Notch in mammary gland development and breast cancer

Notch signaling has been implicated in many processes including cell fate determination and oncogenesis. In mice, the Notch1 and Notch4 genes are both targets for insertion and rearrangement by the mouse mammary tumor virus and these mutations promote epithelial mammary tumorigenesis. Moreover, expression of a constitutively active form of Notch4 in mammary epithelial cells inhibits epithelial differentiation and leads to tumor formation in this organ. These data implicate the Notch pathway in breast tumorigenesis and provide the foundation for future experiments that will aid in our understanding of the role of Notch in human breast cancer development. Here, we review studies of mammary tumorigenesis induced by Notch in mouse and in vitro culture models providing evidence that Notch activation is a causal factor in human breast cancer.     

Key signalling nodes in mammary gland development and cancer. Mitogen-activated protein kinase signalling in experimental models of breast cancer progression and in mammary gland development

Seven classes of mitogen-activated protein kinase (MAPK) intracellular signalling cascades exist, four of which are implicated in breast disease and function in mammary epithelial cells. These are the extracellular regulated kinase (ERK)1/2 pathway, the ERK5 pathway, the p38 pathway and the c-Jun N-terminal kinase (JNK) pathway. In some forms of human breast cancer and in many experimental models of breast cancer progression, signalling through the ERK1/2 pathway, in particular, has been implicated as being important. We review the influence of ERK1/2 activity on the organised three-dimensional association of mammary epithelial cells, and in models of breast cancer cell invasion. We assess the importance of epidermal growth factor receptor family signalling through ERK1/2 in models of breast cancer progression and the influence of ERK1/2 on its substrate, the oestrogen receptor, in this context. In parallel, we consider the importance of these MAPK-centred signalling cascades during the cycle of mammary gland development. Although less extensively studied, we highlight the instances of signalling through the p38, JNK and ERK5 pathways involved in breast cancer progression and mammary gland development.     

The role of maintenance proteins in the preservation of epithelial cell identity during mammary gland remodeling and breast cancer initiation

During normal postnatal mammary gland development and adUlt remodeling related to tne menstrual cycle, pregnancy, and lactation, ovarian hormones and peptide growth factors contribute to the delineation of a definite epithelial cell identity. This identity is maintained during cell replication in a heritable but DNAindependent manner. The preservation of cell identity is fundamental, especially when cells must undergo changes in response to intrinsic and extrinsic signals. The maintenance proteins, which are required for cell identity preservation, act epigenetically by regulating gene expression through DNA methylation, histone modification, and chromatin remodeling. Among the maintenance proteins, the Trithorax （TrxG） and Polycomb （PcG） group proteins are the best characterized. In this review, we summarize the structures and activities of the TrxG and PcG complexes and describe their pivotal roles in nuclear estrogen receptor activity. In addition, we provide evidence that perturbations in these epigenetic regulators are involved in disrupting epithelial cell identity, mammary gland remodeling, and breast cancer initiation.     

The breast proto-oncogene,HRGalpha regulates epithelial proliferation and lobuloalveolar development in the mouse mammary gland

Members of the EGF family of growth factors play critical roles during normal and neoplastic breast development. EGF family member HRGalpha is the only HRG1 isoform expressed in the mouse mammary gland and our previous experiments suggest that HRG1 has a unique role in mammary development. To determine the function of HRGalpha activity during mouse mammary gland development, we generated a HRGalpha-deficient mouse strain. Unlike mice with HRG1 or isoform specific HRGbeta gene deletions, HRGalpha-null mice survive to adulthood. HRGalpha-deficient mice display pronounced defects in mammary gland lobuloalveolar development at 17 days of pregnancy and 3 days post-partum. Terminal and lateral ductal alveoli were condensed and alveolar outgrowth during pregnancy was severely impaired. A dramatic reduction in beta-casein expression accompanied defective alveolar development in the HRGalpha-null mice, as determined by in situ hybridization and Northern blot analysis of HRGalpha-deficient mammary glands at 3 days post-partum. Expression of the milk-protein genes WAP and alpha-lactalbumin was not adversely affected. In situ incorporation of BrdU demonstrated that epithelial proliferation was significantly curtailed in mammary glands of HRGalpha-deficient mice at 17 days post-coitus and 3 days post-partum. These results demonstrate that HRGalpha is an important mammary gland mitogen regulating alveolar development and lactogenesis.     

