammary biology: Although TGF-β orchestrates tissue composition and critical controls during mammary development, its subversion during cancer progressively undermines homeostasis and actively drives malignancy.     

Detection of RASSF1A and RAR? Hypermethylation in Serum DNA from Breast Cancer Patients

Breast cancer is fast emerging as the leading cancer amongst females, especially in young females in metropolitan cities in India. The epigenetic alterations involved in the onset and progression of breast cancer may serve as biomarkers for early detection and prognosis of the disease. Furthermore, using body fluids such as serum offers a non-invasive method to procure multiple samples for such analyses. In this study, we examined methylation status of two normally unmethylated but biologically significant cancer genes, RAS association domain family protein 1A (RASSF1A) and Retionic acid receptor ? (RAR?) by Methylation Specific PCR (MSP) in invasive ductal carcinomas of the breast and paired serum DNA. RASSF1A was found to be methylated in 17 of 20 (85%) breast tumors; while sera from 15 of 20 (75%) of the patients showed concordant methylated RASSF1A, with a sensitivity of 88%. RAR? was methylated in 2/20 (10%) breast tumors. A gene unmethylated in the tumor DNA was always found to be unmethylated in the matched serum DNA for both RASSF1A and RAR? genes; hence specificity was 100%. Immunohistochemical analysis of RAR? protein in 15 breast carcinoma patients harboring unmethylated RAR? in tumors and serum DNA showed the expression of RAR? protein in tumors and paired normal breast tissues, confirming the MSP findings, suggesting that RAR? promoter is functional in these cases. This study underscores the potential utility of DNA methylation based screening of serum, a readily accessible body fluid, as a surrogate marker for early detection of breast cancer.      

United States Acculturation and Cancer Patients’ End-of-Life Care

Background

Culture shapes how people understand illness and death, but few studies examine whether acculturation influences patients’ end-of-life treatment preferences and medical care.

Methods and Findings

In this multi-site, prospective, longitudinal cohort study of terminally-ill cancer patients and their caregivers (n = 171 dyads), trained interviewers administered the United States Acculturation Scale (USAS). The USAS is a 19-item scale developed to assess the degree of “Americanization” in first generation or non-US born caregivers of terminally-ill cancer patients. We evaluated the internal consistency, concurrent, criterion, and content validity of the USAS. We also examined whether caregivers’ USAS scores predicted patients’ communication, treatment preferences, and end-of-life medical care in multivariable models that corrected for significant confounding influences (e.g. education, country of origin, English proficiency). The USAS measure was internally consistent (Cronbach α = 0.98); and significantly associated with US birthplace (r = 0.66, P<0.0001). USAS scores were predictive of patients’ preferences for prognostic information (AOR = 1.31, 95% CI:1.00–1.72), but not comfort asking physicians’ questions about care (AOR 1.23, 95% CI:0.87–1.73). They predicted patients’ preferences for feeding tubes (AOR = 0.68, 95% CI:0.49–0.99) and wish to avoid dying in an intensive care unit (AOR = 1.36, 95% CI:1.05–1.76). Scores indicating greater acculturation were also associated with increased odds of patient participation in clinical trials (AOR = 2.20, 95% CI:1.28–3.78), compared with lower USAS scores, and greater odds of patients receiving chemotherapy (AOR = 1.59, 95% CI:1.20–2.12).

Conclusion

The USAS is a reliable and valid measure of “Americanization” associated with advanced cancer patients’ end-of-life preferences and care. USAS scores indicating greater caregiver acculturation were associated with increased odds of patient participation in cancer treatment (chemotherapy, clinical trials) compared with lower scores. Future studies should examine the effects of acculturation on end-of-life care to identify patient and provider factors that explain these effects and targets for future interventions to improve care (e.g., by designing more culturally-competent health education materials).     

