Humanization of the mouse mammary gland by replacement of the luminal layer with genetically engineered preneoplastic human cells.

PubMed

Verbeke, Stephanie; Richard, Elodie; Monceau, Elodie; Schmidt, Xenia; Rousseau, Benoit; Velasco, Valerie; Bernard, David; Bonnefoi, Herve; MacGrogan, Gaetan; Iggo, Richard D

2014-12-20

The cell of origin for estrogen receptor α-positive (ERα+) breast cancer is probably a luminal stem cell in the terminal duct lobular units. To model these cells, we have used the murine myoepithelial layer in the mouse mammary ducts as a scaffold upon which to build a human luminal layer. To prevent squamous metaplasia, a common artifact in genetically-engineered breast cancer models, we sought to limit activation of the epidermal growth factor receptor (EGFR) during in vitro cell culture before grafting the cells. Human reduction mammoplasty cells were grown in vitro in WIT medium. Epidermal growth factor in the medium was replaced with amphiregulin and neuregulin to decrease activation of EGFR and increase activation of EGFR homologs 3 and 4 (ERBB3 and ERBB4). Lentiviral vectors were used to express oncogenic transgenes and fluorescent proteins. Human mammary epithelial cells were mixed with irradiated mouse fibroblasts and Matrigel, then injected through the nipple into the mammary ducts of immunodeficient mice. Engrafted cells were visualized by stereomicroscopy for fluorescent proteins and characterized by histology and immunohistochemistry. Growth of normal mammary epithelial cells in conditions favoring ERBB3/4 signaling prevented squamous metaplasia in vitro. Normal human cells were quickly lost after intraductal injection, but cells infected with lentiviruses expressing CCND1, MYC, TERT, BMI1 and a short-hairpin RNA targeting TP53 were able to engraft and progressively replace the luminal layer in the mouse mammary ducts, resulting in the formation of an extensive network of humanized ducts. Despite expressing multiple oncogenes, the human cells formed a morphologically normal luminal layer. Expression of a single additional oncogene, PIK3CA-H1047R, converted the cells into invasive cancer cells. The resulting tumors were ERα+, Ki67+ luminal B adenocarcinomas that were resistant to treatment with fulvestrant. Injection of preneoplastic human mammary

Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells

PubMed Central

Rosenfield, Sonia M.; Bowden, Emma T.; Cohen-Missner, Shani; Gibby, Krissa A.; Ory, Virginie; Henke, Ralf T.; Riegel, Anna T.; Wellstein, Anton

2012-01-01

Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development. PMID:23077670

Single-cell RNA-Seq reveals cell heterogeneity and hierarchy within mouse mammary epithelia.

PubMed

Sun, Heng; Miao, Zhengqiang; Zhang, Xin; Chan, Un In; Su, Sek Man; Guo, Sen; Wong, Chris Koon Ho; Xu, Xiaoling; Deng, Chu-Xia

2018-06-01

The mammary gland is very intricately and well organized into distinct tissues, including epithelia, endothelia, adipocytes, and stromal and immune cells. Many mammary gland diseases, such as breast cancer, arise from abnormalities in the mammary epithelium, which is mainly composed of two distinct lineages, the basal and luminal cells. Because of the limitation of traditional transcriptome analysis of bulk mammary cells, the hierarchy and heterogeneity of mammary cells within these two lineages remain unclear. To this end, using single-cell RNA-Seq coupled with FACS analysis and principal component analysis, we determined gene expression profiles of mammary epithelial cells of virgin and pregnant mice. These analyses revealed a much higher heterogeneity among the mammary cells than has been previously reported and enabled cell classification into distinct subgroups according to signature gene markers present in each group. We also identified and verified a rare CDH5 + cell subpopulation within a basal cell lineage as quiescent mammary stem cells (MaSCs). Moreover, using pseudo-temporal analysis, we reconstructed the developmental trajectory of mammary epithelia and uncovered distinct changes in gene expression and in biological functions of mammary cells along the developmental process. In conclusion, our work greatly refines the resolution of the cellular hierarchy in developing mammary tissues. The discovery of CDH5 + cells as MaSCs in these tissues may have implications for our understanding of the initiation, development, and pathogenesis of mammary tumors. © 2018 Sun et al.

Dicer in Mammary Tumor Stem Cell Maintenance

DTIC Science & Technology

2006-03-01

we are cloning small RNAs from mammary stem cells in order to determine the regulatory niches that miRNAs may fill in this cell type. Our ultimate goal is to assess the role of Dicer in mammary tumor stem cell maintenance.

Association of surgical approach with complication rate, progression-free survival time, and disease-specific survival time in cats with mammary adenocarcinoma: 107 cases (1991-2014).

PubMed

Gemignani, Francesco; Mayhew, Philipp D; Giuffrida, Michelle A; Palaigos, Jason; Runge, Jeffrey J; Holt, David E; Robertson, Nicholas A; Seguin, Bernard; Walker, Meaghan; Singh, Ameet; Liptak, Julius M; Romanelli, Giorgio; Martano, Marina; Boston, Sarah E; Lux, Cassie; Busetto, Roberto; Culp, William T N; Skorupski, Katherine A; Burton, Jenna H

2018-06-01

OBJECTIVE To evaluate potential associations between surgical approach and complication rate, progression-free survival time, and disease-specific survival time in cats with mammary adenocarcinoma. DESIGN Retrospective case series. ANIMALS 107 client-owned cats. PROCEDURES Medical records of cats that underwent surgical excision of mammary adenocarcinoma by means of a unilateral or bilateral (staged or single-session) mastectomy at 9 hospitals between 1991 and 2014 were reviewed. Relevant clinicopathologic data and details of surgical and adjuvant treatments were recorded. Outcome data were obtained, including postoperative complications, progression-free survival time, and disease-specific survival time. RESULTS Complications occurred in 12 of 61 (19.7%) cats treated with unilateral mastectomy, 5 of 14 (35.7%) cats treated with staged bilateral mastectomy, and 13 of 32 (40.6%) cats treated with single-session bilateral mastectomy. Complications were significantly more likely to occur in cats undergoing bilateral versus unilateral mastectomy. Median progression-free survival time was longer for cats treated with bilateral mastectomy (542 days) than for cats treated with unilateral mastectomy (289 days). Significant risk factors for disease progression included unilateral mastectomy, tumor ulceration, lymph node metastasis, and tumors arising in the fourth mammary gland. Significant risk factors for disease-specific death included lymph node metastasis and development of regional or distant metastasis. Among cats that did not develop metastasis, unilateral mastectomy was a significant risk factor for disease-specific death. Treatment with chemotherapy was associated with a significantly decreased risk of disease-specific death. CONCLUSIONS AND CLINICAL RELEVANCE Results supported bilateral mastectomy for the treatment of mammary adenocarcinoma in cats to improve progression-free and disease-specific survival time. Performing bilateral mastectomy in a staged fashion

Establishment of mammary gland model in vitro: culture and evaluation of a yak mammary epithelial cell line.

PubMed

Fu, Mei; Chen, Yabing; Xiong, Xianrong; Lan, Daoliang; Li, Jian

2014-01-01

This study aimed to establish yak mammary epithelial cells (YMECs) for an in vitro model of yak mammary gland biology. The primary culture of YMECs was obtained from mammary gland tissues of lactating yak and then characterized using immunocytochemistry, RT-PCR, and western blot analysis. Whether foreign genes could be transfected into the YMECs were examined by transfecting the EGFP gene into the cells. Finally, the effect of Staphylococcus aureus infection on YMECs was determined. The established YMECs retained the mammary epithelial cell characteristics. A spontaneously immortalized yak mammary epithelial cell line was established and could be continuously subcultured for more than 60 passages without senescence. The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing. The cells expressed more antimicrobial peptides upon S.aureus invasion. Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.

Bovine mammary stem cells: Cell biology meets production agriculture

USDA-ARS?s Scientific Manuscript database

Mammary stem cells (MaSC) provide for net growth, renewal and turnover of mammary epithelial cells, and are therefore potential targets for strategies to increase production efficiency. Appropriate regulation of MaSC can potentially benefit milk yield, persistency, dry period management and tissue ...

Sterol regulatory element-binding proteins are regulators of the sodium/iodide symporter in mammary epithelial cells.

PubMed

Wen, G; Pachner, L I; Gessner, D K; Eder, K; Ringseis, R

2016-11-01

The sodium/iodide symporter (NIS), which is essential for iodide concentration in the thyroid, is reported to be transcriptionally regulated by sterol regulatory element-binding proteins (SREBP) in rat FRTL-5 thyrocytes. The SREBP are strongly activated after parturition and throughout lactation in the mammary gland of cattle and are important for mammary epithelial cell synthesis of milk lipids. In this study, we tested the hypothesis that the NIS gene is regulated also by SREBP in mammary epithelial cells, in which NIS is functionally expressed during lactation. Regulation of NIS expression and iodide uptake was investigated by means of inhibition, silencing, and overexpression of SREBP and by reporter gene and DNA-binding assays. As a mammary epithelial cell model, the human MCF-7 cell line, a breast adenocarcinoma cell line, which shows inducible expression of NIS by all-trans retinoic acid (ATRA), and unlike bovine mammary epithelial cells, is widely used to investigate the regulation of mammary gland NIS and NIS-specific iodide uptake, was used. Inhibition of SREBP maturation by treatment with 25-hydroxycholesterol (5 µM) for 48h reduced ATRA (1 µM)-induced mRNA concentration of NIS and iodide uptake in MCF-7 cells by approximately 20%. Knockdown of SREBP-1c and SREBP-2 by RNA interference decreased the mRNA and protein concentration of NIS by 30 to 50% 48h after initiating knockdown, whereas overexpression of nuclear SREBP (nSREBP)-1c and nSREBP-2 increased the expression of NIS in MCF-7 cells by 45 to 60%, respectively, 48h after initiating overexpression. Reporter gene experiments with varying length of NIS promoter reporter constructs revealed that the NIS 5'-flanking region is activated by nSREBP-1c and nSREBP-2 approximately 1.5- and 4.5-fold, respectively, and activation involves a SREBP-binding motif (SRE) at -38 relative to the transcription start site of the NIS gene. Gel shift assays using oligonucleotides spanning either the wild-type or the

Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

PubMed

Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

2016-08-01

The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland. © 2016 International Federation for Cell Biology.

In vivo diagnosis of mammary adenocarcinoma using Raman spectroscopy: an animal model study

NASA Astrophysics Data System (ADS)

Bitar, R. A.; Ribeiro, D. G.; dos Santos, E. A. P.; Ramalho, L. N. Z.; Ramalho, F. S.; Martin, A. A.; Martinho, H. S.

2010-02-01

Breast cancer is the most frequent cancer type in women Worldwide. Sensitivity and specificity of clinical breast examinations have been estimated from clinical trials to be approximately 54 % and 94 %, respectively. Further, approximately 95 % of all positive breast cancer screenings turn out to be false-positive. The optimal method for early detection should be both highly sensitive to ensure that all cancers are detected, and also highly specific to avoid the humanistic and economic costs associated with false-positive results. In vivo optical spectroscopy techniques, Raman in particular, have been pointed out as promising tools to improve the accuracy of screening mammography. The aim of the present study was to apply FT-Raman spectroscopy to discriminate normal and adenocarcinoma breast tissues of Sprague-Dawley female rats. The study was performed on 32 rats divided in the control (N=5) and experimental (N=27) groups. Histological analysis indicated that mammary hyperplasia, cribriform, papillary and solid adenocarcinomas were found in the experimental group subjects. The spectral collection was made using a commercial FT-Raman Spectrometer (Bruker RFS100) equipped with fiber-optic probe (RamProbe) and the spectral region between 900 and 1800 cm-1 was analyzed. Principal Components Analysis, Cluster Analysis, and Linear Discriminant Analysis with cross-validation were applied as spectral classification algorithm. As concluding remarks it is show that normal and adenocarcinoma tissues discriminations was possible (correct proportion for Transcutaneous collection mode was 80.80% and for "Open Sky" mode was 91.70%); however, a conclusive diagnosis among the four lesion subtypes was not possible.

Misregulation of Stromelysin-1 in Mouse Mammary Tumor Cells Accompanies Acquisition of Stromelysin-1 dependent Invasive Properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lochter, A.; Srebrow, A.; Sympson, C.J.

1997-02-21

Stromelysin-1 is a member of the metalloproteinase family of extracellular matrix-degrading enzymes that regulates tissue remodeling. We previously established a transgenic mouse model in which rat stromelysin-1 targeted to the mammary gland augmented expression of endogenous stromelysin-1, disrupted functional differentiation, and induced mammary tumors. A cell line generated from an adenocarcinoma in one of these animals and a previously described mammary tumor cell line generated in culture readily invaded both a reconstituted basement membrane and type I collagen gels, whereas a nonmalignant, functionally normal epithelial cell line did not. Invasion of Matrigel by tumor cells was largely abolished by metalloproteinasemore » inhibitors, but not by inhibitors of other proteinase families. Inhibition experiments with antisense oligodeoxynucleotides revealed that Matrigel invasion of both cell lines was critically dependent on stromelysin-1 expression. Invasion of collagen, on the other hand, was reduced by only 40-50%. Stromelysin-1 was expressed in both malignant and nonmalignant cells grown on plastic substrata. Its expression was completely inhibited in nonmalignant cells, but up-regulated in tumor cells, in response to Matrigel. Thus misregulation of stromelysin-1 expression appears to be an important aspect of mammary tumor cell progression to an invasive phenotype. The matrix metalloproteinases (MMPs) are a family of extracellular matrix (ECM)-degrading enzymes that have been implicated in a variety of normal developmental and pathological processes, including tumorigenesis. The MMP family comprises at least 15 members with different, albeit overlapping, substrate specificities. During activation of latent MMPs, their propeptides are cleaved and they are converted to a lower molecular weight form by other enzymes, including serine proteinases, and by autocatalytic cleavage. Among the MMPs, stromelysin-1 (SL1) possesses the broadest substrate specificity

Hormonally active doses of isoflavone aglycones promote mammary and endometrial carcinogenesis and alter the molecular tumor environment in Donryu rats.

PubMed

Kakehashi, Anna; Tago, Yoshiyuki; Yoshida, Midori; Sokuza, Yui; Wei, Min; Fukushima, Shoji; Wanibuchi, Hideki

2012-03-01

Our research is focused on modifying effects of an isoflavone aglycones (IAs)-rich extract at a hormonally active dose of 150 mg/kg body weight/day on mammary and endometrial carcinogenesis in female Donryu rats. IA administered for 2 weeks in a phytoestrogen-low diet exerted estrogenic activity and induced cell proliferation in the uterus of ovariectomized rats. Furthermore, administration for 4 weeks resulted in elevation of cell proliferation in the mammary glands of 7,12-dimethylbenz[a]anthracene (DMBA)-treated animals. Forty weeks of postpubertal administration of IA to 5-week-old rats after initiation of mammary and endometrial carcinogenesis with DMBA and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) caused significant increase of incidence and multiplicity of mammary adenocarcinoma, multiplicities of endometrial atypical hyperplasia, adenomatous polyps, and an increased trend of uterine adenocarcinomas. Liquid chromatography with tandem mass spectrometry and immunohistochemical analyses revealed significant elevation of tumorigenesis-related proteins such as S100 calcium-binding protein A8, kininogen 1, and annexins 1 and 2 in mammary adenocarcinomas and cadherin EGF LAG seven-pass G-type receptor 2, DEAD box polypeptide 1, and cysteine- and glycine-rich protein 1 in uterine proliferative lesions of IA-treated animals. Those changes are likely to be related to modulation of estrogen receptor (ER), AP1, nuclear factor-kappa B, and actin signaling pathways. Our results indicate that the postpubertal exposure of Donryu rats to IA at an estrogenic dose results in promotion of mammary and uterine carcinogenesis induced by DMBA and ENNG, which might be related to the activation of ER-dependent signaling and alteration of the molecular tumor environment in the mammary gland and endometrium.

Vulvar apocrine adenocarcinoma: a case with nodal metastasis and intranodal mucinous differentiation.

PubMed

Alsaad, Khaled O; Obaidat, Nidal; Dube, Valerie; Chapman, William; Ghazarian, Danny

2009-01-01

Primary vulvar adenocarcinomas are rare tumors, and their histogenesis is not fully understood. They are classified into extramammary Paget's disease, sweat gland carcinomas, and "breast-like" adenocarcinomas of the vulva. The latter resemble adenocarcinomas arising in the breast morphologically and immunophenotypically. Rare cases of adenocarcinoma with apocrine features have been reported, and whether these neoplasms originate from the "native apocrine" sweat glands or from "anogenital mammary-like" glands are still debatable. The presence of normal mammary-like glands in the vicinity of the tumor, the transitional malignant morphological features from normal mammary-like glands and the tumor, the breast-like histological features of the tumor, and the expression of estrogen and progesterone receptors generally suggest an origin from anogenital mammary-like glands. Absence of these features points toward native apocrine sweat glands as the source of these neoplasms. In this report, we present a patient who was initially diagnosed with Paget's disease of the right vulva, which was treated by hemi-vulvectomy, and who later presented with primary vulvar apocrine adenocarcinoma with metastasis to the inguinal lymph nodes and intranodal mucinous/colloidal differentiation: a feature, to the best of our knowledge, not reported before. We also reviewed the histogenesis of the vulvar adenocarcinomas, with emphasis on the morphological features that separate the tumors arising from the anogenital mammary-like glands in the vulva from those arising from the native vulvar sweat glands.

A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

PubMed Central

Mink, S; Härtig, E; Jennewein, P; Doppler, W; Cato, A C

1992-01-01

Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites. Images PMID:1328867

SOCS3 promotes apoptosis of mammary differentiated cells.

PubMed

Le Provost, Fabienne; Miyoshi, Keiko; Vilotte, Jean-Luc; Bierie, Brian; Robinson, Gertraud W; Hennighausen, Lothar

2005-12-30

Growth and function of the mammary gland is regulated by cytokines and modulated by suppressor of cytokine signalling (SOCS) proteins. In vitro experiments demonstrated that SOCS3 can inhibit PRL induction of milk protein gene expression and STAT5 activation. We explored the SOCS3 expression pattern during mouse mammary development and its regulation by PRL and GH in wild-type and STAT5a-null mammary tissue. Our results suggest that, in vivo, PRL stimulates SOCS3 expression in stromal adipocytes, independently of STAT5a stimulation. In mammary epithelial cells, SOCS3 expression appears to be related to STAT3 activation. Together, our results are consistent with a role of SOCS3 in the mammary gland by promoting apoptosis of differentiated cells (adipocytes during gestation and epithelial cells during involution).