Role of JNK in mammary gland development and breast cancer

cJun NH(2)-terminal kinase (JNK) signaling has been implicated in the developmental morphogenesis of epithelial organs. In this study, we employed a compound deletion of the murine Jnk1 and Jnk2 genes in the mammary gland to evaluate the requirement for these ubiquitously expressed genes in breast development and tumorigenesis. JNK1/2 was not required for breast epithelial cell proliferation or motility. However, JNK1/2 deficiency caused increased branching morphogenesis and defects in the clearance of lumenal epithelial cells. In the setting of breast cancer development, JNK1/2 deficiency significantly increased tumor formation. Together, these findings established that JNK signaling is required for normal mammary gland development and that it has a suppressive role in mammary tumorigenesis.     

Role of dietary fatty acids in mammary gland development and breast cancer

Breast cancer is the most common cancer among women worldwide. Estimates suggest up to 35% of cases may be preventable through diet and lifestyle modification. Growing research on the role of fats in human health suggests that early exposure in life to specific fatty acids, when tissues are particularly sensitive to their environment, can have long-term health impacts. The present review examines the role of dietary fat in mammary gland development and breast cancer throughout the lifecycle. Overall, n-3 polyunsaturated fatty acids have promising cancer-preventive effects when introduced early in life, and warrant further research to elucidate the mechanisms of action.     

Intrauterine environment, mammary gland mass and breast cancer risk

Two intimately linked hypotheses on breast cancer etiology are described. The main postulate of the first hypothesis is that higher levels of pregnancy estrogens and other hormones favor the generation of a higher number of susceptible stem cells with compromised genomic stability. The second hypothesis postulates that the mammary gland mass, as a correlate of the number of cells susceptible to transformation, is an important determinant of breast cancer risk. A simple integrated etiological model for breast cancer is presented and it is indicated that the model accommodates most epidemiological aspects of breast cancer occurrence and natural history.     

Intrauterine environment,mammary gland mass and breast cancer risk

Two intimately linked hypotheses on breast cancer etiology are described. The main postulate of the first hypothesis is that higher levels of pregnancy estrogens and other hormones favor the generation of a higher number of susceptible stem cells with compromised genomic stability. The second hypothesis postulates that the mammary gland mass, as a correlate of the number of cells susceptible to transformation, is an important determinant of breast cancer risk. A simple integrated etiological model for breast cancer is presented and it is indicated that the model accommodates most epidemiological aspects of breast cancer occurrence and natural history.     

ERalpha-CITED1 co-regulated genes expressed during pubertal mammary gland development: implications for breast cancer prognosis

Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.     

Aluminium chloride promotes tumorigenesis and metastasis in normal murine mammary gland epithelial cells

Aluminium salts, present in many industrial products of frequent use like antiperspirants, anti‐acid drugs, food additives and vaccines, have been incriminated in contributing to the rise in breast cancer incidence in Western societies. However, current experimental evidence supporting this hypothesis is limited. For example, no experimental evidence that aluminium promotes tumorigenesis in cultured mammary epithelial cells exists. We report here that long‐term exposure to concentrations of aluminium—in the form of aluminium chloride (AlCl₃)—in the range of those measured in the human breast, transform normal murine mammary gland (NMuMG) epithelial cells in vitro as revealed by the soft agar assay. Subcutaneous injections into three different mouse strains with decreasing immunodeficiency, namely, NOD SCID gamma (NSG), NOD SCID or nude mice, revealed that untreated NMuMG cells form tumors and metastasize, to a limited extent, in the highly immunodeficient and natural killer (NK) cell deficient NSG strain, but not in the less permissive and NK cell competent NOD SCID or nude strains. In contrast, NMuMG cells transformed in vitro by AlCl₃ form large tumors and metastasize in all three mouse models. These effects correlate with a mutagenic activity of AlCl₃. Our findings demonstrate for the first time that concentrations of aluminium in the range of those measured in the human breast fully transform cultured mammary epithelial cells, thus enabling them to form tumors and metastasize in well‐established mouse cancer models. Our observations provide experimental evidence that aluminium salts could be environmental breast carcinogens.     