Angiogenic Activity of Breast Cancer Patients’ Monocytes Reverted by Combined Use of Systems Modeling and Experimental Approaches

Angiogenesis plays a key role in tumor growth and cancer progression. TIE-2-expressing monocytes (TEM) have been reported to critically account for tumor vascularization and growth in mouse tumor experimental models, but the molecular basis of their pro-angiogenic activity are largely unknown. Moreover, differences in the pro-angiogenic activity between blood circulating and tumor infiltrated TEM in human patients has not been established to date, hindering the identification of specific targets for therapeutic intervention. In this work, we investigated these differences and the phenotypic reversal of breast tumor pro-angiogenic TEM to a weak pro-angiogenic phenotype by combining Boolean modelling and experimental approaches. Firstly, we show that in breast cancer patients the pro-angiogenic activity of TEM increased drastically from blood to tumor, suggesting that the tumor microenvironment shapes the highly pro-angiogenic phenotype of TEM. Secondly, we predicted in silico all minimal perturbations transitioning the highly pro-angiogenic phenotype of tumor TEM to the weak pro-angiogenic phenotype of blood TEM and vice versa. In silico predicted perturbations were validated experimentally using patient TEM. In addition, gene expression profiling of TEM transitioned to a weak pro-angiogenic phenotype confirmed that TEM are plastic cells and can be reverted to immunological potent monocytes. Finally, the relapse-free survival analysis showed a statistically significant difference between patients with tumors with high and low expression values for genes encoding transitioning proteins detected in silico and validated on patient TEM. In conclusion, the inferred TEM regulatory network accurately captured experimental TEM behavior and highlighted crosstalk between specific angiogenic and inflammatory signaling pathways of outstanding importance to control their pro-angiogenic activity. Results showed the successful in vitro reversion of such an activity by perturbation of in silico predicted target genes in tumor derived TEM, and indicated that targeting tumor TEM plasticity may constitute a novel valid therapeutic strategy in breast cancer.     

Heterogeneity of ERα and ErbB2 Status in Cell Lines and Circulating Tumor Cells of Metastatic Breast Cancer Patients

Hormone therapy and anti-ErbB2 therapies are prescribed according to the hormone receptor [estrogen receptor α (ERα)/progesterone receptor] and ErbB2 status of the initial tumor, but it appears that circulating tumor cells (CTCs) and, consequently, the metastatic cells may have a different receptor status. As an attempt to meet the crucial need for identification of the subpopulation of patients that will benefit from more individualized therapies, rapidly evolving therapies should allow a profiling of the tumors and/or of the CTCs. We established a triple fluorescence staining using eight cell lines to visualize the CTCs (cytokeratin detection) and then to define their individual ERα and ErbB2 status. Afterward, we used this method for blood samples from 26 metastatic breast cancer patients. We identified major differences of ERα levels between the cell lines and even within one cell line. For the metastatic patients, we detected and characterized CTCs in 38.5% of the patients with a total of 92 CTCs. We could demonstrate that at least 69.6% of the CTCs exhibit an ERα and/or ErbB2 status different from the status of the primary tumor and that the CTCs from only 30% of the patients had no change of receptor status. Strikingly, heterogeneities of the status, aggregation, and size clearly appear within the CTCs. The data we generated outline the importance of a profiling not only of tumors but also of CTCs to establish individualized treatments. CTCs may then appear as new prognosis and treatment marker for both metastatic and adjuvant breast cancers.     