Cutaneous metastases of a mammary carcinoma in a llama.

PubMed Central

Leichner, T L; Turner, O; Mason, G L; Barrington, G M

2001-01-01

An 8-year-old, female llama was evaluated for nonhealing, ulcerative, cutaneous lesions, which also involved the mammary gland. Biopsies of the lesions distant from and within the mammary gland area revealed an aggressive carcinoma. The tumor was confirmed at necropsy to be a mammary gland adenocarcinoma with cutaneous metastasis. Images Figure 1. PMID:11265189

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Booth, Brian W., E-mail: brbooth@clemson.edu; Institute for Biological Interfaces of Engineering, Clemson University, Clemson, SC 29634; Boulanger, Corinne A.

2010-02-01

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D {beta}-geo (CD{beta}geo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CD{beta}geo cells and that the mitogen activated protein kinase signalingmore » pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG{sup -/-} mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.« less

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics.

PubMed

Booth, Brian W; Boulanger, Corinne A; Anderson, Lisa H; Jimenez-Rojo, Lucia; Brisken, Cathrin; Smith, Gilbert H

2010-02-01

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D beta-geo (CDbetageo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CDbetageo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG(-/-) mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro. Copyright 2009 Elsevier Inc. All rights reserved.

Lgr6 labels a rare population of mammary gland progenitor cells that are able to originate luminal mammary tumours

PubMed Central

Messal, Hendrik A.; Andersson, Agneta B.; Ruiz, E. Josue; Gerling, Marco; Douagi, Iyadh; Spencer-Dene, Bradley; Musch, Alexandra; Mitter, Richard; Bhaw, Leena; Stone, Richard; Bornhorst, Dorothee; Sesay, Abdul K.; Jonkers, Jos; Stamp, Gordon; Malanchi, Ilaria; Toftgård, Rune; Behrens, Axel

2018-01-01

The mammary gland is composed of a complex cellular hierarchy with unusual postnatal plasticity. The identities of stem/progenitor cell populations, as well as tumour-initiating cells that give rise to breast cancer, are incompletely understood. Here we show that Lgr6 marks rare populations of cells in both basal and luminal mammary gland compartments in mice. Lineage tracing analysis showed that Lgr6+ cells are unipotent progenitors, which expand clonally during puberty but diminish in adulthood. In pregnancy or upon stimulation with ovarian hormones, adult Lgr6+ cells regained proliferative potency and their progeny formed alveoli over repeated pregnancies. Oncogenic mutations in Lgr6+ cells resulted in expansion of luminal cells, culminating in mammary gland tumours. Conversely, depletion of Lgr6+ cells in the MMTV-PyMT model of mammary tumourigenesis significantly impaired tumour growth. Thus, Lgr6 marks mammary gland progenitor cells that can initiate tumours, and cells of luminal breast tumours required for efficient tumour maintenance. PMID:27798604

Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis

PubMed Central

Teoh-Fitzgerald, ML; Fitzgerald, MP; Zhong, W; Askeland, RW; Domann, FE

2013-01-01

Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging

Leptin expression in human mammary epithelial cells and breast milk.

PubMed

Smith-Kirwin, S M; O'Connor, D M; De Johnston, J; Lancey, E D; Hassink, S G; Funanage, V L

1998-05-01

Leptin has recently been shown to be produced by the human placenta and potentially plays a role in fetal and neonatal growth. Many functions of the placenta are replaced by the mammary gland in terms of providing critical growth factors for the newborn. In this study, we show that leptin is produced by human mammary epithelial cells as revealed by RT/PCR analysis of total RNA from mammary gland and immunohistochemical staining of breast tissue, cultured mammary epithelial cells, and secretory epithelial cells present in human milk. We also verify that immunoreactive leptin is present in whole milk at 30- to 150-fold higher concentrations than skim milk. We propose that leptin is secreted by mammary epithelial cells in milk fat globules, which partition into the lipid portion of breast milk.

SOX10-positive salivary gland tumors: a growing list, including mammary analogue secretory carcinoma of the salivary gland, sialoblastoma, low-grade salivary duct carcinoma, basal cell adenoma/adenocarcinoma, and a subgroup of mucoepidermoid carcinoma.

PubMed

Hsieh, Min-Shu; Lee, Yi-Hsuan; Chang, Yih-Leong

2016-10-01

Transcription factor SRY-related HMG-box 10 (SOX10) is an important marker for melanocytic, schwannian, myoepithelial, and some salivary gland tumors. The aim of this study was to investigate SOX10 expression more thoroughly in the salivary gland neoplasms, including mammary analogue secretory carcinoma and hyalinizing clear cell carcinoma harboring specific genetic rearrangements. A new rabbit monoclonal anti-SOX10 antibody (clone EP268) was used to examine SOX10 expression in 14 different types of salivary gland tumors. We found that acinic cell carcinoma (AciCC), adenoid cystic carcinoma, mammary analogue secretory carcinoma (MASC), epithelial-myoepithelial carcinoma, low-grade salivary duct carcinoma, sialoblastoma, basal cell adenocarcinoma, basal cell adenoma, and pleomorphic adenoma were SOX10 positive. Salivary duct carcinoma, lymphoepithelial carcinoma, hyalinizing clear cell carcinoma, and oncocytoma were SOX10 negative. Earlier, mucoepidermoid carcinoma (MEC) was considered a SOX10-negative tumor. This study identified a subgroup of SOX10-positive MEC cases with characteristic polygonal epithelial cells, pale-to-eosinophilic cytoplasm, and colloid-like dense eosinophilic material. Our data show SOX10 expression can be observed in salivary gland tumors with either one of the 4 cell types: acinic cells, cuboidal ductal cells with low-grade cytologic features, basaloid cells, and myoepithelial cells. In this article we thoroughly evaluated SOX10 expression in salivary gland tumors. SOX10 is useful in the differential diagnosis between myoepithelial carcinoma with clear cell features and hyalinizing clear cell carcinoma. It can also be used to discriminate low-grade salivary duct carcinoma from high-grade ones. Pathologists should be cautious with the interpretation of SOX10 positivity in salivary gland tumors, and correlation with histologic feature is mandatory. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanical strain induces involution-associated events in mammary epithelial cells

PubMed Central

Quaglino, Ana; Salierno, Marcelo; Pellegrotti, Jesica; Rubinstein, Natalia; Kordon, Edith C

2009-01-01

Background Shortly after weaning, a complex multi-step process that leads to massive epithelial apoptosis is triggered by tissue local factors in the mouse mammary gland. Several reports have demonstrated the relevance of mechanical stress to induce adaptive responses in different cell types. Interestingly, these signaling pathways also participate in mammary gland involution. Then, it has been suggested that cell stretching caused by milk accumulation after weaning might be the first stimulus that initiates the complete remodeling of the mammary gland. However, no previous report has demonstrated the impact of mechanical stress on mammary cell physiology. To address this issue, we have designed a new practical device that allowed us to evaluate the effects of radial stretching on mammary epithelial cells in culture. Results We have designed and built a new device to analyze the biological consequences of applying mechanical stress to cells cultured on flexible silicone membranes. Subsequently, a geometrical model that predicted the percentage of radial strain applied to the elastic substrate was developed. By microscopic image analysis, the adjustment of these calculations to the actual strain exerted on the attached cells was verified. The studies described herein were all performed in the HC11 non-tumorigenic mammary epithelial cell line, which was originated from a pregnant BALB/c mouse. In these cells, as previously observed in other tissue types, mechanical stress induced ERK1/2 phosphorylation and c-Fos mRNA and protein expression. In addition, we found that mammary cell stretching triggered involution associated cellular events as Leukemia Inhibitory Factor (LIF) expression induction, STAT3 activation and AKT phosphorylation inhibition. Conclusion Here, we show for the first time, that mechanical strain is able to induce weaning-associated events in cultured mammary epithelial cells. These results were obtained using a new practical and affordable device

The Origin and Significance of Mammary Intraductal Foam Cells

DTIC Science & Technology

2005-09-01

hematopoeitic origin developed in mammary tissue with both benign and malignant differentiation, depending on environmental cues. Progression of the cells...contribution of hematopoeitic precursors to the heterogeneity of cell types in benign and malignant mammary tissue.

Pueraria mirifica Exerts Estrogenic Effects in the Mammary Gland and Uterus and Promotes Mammary Carcinogenesis in Donryu Rats

PubMed Central

Kakehashi, Anna; Yoshida, Midori; Tago, Yoshiyuki; Ishii, Naomi; Okuno, Takahiro; Gi, Min; Wanibuchi, Hideki

2016-01-01

Pueraria mirifica (PM), a plant whose dried and powdered tuberous roots are now widely used in rejuvenating preparations to promote youthfulness in both men and women, may have major estrogenic influence. In this study, we investigated modifying effects of PM at various doses on mammary and endometrial carcinogenesis in female Donryu rats. Firstly, PM administered to ovariectomized animals at doses of 0.03%, 0.3%, and 3% in a phytoestrogen-low diet for 2 weeks caused significant increase in uterus weight. Secondly, a 4 week PM application to non-operated rats at a dose of 3% after 7,12-dimethylbenz[a]anthracene (DMBA) initiation resulted in significant elevation of cell proliferation in the mammary glands. In a third experiment, postpubertal administration of 0.3% (200 mg/kg body weight (b.w.)/day) PM to 5-week-old non-operated animals for 36 weeks following initiation of mammary and endometrial carcinogenesis with DMBA and N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG), respectively, resulted in significant increase of mammary adenocarcinoma incidence. A significant increase of endometrial atypical hyperplasia multiplicity was also observed. Furthermore, PM at doses of 0.3%, and more pronouncedly, at 1% induced dilatation, hemorrhage and inflammation of the uterine wall. In conclusion, postpubertal long-term PM administration to Donryu rats exerts estrogenic effects in the mammary gland and uterus, and at a dose of 200 mg/kg b.w./day was found to promote mammary carcinogenesis initiated by DMBA. PMID:27827907

Pueraria mirifica Exerts Estrogenic Effects in the Mammary Gland and Uterus and Promotes Mammary Carcinogenesis in Donryu Rats.

PubMed

Kakehashi, Anna; Yoshida, Midori; Tago, Yoshiyuki; Ishii, Naomi; Okuno, Takahiro; Gi, Min; Wanibuchi, Hideki

2016-11-04

Pueraria mirifica (PM), a plant whose dried and powdered tuberous roots are now widely used in rejuvenating preparations to promote youthfulness in both men and women, may have major estrogenic influence. In this study, we investigated modifying effects of PM at various doses on mammary and endometrial carcinogenesis in female Donryu rats. Firstly, PM administered to ovariectomized animals at doses of 0.03%, 0.3%, and 3% in a phytoestrogen-low diet for 2 weeks caused significant increase in uterus weight. Secondly, a 4 week PM application to non-operated rats at a dose of 3% after 7,12-dimethylbenz[a]anthracene (DMBA) initiation resulted in significant elevation of cell proliferation in the mammary glands. In a third experiment, postpubertal administration of 0.3% (200 mg/kg body weight (b.w.)/day) PM to 5-week-old non-operated animals for 36 weeks following initiation of mammary and endometrial carcinogenesis with DMBA and N -ethyl- N '-nitro- N -nitrosoguanidine (ENNG), respectively, resulted in significant increase of mammary adenocarcinoma incidence. A significant increase of endometrial atypical hyperplasia multiplicity was also observed. Furthermore, PM at doses of 0.3%, and more pronouncedly, at 1% induced dilatation, hemorrhage and inflammation of the uterine wall. In conclusion, postpubertal long-term PM administration to Donryu rats exerts estrogenic effects in the mammary gland and uterus, and at a dose of 200 mg/kg b.w./day was found to promote mammary carcinogenesis initiated by DMBA.

STAT signaling in mammary gland differentiation, cell survival and tumorigenesis.

PubMed

Haricharan, S; Li, Y

2014-01-25

The mammary gland is a unique organ that undergoes extensive and profound changes during puberty, menstruation, pregnancy, lactation and involution. The changes that take place during puberty involve large-scale proliferation and invasion of the fat-pad. During pregnancy and lactation, the mammary cells are exposed to signaling pathways that inhibit apoptosis, induce proliferation and invoke terminal differentiation. Finally, during involution the mammary gland is exposed to milk stasis, programmed cell death and stromal reorganization to clear the differentiated milk-producing cells. Not surprisingly, the signaling pathways responsible for bringing about these changes in breast cells are often subverted during the process of tumorigenesis. The STAT family of proteins is involved in every stage of mammary gland development, and is also frequently implicated in breast tumorigenesis. While the roles of STAT3 and STAT5 during mammary gland development and tumorigenesis are well studied, others members, e.g. STAT1 and STAT6, have only recently been observed to play a role in mammary gland biology. Continued investigation into the STAT protein network in the mammary gland will likely yield new biomarkers and risk factors for breast cancer, and may also lead to novel prophylactic or therapeutic strategies against breast cancer. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

STAT signaling in mammary gland differentiation, cell survival and tumorigenesis

PubMed Central

Haricharan, S; Li, Y

2013-01-01

The mammary gland is a unique organ that undergoes extensive and profound changes during puberty, menstruation, pregnancy, lactation and involution. The changes that take place during puberty involve large-scale proliferation and invasion of the fat-pad. During pregnancy and lactation, the mammary cells are exposed to signaling pathways that inhibit apoptosis, induce proliferation and invoke terminal differentiation. Finally, during involution the mammary gland is exposed to milk stasis, programed cell death and stromal reorganization to clear the differentiated milk-producing cells. Not surprisingly, the signaling pathways responsible for bringing about these changes in breast cells are often subverted during the process of tumorigenesis. The STAT family of proteins is involved in every stage of mammary gland development, and is also frequently implicated in breast tumorigenesis. While the roles of STAT3 and STAT5 during mammary gland development and tumorigenesis are well studied, others members, e.g. STAT1 and STAT6, have only recently been observed to play a role in mammary gland biology. Continued investigation into the STAT protein network in the mammary gland will likely yield new biomarkers and risk factors for breast cancer, and may also lead to novel prophylactic or therapeutic strategies against breast cancer. PMID:23541951

Ganoderma lucidum total triterpenes induce apoptosis in MCF-7 cells and attenuate DMBA induced mammary and skin carcinomas in experimental animals.

PubMed

Smina, T P; Nitha, B; Devasagayam, T P A; Janardhanan, K K

2017-01-01

Ganoderma lucidum total triterpenes were evaluated for its apoptosis-inducing and anti-cancer activities. Cytotoxicity and pro-apoptotic effect of total triterpenes were evaluated in human breast adenocarcinoma (MCF-7) cell line using MTT assay and DNA fragmentation analysis. Total triterpenes induced apoptosis in MCF-7 cells by down-regulating the levels of cyclin D1, Bcl-2, Bcl-xL and also by up-regulating the levels of Bax and caspase-9. Anti-carcinogenicity of total triterpenes was analysed using dimethyl benz [a] anthracene (DMBA) induced skin papilloma and mammary adenocarcinoma in Swiss albino mice and Wistar rats respectively. Topical application of 5mg, 10mg and 20mg total triterpenes reduced the incidence of skin papilloma by 62.5, 37.5 and 12.5% respectively. Incidence of the mammary tumour was also reduced significantly by 33.33, 66.67 and 16.67% in 10, 50 and 100mg/kg b.wt. total triterpenes treated animals respectively. Total triterpenes were also found to reduce the average number of tumours per animal and extended the tumour latency period in both the models. The results indicate the potential cytotoxicity and anti-cancerous activity of total triterpenes, there by opens up a path to the development of a safe and successive chemo preventive agent of natural origin. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of ω-3 and ω-9 fatty acid rich oils on lipoxygenases and cyclooxygenases enzymes and on the growth of a mammary adenocarcinoma model.

PubMed

Comba, Andrea; Maestri, Damian M; Berra, María A; Garcia, Carolina Paola; Das, Undurti N; Eynard, Aldo R; Pasqualini, María E

2010-10-08

Nutritional factors play a major role in cancer initiation and development. Dietary polyunsaturated fatty acids (PUFAs) have the ability to induce modifications in the activity of lipoxygenase (LOX) and cyclooxygenase (COX) enzymes that affect tumour growth. We studied the effect of two diets enriched in 6% Walnut and Peanut oils that are rich in ω-3 and ω9 PUFAs respectively on a murine mammary gland adenocarcinoma as compared with the control (C) that received commercial diet. Peanut oil enriched diet induced an increase in membrane arachidonic acid (AA) content and the cyclooxygenase enzyme derived 12-HHT (p < 0.05) and simultaneously showed decrease in 12-LOX, 15-LOX-2, 15-LOX-1 and PGE activities (p < 0.05) that corresponded to higher apoptosis and lower mitosis seen in this group (p < 0.05). Furthermore, Peanut oil group showed lower T-cell infiltration (p < 0.05), number of metastasis (p < 0.05) and tumour volume (p < 0.05) and longer survival rate compared to other groups. The results of the present study showed that Peanut oil-enriched diet protects against mammary cancer development by modulating tumour membrane fatty acids composition and LOX and COX enzyme activities.

A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells

NASA Astrophysics Data System (ADS)

Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.

1981-05-01

Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

Human Breast Cancer Cells Are Redirected to Mammary Epithelial Cells upon Interaction with the Regenerating Mammary Gland Microenvironment In-Vivo

PubMed Central

Bussard, Karen M.; Smith, Gilbert H.