The Septin 9 (MSF) gene is amplified and overexpressed in mouse mammary gland adenocarcinomas and human breast cancer cell lines

The expression of polyomavirus middle T antigen under the control of the mouse mammary tumor virus promoter in transgenic mice results in the induction of aggressive mammary gland adenocarcinomas at an early age. We screened 26 tumors for chromosomal aneuploidies using SKY and CGH. In 70% of the tumor samples we could detect high-level copy number gains, which mapped to chromosome band 11E2, a region orthologous to human 17q25.3. We then identified a bacterial artificial chromosome clone that labeled double-minute chromosomes found in the tumor metaphases. This bacterial artificial chromosome clone showed sequence homology to a member of the septin gene family. Real-time PCR analysis revealed a consistently increased expression of septin 9 (Sept9), not only in polyomavirus middle T antigen-induced, but in a wide variety of mouse models of breast cancer. Six of 9 human tumor cell lines also revealed elevated expression levels of Sept9. The family of septin genes is involved in a plethora of cellular processes, including cytokinesis in yeast and vesicle transport, and possesses GTPase activity. We identified down-regulation of Thsp1- and Bax-regulated apoptotic response in those tumors with Sept9 overexpression, an effect that could be reversed by inhibiting Sept9 expression using transfection with small interference RNA. Our results now suggest that signaling via members of the septin family plays a novel and common role in breast tumorigenesis.     

Lipidomic approach to identify patterns in phospholipid profiles and define class differences in mammary epithelial and breast cancer cells

Breast cancer is the leading cause of cancer-related deaths in women. Altered cellular functions of cancer cells lead to uncontrolled cellular growth and morphological changes. Cellular biomembranes are intimately involved in the regulation of cell signaling; however, they remain largely understudied. Phospholipids (PLs) are the main constituents of biological membranes and play important functional, structural and metabolic roles. The aim of this study was to establish if patterns in the PL profiles of mammary epithelial cells and breast cancer cells differ in relation to degree of differentiation and metastatic potential. For this purpose, PLs were analyzed using a lipidomic approach. In brief, PLs were extracted using Bligh and Dyer method, followed by a separation of PL classes by thin layer chromatography, and subsequent analysis by mass spectrometry (MS). Differences and similarities were found in the relative levels of PL content between mammary epithelial and breast cancer cells and between breast cancer cells with different levels of aggressiveness. When compared to the total PL content, phosphatidylcholine levels were reduced and lysophosphatydilcholines increased in the more aggressive cancer cells; while phosphatidylserine levels remained unchanged. MS analysis showed alterations in the classes of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, and phosphatidylinositides. In particular, the phosphatidylinositides, which are signaling molecules that affect proliferation, survival, and migration, showed dramatic alterations in their profile, where an increase of phosphatdylinositides saturated fatty acids chains and a decrease in C20 fatty acids in cancer cells compared with mammary epithelial cells was observed. At present, information about PL changes in cancer progression is lacking. Therefore, these data will be useful as a starting point to define possible PLs with prospective as biomarkers and disclose metabolic pathways with potential for therapy.     