Cell–Matrix Interactions in Mammary Gland Development and Breast Cancer

The mammary gland is an organ that at once gives life to the young, but at the same time poses one of the greatest threats to the mother. Understanding how the tissue develops and functions is of pressing importance in determining how its control mechanisms break down in breast cancer. Here we argue that the interactions between mammary epithelial cells and their extracellular matrix (ECM) are crucial in the development and function of the tissue. Current strategies for treating breast cancer take advantage of our knowledge of the endocrine regulation of breast development, and the emerging role of stromal–epithelial interactions (Fig. 1). Focusing, in addition, on the microenvironmental influences that arise from cell–matrix interactions will open new opportunities for therapeutic intervention. We suggest that ultimately a three-pronged approach targeting endocrine, growth factor, and cell-matrix interactions will provide the best chance of curing the disease.Cellular interactions with the ECM are one of the defining features of metazoans (Huxley-Jones et al. 2007). Matrix proteins are among the most abundant in the body, and are integral components of cell regulation and developmental programs operating in all tissues. They provide structure and support to tissues, and they interact with cells through diverse receptors to guide development, patterning, and cell fate decisions (Streuli 2009). Together with cytokines and growth factors, and cell–cell interactions, the ECM determines whether cells survive, proliferate, differentiate, or migrate, and it influences cell shape and polarity (Streuli and Akhtar 2009). Cell–ECM interactions also are central in the assembly of the matrix itself, and in determining ECM organization and rigidity (Kadler et al. 2008; Kass et al. 2007). The cell–matrix interface is therefore pivotal in controlling both cell function and tissue structure, which together build organs into operational structures. Thus, elucidating precisely how the matrix directs cell phenotype is crucial for understanding mechanisms of development and disease.Mammary gland tissue contains epithelium and stroma (​(Fig.Fig. 2). Mammary epithelial cells (MEC) form collecting ducts and, in pregnancy and lactation, milk-secreting alveoli (or lobules). The mammary epithelium is bilayered, with the inner luminal cells facing a central apical cavity and surrounded by the outer basal, myoepithelial cells. It also harbors stem and progenitor cells, which are the source of both luminal and myoepithelial cells (Visvader 2009). The epithelium is ensheathed by one of the main types of ECM, basement membrane (BM), which separates epithelium from stroma, and profoundly influences the development and biology of the gland (Streuli 2003). The stroma includes fibrous connective tissue ECM proteins, and a wide variety of cell types, including inter- and intralobular fibroblasts, adipocytes, endothelial cells, and innate immune cells (both macrophages and mast cells). The stroma is the support network for the epithelium, providing both nutrients and blood supply, and immune defenses, as well as physical structure to the gland. Importantly, each of the different stromal cell types secrete instructive signals that are crucial for various aspects of the development and function of the epithelium (Sternlicht 2006).Open in a separate windowFigure 1.Mammary gland development. Whole mounts of (A) virgin and (B) mid-pregnant mouse mammary gland. The thin, branched epithelial ducts that are characteristic of nonpregnant gland undergo dramatic alterations in pregnancy, when new types of epithelial structures, the milk-producing alveoli, emerge. The huge amount of proliferation that accompanies this change occurs in a discrete and controlled fashion. The formation of ducts and alveoli is under three types of environmental control. The first is long-range endocrine hormones, which includes estrogen, progesterone, glucocorticoids, and prolactin. The second is locally acting growth factors, which arise from stromal–epithelial conversation, and includes amphiregulin, FGF, HGF, and IGF. Finally, microenvironmental adhesive signals from adjacent cells (e.g., via cadherins) and from the ECM (e.g., integrin) have an equally central role in all aspects of mammary development and function. Importantly, the proliferation that occurs in breast cancer is not well controlled, indicating not only defects in growth signaling, but also in cellular organization. Chronologically, breast cancer drugs were initially developed against endocrine regulators, e.g., estrogen, and more recently against the stromal/epithelial regulators, e.g., receptor tyrosine kinases. A complete control of the disease will only happen when therapies targeting the microenvironmental adhesion breast regulators, e.g., cell–matrix interactions, are formulated, and used in combination.Open in a separate windowFigure 2.Ducts and alveoli in early pregnancy. Transverse section of ducts surrounded by a thick layer of collagenous (stromal) connective tissue containing fibroblasts and the fat pad. Also visible are small alveoli, which fill the fat pad by the time the gland lactates, but note that they are not surrounded collagen. A capillary is evident, and macrophages and mast cells are also present, though they require specific staining to visualize. A basement membrane is present directly at the basal surface of both ductal and alveolar epithelium (see Fig. 3).BMs surround three cell types in the mammary gland: the epithelium, the endothelium of the vasculature, and adipocytes (Fig. 3). These ECMs are thin, ∼100-nm thick sheets of glycoproteins and proteoglycans, which are constructed around an assembled polymer of laminins and a cross-linked network of collagen IV fibrils (Yurchenco and Patton 2009). Laminins form αβγ trimers, and in the breast at least four distinct isoforms are present: laminin-111, -322, and -511 and -521 (previously known as LM-1, 5, 10, and 11) (Aumailley et al. 2005; Prince et al. 2002). Similarly, BM proteoglycans are diverse and show complexity in their GAG chain modifications that vary with development of the mammary gland, though the major species is perlecan (Delehedde et al. 2001). BM proteins interact with MEC via integrins and transmembrane proteoglycans dystroglycan and syndecan, which all couple to the cytoskeleton and assemble signaling platforms to control cell fate (Barresi and Campbell 2006; Morgan et al. 2007). The best-studied MEC BM receptors are integrins, which are αβ heterodimers: they include receptors for collagen (α1β1 and α2β1), LM-111, -511, -521 (α3β1, α6β1, and α6β4), LM-322 (α3β1 and α6β4), and in some MECs fibronectin and vitronectin (α5β1 and β3 integrins) (Naylor and Streuli 2006). BM proteoglycans have a further signaling role via their capacity to bind growth factors and cytokines: They act both as a reservoir and a delivery vehicle to GF receptors, thereby controlling the passage of GFs across the BM (Iozzo 2005). Because of these diverse roles, the BM is a dominant regulator of the mammary epithelial phenotype.Open in a separate windowFigure 3.Alveolar and ductal architecture of breast epithelia shown through fluorescence and histological images. (A) An alveolus from a lactating mammary gland, showing luminal epithelial cells with cell–cell adhesion junctions (green, E-cadherin) and cell–matrix interactions (red, laminin-111). The central lumen is where milk collects. (B) The duct of a nonpregnant gland is stained with an antibody to laminin (brown) and counterstained with hematoxylin. Note that the laminin-containing basement membrane surrounds the ductal epithelial cells, and outside this lie collagenous connective tissue and adipocytes. Figure B courtesy of Dr. Rama Khokha.Apart from the endothelium and adipocytes, which contact BMs, the mammary stromal cells are mostly solitary and embedded within a fibrous ECM. Stromal matrix components include collagens type I and III, proteoglycans and hyaluronic acid, fibronectin and tenascins, and the composition varies with development and pregnancy (Schedin et al. 2004). Not a great deal is known about the specific interactions between breast stromal cells and their ECM, or how the matrix composition and density determines stromal cell function. However, it is becoming evident that the stromal matrix exerts a powerful influence on malignant breast epithelial cells, which invade the stroma and are further transformed by exposure to this distinct microenvironment (Kumar and Weaver 2009; Streuli 2006).In this article we focus on cell–matrix interactions within mammary epithelium, and reveal known and possible mechanisms for its control on ductal development, alveolar function, and cancer progression.     