2012-01-01

Breast cancer is the second leading cause of cancer deaths in the United States. At present, the etiology of breast cancer is unknown; however the possibility of a distinct cell of origin, i.e. a cancer stem cell, is a heavily investigated area of research. Influencing signals from the tissue niche are known to affect stem cells. Literature has shown that cancer cells lose their tumorigenic potential and display ‘normal’ behavior when placed into ‘normal’ ontogenic environments. Therefore, it may be the case that the tissue microenvironment is able to generate signals to redirect cancer cell fate. Previously, we showed that pluripotent human embryonal carcinoma cells could be redirected by the regenerating mammary gland microenvironment to contribute epithelial progeny for ‘normal’ gland development in-vivo. Here, we show that that human metastatic, non-metastatic, and metastasis-suppressed breast cancer cells proliferate and contribute to normal mammary gland development in-vivo without tumor formation. Immunochemistry for human-specific mitochondria, keratin 8 and 14, as well as human-specific milk proteins (alpha-lactalbumin, impregnated transplant hosts) confirmed the presence of human cell progeny. Features consistent with normal mammary gland development as seen in intact hosts (duct, lumen formation, development of secretory acini) were recapitulated in both primary and secondary outgrowths from chimeric implants. These results suggest the dominance of the tissue microenvironment over cancer cell fate. This work demonstrates that cultured human breast cancer cells (metastatic and non-metastatic) respond developmentally to signals generated by the mouse mammary gland microenvironment during gland regeneration in-vivo. PMID:23155468

Phenotypic conversion of human mammary carcinoma cells by autocrine human growth hormone

PubMed Central

Mukhina, Svetlana; Mertani, Hichem C.; Guo, Ke; Lee, Kok-Onn; Gluckman, Peter D.; Lobie, Peter E.

2004-01-01

We report here that autocrine production of human growth hormone (hGH) results in a phenotypic conversion of mammary carcinoma cells such that they exhibit the morphological and molecular characteristics of a mesenchymal cell, including expression of fibronectin and vimentin. Autocrine production of hGH resulted in reduced plakoglobin expression and relocalization of E-cadherin to the cytoplasm, leading to dissolution of cell-cell contacts and decreased cell height. These phenotypic changes were accompanied by an increase in cell motility, elevated activity of specific matrix metalloproteinases, and an acquired ability to invade a reconstituted basement membrane. Forced expression of plakoglobin significantly decreased mammary carcinoma cell migration and invasion stimulated by autocrine hGH. In vivo, autocrine hGH stimulated local invasion of mammary carcinoma cells concomitant with a prominent stromal reaction in comparison with well delineated and capsulated growth of mammary carcinoma cells lacking autocrine production of hGH. Thus, autocrine production of hGH by mammary carcinoma cells is sufficient for generation of an invasive phenotype. Therapeutic targeting of autocrine hGH may provide a mechanistic approach to prevent metastatic extension of human mammary carcinoma. PMID:15353581

Physiologically activated mammary fibroblasts promote postpartum mammary cancer

PubMed Central

Guo, Qiuchen; Burchard, Julja; Spellman, Paul

2017-01-01

Women diagnosed with breast cancer within 5 years of childbirth have poorer prognosis than nulliparous or pregnant women. Weaning-induced breast involution is implicated, as the collagen-rich, immunosuppressive microenvironment of the involuting mammary gland is tumor promotional in mice. To investigate the role of mammary fibroblasts, isolated mammary PDGFRα+ cells from nulliparous and postweaning mice were assessed for activation phenotype and protumorigenic function. Fibroblast activation during involution was evident by increased expression of fibrillar collagens, lysyl oxidase, Tgfb1, and Cxcl12 genes. The ability of mammary tumors to grow in an isogenic, orthotopic transplant model was increased when tumor cells were coinjected with involution-derived compared with nulliparous-derived mammary fibroblasts. Mammary tumors in the involution-fibroblast group had increased Ly6C+ monocytes at the tumor border, and decreased CD8+ T cell infiltration and tumor cell death. Ibuprofen treatment suppressed involution-fibroblast activation and tumor promotional capacity, concurrent with decreases in tumor Ly6C+ monocytes, and increases in intratumoral CD8+ T cell infiltration, granzyme levels, and tumor cell death. In total, our data identify a COX/prostaglandin E2 (PGE2)–dependent activated mammary fibroblast within the involuting mammary gland that displays protumorigenic, immunosuppressive activity, identifying fibroblasts as potential targets for the prevention and treatment of postpartum breast cancer. PMID:28352652

Progesterone facilitates chromosome instability (aneuploidy) in p53 null normal mammary epithelial cells

NASA Technical Reports Server (NTRS)

Goepfert, T. M.; McCarthy, M.; Kittrell, F. S.; Stephens, C.; Ullrich, R. L.; Brinkley, B. R.; Medina, D.

2000-01-01

Mammary epithelial cells from p53 null mice have been shown recently to exhibit an increased risk for tumor development. Hormonal stimulation markedly increased tumor development in p53 null mammary cells. Here we demonstrate that mammary tumors arising in p53 null mammary cells are highly aneuploid, with greater than 70% of the tumor cells containing altered chromosome number and a mean chromosome number of 56. Normal mammary cells of p53 null genotype and aged less than 14 wk do not exhibit aneuploidy in primary cell culture. Significantly, the hormone progesterone, but not estrogen, increases the incidence of aneuploidy in morphologically normal p53 null mammary epithelial cells. Such cells exhibited 40% aneuploidy and a mean chromosome number of 54. The increase in aneuploidy measured in p53 null tumor cells or hormonally stimulated normal p53 null cells was not accompanied by centrosome amplification. These results suggest that normal levels of progesterone can facilitate chromosomal instability in the absence of the tumor suppressor gene, p53. The results support the emerging hypothesis based both on human epidemiological and animal model studies that progesterone markedly enhances mammary tumorigenesis.

Brca1 regulates in vitro differentiation of mammary epithelial cells.

PubMed

Kubista, Marion; Rosner, Margit; Kubista, Ernst; Bernaschek, Gerhard; Hengstschläger, Markus

2002-07-18

Murine Brca1 is widely expressed during development in different tissues. Why alterations of BRCA1 lead specifically to breast and ovarian cancer is currently not clarified. Here we show that Brca1 protein expression is upregulated during mammary epithelial differentiation of HC11 cells, during differentiation of C2C12 myoblasts into myotubes and during neuronal differentiation of N1E-115 cells. Ectopic overexpression of BRCA1 and downregulation of endogenous Brca1 expression specifically affect the regulation of mammary epithelial cell differentiation. Accelerated mammary epithelial cell differentiation upon high ectopic BRCA1 expression is not a consequence of the anti-proliferative capacity of this tumor suppressor and independent of functional p53. Overexpression of the BRCA1 variant lacking the large central exon 11 has no effects on mammary epithelial cell differentiation. These data provide new insights into the cellular role of Brca1.

Ethanol Extract of Haliclona sp. Improved Histological Grade of Mammary Adenocarcinoma in C3H Mice

NASA Astrophysics Data System (ADS)

Murwani, R.; Trianto, A.; Wijayanti, E.; Ridlo, A.; Susilaningsih, N.

2018-02-01

The sponge Haliclona sp. contains secondary metabolites belong to alkaloids which are cytotoxic to the human tumor. The following research was conducted to study the effect of a graded level of Haliclona sp. extract on mammary adenocarcinoma in C3H mice. Haliclona sp. was obtained from Bandengan water in Jepara, and the crude extract was prepared by maceration with ethanol. Fifteen C3H mice with an initial weight of 20-25 gram were assigned into control, H-1, and H-2 groups. Control, H1, and H2 groups each received the ethanol extract of 0, 0.15, and 1.5 mg per mouse per day respectively for two weeks. Cancer cells were introduced to all groups from a cancerous donor mouse. The donor cancer cells were injected into each mouse via left or right axilla and allowed to grow. The cancer mass was removed and processed for histological examination, and the cancer growth was determined according to Elston and Ellis criteria. The result showed that histological grade of cancer mass from control group was in grade 2 or differentiated moderately. The histological grade of cancer mass from H-1 and H-2 groups were in grade 1 or similar to a normal cell. Analyses of the data by Kruskal-Wallis showed a significant difference (p<0,05) between control and treated groups. No significant difference was found between H-1 and H-2 groups. The results suggest the potential of active substances in the ethanol extract of Haliclona sp as an anti-cancer drug.

In vitro expansion of the mammary stem/progenitor cell population by xanthosinetreatment

USDA-ARS?s Scientific Manuscript database

Background: Mammary stem cells are critical for growth and maintenance of the mammary gland and therefore of considerable interest for improving productivity and efficiency of dairy animals. Xanthosine (Xs) treatment has been demonstrated to promote expansion of putative mammary stem cells in vivo ...

Mammary Stem Cells: Premise, Properties, and Perspectives.

PubMed

Lloyd-Lewis, Bethan; Harris, Olivia B; Watson, Christine J; Davis, Felicity M

2017-08-01

Adult mammary stem cells (MaSCs) drive postnatal organogenesis and remodeling in the mammary gland, and their longevity and potential have important implications for breast cancer. However, despite intense investigation the identity, location, and differentiation potential of MaSCs remain subject to deliberation. The application of genetic lineage-tracing models, combined with quantitative 3D imaging and biophysical methods, has provided new insights into the mammary epithelial hierarchy that challenge classical definitions of MaSC potency and behaviors. We review here recent advances - discussing fundamental unresolved properties of MaSC potency, dynamics, and plasticity - and point to evolving technologies that promise to shed new light on this intractable debate. Elucidation of the physiological mammary differentiation hierarchy is paramount to understanding the complex heterogeneous breast cancer landscape. Copyright © 2017 Elsevier Ltd. All rights reserved.

Notch3 marks clonogenic mammary luminal progenitor cells in vivo.

PubMed

Lafkas, Daniel; Rodilla, Veronica; Huyghe, Mathilde; Mourao, Larissa; Kiaris, Hippokratis; Fre, Silvia

2013-10-14

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive "triple negative" human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2(SAT) transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells.

Notch3 marks clonogenic mammary luminal progenitor cells in vivo

PubMed Central

Lafkas, Daniel; Rodilla, Veronica; Huyghe, Mathilde; Mourao, Larissa; Kiaris, Hippokratis

2013-01-01

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive “triple negative” human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2SAT transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells. PMID:24100291

Mammary stem cells and the differentiation hierarchy: current status and perspectives

PubMed Central

Visvader, Jane E.; Stingl, John

2014-01-01

The mammary epithelium is highly responsive to local and systemic signals, which orchestrate morphogenesis of the ductal tree during puberty and pregnancy. Based on transplantation and lineage tracing studies, a hierarchy of stem and progenitor cells has been shown to exist among the mammary epithelium. Lineage tracing has highlighted the existence of bipotent mammary stem cells (MaSCs) in situ as well as long-lived unipotent cells that drive morphogenesis and homeostasis of the ductal tree. Moreover, there is accumulating evidence for a heterogeneous MaSC compartment comprising fetal MaSCs, slow-cycling cells, and both long-term and short-term repopulating cells. In parallel, diverse luminal progenitor subtypes have been identified in mouse and human mammary tissue. Elucidation of the normal cellular hierarchy is an important step toward understanding the “cells of origin” and molecular perturbations that drive breast cancer. PMID:24888586

The Analysis of Cell Population Dynamics in Mammary Gland Development and Tumorigenesis

DTIC Science & Technology

2005-08-01

AD Award Number: DAMD17-03-1-0498 TITLE: The Analysis of Cell Population Dynamics in Mammary Gland Development and Tumorigenesis PRINCIPAL...Summary 1 Aug 2004 - 31 Jul 2005 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER The Analysis of Cell Population Dynamics in Mammary Gland Development and...STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The mammary gland is made up of several epithelial cell

Dendritic cells in Barrett's esophagus and esophageal adenocarcinoma.

PubMed

Bobryshev, Yuri V; Tran, Dinh; Killingsworth, Murray C; Buckland, Michael; Lord, Reginald V N

2009-01-01

Like other premalignant conditions that develop in the presence of chronic inflammation, the development and progression of Barrett's esophagus is associated with the development of an immune response, but how this immune response is regulated is poorly understood. A comprehensive literature search failed to find any report of the presence of dendritic cells in Barrett's intestinal metaplasia and esophageal adenocarcinoma and this prompted our study. We used immunohistochemical staining and electron microscopy to examine whether dendritic cells are present in Barrett's esophagus and esophageal adenocarcinoma. Immunohistochemical staining with CD83, a specific marker for dendritic cells, was performed on paraffin-embedded sections of Barrett's intestinal metaplasia (IM, n = 12), dysplasia (n = 11) and adenocarcinoma (n = 14). CD83+ cells were identified in the lamina propria surrounding intestinal type glands in Barrett's IM, dysplasia, and cancer tissues. Computerized quantitative analysis showed that the numbers of dendritic cells were significantly higher in cancer tissues. Double immunostaining with CD83, CD20, and CD3, and electron microscopy demonstrated that dendritic cells are present in Barrett's esophagus and form clusters with T cells and B cells directly within the lamina propria. These findings demonstrate that dendritic cells are present in Barrett's tissues, with a significant increase in density in adenocarcinoma compared to benign Barrett's esophagus. Dendritic cells may have a role in the pathogenesis and immunotherapy treatment of Barrett's esophagus and adenocarcinoma.

Mammary molecular portraits reveal lineage-specific features and progenitor cell vulnerabilities.

PubMed

Casey, Alison E; Sinha, Ankit; Singhania, Rajat; Livingstone, Julie; Waterhouse, Paul; Tharmapalan, Pirashaanthy; Cruickshank, Jennifer; Shehata, Mona; Drysdale, Erik; Fang, Hui; Kim, Hyeyeon; Isserlin, Ruth; Bailey, Swneke; Medina, Tiago; Deblois, Genevieve; Shiah, Yu-Jia; Barsyte-Lovejoy, Dalia; Hofer, Stefan; Bader, Gary; Lupien, Mathieu; Arrowsmith, Cheryl; Knapp, Stefan; De Carvalho, Daniel; Berman, Hal; Boutros, Paul C; Kislinger, Thomas; Khokha, Rama

2018-06-19

The mammary epithelium depends on specific lineages and their stem and progenitor function to accommodate hormone-triggered physiological demands in the adult female. Perturbations of these lineages underpin breast cancer risk, yet our understanding of normal mammary cell composition is incomplete. Here, we build a multimodal resource for the adult gland through comprehensive profiling of primary cell epigenomes, transcriptomes, and proteomes. We define systems-level relationships between chromatin-DNA-RNA-protein states, identify lineage-specific DNA methylation of transcription factor binding sites, and pinpoint proteins underlying progesterone responsiveness. Comparative proteomics of estrogen and progesterone receptor-positive and -negative cell populations, extensive target validation, and drug testing lead to discovery of stem and progenitor cell vulnerabilities. Top epigenetic drugs exert cytostatic effects; prevent adult mammary cell expansion, clonogenicity, and mammopoiesis; and deplete stem cell frequency. Select drugs also abrogate human breast progenitor cell activity in normal and high-risk patient samples. This integrative computational and functional study provides fundamental insight into mammary lineage and stem cell biology. © 2018 Casey et al.

DNMT1 is essential for mammary and cancer stem cell maintenance and tumorigenesis

PubMed Central

Pathania, Rajneesh; Ramachandran, Sabarish; Elangovan, Selvakumar; Padia, Ravi; Yang, Pengyi; Cinghu, Senthilkumar; Veeranan-Karmegam, Rajalakshmi; Arjunan, Pachiappan; Gnana-Prakasam, Jaya P.; Fulzele, Sadanand; Pei, Lirong; Chang, Chang-Sheng; Choi, Hyeon; Shi, Huidong; Manicassamy, Santhakumar; Prasad, Puttur D.; Sharma, Suash; Ganapathy, Vadivel; Jothi, Raja; Thangaraju, Muthusamy

2015-01-01

Mammary stem/progenitor cells (MaSCs) maintain self-renewal of the mammary epithelium during puberty and pregnancy. DNA methylation provides a potential epigenetic mechanism for maintaining cellular memory during self-renewal. Although DNA methyltransferases (DNMTs) are dispensable for embryonic stem cell maintenance, their role in maintaining MaSCs and cancer stem cells (CSCs) in constantly replenishing mammary epithelium is unclear. Here we show that DNMT1 is indispensable for MaSC maintenance. Furthermore, we find that DNMT1 expression is elevated in mammary tumors, and mammary gland-specific DNMT1 deletion protects mice from mammary tumorigenesis by limiting the CSC pool. Through genome-scale methylation studies, we identify ISL1 as a direct DNMT1 target, hypermethylated and downregulated in mammary tumors and CSCs. DNMT inhibition or ISL1 expression in breast cancer cells limits CSC population. Altogether, our studies uncover an essential role for DNMT1 in MaSC and CSC maintenance and identify DNMT1-ISL1 axis as a potential therapeutic target for breast cancer treatment. PMID:25908435

DNMT1 is essential for mammary and cancer stem cell maintenance and tumorigenesis.

PubMed

Pathania, Rajneesh; Ramachandran, Sabarish; Elangovan, Selvakumar; Padia, Ravi; Yang, Pengyi; Cinghu, Senthilkumar; Veeranan-Karmegam, Rajalakshmi; Arjunan, Pachiappan; Gnana-Prakasam, Jaya P; Sadanand, Fulzele; Pei, Lirong; Chang, Chang-Sheng; Choi, Jeong-Hyeon; Shi, Huidong; Manicassamy, Santhakumar; Prasad, Puttur D; Sharma, Suash; Ganapathy, Vadivel; Jothi, Raja; Thangaraju, Muthusamy

2015-04-24

Mammary stem/progenitor cells (MaSCs) maintain self-renewal of the mammary epithelium during puberty and pregnancy. DNA methylation provides a potential epigenetic mechanism for maintaining cellular memory during self-renewal. Although DNA methyltransferases (DNMTs) are dispensable for embryonic stem cell maintenance, their role in maintaining MaSCs and cancer stem cells (CSCs) in constantly replenishing mammary epithelium is unclear. Here we show that DNMT1 is indispensable for MaSC maintenance. Furthermore, we find that DNMT1 expression is elevated in mammary tumours, and mammary gland-specific DNMT1 deletion protects mice from mammary tumorigenesis by limiting the CSC pool. Through genome-scale methylation studies, we identify ISL1 as a direct DNMT1 target, hypermethylated and downregulated in mammary tumours and CSCs. DNMT inhibition or ISL1 expression in breast cancer cells limits CSC population. Altogether, our studies uncover an essential role for DNMT1 in MaSC and CSC maintenance and identify DNMT1-ISL1 axis as a potential therapeutic target for breast cancer treatment.

Characterization of immortalized human mammary epithelial cell line HMEC 2.6.