Expression of activated oncogenes in the murine mammary gland: transgenic models for human breast cancer

Breast cancer is the leading cause of death among non-smoking women and thus has been the focus of intensive research. It has been generally accepted that the deregulation of oncogenes or their regulators play a pivotal role in progression of this prevalent disease. For example, amplification and overexpression of a number of oncogenes has been observed in a proportion of primary breast cancer biopsies. More recently, there has also been reports of inactivation tumor supressor genes in human breast cancer. While there is compelling evidence for a role of these genes in breast cancer tumor progression due to limitations inherent in these studies it is difficult to establish a direct causal association between expression of a certain oncogene and tumor progression. For this reason many groups have employed the transgenic mouse as a model system to directly study effects of oncogene expression in the murine mammary gland. This review will attempt to highlight some of the important lessons and potential applications that have emerged from the study of oncogene expression in the mammary epithelium of transgenic mice. The utility of the transgenic system to assess the transforming potential of oncogenes, to investigate the multi-step nature of malignant progression, and to be used as models for therapeutic intervention will be discussed.     

The role of GABA-ergic system in human mammary gland pathology and in growth of transplantable murine mammary cancer

In this paper we described the results of our studies on the baclophen (gamma aminobutyric acid (GABA)-B receptor agonist) inhibitory effect on the growth of experimental mammary cancer 16/C in mice and on the estimation of GABA level and GAD (glutamine acid decarboxylase--the key enzyme in GABA synthesis) activity after this treatment in mice. The experimental data are confronted with the estimation of GABA level and GAD activity in human mammary gland material taken from the patients with benign breast tumors of different pathological and age related hormonal stages. A significant inhibition of 16/C tumor growth in treated with baclophen mice was observed. Mean GABA level and GAD activity were significantly higher both in tumor and in normal tissue of baclophen treated mice in comparison to control animals. The results of clinical studies have shown that the lowest GABA level and GAD activity in tumor and normal mammary gland tissue was detected in patients in peri-menopausal stage. Both, in human and mouse material, the GABA level and GAD activity were higher in tumor than in normal tissue and there was a clear positive correlation between GABA level and GAD activity in both tissues studied. GABA level and GAD activity in tumor and in normal tissue were lower in patients with dysplasia than in patients with fibroadenoma. Considering our results, namely an inhibitory effect of GABA receptor agonist on mammary cancer growth and the correlation between GABA level and the stage of breast pathology and/or hormonal activity, it seems probable that GABA-ergic system is involved in hormonal regulation and pathogenesis of breast cancer.     

The seventh ENBDC workshop on methods in mammary gland development and cancer

The seventh annual meeting of the European Network of Breast Development and Cancer Laboratories, held in Weggis, Switzerland, in April 2015, was focused on techniques for the study of normal and cancer stem cells, cell fate decisions, cancer initiation and progression.     

Constitutive activation of nuclear factor-κB is preferentially involved in the proliferation of basal-like subtype breast cancer cell lines

Constitutive nuclear factor (NF)-κB activation is thought to be involved in survival, invasion, and metastasis in various types of cancers. However, neither the subtypes of breast cancer cells with constitutive NF-κB activation nor the molecular mechanisms leading to its constitutive activation have been clearly defined. Here, we quantitatively analyzed basal NF-κB activity in 35 human breast cancer cell lines and found that most of the cell lines with high constitutive NF-κB activation were categorized in the estrogen receptor negative, progesterone receptor negative, ERBB2 negative basal-like subtype, which is the most malignant form of breast cancer. Inhibition of constitutive NF-κB activation by expression of IκBα super-repressor reduced proliferation of the basal-like subtype cell lines. Expression levels of mRNA encoding NF-κB-inducing kinase (NIK) were elevated in several breast cancer cell lines, and RNA interference-mediated knockdown of NIK reduced NF-κB activation in a subset of the basal-like subtype cell lines with upregulated NIK expression. Taken together, these results suggest that constitutive NF-κB activation, partially dependent on NIK, is preferentially involved in proliferation of basal-like subtype breast cancer cells and may be a useful therapeutic target for this subtype of cancer. ( Cancer Sci 2009; 100: 1668–1674)