Relationships Between Element Contents in Polish Children’s and Adolescents’ Hair

Environment, sex, and age are the main factors which determine the elemental composition of hair. The objective of the study is to determine the contents of calcium (Ca), magnesium (Mg), zinc (Zn), copper (Cu), iron (Fe), lead (Pb), and cadmium (Cd) in girls’ and boys’ hair in five age groups (within 1–19-year range) corresponding to successive human ontogenesis phases as well as to evaluate the relationships between these elements. Quantitative analysis has been carried out using atomic absorption spectrometry (AAS). Experimental results were analyzed using classic and principal component (PCA) statistical analyses. In particular, differences between contents of particularly Ca, Mg, and Zn in girls’ and boys’ hair were found, and substantial differences between age groups were stated. In general, larger amounts of Ca, Mg, and Zn as compared to boys’ hair have been observed for girls’ hair and higher toxic element (Pb, Cd) contents for boys were measured in some age groups. An increasing trend was found for bioelements (Ca, Mg, Zn) both for girls and boys in all age groups, while for Cu and Fe content, changes are insignificant and even decreasing for teenagers. The most frequently correlating element pairs are Ca–Mg, Ca–Zn, Mg–Zn, and Pb–Cd. Classic and PCA statistics show, in general, a satisfactory consistence. The elemental composition of hair varies depending on the gender and age of children and young people.     