PubMed

Joshi, Pooja S; Modur, Vishnu; Cheng, JiMing; Robinson, Kathy; Rao, Krishna

2017-10-01

Primary human mammary epithelial cells have a limited life span which makes it difficult to study them in vitro for most purposes. To overcome this problem, we have developed a cell line that was immortalized using defined genetic elements, and we have characterized this immortalized non-tumorigenic human mammary epithelial cell line to establish it as a potential model system. human mammary epithelial cells were obtained from a healthy individual undergoing reduction mammoplasty at SIU School of Medicine. The cells were transduced with CDK4R24C followed by transduction with human telomerase reverse transcriptase. Post all manipulation, the cells displayed a normal cell cycle phase distribution and were near diploid in nature, which was confirmed by flow cytometry and karyotyping. In vitro studies showed that the cells were anchorage dependent and were non-invasive in nature. The cell line expressed basal epithelial markers such as cytokeratin 7, CD10, and p63 and was negative for the expression of estrogen receptor and progesterone receptor. Upon G-band karyotyping, the cell line displayed the presence of a few cytogenic abnormalities, including trisomy 20 and trisomy 7, which are also commonly present in other immortalized mammary cell lines. Furthermore, the benign nature of these cells was confirmed by multiple in vitro and in vivo experiments. Therefore, we think that this cell line could serve as a good model to understand the molecular mechanisms involved in the development and progression of breast cancer and to also assess the effect of novel therapeutics on human mammary epithelial cells.

A novel approach for the generation of genetically modified mammary epithelial cell cultures yields new insights into TGFβ signaling in the mammary gland

PubMed Central

2010-01-01

Introduction Molecular dissection of the signaling pathways that underlie complex biological responses in the mammary epithelium is limited by the difficulty of propagating large numbers of mouse mammary epithelial cells, and by the inability of ribonucleic acid interference-based knockdown approaches to fully ablate gene function. Here we describe a method for the generation of conditionally immortalized mammary epithelial cells with defined genetic defects, and we show how such cells can be used to investigate complex signal transduction processes using the transforming growth factor beta (TGFβ)/Smad pathway as an example. Methods We intercrossed the previously described H-2Kb-tsA58 transgenic mouse (Immortomouse), which expresses a temperature-sensitive mutant of the simian virus-40 large T-antigen (tsTAg), with mice of differing Smad genotypes. Conditionally immortalized mammary epithelial cell cultures were derived from the virgin mammary glands of offspring of these crosses and were used to assess the Smad dependency of different biological responses to TGFβ. Results IMECs could be propagated indefinitely at permissive temperatures and had a stable epithelial phenotype, resembling primary mammary epithelial cells with respect to several criteria, including responsiveness to TGFβ. Using this panel of cells, we demonstrated that Smad3, but not Smad2, is necessary for TGFβ-induced apoptotic, growth inhibitory and epithelial-to-mesenchymal transition responses, whereas either Smad2 or Smad3 can support TGFβ-induced invasion as long as a threshold level of total Smad is exceeded. Conclusions The present work demonstrates the practicality and utility of generating conditionally immortalized mammary epithelial cell lines from genetically modified Immortomice for detailed investigation of complex signaling pathways in the mammary epithelium. PMID:20942910

Proliferation of human mammary cancer cells exposed to 27-hydroxycholesterol

PubMed Central

CRUZ, PAMELA; TORRES, CRISTIAN; RAMÍREZ, MARÍA EUGENIA; EPUÑÁN, MARÍA JOSÉ; VALLADARES, LUIS EMILIO; SIERRALTA, WALTER DANIEL

2010-01-01

The aim of the present study was to identify the possible mechanisms by which certain estradiol receptor (ER)-positive mammary tumor cells remain resistant to treatment with anti-estrogens or inhibitors of local estradiol (E2) production. To this end, we compared the proliferative effects on mammary cancer cells of the novel selective ER modulator 27-hydroxycholesterol (27OHC) to those of E2, and evaluated their inhibition by ICI 182,780 (ICI). Analysis of the effects on the cell cycle of 27OHC and E2 in the absence or presence of ICI was conducted. In ER-positive mammary tumor cells, we detected the blocking of 27OHC proliferation-stimulatory activity by simvastatin, as well as the inhibition of E2-stimulated proliferation by an α-fetoprotein-derived cyclic nonapeptide. The effects reported herein may be extrapolated to infiltrating mammary cancer, where the activity of local macrophages may stimulate tumor growth. We suggest that increased breast cancer growth in obese patients may be related to increased 27OHC circulatory levels. PMID:22993572

Proliferation of human mammary cancer cells exposed to 27-hydroxycholesterol.

PubMed

Cruz, Pamela; Torres, Cristian; Ramírez, María Eugenia; Epuñán, María José; Valladares, Luis Emilio; Sierralta, Walter Daniel

2010-05-01

The aim of the present study was to identify the possible mechanisms by which certain estradiol receptor (ER)-positive mammary tumor cells remain resistant to treatment with anti-estrogens or inhibitors of local estradiol (E(2)) production. To this end, we compared the proliferative effects on mammary cancer cells of the novel selective ER modulator 27-hydroxycholesterol (27OHC) to those of E(2), and evaluated their inhibition by ICI 182,780 (ICI). Analysis of the effects on the cell cycle of 27OHC and E(2) in the absence or presence of ICI was conducted. In ER-positive mammary tumor cells, we detected the blocking of 27OHC proliferation-stimulatory activity by simvastatin, as well as the inhibition of E(2)-stimulated proliferation by an α-fetoprotein-derived cyclic nonapeptide. The effects reported herein may be extrapolated to infiltrating mammary cancer, where the activity of local macrophages may stimulate tumor growth. We suggest that increased breast cancer growth in obese patients may be related to increased 27OHC circulatory levels.

5-Fluorouracil may enrich cancer stem cells in canine mammary tumor cells in vitro.

PubMed

Zhou, Bin; Jin, Yipeng; Zhang, Di; Lin, Degui

2018-05-01

Mammary gland carcinomas are the most common neoplasms in women and unsterilized female dogs. Owing to the existence of cancer stem cells (CSCs), chemotherapy is not able to cure these types of diseases completely. A number of studies have demonstrated that CSCs are resistant to chemotherapeutic drugs, but whether canine mammary tumor cells that have acquired resistance to 5-fluorouracil (5-FU) exhibited properties of CSCs remains unknown. The aim of the present study was to investigate whether 5-fluorouracil-resistant canine mammary tumor cells exhibited properties of CSCs. CSCs were analyzed using western blot assays, ultra-low attachment sphere cultures, flow cytometry and migration (wound healing and Transwell) assays. The results indicated that, compared with parental cells, proteins associated with the Wnt/β-catenin signaling pathway and aldehyde dehydrogenase 1 were overexpressed, the number and size of spheres in the 5-FU-resistant cells were increased, the ratio of CD44 + /CD24 -/low cells was increased and the migratory ability was improved in vitro compared with the 5-FU-susceptible cells. In conclusion, stimulation with chemotherapeutic drugs including 5-FU is a good method for increasing the proportion of canine mammary tumor stem cells in vitro , which may provide further understanding of chemotherapeutic methods and CSCs.

Comparison of the transcriptpmes of long-tern label retaining-cells and C cells microdissected from mammary epithelium: an initial study to character potential stem/progenitor cells

USDA-ARS?s Scientific Manuscript database

Mammary stem cells (MaSC) account for the cell lineage of mammary epithelia and provide for mammary growth, development and tissue homeostasis. The presence of MaSC was clearly demonstrated by the generation of an entire mammary gland from a single cell implanted into epithelium-ablated mammary fat...

Sequestration of human cytomegalovirus by human renal and mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Twite, Nicolas; Andrei, Graciela; Kummert, Caroline

2014-07-15

Urine and breast milk represent the main routes of human cytomegalovirus (HCMV) transmission but the contribution of renal and mammary epithelial cells to viral excretion remains unclear. We observed that kidney and mammary epithelial cells were permissive to HCMV infection and expressed immediate early, early and late antigens within 72 h of infection. During the first 24 h after infection, high titers of infectious virus were measured associated to the cells and in culture supernatants, independently of de novo synthesis of virus progeny. This phenomenon was not observed in HCMV-infected fibroblasts and suggested the sequestration and the release of HCMVmore » by epithelial cells. This hypothesis was supported by confocal and electron microscopy analyses. The sequestration and progressive release of HCMV by kidney and mammary epithelial cells may play an important role in the excretion of the virus in urine and breast milk and may thereby contribute to HCMV transmission. - Highlights: • Primary renal and mammary epithelial cells are permissive to HCMV infection. • HCMV is sequestered by epithelial cells and this phenomenon does not require viral replication. • HCMV sequestration by epithelial cells is reduced by antibodies and IFN-γ.« less

Un(MaSC)ing Stem Cell Dynamics in Mammary Branching Morphogenesis.

PubMed

Greenwood, Erin; Wrenn, Emma D; Cheung, Kevin J

2017-02-27

The properties of stem cells that participate in mammary gland branching morphogenesis remain contested. Reporting in Nature, Scheele et al. (2017) establish a model for post-pubertal mammary branching morphogenesis in which position-dependent, lineage-restricted stem cells undergo cell mixing in order to contribute to long-term growth. Copyright © 2017 Elsevier Inc. All rights reserved.

Ectodysplasin/NF-κB Promotes Mammary Cell Fate via Wnt/β-catenin Pathway

PubMed Central

Voutilainen, Maria; Lönnblad, Darielle; Shirokova, Vera; Elo, Teresa; Rysti, Elisa; Schmidt-Ullrich, Ruth; Schneider, Pascal; Mikkola, Marja L.

2015-01-01

Mammary gland development commences during embryogenesis with the establishment of a species typical number of mammary primordia on each flank of the embryo. It is thought that mammary cell fate can only be induced along the mammary line, a narrow region of the ventro-lateral skin running from the axilla to the groin. Ectodysplasin (Eda) is a tumor necrosis factor family ligand that regulates morphogenesis of several ectodermal appendages. We have previously shown that transgenic overexpression of Eda (K14-Eda mice) induces formation of supernumerary mammary placodes along the mammary line. Here, we investigate in more detail the role of Eda and its downstream mediator transcription factor NF-κB in mammary cell fate specification. We report that K14-Eda mice harbor accessory mammary glands also in the neck region indicating wider epidermal cell plasticity that previously appreciated. We show that even though NF-κB is not required for formation of endogenous mammary placodes, it is indispensable for the ability of Eda to induce supernumerary placodes. A genome-wide profiling of Eda-induced genes in mammary buds identified several Wnt pathway components as potential transcriptional targets of Eda. Using an ex vivo culture system, we show that suppression of canonical Wnt signalling leads to a dose-dependent inhibition of supernumerary placodes in K14-Eda tissue explants. PMID:26581094

Silencing of Kv4.1 potassium channels inhibits cell proliferation of tumorigenic human mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jang, Soo Hwa; Choi, Changsun; Hong, Seong-Geun

2009-06-26

Potassium channel activity has been shown to facilitate cell proliferation in cancer cells. In the present study, the role of Kv4.1 channels in immortal and tumorigenic human mammary epithelial cells was investigated. Kv4.1 protein expression was positively correlated with tumorigenicity. Moreover, transfection with siRNAs targeting Kv4.1 mRNA suppressed proliferation of tumorigenic mammary epithelial cells. Experiments using mRNA isolated from human breast cancer tissues revealed that the level of Kv4.1 mRNA expression varied depending on the stage of the tumor. Kv4.1 protein expression increased during stages T2 and T3 compared to normal tissue. These results demonstrated that Kv4.1 plays a rolemore » in proliferation of tumorigenic human mammary epithelial cells. In addition, elevated Kv4.1 expression may be useful as a diagnostic marker for staging mammary tumors and selective blockers of Kv4.1 may serve to suppress tumor cell proliferation.« less

Luminal epithelial cells within the mammary gland can produce basal cells upon oncogenic stress.

PubMed

Hein, S M; Haricharan, S; Johnston, A N; Toneff, M J; Reddy, J P; Dong, J; Bu, W; Li, Y

2016-03-17

In the normal mammary gland, the basal epithelium is known to be bipotent and can generate either basal or luminal cells, whereas the luminal epithelium has not been demonstrated to contribute to the basal compartment in an intact and normally developed mammary gland. It is not clear whether cellular heterogeneity within a breast tumor results from transformation of bipotent basal cells or from transformation and subsequent basal conversion of the more differentiated luminal cells. Here we used a retroviral vector to express an oncogene specifically in a small number of the mammary luminal epithelial cells and tested their potential to produce basal cells during tumorigenesis. This in-vivo lineage-tracing work demonstrates that luminal cells are capable of producing basal cells on activation of either polyoma middle T antigen or ErbB2 signaling. These findings reveal the plasticity of the luminal compartment during tumorigenesis and provide an explanation for cellular heterogeneity within a cancer.

Luminal Epithelial Cells within the Mammary Gland Can Produce Basal Cells upon Oncogenic Stress

PubMed Central

Hein, Sarah M.; Haricharan, Svasti; Johnston, Alyssa N.; Toneff, Michael J.; Reddy, Jay P.; Dong, Jie; Bu, Wen; Li, Yi

2015-01-01

In the normal mammary gland, the basal epithelium is known to be bi-potent and can generate either basal or luminal cells, whereas the luminal epithelium has not been demonstrated to contribute to the basal compartment in an intact and normally developed mammary gland. It is not clear whether cellular heterogeneity within a breast tumor results from transformation of bi-potent basal cells or from transformation and subsequent basal conversion of the more differentiated luminal cells. Here, we used a retroviral vector to express an oncogene specifically in a small number of the mammary luminal epithelial cells and tested their potential to produce basal cells during tumorigenesis. This in vivo lineage tracing work demonstrates that luminal cells are capable of producing basal cells upon activation of either Polyoma Middle T antigen (PyMT) or ErbB2 signaling. These findings reveal the plasticity of the luminal compartment during tumorigenesis and provide an explanation for cellular heterogeneity within a cancer. PMID:26096929

The Mammary Stem Cell Hierarchy: A Looking Glass into Heterogeneous Breast Cancer Landscapes

PubMed Central

Sreekumar, Amulya; Roarty, Kevin; Rosen, Jeffrey M.

2015-01-01

The mammary gland is a dynamic organ that undergoes extensive morphogenesis during the different stages of embryonic development, puberty, estrus, pregnancy, lactation and involution. Systemic and local cues underlie this constant tissue remodeling and act by eliciting an intricate pattern of responses in the mammary epithelial and stromal cells. Decades of studies utilizing methods such as transplantation and lineage tracing have identified a complex hierarchy of mammary stem cells, progenitors and differentiated epithelial cells that fuel mammary epithelial development. Importantly, these studies have extended our understanding of the molecular crosstalk between cell types, and signaling pathways maintaining normal homeostasis that often are deregulated during tumorigenesis. While several questions remain, this research has many implications for breast cancer. Fundamental among these are the identification of the cells of origin for the multiple subtypes of breast cancer and the understanding of tumor heterogeneity. A deeper understanding of these critical questions will unveil novel breast cancer drug targets and treatment paradigms. In this review, we provide a current overview of normal mammary development and tumorigenesis from a stem cell perspective. PMID:26206777

TRIENNIAL LACTATION SYMPOSIUM/BOLFA: Plasticity of mammary development in the prepubertal bovine mammary gland.

PubMed

Akers, R M

2017-12-01

Although peripubertal mammary development represents only a small fraction of the total mass of mammary parenchyma present in the udder at the end of gestation and into lactation, there is increasing evidence that the tissue foundations created in early life can affect future mammary development and function. Studies on expression of estrogen and progesterone receptors seem to confirm the relevance of these steroids in prepubertal mammary development, but connections with other growth factors, hormones, and local tissue factors remain elusive. Enhanced preweaning feeding in the bovine appears to enhance the capacity of mammary tissue to response to mammogenic stimulation. This suggests the possibility that improved early nutrition might allow for creation of stem or progenitor cell populations to better support the massive ductal growth and lobulo-alveolar development during gestation. Increasing evidence that immune cells are involved in mammary development suggests there are unexpected and poorly understood connections between the immune system and mammary development. This is nearly unexplored in ruminants. Development of new tools to identify, isolate, and characterize cell populations within the developing bovine mammary gland offer the possibility of identifying and perhaps altering populations of mammary stem cells or selected progenitor cells to modulate mammary development and, possibly, mammary function.

An autologous dendritic cell canine mammary tumor hybrid-cell fusion vaccine.

PubMed

Bird, R Curtis; Deinnocentes, Patricia; Church Bird, Allison E; van Ginkel, Frederik W; Lindquist, Joni; Smith, Bruce F

2011-01-01

Mammary cancer is among the most prevalent canine tumors and frequently resulting in death due to metastatic disease that is highly homologous to human breast cancer. Most canine tumors fail to raise effective immune reactions yet, some spontaneous remissions do occur. Hybrid canine dendritic cell-tumor cell fusion vaccines were designed to enhance antigen presentation and tumor immune recognition. Peripheral blood-derived autologous dendritic cell enriched populations were isolated from dogs based on CD11c(+) expression and fused with canine mammary tumor (CMT) cells for vaccination of laboratory Beagles. These hybrid cells were injected into popliteal lymph nodes of normal dogs, guided by ultrasound, and included CpG-oligonucleotide adjuvants. Three rounds of vaccination were delivered. Significant IgG responses were observed in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized shared CMT antigens as was demonstrated by IgG-recognition of three unrelated/independently derived CMT cell lines, and recognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype response was observed after two vaccinations in most dogs. Neither significant cytotoxic T cell responses were detected, nor adverse or side-effects due to vaccination or due to the induced immune responses noted. These data provide proof-of-principle for this cancer vaccine strategy and demonstrate the presence of shared CMT antigens that promote immune recognition of mammary cancer.

Canonical Wnt Signaling as a Specific Marker of Normal and Tumorigenic Mammary Stem Cells

DTIC Science & Technology

2010-02-01

for mammary stem cells and be a target for transformation that results in the formation of aggressive mammary tumors. Breast cancer stem cells, Wnt...tumorigenesis, and human breast cancer. In addition, increasing evidence suggests that tumors arise from either normal stem or progenitor cells...population of mammary tumor cells that are CD24+/CD49++. Since Wnt pathway activation occurs in human breast cancer and is required for

Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina, D.; Oborn, C.J.; Li, M.L.

1987-09-01

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appearedmore » to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.« less

Mutational landscape of goblet cell carcinoids and adenocarcinoma ex goblet cell carcinoids of the appendix is distinct from typical carcinoids and colorectal adenocarcinomas.

PubMed

Johncilla, Melanie; Stachler, Matthew; Misdraji, Joseph; Lisovsky, Mikhail; Yozu, Masato; Lindeman, Neal; Lauwers, Gregory Y; Odze, Robert D; Srivastava, Amitabh

2018-02-08

There is limited data on the spectrum of molecular alterations in goblet cell carcinoids and adenocarcinoma ex goblet cell carcinoids of the appendix. We used next generation sequencing to determine mutations of potential pathogenetic and therapeutic significance in this rare group of tumors. Adequate DNA was successfully extracted in 34/46 cases and the final group included 18 goblet cell carcinoids and 16 adenocarcinoma ex goblet cell carcinoids. Illumina TruSeq™ was used for sequencing exons of a custom 282 gene panel using an Illumina HiSeq 2000. All cases had a minimum coverage depth of at least 50 reads. After filtering through the Exome Sequencing Project, the number of mutations per case ranged from 0-9 (mean:3). The mutational burden in adenocarcinoma ex goblet cell carcinoids was significantly higher than goblet cell carcinoids (mean 5 vs. 3; p < 0.05) but the spectrum of alterations overlapped between the two groups. The most frequent mutations included ARID1A (4/34), ARID2 (4/34), CDH1 (4/34), RHPN2 (4/34), and MLL2 (3/34). Some mutations typically seen in conventional colorectal adenocarcinomas were also identified but with much lower frequency (APC :4/34; KRAS :2/34). MLL2 and KRAS mutations were only seen in adenocarcinoma ex goblet cell carcinoids and TP53 mutations were limited to poorly differentiated adenocarcinoma ex goblet cell carcinoids (2/34). Copy number changes could be evaluated in 15/34 cases and showed low copy number gains in CDKN1B (6/15) and NFKBIA (6/15), among others. The overlapping molecular alterations suggest that goblet cell carcinoids and adenocarcinoma ex goblet cell carcinoids are best considered two grades of differentiation of the same tumor rather than two distinct histological types. Mutations in TP53, CDH1 and MLL2 mutations were predominantly present in the adenocarcinoma ex goblet cell carcinoid group consistent with transformation to a higher grade lesion. The unique mutational profile also offers an

CLOCK regulates mammary epithelial cell growth and differentiation

PubMed Central

Crodian, Jennifer; Suárez-Trujillo, Aridany; Erickson, Emily; Weldon, Bethany; Crow, Kristi; Cummings, Shelby; Chen, Yulu; Shamay, Avi; Mabjeesh, Sameer J.; Plaut, Karen

2016-01-01

Circadian clocks influence virtually all physiological processes, including lactation. Here, we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late-pregnant wild-type and ClockΔ19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand whether these effects were due, in part, to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with short hairpin RNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P < 0.05). HC11 lines carrying shClock had four-fold higher growth rates (P < 0.05), and the percentage of cells in G1 phase was significantly higher (90.1 ± 1.1% of shClock vs. 71.3 ± 3.6% of WT-HC11) following serum starvation. Quantitative-PCR (qPCR) analysis showed shClock had significant effects (P < 0.0001) on relative expression levels of Ccnd1, Wee1, and Tp63. qPCR analysis of the effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures and cultures treated 96 h with dexamethasone, insulin, and prolactin (differentiated) found levels were reduced by twofold and threefold, respectively (P < 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock. These data support how CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland. PMID:27707717

Mammary stem cells: Novel markers and novel approaches to increase lactation efficiency

USDA-ARS?s Scientific Manuscript database

Mammary stem cells (MaSC) provide for net growth, renewal and turnover of mammary epithelial cells, and are therefore potential targets for strategies to increase production efficiency. Appropriate regulation of MaSC can potentially benefit milk yield, persistency, dry period management and tissue r...

Mena deficiency delays tumor progression and decreases metastasis in polyoma middle-T transgenic mouse mammary tumors.

PubMed

Roussos, Evanthia T; Wang, Yarong; Wyckoff, Jeffrey B; Sellers, Rani S; Wang, Weigang; Li, Jiufeng; Pollard, Jeffrey W; Gertler, Frank B; Condeelis, John S

2010-01-01

The actin binding protein Mammalian enabled (Mena), has been implicated in the metastatic progression of solid tumors in humans. Mena expression level in primary tumors is correlated with metastasis in breast, cervical, colorectal and pancreatic cancers. Cells expressing high Mena levels are part of the tumor microenvironment for metastasis (TMEM), an anatomical structure that is predictive for risk of breast cancer metastasis. Previously we have shown that forced expression of Mena adenocarcinoma cells enhances invasion and metastasis in xenograft mice. Whether Mena is required for tumor progression is still unknown. Here we report the effects of Mena deficiency on tumor progression, metastasis and on normal mammary gland development. To investigate the role of Mena in tumor progression and metastasis, Mena deficient mice were intercrossed with mice carrying a transgene expressing the polyoma middle T oncoprotein, driven by the mouse mammary tumor virus. The progeny were investigated for the effects of Mena deficiency on tumor progression via staging of primary mammary tumors and by evaluation of morbidity. Stages of metastatic progression were investigated using an in vivo invasion assay, intravital multiphoton microscopy, circulating tumor cell burden, and lung metastases. Mammary gland development was studied in whole mount mammary glands of wild type and Mena deficient mice. Mena deficiency decreased morbidity and metastatic dissemination. Loss of Mena increased mammary tumor latency but had no affect on mammary tumor burden or histologic progression to carcinoma. Elimination of Mena also significantly decreased epidermal growth factor (EGF) induced in vivo invasion, in vivo motility, intravasation and metastasis. Non-tumor bearing mice deficient for Mena also showed defects in mammary gland terminal end bud formation and branching. Deficiency of Mena decreases metastasis by slowing tumor progression and reducing tumor cell invasion and intravasation. Mena

Effects of glucose on lactose synthesis in mammary epithelial cells from dairy cow.

PubMed

Lin, Ye; Sun, Xiaoxu; Hou, Xiaoming; Qu, Bo; Gao, Xuejun; Li, Qingzhang

2016-05-26

Lactose, as the primary osmotic component in milk, is the major determinant of milk volume. Glucose is the primary precursor of lactose. However, the effect of glucose on lactose synthesis in dairy cow mammary glands and the mechanism governing this process are poorly understood. Here we showed that glucose has the ability to induce lactose synthesis in dairy cow mammary epithelial cells, as well as increase cell viability and proliferation. A concentration of 12 mM glucose was the optimum concentration to induce cell growth and lactose synthesis in cultured dairy cow mammary epithelial cells. In vitro, 12 mM glucose enhanced lactose content, along with the expression of genes involved in glucose transportation and the lactose biosynthesis pathway, including GLUT1, SLC35A2, SLC35B1, HK2, β4GalT-I, and AKT1. In addition, we found that AKT1 knockdown inhibited cell growth and lactose synthesis as well as expression of GLUT1, SLC35A2, SLC35B1, HK2, and β4GalT-I. Glucose induces cell growth and lactose synthesis in dairy cow mammary epithelial cells. Protein kinase B alpha acts as a regulator of metabolism in dairy cow mammary gland to mediate the effects of glucose on lactose synthesis.

Lgr4 regulates mammary gland development and stem cell activity through the pluripotency transcription factor Sox2.

PubMed

Wang, Ying; Dong, Jie; Li, Dali; Lai, Li; Siwko, Stefan; Li, Yi; Liu, Mingyao

2013-09-01

The key signaling networks regulating mammary stem cells are poorly defined. The leucine-rich repeat containing G protein-coupled receptor (Lgr) family has been implicated in intestinal, gastric, and epidermal stem cell functions. We investigated whether Lgr4 functions in mammary gland development and mammary stem cells. We found that Lgr4(-/-) mice had delayed ductal development, fewer terminal end buds, and decreased side-branching. Crucially, the mammary stem cell repopulation capacity was severely impaired. Mammospheres from Lgr4(-/-) mice showed decreased Wnt signaling. Wnt3a treatment prevented the adverse effects of Lgr4 loss on organoid formation. Chromatin immunoprecipitation analysis indicated that Sox2 expression was controlled by the Lgr4/Wnt/β-catenin/Lef1 pathway. Importantly, Sox2 overexpression restored the in vivo mammary regeneration potential of Lgr4(-/-) mammary stem cells. Therefore, Lgr4 activates Sox2 to regulate mammary development and stem cell functions via Wnt/β-catenin/Lef1. © AlphaMed Press.

Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

PubMed

Jin, Liting; Qu, Ying; Gomez, Liliana J; Chung, Stacey; Han, Bingchen; Gao, Bowen; Yue, Yong; Gong, Yiping; Liu, Xuefeng; Amersi, Farin; Dang, Catherine; Giuliano, Armando E; Cui, Xiaojiang

2018-02-20

Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

Role of peptidylarginine deiminase 2 (PAD2) in mammary carcinoma cell migration.

PubMed

Horibata, Sachi; Rogers, Katherine E; Sadegh, David; Anguish, Lynne J; McElwee, John L; Shah, Pragya; Thompson, Paul R; Coonrod, Scott A

2017-05-26

Penetration of the mammary gland basement membrane by cancer cells is a crucial first step in tumor invasion. Using a mouse model of ductal carcinoma in situ, we previously found that inhibition of peptidylarginine deiminase 2 (PAD2, aka PADI2) activity appears to maintain basement membrane integrity in xenograft tumors. The goal of this investigation was to gain insight into the mechanisms by which PAD2 mediates this process. For our study, we modulated PAD2 activity in mammary ductal carcinoma cells by lentiviral shRNA-mediated depletion, lentiviral-mediated PAD2 overexpression, or PAD inhibition and explored the effects of these treatments on changes in cell migration and cell morphology. We also used these PAD2-modulated cells to test whether PAD2 may be required for EGF-induced cell migration. To determine how PAD2 might promote tumor cell migration in vivo, we tested the effects of PAD2 inhibition on the expression of several cell migration mediators in MCF10DCIS.com xenograft tumors. In addition, we tested the effect of PAD2 inhibition on EGF-induced ductal invasion and elongation in primary mouse mammary organoids. Lastly, using a transgenic mouse model, we investigated the effects of PAD2 overexpression on mammary gland development. Our results indicate that PAD2 depletion or inhibition suppresses cell migration and alters the morphology of MCF10DCIS.com cells. In addition, we found that PAD2 depletion suppresses the expression of the cytoskeletal regulatory proteins RhoA, Rac1, and Cdc42 and also promotes a mesenchymal to epithelial-like transition in tumor cells with an associated increase in the cell adhesion marker, E-cadherin. Our mammary gland organoid study found that inhibition of PAD2 activity suppresses EGF-induced ductal invasion. In vivo, we found that PAD2 overexpression causes hyperbranching in the developing mammary gland. Together, these results suggest that PAD2 plays a critical role in breast cancer cell migration. Our findings that EGF

Expression and function of activin receptors in human endometrial adenocarcinoma cells.

PubMed

Tanaka, Tetsuji; Toujima, Saori; Otani, Tsutomu; Minami, Sawako; Yamoto, Mareo; Umesaki, Naohiko

2003-09-01

Menstrual cycle-dependent expressions of activin A in normal human endometrial tissues have been reported. Expression of activin receptor mRNAs and increased activin A production were also observed in human endometrial adenocarcinoma tissues, suggesting that activin A might enhance cell proliferation and inhibit apoptotic signaling in endometrial cancer cells. In this study, we have examined the effects of activin A on cell proliferation, anticancer drug-induced apoptosis and Fas-mediated apoptosis in 3 differentiated human endometrial adenocarcinoma cell lines, namely HEC-1, HHUA and Ishikawa. Flow cytometric analyses revealed moderate expressions of all 4 types of activin receptor subunits on the cell surfaces of the 3 cell lines. The proliferations of the 3 endometrial cancer cells were completely unaffected by activin A, whereas it suppressed the cell proliferation of a human ovarian endometrioid adenocarcinoma cell line, OVK-18, in a dose-dependent manner. Moreover, activin A did not affect the apoptotic changes in the 3 endometrial adenocarcinoma cells treated with 4 different anticancer drugs, namely CDDP, paclitaxel, etoposide and SN38. The apoptotic changes in HHUA cells treated with anti-Fas IgM were also unaffected by activin A. These results indicate that the increased activin A production in human endometrial adenocarcinoma tissues in vivo may not stimulate carcinoma cell proliferation or inhibit apoptotic signaling in carcinoma cells. Insensitivity to the usual growth suppression signals induced by activin A might be one of the mechanisms of immortality of human endometrial adenocarcinoma cells.

Establishment and characterization of a lactating dairy goat mammary gland epithelial cell line.

PubMed

Tong, Hui-Li; Li, Qing-Zhang; Gao, Xue-Jun; Yin, De-Yun

2012-03-01

To study milk synthesis in dairy goat mammary gland, we had established an in vitro lactating dairy goat mammary epithelial cell (DGMEC) line. Mammary tissues of Guan Zhong dairy goats at 35 d of lactation were dispersed and cultured in a medium containing epithelial growth factor, insulin-like growth factor-1, insulin transferrin serum, and fetal bovine serum. Epithelial cells were enriched by digesting with 0.25% trypsin repeatedly to remove fibroblast cells and were identified as epithelial origin by staining with antibody against cytokeratine 18. The DGMECs displayed monolayer, cobble-stone, epithelial-like morphology, and formed alveoli-like structures and island monolayer aggregates which were the typical characteristics of mammary epithelial cells. A one-half logarithmically growth curve and cytoplasmic lipid droplets in these cells were observed. In this paper, we also studied the lactating function of DGMECs. Results showed that DGMECs could secrete lactose and β-casein. Lactating function of the cells had no obvious change after 48 h treated by insulin, while prolactin could obviously raise the secretion of milk proteins and lactose.

Mammary Stem Cells and Breast Cancer Stem Cells: Molecular Connections and Clinical Implications.

PubMed

Celià-Terrassa, Toni

2018-05-04

Cancer arises from subpopulations of transformed cells with high tumor initiation and repopulation ability, known as cancer stem cells (CSCs), which share many similarities with their normal counterparts. In the mammary gland, several studies have shown common molecular regulators between adult mammary stem cells (MaSCs) and breast cancer stem cells (bCSCs). Cell plasticity and self-renewal are essential abilities for MaSCs to maintain tissue homeostasis and regenerate the gland after pregnancy. Intriguingly, these properties are similarly executed in breast cancer stem cells to drive tumor initiation, tumor heterogeneity and recurrence after chemotherapy. In addition, both stem cell phenotypes are strongly influenced by external signals from the microenvironment, immune cells and supportive specific niches. This review focuses on the intrinsic and extrinsic connections of MaSC and bCSCs with clinical implications for breast cancer progression and their possible therapeutic applications.

Initiation of oncogenic transformation in human mammary epithelial cells by charged particles

NASA Technical Reports Server (NTRS)

Yang, T. C.; Georgy, K. A.; Craise, L. M.; Durante, M.

1997-01-01

Experimental studies have shown that high linear-energy transfer (LET) charged particles can be more effective than x-rays and gamma-rays in inducing oncogenic transformation in cultured cells and tumors in animals. Based on these results, experiments were designed and performed with an immortal human mammary epithelial cell line (H184B5), and several clones transformed by heavy ions were obtained. Cell fusion experiments were subsequently done, and results indicate that the transforming gene(s) is recessive. Chromosome analysis with fluorescence in situ hybridization (FISH) techniques also showed additional translocations in transformed human mammary epithelial cells. In addition, studies with these cell lines indicate that heavy ions can effectively induce deletion, break, and dicentrics. Deletion of tumor suppressor gene(s) and/or formation of translocation through DNA double strand breaks is a likely mechanism for the initiation of oncogenic transformation in human mammary epithelial cells.

Early detection, aggressive therapy: optimizing the management of feline mammary masses.

PubMed

Giménez, Fernanda; Hecht, Silke; Craig, Linden E; Legendre, Alfred M

2010-03-01

This article reviews the incidence, etiology, diagnosis, treatment and prognosis of mammary tumors in cats. Approximately 80% of feline mammary masses are malignant, with adenocarcinoma being the most common tumor type. Early diagnosis is, therefore, essential to improve the prognosis and quality of life of affected cats. Surgery is the most widely used treatment for malignant tumors. However, as mammary tumors are often advanced and metastasis has already occurred by the time of diagnosis, surgery routinely does not provide a cure. Ovariohysterectomy or hormonal therapy are the treatments of choice for fibroadenomatous hyperplasia (the most common benign mass) and usually lead to a successful outcome. Copyright 2010. Published by Elsevier Ltd.

JS-K, a nitric oxide-releasing prodrug, induces breast cancer cell death while sparing normal mammary epithelial cells.

PubMed

McMurtry, Vanity; Saavedra, Joseph E; Nieves-Alicea, René; Simeone, Ann-Marie; Keefer, Larry K; Tari, Ana M

2011-04-01

Targeted therapy with reduced side effects is a major goal in cancer research. We investigated the effects of JS-K, a nitric oxide (NO) prodrug designed to release high levels of NO when suitably activated, on human breast cancer cell lines, on non-transformed human MCF-10A mammary cells, and on normal human mammary epithelial cells (HMECs). Cell viability assay, flow cytometry, electron microscopy, and Western blot analysis were used to study the effects of JS-K on breast cancer and on mammary epithelial cells. After a 3-day incubation, the IC50s of JS-K against the breast cancer cells ranged from 0.8 to 3 µM. However, JS-K decreased the viability of the MCF-10A cells by only 20% at 10-µM concentration, and HMECs were unaffected by 10 µM JS-K. Flow cytometry indicated that JS-K increased the percentages of breast cancer cells under-going apoptosis. Interestingly, flow cytometry indicated that JS-K increased acidic vesicle organelle formation in breast cancer cells, suggesting that JS-K induced autophagy in breast cancer cells. Electron microscopy confirmed that JS-K-treated breast cancer cells underwent autophagic cell death. Western blot analysis showed that JS-K induced the expression of microtubule light chain 3-II, another autophagy marker, in breast cancer cells. However, JS-K did not induce apoptosis or autophagy in normal human mammary epithelial cells. These data indicate that JS-K selectively induces programmed cell death in breast cancer cells while sparing normal mammary epithelial cells under the same conditions. The selective anti-tumor activity of JS-K warrants its further investigation in breast tumors.