γ-Glutamyltransferase and Breast Cancer Risk Beyond Alcohol Consumption and Other Life Style Factors – A Pooled Cohort Analysis

Objective

Elevated γ-Glutamyltransferase serum levels are associated with increased risk of overall cancer incidence and several site-specific malignancies. In the present prospective study we report on the associations of serum γ-Glutamyltransferase with the risk of breast cancer in a pooled population-based cohort considering established life style risk factors.

Methods

Two cohorts were included in the present study, i.e. the Vorarlberg (n = 97,268) and the Malmoe cohort (n = 9,790). Cox proportional hazards regression models were fitted to estimate HRs for risk of breast cancer.

Results

In multivariate analysis adjusted for age, body mass index and smoking status, women with γ-Glutamyltransferase levels in the top quartile were at significantly higher risk for breast cancer compared to women in the lowest quartile (HR 1.21, 95% CI 1.09 to 1.35; p = 0.005). In the subgroup analysis of the Malmoe cohort, γ-Glutamyltransferase remained an independent risk factor for breast cancer when additionally considering alcohol intake. A statistically significant increase in risk was seen in women with γ-Glutamyltransferase-levels in the top versus lowest quartile in a multivariate model adjusted for age, body mass index, smoking status, physical activity, parity, oral contraceptive-use and alcohol consumption (HR 1.37, 95% CI 1.11–1.69, p = 0.006).

Conclusion

Our findings identified γ-Glutamyltransferase as an independent risk factor for breast cancer beyond the consumption of alcohol and other life style risk factors.     

Copper and Zinc in the Serum,Urine, and Hair of Patients with Wilson’s Disease Treated with Penicillamine and Zinc

The purpose of this study was to determine the different levels of copper and zinc in the serum, urine, and scalp hair of patients with Wilson’s disease receiving different, currently accepted methods of treatment to reduce the copper load (penicillamine—group 1, n = 8; zinc—group 2, n = 8; penicillamine+zinc—group 3, n = 8). Blood, urine, and hair samples were collected from the patients. All three treatments resulted in a significant decrease of the serum copper levels. Significantly increased levels of zinc in the serum were detected in the patients in groups 2 and 3 (19.1 and 18.8 μmol/l, respectively; p < 0.05). Copper excretion in the urine significantly increased during its administration to groups 1 and 3 (11.5 and 7.94 μmol/24 h respectively; p < 0.001) due to the effect of penicillamine. The administration of zinc as monotherapy (group 2) or in combination with penicillamine (group 3) led to an increase of its excretion (25.3 and 22.4 μmol/24 h, respectively; p < 0.01). Only an insignificant rise of the copper content in the hair was found in all three groups of patients. The content of zinc in the hair did not differ significantly in any of the groups in comparison with the control group.     

BRCA1 and Estrogen Receptor α Expression Regulation in Breast Cancer Cells

Molecular Biology - BRCA1 (breast cancer 1) protein is involved in the genome stability maintenance participating in homologous recombination-dependent DNA repair. Disruption of BRCA1 functioning...     