Primary mammary mucinous cystadenocarcinoma: cytological and histological findings.

PubMed

Sentani, Kazuhiro; Tashiro, Takashi; Uraoka, Naohiro; Aosaki, Yoriyuki; Yano, Satomi; Takaeko, Fumio; Yasui, Wataru

2012-07-01

Mucinous cystadenocarcinoma (MCA), commonly encountered in the ovary or pancreas, is rare in the breast and was only recently described as a distinct variant of invasive ductal carcinoma of the breast. Only 11 cases of primary mammary MCA have been reported. In this article, we report a case of primary mammary MCA with focus on cytological and histological findings. A 65-year-old female noticed right palpable breast mass. Sonography showed an irregularly shaped 2.8 × 2.4 cm lesion in the upper outer quadrant of the right breast. Fine-needle aspiration cytology was performed on the right breast nodule, and cytopathologic examination suggested an adenocarcinoma composed of tall columnar cells with mucin. A partial mastectomy of the right breast and the axillary lymph nodes dissection was performed. The gross examination revealed a well-demarcated and mucus-filled tumor. Histologically, it had complex papillae, some of which were supported by delicate fibrovascular stroma lined by simple to slightly stratified columnar neoplastic epithelial cells with intracellular mucin, coexisting with MCA in situ and ordinary intraductal carcinoma component (ICC). Immunohistochemically, ICC was HER2-negative and estrogen receptor/progesterone receptor-positive, while MCA was triple negative. MCA might be derived from a metaplasia of ordinary ICC, but its pathogenesis and biologic behavior remains unclear. Despite the invasive nature of mammary MCA, these carcinomas appear to be associated with a good prognosis. The patient has remained well and disease-free for 6 months after the operation. Copyright © 2011 Wiley Periodicals, Inc.

Xanthosine administration does not affect the proportion of epithelial stem cells in bovine mammary tissue, but has a latent negative effect on cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rauner, Gat, E-mail: gat.rauner@mail.huji.ac.il; The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem; Barash, Itamar, E-mail: itamar.barash@mail.huji.ac.il

The challenge in manipulating the proportion of somatic stem cells lies in having to override tissue homeostasis. Xanthosine infusion via the teat canal has been reported to augment the number of label-retaining cells in the mammary gland of 3-month-old bovine calves. To further delineate xanthosine's effect on defined stem cells in the mammary gland of heifers—which are candidates for increased prospective milk production following such manipulation—bovine mammary parenchymal tissue was transplanted and integrated into the cleared mammary fat pad of immunodeficient mice. Xanthosine administration for 14 days did not affect the number of label-retaining cells after 10- and 11-week chases.more » No change in stem cell proportion, analyzed according to CD49f and CD24 expression, was noted. Clone formation and propagation rate of cultured cells, as well as expression of stem cell markers, were also unaffected. In contrast, a latent 50% decrease in bovine mammary cell proliferation rate was observed 11 weeks after xanthosine administration. Tumor development in mice was also limited by xanthosine administration. These effects may have resulted from an initial decrease in expression of the rate-limiting enzyme in guanine synthesis, IMPDH. The data indicate that caution should be exerted when considering xanthosine for stem cell manipulation. - Highlights: • Novel “bovinized“ mouse model for exogenous effects on bovine mammary gland. • Xanthosine did not affect stem cell number/function in bovine mammary gland. • Xanthosine caused an immediate decrease in IMPDH expression in bovine mammary gland. • Xanthosine had latent negative effect on cell proliferation in bovine mammary gland. • Xanthosine administration limited mammary tumor growth.« less

Inhibitory effects of a polypeptide thymic factor on the development of 7,12-dimethylbenz(a)anthragene-induced mammary adenocarcinoma in female rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anisimov, V.N.; Danetskaya, E.V.; Morozov, V.G.

1980-01-01

It has come to be recognized that tumor growth is accompanied by inhibition of cellular immunity and the function of the T lymphocytes. Restitution of T lymphocyte function by means of several pharmacologic agents such as levamisole, phenformin, or epithalamin (an epiphyseal factor) has, in a number of cases, been accompanied by growth inhibition of both spontaneous and induced tumors. In addition, the importance of the thymus in the regulation of T lymphocytes and in antitumor immunity has been recognized. Several indicators point to the fact that the thymus contains physiologically active substances which stimulate T cell-dependent immunity and preventmore » the occurrence of neoplasms. These considerations have led to attempts at isolation of active thymic factors and studies on their effects on the appearance and growth of tumors. Previously, a thymic factor - thymarin - had been isolated which imparted immunocompetence to the T lymphocytes. This factor differs from other thymic preparations, including thymosine, in terms of a number of physicochemical characteristics and is a polypeptide with a molecular weight of 5000. This study is concerned with its effects on tumor development - mammary gland adenocarcinoma induced in animals with a chemical carcinogen.« less

Retinoids, retinoid analogs, and lactoferrin interact and differentially affect cell viability of 2 bovine mammary cell types in vitro.

PubMed

Wang, Y; Baumrucker, C R

2010-07-01

Two bovine mammary cell types (BME-UV1 and MeBo cells) were used to evaluate the effect of natural retinoids, retinoid analogs, and bovine lactoferrin (bLf) on cell viability in vitro. Experiments with Alamar Blue showed a linear relationship between fluorescence and cell viability index. The BME-UV1 cells exhibited twice the metabolic activity but required half the doubling time of the MeBo cells. The BME-UV1 cells were very sensitive to all-trans retinoic acid (atRA) inhibition of cell viability (P<0.05) and exhibited a dose-dependent inhibition with 9-cisRA (9cRA; P<0.05). The MeBo cells exhibited some inhibition with these natural ligands (P<0.05), but they were not as sensitive. The addition of bLf had similar inhibitory effects (P<0.05) on cell viability of the 2 mammary cell types. Applications of RA receptor (RAR) agonist indicated that the stimulation of the RAR in both mammary cell types was highly effective in inhibition of cell viability (P<0.05), whereas the application of an RAR antagonist stimulated MeBo cell viability (P<0.05) and inhibited BME-UV1 cell viability (P<0.05). Finally, the use of the RAR antagonist in conjunction with bLf indicated a rescue of the bLf effect in the MeBo cells, suggesting that bLf is acting through the RAR receptor. Conversely, bLf reverted inhibition of cell viability by 9cRA in the BME-UV1 cell type (P<0.05). We conclude that RAR interaction in bovine mammary cell types regulates cell viability in vitro; we hypothesize that the natural ligands mediate regulation of bovine mammary cell viability in vivo and that bLf can either enhance or reverse the retinoid-induced inhibition of cell viability, depending on the type of bovine mammary cell studied.

Calmodulin-mediated activation of Akt regulates survival of c-Myc-overexpressing mouse mammary carcinoma cells.

PubMed

Deb, Tushar B; Coticchia, Christine M; Dickson, Robert B

2004-09-10

c-Myc-overexpressing mammary epithelial cells are proapoptotic; their survival is strongly promoted by epidermal growth factor (EGF). We now demonstrate that EGF-induced Akt activation and survival in transgenic mouse mammary tumor virus-c-Myc mouse mammary carcinoma cells are both calcium/calmodulin-dependent. Akt activation is abolished by the phospholipase C-gamma inhibitor U-73122, by the intracellular calcium chelator BAPTA-AM, and by the specific calmodulin antagonist W-7. These results implicate calcium/calmodulin in the activation of Akt in these cells. In addition, Akt activation by serum and insulin is also inhibited by W-7. EGF-induced and calcium/calmodulin-mediated Akt activation occurs in both tumorigenic and non-tumorigenic mouse and human mammary epithelial cells, independent of their overexpression of c-Myc. These results imply that calcium/calmodulin may be a common regulator of Akt activation, irrespective of upstream receptor activator, mammalian species, and transformation status in mammary epithelial cells. However, only c-Myc-overexpressing mouse mammary carcinoma cells (but not normal mouse mammary epithelial cells) undergo apoptosis in the presence of the calmodulin antagonist W-7, indicating the vital selective role of calmodulin for survival of these cells. Calcium/calmodulin-regulated Akt activation is mediated directly by neither calmodulin kinases nor phosphatidylinositol 3-kinase (PI-3 kinase). Pharmacological inhibitors of calmodulin kinase kinase and calmodulin kinases II and III do not inhibit EGF-induced Akt activation, and calmodulin antagonist W-7 does not inhibit phosphotyrosine-associated PI-3 kinase activation. Akt is, however, co-immunoprecipitated with calmodulin in an EGF-dependent manner, which is inhibited by calmodulin antagonist W-7. We conclude that calmodulin may serve a vital regulatory function to direct the localization of Akt to the plasma membrane for its activation by PI-3 kinase.

GosB Inhibits Triacylglycerol Synthesis and Promotes Cell Survival in Mouse Mammary Epithelial Cells.

PubMed

Xu, Gaoxiao; Duan, Saixing; Hou, Jianye; Wei, Zhongxin; Zhao, Guangwei

2017-01-01

It has been demonstrated that the activator protein related transcription factor Finkel-Biskis-Jinkins murine osteosarcoma B (GosB) is involved in preadipocyte differentiation and triacylglycerol synthesis. However, the role of GosB in regulating the synthesis of milk fatty acid in mouse mammary glands remains unclear. This research uncovered potentially new roles of GosB in suppressing milk fatty acid synthesis. Results revealed that GosB had the highest expression in lung tissue and showed a higher expression level during nonlactation than during lactation. GosB inhibited the expression of fatty acid synthase (FASN) , stearoyl-CoA desaturase (SCD) , fatty acid binding protein 4 (FABP4) , diacylglycerol acyltransferase 1 (DGAT1) , perilipin 2 (PLIN2) , perilipin 3 (PLIN3) , and C/EBPα in mouse mammary gland epithelial cells (MEC). In addition, GosB reduced cellular triglyceride content and the accumulation of lipid droplets; in particular, GosB enhanced saturated fatty acid concentration (C16:0 and C18:0). The PPAR γ agonist, rosiglitazone (ROSI), promoted apoptosis and inhibited cell proliferation. GosB increased the expression of Bcl-2 and protected MEC from ROSI-induced apoptosis. Furthermore, MECs were protected from apoptosis through the GosB regulation of intracellular calcium concentrations. These findings suggest that GosB may regulate mammary epithelial cells milk fat synthesis and apoptosis via PPAR γ in mouse mammary glands.

Different mechanisms lead to the angiogenic process induced by three adenocarcinoma cell lines.

PubMed

Davel, Lilia E; Rimmaudo, Laura; Español, Alejandro; de la Torre, Eulalia; Jasnis, María Adela; Ribeiro, María Laura; Gotoh, Tomomi; de Lustig, Eugenia Sacerdote; Sales, María Elena

2004-01-01

Neoangiogenesis is essential for tumor and metastasis growth, but this complex process does not follow the same activation pathway, at least in tumor cell lines originated from different murine mammary adenocarcinomas. LMM3 cells were the most potent to stimulate new blood vessel formation. This response was significantly reduced by preincubating cells with indomethacin and NS-398, non-selective cyclooxygenase (COX) and COX-2 selective inhibitors, respectively. COX-1 and COX-2 isoenzymes were both highly expressed in LMM3 cells, and we observed that indomethacin was more effective than NS-398 to inhibit prostaglandin E2 (PGE2) synthesis. In addition, nitric oxide synthase (NOS) inhibitors, Nomega monomethyl L-arginine and aminoguanidine, also reduced LMM3-induced angiogenesis and nitric oxide (NO) synthesis as well. NOS2 > NOS3 proteins and arginase II isoform were detected in LMM3 cells by Western blot. The latter enzyme was also involved in the LMM3 neovascular response, since the arginase inhibitor, Nomega hydroxy L-arginine reduced the angiogenic cascade. On the other hand, parental LM3 cells were able to stimulate neovascularization via COX-1 and arginase products since only indomethacin and Nomega hydroxy L-arginine, which diminished PGE2 and urea synthesis, respectively, also reduced angiogenesis. In turn, LM2 cells angiogenic response could be due in fact to PGE2-induced VEGF liberation that stimulated neoangiogenesis at very low levels of NO.

Amplification of tumor inducing putative cancer stem cells (CSCs) by vitamin A/retinol from mammary tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Rohit B.; Wang, Qingde; Khillan, Jaspal S., E-mail: khillan@pitt.edu

Highlights: •Vitamin A supports self renewal of putative CSCs from mammary tumors. •These cells exhibit impaired retinol metabolism into retinoic acid. •CSCs from mammary tumors differentiate into mammary specific cell lineages. •The cells express mammary stem cell specific CD29 and CD49f markers. •Putative CSCs form highly metastatic tumors in NOD SCID mouse. -- Abstract: Solid tumors contain a rare population of cancer stem cells (CSCs) that are responsible for relapse and metastasis. The existence of CSC however, remains highly controversial issue. Here we present the evidence for putative CSCs from mammary tumors amplified by vitamin A/retinol signaling. The cells exhibitmore » mammary stem cell specific CD29{sup hi}/CD49f{sup hi}/CD24{sup hi} markers, resistance to radiation and chemo therapeutic agents and form highly metastatic tumors in NOD/SCID mice. The cells exhibit indefinite self renewal as cell lines. Furthermore, the cells exhibit impaired retinol metabolism and do not express enzymes that metabolize retinol into retinoic acid. Vitamin A/retinol also amplified putative CSCs from breast cancer cell lines that form highly aggressive tumors in NOD SCID mice. The studies suggest that high purity putative CSCs can be isolated from solid tumors to establish patient specific cell lines for personalized therapeutics for pre-clinical translational applications. Characterization of CSCs will allow understanding of basic cellular and molecular pathways that are deregulated, mechanisms of tumor metastasis and evasion of therapies that has direct clinical relevance.« less

Expression of novel, putative stem cell markers in prepubertal and lactating mammary glands of bovine

USDA-ARS?s Scientific Manuscript database

Mammary stem cells (MaSC) are essential for growth and maintenance of the mammary epithelium. Two main phases of mammary growth include ductal elongation prior to puberty and lobulo-alveolar growth and development during pregnancy. Some studies have utilized morphological characteristics and retenti...

Canonical Wnt Signaling as a Specific Mark of Normal and Tumorigenic Mammary Stem Cells

DTIC Science & Technology

2011-02-01

aggressive mammary tumors. 15. SUBJECT TERMS Breast cancer stem cells, Wnt signaling, canonical Wnt signaling, B-catenin, normal stem cells, adult stem...Wnt pathway is associated with abnormal mouse mammary development, tumorigenesis, and human breast cancer. In addition, increasing evidence suggests...activation occurs in human breast cancer and is required for proliferation of various other stem cell compartments, addressing how Wnt signaling promotes

Regulation of exosome release from mammary epithelial and breast cancer cells - a new regulatory pathway.

PubMed

Riches, Andrew; Campbell, Elaine; Borger, Eva; Powis, Simon

2014-03-01

Exosomes are small 50-100nm sized extracellular vesicles released from normal and tumour cells and are a source of a new intercellular communication pathway. Tumour exosomes promote tumour growth and progression. What regulates the release and homoeostatic levels of exosomes, in cancer, in body fluids remains undefined. We utilised a human mammary epithelial cell line (HMEC B42) and a breast cancer cell line derived from it (B42 clone 16) to investigate exosome production and regulation. Exosome numbers were quantified using a Nanosight LM10 and measured in culture supernatants in the absence and presence of exosomes in the medium. Concentrated suspensions of exosomes from the normal mammary epithelial cells, the breast cancer cells and bladder cancer cells were used. The interaction of exosomes with tumour cells was also investigated using fluorescently labelled exosomes. Exosome release from normal human mammary epithelial cells and breast cancer cells is regulated by the presence of exosomes, derived from their own cells, in the extracellular environment of the cells. Exosomes from normal mammary epithelial cells also inhibit exosome secretion by breast cancer cells, which occurs in a tissue specific manner. Labelled exosomes from mammary epithelial cells are internalised into the tumour cells implicating a dynamic equilibrium and suggesting a mechanism for feedback control. These data suggest a previously unknown novel feedback regulatory mechanism for controlling exosome release, which may highlight a new therapeutic approach to controlling the deleterious effects of tumour exosomes. This regulatory mechanism is likely to be generic to other tumours. Copyright © 2014 Elsevier Ltd. All rights reserved.

The relevance and implications of signet-ring cell adenocarcinoma of the oesophagus.

PubMed

Bleaney, Christopher William; Barrow, Mickhaiel; Hayes, Stephen; Ang, Yeng

2018-03-01

To review the current understanding of signet-ring type oesophageal adenocarcinoma including evidence for prognosis. We conducted a literature search of nine healthcare literature databases for articles detailing the biology and clinical outcomes of signet-ring cell adenocarcinoma of the oesophagus. The impact of signet-ring cell morphology was analysed and detailed in written text and tabular format. Current understanding of the biology of signet-ring cell adenocarcinoma of the oesophagus was summarised. Signet-ring cell carcinoma was represented in 7.61% of the 18 989 cases of oesophageal carcinoma reviewed in multiple studies. The presence of signet-ring cells conferred a worse prognosis and these tumours responded differently to conventional treatments as compared with typical adenocarcinoma. Little is known about the biological features of signet-ring cell adenocarcinoma of the oesophagus. Work in gastric lesions has identified potential targets for future treatments such as CDH1 and RHOA genes. Categorisation of signet-ring cell carcinomas by the proportion of signet-ring cells within tumours differs among clinicians despite WHO criteria for classification. The current UK guidelines for histopathological reporting of oesophageal tumours do not emphasise the importance of identifying signet-ring cells. The presence of signet-ring cells in oesophageal adenocarcinomas leads to poorer clinical outcomes. Current understanding of signet-ring cell biology in oesophageal cancer is limited. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Aluminium chloride promotes anchorage-independent growth in human mammary epithelial cells.

PubMed

Sappino, André-Pascal; Buser, Raphaële; Lesne, Laurence; Gimelli, Stefania; Béna, Frédérique; Belin, Dominique; Mandriota, Stefano J

2012-03-01

Aluminium salts used as antiperspirants have been incriminated as contributing to breast cancer incidence in Western societies. To date, very little or no epidemiological or experimental data confirm or infirm this hypothesis. We report here that in MCF-10A human mammary epithelial cells, a well-established normal human mammary epithelial cell model, long-term exposure to aluminium chloride (AlCl(3) ) concentrations of 10-300 µ m, i.e. up to 100 000-fold lower than those found in antiperspirants, and in the range of those recently measured in the human breast, results in loss of contact inhibition and anchorage-independent growth. These effects were preceded by an increase of DNA synthesis, DNA double strand breaks (DSBs), and senescence in proliferating cultures. AlCl(3) also induced DSBs and senescence in proliferating primary human mammary epithelial cells. In contrast, it had no similar effects on human keratinocytes or fibroblasts, and was not detectably mutagenic in bacteria. MCF-10A cells morphologically transformed by long-term exposure to AlCl(3) display strong upregulation of the p53/p21(Waf1) pathway, a key mediator of growth arrest and senescence. These results suggest that aluminium is not generically mutagenic, but similar to an activated oncogene, it induces proliferation stress, DSBs and senescence in normal mammary epithelial cells; and that long-term exposure to AlCl(3) generates and selects for cells able to bypass p53/p21(Waf1) -mediated cellular senescence. Our observations do not formally identify aluminium as a breast carcinogen, but challenge the safety ascribed to its widespread use in underarm cosmetics. Copyright © 2012 John Wiley & Sons, Ltd.

Antiproliferative Effects of Oxytocin and Desmopressin on Canine Mammary Cancer Cells

PubMed Central

Benavente, Micaela Andrea; Bianchi, Carolina Paula; Imperiale, Fernanda; Aba, Marcelo Alfredo

2016-01-01

Neoplasms of the mammary gland represent the most frequent tumor type in the female dog, and according to the histologic criteria, approximately 50% of them are malignant. In the most aggressive cases of mammary cancer, surgery is not enough to warrant a favorable outcome, and adjuvant therapies are needed to improve the patient’s overall survival. The aim of the present study was to evaluate the effects of two peptides on proliferation of a canine mammary cancer cell line derived from a simple carcinoma. The cell line CMT-U27 was grown in 96-well plates, at two cell densities (4 × 103 and 8 × 103 cells/well). Cultures were treated with oxytocin (OT) or desmopressin at five concentrations (10, 50, 100, 500, and 1000 nM). After 72 h of incubation, cell proliferation was determined by the MTT assay. Results showed that with 4 × 103 cells/well, OT at 50, 500, and 1000 nM was growth inhibitory for the cells, being statistically significant at 1000 nM. On the contrary, no antiproliferative effect was observed with 10 or 100 nM. At 8 × 103 cells/well, OT showed a significant antiproliferative effect only with the highest concentration (1000 nM). Desmopressin at 4 × 103 cells/well decreased cell viability at concentrations of 50, 100, 500, and 1000 nM (statistically significant with the highest concentration), while no effect was observed with 10 nM. With 8 × 103 cells/well, this peptide reduced cell growth at 100, 500, and 1000 nM. In conclusion, we suggest that these peptides may be potential and promising compounds for the treatment of dogs with simple carcinomas of the mammary gland. In vivo studies are required to confirm this hypothesis. PMID:28083539

Antiproliferative Effects of Oxytocin and Desmopressin on Canine Mammary Cancer Cells.

PubMed

Benavente, Micaela Andrea; Bianchi, Carolina Paula; Imperiale, Fernanda; Aba, Marcelo Alfredo

2016-01-01

Neoplasms of the mammary gland represent the most frequent tumor type in the female dog, and according to the histologic criteria, approximately 50% of them are malignant. In the most aggressive cases of mammary cancer, surgery is not enough to warrant a favorable outcome, and adjuvant therapies are needed to improve the patient's overall survival. The aim of the present study was to evaluate the effects of two peptides on proliferation of a canine mammary cancer cell line derived from a simple carcinoma. The cell line CMT-U27 was grown in 96-well plates, at two cell densities (4 × 10 3 and 8 × 10 3 cells/well). Cultures were treated with oxytocin (OT) or desmopressin at five concentrations (10, 50, 100, 500, and 1000 nM). After 72 h of incubation, cell proliferation was determined by the MTT assay. Results showed that with 4 × 10 3 cells/well, OT at 50, 500, and 1000 nM was growth inhibitory for the cells, being statistically significant at 1000 nM. On the contrary, no antiproliferative effect was observed with 10 or 100 nM. At 8 × 10 3 cells/well, OT showed a significant antiproliferative effect only with the highest concentration (1000 nM). Desmopressin at 4 × 10 3 cells/well decreased cell viability at concentrations of 50, 100, 500, and 1000 nM (statistically significant with the highest concentration), while no effect was observed with 10 nM. With 8 × 10 3 cells/well, this peptide reduced cell growth at 100, 500, and 1000 nM. In conclusion, we suggest that these peptides may be potential and promising compounds for the treatment of dogs with simple carcinomas of the mammary gland. In vivo studies are required to confirm this hypothesis.

Wnt proteins are self-renewal factors for mammary stem cells and promote their long-term expansion in culture.

PubMed

Zeng, Yi Arial; Nusse, Roel

2010-06-04

Adult stem cells have the ability to self-renew and to generate specialized cells. Self-renewal is dependent on extrinsic niche factors but few of those signals have been identified. In addition, stem cells tend to differentiate in the absence of the proper signals and are therefore difficult to maintain in cell culture. The mammary gland provides an excellent system to study self-renewal signals, because the organ develops postnatally, arises from stem cells, and is readily generated from transplanted cells. We show here that adult mammary glands contain a Wnt-responsive cell population that is enriched for stem cells. In addition, stem cells mutant for the negative-feedback regulator Axin2 and therefore sensitized to Wnt signals have a competitive advantage in mammary gland reconstitution assays. In cell culture experiments, exposure to purified Wnt protein clonally expands mammary stem cells for many generations and maintains their ability to generate functional glands in transplantation assays. We conclude that Wnt proteins serve as rate-limiting self-renewal signals acting directly on mammary stem cells. Copyright 2010 Elsevier Inc. All rights reserved.

Relative quantification of beta-casein expression in primary goat mammary epithelial cell lines.

PubMed

Ogorevc, J; Dovč, P

2015-04-15

Primary mammary epithelial cell cultures were established from mammary tissue of lactating and non-lactating goats to assess the expression of beta-casein (CSN2) in vitro. Primary cell cultures were established by enzymatic digestion of mammary tissue and characterized using antibodies against cytokeratin 14, cytokeratin 18, and vimentin. The established primary cell lines in the second passage were grown in basal medium on plastic and in hormone-supplemented (lactogenic) medium on plastic and on an extracellular matrix-covered surface, respectively. CSN2 gene expression was evaluated using quantitative reverse transcription PCR. The presence of CSN2 transcripts was detected in all samples, including cells originating from non-lactating goat, grown in basal medium. The presence of CSN2 protein was confirmed using immunofluorescence. Response to the hormonal treatment and cell morphology differed between the cell lines and treatments. In 2 cell lines supplemented with lactogenic hormones in the medium, CSN2 expression was increased, while CSN2 levels in one of the cell lines remained constant, regardless of the treatment. Addition of extracellular matrix showed positive effects on CSN2 transcription activity in 1 of the cell lines, while in the other 2 showed no statistically significant effects. CSN2 expression appeared to depend on subtle differences in physiological state of the starting tissue material, growth conditions, cell types present in the culture, and methods used for cell culture establishment. Further studies are necessary to identify factors that determine hormone-responsiveness and transcriptional activity of milk protein genes in goat primary mammary cell cultures.

Understanding Collagen Organization in Breast Tumors to Predict and Prevent Metastasis

DTIC Science & Technology

2015-11-01

and their mechanism of action. We have explored stromal effects of macrophages in the E0771 murine mammary adenocarcinoma grown in the mammary fat ...and impacted tumor progression in breast tumor cell lines grown in the mammary fat pad (Figure 2 in Madden K, et al. 2013, Figure 9 here). We then...expression of TNF-α. To test this hypothesis, we grew mammary fat pad (MFP) tumors using a breast tumor cell line (E0771, a mammary adenocarcinoma derived from

Monitoring of Breast Tumor Response to Local Chemotherapeutic Agent Delivered by Biodegradable Fibers

DTIC Science & Technology

2006-05-01

mammary adenocarcinomas : one group received a radiation therapy during air inhalation and the other group was treated by irradiation during oxygen...procedures The tumor line was rat mammary adenocarcinomas 13762NF (cells originally provided by the Division of Cancer Therapeutics, NCI), and the...and Tumor Model Rat mammary 13762NF adenocarcinomas (original obtained from the Division of Cancer Therapeutics, NIH, Bethesda, Maryland) were

SDF-1 in Mammary Fibroblasts of Bovine with Mastitis Induces EMT and Inflammatory Response of Epithelial Cells.

PubMed

He, Guiliang; Ma, Mengru; Yang, Wei; Wang, Hao; Zhang, Yong; Gao, Ming-Qing

2017-01-01

Fibroblasts constitute the majority of the stromal cells within bovine mammary gland, yet the functional contributions of these cells to mastitis and fibrosis and the mechanism are poorly understood. In this study, we demonstrate that inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis had different expression pattern regarding to several extracellular matrix (ECM) proteins, chemokines and cytokines compared to normal fibroblasts (NFs) from dairy cows during lactation. The INFs induced epithelial-mesenchymal transition (EMT) and inflammatory responses of mammary epithelial cells in a vitro co-culture model. These functional contributions of INFs to normal epithelial cells were mediated through their ability to secrete stromal cell-derived factor 1 (SDF-1). SDF-1 was highly secreted/expressed by INFs, lipopolysaccharide (LPS) -treated NFs, lipoteichoic acid (LTA) -treated NFs, as well as mastitic tissue compared to their counterparts. Exogenous SDF-1 promoted EMT on epithelial cells through activating NF-κB pathway, induced inflammation response and inhibited proliferation of epithelial cells. In addition, SDF-1 was able to induce mastitis and slight fibrosis of mouse mammary gland, which was attenuated by a specific inhibitor of the receptor of SDF-1. Our findings indicate that stromal fibroblasts within mammary glands with mastitis contribute to EMT and inflammatory responses of epithelial cells through the secretion of SDF-1, which could result in the inflammation spread and fibrosis within mammary gland.

Primary non-clear-cell adenocarcinoma of the vagina in a diethylstilbestrol exposed woman.

PubMed

Patel, Samit A; Sunde, Jan

2014-04-01

A 54-year-old woman with a history of in-utero diethylstilbestrol (DES) exposure, who had a prior hysterectomy for symptomatic leiomyomata and dysmenorrhea, presented for vaginal bleeding. Vaginal biopsies showed a non-clear-cell adenocarcinoma, and the patient was subsequently treated with radiation therapy. We present a case of primary vaginal non-clear-cell adenocarcinoma in a patient with in-utero DES exposure. Continued monitoring of older DES-exposed women for vaginal lesions is warranted because of reported cases of non-clear-cell adenocarcinoma and persistent risk of clear cell adenocarcinoma. Reprint & Copyright © 2014 Association of Military Surgeons of the U.S.

Bone morphogenetic protein-4 strongly potentiates growth factor-induced proliferation of mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Montesano, Roberto; Sarkoezi, Rita; Schramek, Herbert

2008-09-12

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that elicit pleiotropic effects on biological processes such as cell proliferation, cell differentiation and tissue morphogenesis. With respect to cell proliferation, BMPs can exert either mitogenic or anti-mitogenic activities, depending on the target cells and their context. Here, we report that in low-density cultures of immortalized mammary epithelial cells, BMP-4 did not stimulate cell proliferation by itself. However, when added in combination with suboptimal concentrations of fibroblast growth factor (FGF)-2, FGF-7, FGF-10, epidermal growth factor (EGF) or hepatocyte growth factor (HGF), BMP-4 potently enhanced growth factor-induced cell proliferation. These results reveal a hithertomore » unsuspected interplay between BMP-4 and growth factors in the regulation of mammary epithelial cell proliferation. We suggest that the ability of BMP-4 to potentiate the mitogenic activity of multiple growth factors may contribute to mammary gland ductal morphogenesis as well as to breast cancer progression.« less

Influence of tangeretin on tamoxifen's therapeutic benefit in mammary cancer.

PubMed

Bracke, M E; Depypere, H T; Boterberg, T; Van Marck, V L; Vennekens, K M; Vanluchene, E; Nuytinck, M; Serreyn, R; Mareel, M M

1999-02-17

Tamoxifen and the citrus flavonoid tangeretin exhibit similar inhibitory effects on the growth and invasive properties of human mammary cancer cells in vitro; furthermore, the two agents have displayed additive effects in vitro. In this study, we examined whether tangeretin would enhance tamoxifen's therapeutic benefit in vivo. Female nude mice (n = 80) were inoculated subcutaneously with human MCF-7/6 mammary adenocarcinoma cells. Groups of 20 mice were treated orally by adding the following substances to their drinking water: tamoxifen (3 x 10(-5) M), tangeretin (1 x 10(-4) M), tamoxifen plus tangeretin (3 x 10(-5) M plus 1 x 10(-4) M), or solvent. Oral treatment of mice with tamoxifen resulted in a statistically significant inhibition of tumor growth compared with solvent treatment (two-sided P = .001). Treatment with tangeretin did not inhibit tumor growth, and addition of this compound to drinking water with tamoxifen completely neutralized tamoxifen's inhibitory effect. The median survival time of tumor-bearing mice treated with tamoxifen plus tangeretin was reduced in comparison with that of mice treated with tamoxifen alone (14 versus 56 weeks; two-sided P = .002). Tangeretin (1 x 10(-6) M or higher) inhibited the cytolytic effect of murine natural killer cells on MCF-7/6 cells in vitro, which may explain why tamoxifen-induced inhibition of tumor growth in mice is abolished when tangeretin is present in drinking water. We describe an in vivo model to study potential interference of dietary compounds, such as flavonoids, with tamoxifen, which could lead to reduced efficacy of adjuvant therapy. In our study, the tumor growth-inhibiting effect of oral tamoxifen was reversed upon addition of tangeretin to the diet. Our data argue against excessive consumption of tangeretin-added products and supplements by patients with mammary cancer during tamoxifen treatment.

PGE2 /EP4 Signaling Controls the Transfer of the Mammary Stem Cell State by Lipid Rafts in Extracellular Vesicles.

PubMed

Lin, Meng-Chieh; Chen, Shih-Yin; Tsai, Ho-Min; He, Pei-Lin; Lin, Yen-Chun; Herschman, Harvey; Li, Hua-Jung

2017-02-01

Prostaglandin E 2 (PGE 2 )-initiated signaling contributes to stem cell homeostasis and regeneration. However, it is unclear how PGE 2 signaling controls cell stemness. This study identifies a previously unknown mechanism by which PGE 2 /prostaglandin E receptor 4 (EP 4 ) signaling regulates multiple signaling pathways (e.g., PI3K/Akt signaling, TGFβ signaling, Wnt signaling, EGFR signaling) which maintain the basal mammary stem cell phenotype. A shift of basal mammary epithelial stem cells (MaSCs) from a mesenchymal/stem cell state to a non-basal-MaSC state occurs in response to prostaglandin E receptor 4 (EP 4 ) antagonism. EP 4 antagonists elicit release of signaling components, by controlling their trafficking into extracellular vesicles/exosomes in a lipid raft/caveolae-dependent manner. Consequently, EP 4 antagonism indirectly inactivates, through induced extracellular vesicle/exosome release, pathways required for mammary epithelial stem cell homeostasis, e.g. canonical/noncanonical Wnt, TGFβ and PI3K/Akt pathways. EP 4 antagonism causes signaling receptors and signaling components to shift from non-lipid raft fractions to lipid raft fractions, and to then be released in EP 4 antagonist-induced extracellular vesicles/exosomes, resulting in the loss of the stem cell state by mammary epithelial stem cells. In contrast, luminal mammary epithelial cells can acquire basal stem cell properties following ingestion of EP 4 antagonist-induced stem cell extracellular vesicles/exosomes, and can then form mammary glands. These findings demonstrate that PGE 2 /EP 4 signaling controls homeostasis of mammary epithelial stem cells through regulating extracellular vesicle/exosome release. Reprogramming of mammary epithelial cells can result from EP 4 -mediated stem cell property transfer by extracellular vesicles/exosomes containing caveolae-associated proteins, between mammary basal and luminal epithelial cells. Stem Cells 2017;35:425-444. © 2016 The Authors STEM CELLS

Increased risk of gastric adenocarcinoma after treatment of primary gastric diffuse large B-cell lymphoma.

PubMed

Inaba, Koji; Kushima, Ryoji; Murakami, Naoya; Kuroda, Yuuki; Harada, Ken; Kitaguchi, Mayuka; Yoshio, Kotaro; Sekii, Shuhei; Takahashi, Kana; Morota, Madoka; Mayahara, Hiroshi; Ito, Yoshinori; Sumi, Minako; Uno, Takashi; Itami, Jun

2013-10-26

There have been sporadic reports about synchronous as well as metachronous gastric adenocarcinoma and primary gastric lymphoma. Many reports have dealt with metachronous gastric adenocarcinoma in mucosa-associated lymphoid tissue lymphoma of stomach. But to our knowledge, there have been no reports that document the increased incidence of metachronous gastric adenocarcinoma in patients with gastric diffuse large B-cell lymphoma. This retrospective study was conducted to estimate the incidence of metachronous gastric adenocarcinoma after primary gastric lymphoma treatment, especially in diffuse large B-cell lymphoma. The retrospective cohort study of 139 primary gastric lymphoma patients treated with radiotherapy at our hospital. Mean observation period was 61.5 months (range: 3.7-124.6 months). Patients profile, characteristics of primary gastric lymphoma and metachronous gastric adenocarcinoma were retrieved from medical records. The risk of metachronous gastric adenocarcinoma was compared with the risk of gastric adenocarcinoma in Japanese population. There were 10 (7.2%) metachronous gastric adenocarcinoma patients after treatment of primary gastric lymphomas. It was quite high risk compared with the risk of gastric carcinoma in Japanese population of 54.7/100,000. Seven patients of 10 were diffuse large B-cell lymphoma and other 3 patients were mixed type of diffuse large B-cell lymphoma and mucosa associated lymphoid tissue lymphoma. Four patients of 10 metachronous gastric adenocarcinomas were signet-ring cell carcinoma and two patients died of gastric adenocarcinoma. Metachronous gastric adenocarcinoma may have a more malignant potential than sporadic gastric adenocarcinoma. Old age, Helicobacter pylori infection and gastric mucosal change of chronic gastritis and intestinal metaplasia were possible risk factors for metachronous gastric adenocarcinoma. There was an increased risk of gastric adenocarcinoma after treatment of primary gastric lymphoma

Tammar wallaby mammary cathelicidins are differentially expressed during lactation and exhibit antimicrobial and cell proliferative activity.