Development and Validation of Nomograms for Predicting Overall and Breast Cancer–Specific Survival in Young Women with Breast Cancer: A Population-Based Study

INTRODUCTION: The objective of current study was to develop and validate comprehensive nomograms for predicting the survival of young women with breast cancer. METHODS: Women aged <40 years diagnosed with invasive breast cancer between 1990 and 2010 were selected from the Surveillance, Epidemiology, and End Results database and randomly divided into training (n = 12,465) and validation (n = 12,424) cohorts. A competing-risks model was used to estimate the probability of breast cancer–specific survival (BCSS). We identified and integrated significant prognostic factors for overall survival (OS) and BCSS to construct nomograms. The performance of the nomograms was assessed with respect to calibration, discrimination, and risk group stratification. RESULTS: The entire cohort comprised 24,889 patients. The 5- and 10-year probabilities of breast cancer–specific mortality were 11.6% and 20.5%, respectively. Eight independent prognostic factors for both OS and BCSS were identified and integrated for the construction of the nomograms. The calibration curves showed optimal agreement between the predicted and observed probabilities. The C-indexes of the nomograms in the training cohort were higher than those of the TNM staging system for predicting OS (0.724 vs 0.694; P < .001) and BCSS (0.733 vs 0.702; P < .001). Additionally, significant differences in survival were observed in patients stratified into different risk groups within respective TNM categories. CONCLUSIONS: We developed and validated novel nomograms that can accurately predict OS and BCSS in young women with breast cancer. These nomograms may help clinicians in making decisions on an individualized basis.     

Treatment Strategies and Survival of Older Breast Cancer Patients – An International Comparison between the Netherlands and Ireland

ObjectivesForty percent of breast cancers occur among older patients. Unfortunately, there is a lack of evidence for treatment guidelines for older breast cancer patients. The aim of this study is to compare treatment strategy and relative survival for operable breast cancer in the elderly between The Netherlands and Ireland.ResultsOverall, 41,055 patients from The Netherlands and 5,826 patients from Ireland were included. Overall, more patients received guideline-adherent locoregional treatment in The Netherlands, overall (80% vs. 68%, adjusted p<0.001), stage I (83% vs. 65%, p<0.001), stage II (80% vs. 74%, p<0.001) and stage III (74% vs. 57%, P<0.001) disease. On the other hand, more systemic treatment was provided in Ireland, where endocrine therapy was prescribed to 92% of hormone receptor-positive patients, compared to 59% in The Netherlands. In The Netherlands, only 6% received chemotherapy, as compared 24% in Ireland. But relative survival was poorer in Ireland (5 years relative survival 89% vs. 83%), especially in stage II (87% vs. 85%) and stage III (61% vs. 58%) patients.ConclusionTreatment for older breast cancer patients differed significantly on all treatment modalities between The Netherlands and Ireland. More locoregional treatment was provided in The Netherlands, and more systemic therapy was provided in Ireland. Relative survival for Irish patients was worse than for their Dutch counterparts. This finding should be a strong recommendation to study breast cancer treatment and survival internationally, with the ultimate goal to equalize the survival rates for breast cancer patients across Europe.     

Assessing Plasma Levels of Selenium,Copper, Iron and Zinc in Patients of Parkinson’s Disease

Trace elements have been recognized to play an important role in the development of Parkinson’s disease (PD). However, it is difficult to precisely identify the relationship between these elements and the progression of PD because of an insufficient number of patients. In this study, quantifications of selenium (Se), copper (Cu), iron (Fe) and zinc (Zn) by atomic absorption spectrophotometry were performed in plasma from 238 PD patients and 302 controls recruited from eastern China, which is so far the largest cohort of PD patients and controls for measuring plasma levels of these elements. We found that plasma Se and Fe concentrations were significantly increased whereas Cu and Zn concentrations decreased in PD patients as compared with controls. Meanwhile, these four elements displayed differential changes with regard to age. Linear and logistic regression analyses revealed that both Fe and Zn were negatively correlated with age in PD patients. Association analysis suggests that lower plasma Se and Fe levels may reduce the risk for PD, whereas lower plasma Zn is probably a PD risk factor. Finally, a model was generated to predict PD patients based on the plasma concentrations of these four trace elements as well as other features such as sex and age, which achieved an accuracy of 80.97±1.34% using 10-fold cross-validation. In summary, our data provide new insights into the roles of Se, Cu, Fe and Zn in PD progression.