PubMed

Wanyonyi, Stephen S; Sharp, Julie A; Khalil, Elie; Lefevre, Christophe; Nicholas, Kevin R

2011-11-01

Cathelicidins secreted in milk may be central to autocrine feedback in the mammary gland for optimal development in addition to conferring innate immunity to both the mammary gland and the neonate. This study exploits the unique reproductive strategy of the tammar wallaby (Macropus eugenii) model to analyse differential splicing of cathelicidin genes and to evaluate the bactericidal activity and effect of the protein on mammary epithelial cell proliferation. Two linear peptides, Con73 and Con218, derived from the heterogeneous carboxyl end of cathelicidin transcripts, MaeuCath1 and MaeuCath7 respectively, were evaluated for antimicrobial activity. Both Con73 and Con218 significantly inhibited the growth of Staphylococcus aureus, Pseudomonas aureginosa, Enterococcus faecalis and Salmonella enterica. In addition both MaeuCath1 and MaeuCath7 stimulated proliferation of primary tammar wallaby mammary epithelial cells (WallMEC). Lactation-phase specific alternate spliced transcripts were determined for MaeuCath1 showing utilisation of both antimicrobial and proliferative functions are required by the mammary gland and the suckled young. The study has shown for the first time that temporal regulation of milk cathelicidins may be crucial in antimicrobial protection of the mammary gland and suckled young and mammary cell proliferation. Copyright © 2011 Elsevier Inc. All rights reserved.

F-prostanoid receptor regulation of fibroblast growth factor 2 signaling in endometrial adenocarcinoma cells.

PubMed

Sales, Kurt J; Boddy, Sheila C; Williams, Alistair R W; Anderson, Richard A; Jabbour, Henry N

2007-08-01

Prostaglandin (PG) F(2alpha) is a potent bioactive lipid in the female reproductive tract, and exerts its function after coupling with its heptahelical G-protein-coupled receptor [F-series-prostanoid (FP) receptor] to initiate cell signaling and target gene transcription. In the present study, we found elevated expression of fibroblast growth factor (FGF) 2, FGF receptor 1 (FGFR1), and FP receptor, colocalized within the neoplastic epithelial cells of endometrial adenocarcinomas. We investigated a role for PGF(2alpha)-FP receptor interaction in modulating FGF2 expression and signaling using an endometrial adenocarcinoma cell line stably expressing the FP receptor to the levels detected in endometrial adenocarcinomas (FPS cells) and endometrial adenocarcinoma tissue explants. PGF(2alpha)-FP receptor activation rapidly induced FGF2 mRNA expression, and elevated FGF2 protein expression and secretion into the culture medium in FPS cells and endometrial adenocarcinoma explants. The effect of PGF(2alpha) on the expression and secretion of FGF2 could be abolished by treatment of FPS cells and endometrial tissues with an FP receptor antagonist (AL8810) and inhibitor of ERK (PD98059). Furthermore, we have shown that FGF2 can promote the expression of FGF2 and cyclooxygenase-2, and enhance proliferation of endometrial adenocarcinoma cells via the FGFR1 and ERK pathways, thereby establishing a positive feedback loop to regulate neoplastic epithelial cell function in endometrial adenocarcinomas.

Lactation-induced WAP-SV40 Tag transgene expression in C57BL/6J mice leads to mammary carcinoma.

PubMed

Hüsler, M R; Kotopoulis, K A; Sundberg, J P; Tennent, B J; Kunig, S V; Knowles, B B

1998-07-01

Two transgenic lineages were generated by directing the expression of SV40 T antigen to the mammary gland of inbred C57BL/6J mice using the whey acidic protein (WAP) promoter. In one lineage, WAPTag 1, multiparous female mice developed mammary adenocarcinoma with an average latency period of 13 months. The histopathological phenotype was heterogeneous, tumours occurred in a stochastic fashion, normal tissue was located next to neoplastic tissue, the mammary tumours usually developed and were remarkably similar to that observed in human cases. In addition, male and virgin females developed a poorly differentiated SV40 T antigen-positive soft tissue sarcoma, also at 13 months of age. In the other lineage, WAPTag 3, some parous females developed mammary tumours, but most mice succumbed to osteosarcomas arising from the os petrosum at 5.5 to 6 months of age and on necropsy, renal adenocarcinomas were also found. Appearance of these unexpected tumour types demonstrates the non-specific expression of SV40 Tag under the control of the WAP promoter. The expression of SV40 Tag in mammary glands at different stages of development was also examined, and only actively lactating glands were positive. This suggests that the abundant cyclic synthesis of SV40 Tag associated with pregnancy is required for mammary tumorigenesis in these lineages.

NFAT5 promotes proliferation and migration of lung adenocarcinoma cells in part through regulating AQP5 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Kai, E-mail: gk161@163.com; Department of Respiration, 161th Hospital, PLA, Wuhan 430015; Jin, Faguang, E-mail: jinfag@fmmu.edu.cn

2015-09-25

The osmoregulated transcription factor nuclear factor of activated T-cells 5(NFAT5), has been found to play important roles in the development of many kinds of human cancers, including breast cancer, colon carcinoma, renal cell carcinoma and melanoma. The aim of the present study was to determine whether NFAT5 is involved in the proliferation and migration of lung adenocarcinoma cells. We found that NFAT5 was upregulated in lung adenocarcinoma cells and knockdown of NFAT5 decreased proliferation and migration of the cells, accompanied by a significant reduction in the expression of AQP5. AQP5 was upregulated in lung adenocarcinoma cells and knockdown of AQP5more » also inhibited proliferation and migration of the cells as knockdown of NFAT5 did. Moreover, overexpression of NFAT5 promoted proliferation and migration of lung adenocarcinoma cells, accompanied by a significant increase in the expression of AQP5. These results indicate that NFAT5 plays important roles in proliferation and migration of human lung adenocarcinoma cells through regulating AQP5 expression, providing a new therapeutic option for lung adenocarcinoma therapy. - Highlights: • NFAT5 expression is higher in lung adenocarcinoma cells compared with normal cells. • NFAT5 knockdown decreases proliferation and migration of lung adenocarcinoma cells. • Knockdown of NFAT5 reduces AQP5 expression in human lung adenocarcinoma cells. • Overexpression of NFAT5 promotes proliferation and migration of lung adenocarcinoma cells. • Overexpression of NFAT5 increases AQP5 expression in human lung adenocarcinoma cells.« less

Stromal adipocyte enhancer-binding protein (AEBP1) promotes mammary epithelial cell hyperplasia via proinflammatory and hedgehog signaling.

PubMed

Holloway, Ryan W; Bogachev, Oleg; Bharadwaj, Alamelu G; McCluskey, Greg D; Majdalawieh, Amin F; Zhang, Lei; Ro, Hyo-Sung

2012-11-09

Disruption of mammary stromal-epithelial communication leads to aberrant mammary gland development and induces mammary tumorigenesis. Macrophages have been implicated in carcinogenesis primarily by creating an inflammatory microenvironment, which promotes growth of the adjacent epithelial cells. Adipocyte enhancer-binding protein 1 (AEBP1), a novel proinflammatory mediator, promotes macrophage inflammatory responsiveness by inducing NF-κB activity, which has been implicated in tumor cell growth and survival by aberrant sonic hedgehog (Shh) expression. Here, we show that stromal macrophage AEBP1 overexpression results in precocious alveologenesis in the virgin AEBP1 transgenic (AEBP1(TG)) mice, and the onset of ductal hyperplasia was accelerated in AEBP1(TG) mice fed a high fat diet, which induces endogenous AEBP1 expression. Transplantation of AEBP1(TG) bone marrow cells into non-transgenic (AEBP1(NT)) mice resulted in alveolar hyperplasia with up-regulation of NF-κB activity and TNFα expression as displayed in the AEBP1(TG) mammary macrophages and epithelium. Shh expression was induced in AEBP1(TG) macrophages and RAW264.7 macrophages overexpressing AEBP1. The Shh target genes Gli1 and Bmi1 expression was induced in the AEBP1(TG) mammary epithelium and HC11 mammary epithelial cells co-cultured with AEBP1(TG) peritoneal macrophages. The conditioned AEBP1(TG) macrophage culture media promoted NF-κB activity and survival signal, Akt activation, in HC11 cells, whereas such effects were abolished by TNFα neutralizing antibody treatment. Furthermore, HC11 cells displayed enhanced proliferation in response to AEBP1(TG) macrophages and their conditioned media. Our findings highlight the role of AEBP1 in the signaling pathways regulating the cross-talk between mammary epithelium and stroma that could predispose the mammary tissue to tumorigenesis.

Stromal Adipocyte Enhancer-binding Protein (AEBP1) Promotes Mammary Epithelial Cell Hyperplasia via Proinflammatory and Hedgehog Signaling*

PubMed Central

Holloway, Ryan W.; Bogachev, Oleg; Bharadwaj, Alamelu G.; McCluskey, Greg D.; Majdalawieh, Amin F.; Zhang, Lei; Ro, Hyo-Sung

2012-01-01

Disruption of mammary stromal-epithelial communication leads to aberrant mammary gland development and induces mammary tumorigenesis. Macrophages have been implicated in carcinogenesis primarily by creating an inflammatory microenvironment, which promotes growth of the adjacent epithelial cells. Adipocyte enhancer-binding protein 1 (AEBP1), a novel proinflammatory mediator, promotes macrophage inflammatory responsiveness by inducing NF-κB activity, which has been implicated in tumor cell growth and survival by aberrant sonic hedgehog (Shh) expression. Here, we show that stromal macrophage AEBP1 overexpression results in precocious alveologenesis in the virgin AEBP1 transgenic (AEBP1TG) mice, and the onset of ductal hyperplasia was accelerated in AEBP1TG mice fed a high fat diet, which induces endogenous AEBP1 expression. Transplantation of AEBP1TG bone marrow cells into non-transgenic (AEBP1NT) mice resulted in alveolar hyperplasia with up-regulation of NF-κB activity and TNFα expression as displayed in the AEBP1TG mammary macrophages and epithelium. Shh expression was induced in AEBP1TG macrophages and RAW264.7 macrophages overexpressing AEBP1. The Shh target genes Gli1 and Bmi1 expression was induced in the AEBP1TG mammary epithelium and HC11 mammary epithelial cells co-cultured with AEBP1TG peritoneal macrophages. The conditioned AEBP1TG macrophage culture media promoted NF-κB activity and survival signal, Akt activation, in HC11 cells, whereas such effects were abolished by TNFα neutralizing antibody treatment. Furthermore, HC11 cells displayed enhanced proliferation in response to AEBP1TG macrophages and their conditioned media. Our findings highlight the role of AEBP1 in the signaling pathways regulating the cross-talk between mammary epithelium and stroma that could predispose the mammary tissue to tumorigenesis. PMID:22995915

Trefoil Factor 3 as a Novel Biomarker to Distinguish Between Adenocarcinoma and Squamous Cell Carcinoma

PubMed Central

Wang, Xiao-Nan; Wang, Shu-Jing; Pandey, Vijay; Chen, Ping; Li, Qing; Wu, Zheng-Sheng; Wu, Qiang; Lobie, Peter E.

2015-01-01

Abstract In carcinoma, such as of the lung, the histological subtype is important to select an appropriate therapeutic strategy for patients. However, carcinomas with poor differentiation cannot always be distinguished on the basis of morphology alone nor on clinical findings. Hence, delineation of poorly differentiated adenocarcinoma and squamous cell carcinoma, the 2 most common epithelial-origin carcinomas, is pivotal for selection of optimum therapy. Herein, we explored the potential utility of trefoil factor 3 (TFF3) as a biomarker for primary lung adenocarcinoma and extrapulmonary adenocarcinomas derived from different organs. We observed that 90.9% of lung adenocarcinomas were TFF3-positive, whereas no expression of TFF3 was observed in squamous cell carcinomas. The subtype of lung carcinoma was confirmed by four established biomarkers, cytokeratin 7 and thyroid transcription factor 1 for adenocarcinoma and P63 and cytokeratin 5/6 for squamous cell carcinoma. Furthermore, expression of TFF3 mRNA was observed by quantitative PCR in all of 11 human lung adenocarcinoma cell lines and highly correlated with markers of the adenocarcinomatous lineage. In contrast, little or no expression of TFF3 was observed in 4 lung squamous cell carcinoma cell lines. By use of forced expression, or siRNA-mediated depletion of TFF3, we determined that TFF3 appeared to maintain rather than promote glandular differentiation of lung carcinoma cells. In addition, TFF3 expression was also determined in adenocarcinomas from colorectum, stomach, cervix, esophagus, and larynx. Among all these extrapulmonary carcinomas, 93.7% of adenocarcinomas exhibited TFF3 positivity, whereas only 2.9% of squamous cell carcinomas were TFF3-positive. Totally, 92.9% of both pulmonary and extrapulmonary adenocarcinomas exhibited TFF3 positivity, whereas only 1.5% of squamous cell carcinomas were TFF3-positive. In conclusion, TFF3 is preferentially expressed in adenocarcinoma and may function as an

Roles of Breast Cancer Susceptibility Genes BRCA’s in Mammary Epithelial Cell Differentiation

DTIC Science & Technology

2006-03-01

1-0322 TITLE: Roles of Breast Cancer Susceptibility Genes BRCA’s in Mammary Epithelial Cell Differentiation PRINCIPAL...Summary 3. DATES COVERED (From - To) 1 MAR 2005 - 28 FEB 2006 4. TITLE AND SUBTITLE Roles of Breast Cancer Susceptibility Genes BRCA’s in Mammary...reverse the phenotype of differentiation-defective breast cancer cells bearing reduced BRCA1 functions. This result implies BRCA1 is involved in

Mammary and extramammary Paget's disease*

PubMed Central

Lopes, Lauro Lourival; Lopes, Ione Maria Ribeiro Soares; Lopes, Lauro Rodolpho Soares; Enokihara, Milvia M. S. S.; Michalany, Alexandre Osores; Matsunaga, Nobuo

2015-01-01

Paget's disease, described by Sir James Paget in 1874, is classified as mammary and extramammary. The mammary type is rare and often associated with intraductal cancer (93-100% of cases). It is more prevalent in postmenopausal women and it appears as an eczematoid, erythematous, moist or crusted lesion, with or without fine scaling, infiltration and inversion of the nipple. It must be distinguished from erosive adenomatosis of the nipple, cutaneous extension of breast carcinoma, psoriasis, atopic dermatitis, contact dermatitis, chronic eczema, lactiferous ducts ectasia, Bowen's disease, basal cell carcinoma, melanoma and intraductal papilloma. Diagnosis is histological and prognosis and treatment depend on the type of underlying breast cancer. Extramammary Paget's disease is considered an adenocarcinoma originating from the skin or skin appendages in areas with apocrine glands. The primary location is the vulvar area, followed by the perianal region, scrotum, penis and axillae. It starts as an erythematous plaque of indolent growth, with well-defined edges, fine scaling, excoriations, exulcerations and lichenification. In most cases it is not associated with cancer, although there are publications linking it to tumors of the vulva, vagina, cervix and corpus uteri, bladder, ovary, gallbladder, liver, breast, colon and rectum. Differential diagnoses are candidiasis, psoriasis and chronic lichen simplex. Histopathology confirms the diagnosis. Before treatment begins, associated malignancies should be investigated. Surgical excision and micrographic surgery are the best treatment options, although recurrences are frequent. PMID:25830993

Canine mammary minute oncocytomas with neuroendocrine differentiation associated with multifocal acinar cell oncocytic metaplasia.

PubMed

Nagahara, Rei; Kimura, Masayuki; Itahashi, Megu; Sugahara, Go; Kawashima, Masashi; Murayama, Hirotada; Yoshida, Toshinori; Shibutani, Makoto

2016-11-01

Two solitary and minute tumors of 1 and 1.5 mm diameter were identified by microscopy in the left fourth mammary gland of a 13-year-old female Labrador Retriever dog, in addition to multiple mammary gland tumors. The former tumors were well circumscribed and were composed of small-to-large polyhedral neoplastic oncocytes with finely granular eosinophilic cytoplasm, and were arranged in solid nests separated by fine fibrovascular septa. Scattered lumina of variable sizes containing eosinophilic secretory material were evident. Cellular atypia was minimal, and no mitotic figures were visible. One tumor had several oncocytic cellular foci revealing cellular transition, with perivascular pseudorosettes consisting of columnar epithelial cells surrounding the fine vasculature. Scattered foci of mammary acinar cell hyperplasia showing oncocytic metaplasia were also observed. Immunohistochemically, the cytoplasm of neoplastic cells of the 2 microtumors showed diffuse immunoreactivity to anti-cytokeratin antibody AE1/AE3, and finely granular immunoreactivity for 60-kDa heat shock protein, mitochondrial membrane ATP synthase complex V beta subunit, and chromogranin A. One tumor also had oncocytic cellular foci forming perivascular pseudorosettes showing cellular membrane immunoreactivity for neural cell adhesion molecule. The tumors were negative for smooth muscle actin, neuron-specific enolase, vimentin, desmin, S100, and synaptophysin. Ultrastructural observation confirmed the abundant mitochondria in the cytoplasm of both neoplastic and hyperplastic cells, the former cells also having neuroendocrine granule-like electron-dense bodies. From these results, our case was diagnosed with mammary oncocytomas accompanied by neuroendocrine differentiation. Scattered foci of mammary oncocytosis might be related to the multicentric occurrence of these oncocytomas. © 2016 The Author(s).

Isolation, purification, culture and characterisation of myoepithelial cells from normal and neoplastic canine mammary glands using a magnetic-activated cell sorting separation system.

PubMed

Sánchez-Céspedes, R; Maniscalco, L; Iussich, S; Martignani, E; Guil-Luna, S; De Maria, R; Martín de Las Mulas, J; Millán, Y

2013-08-01

Mammary gland tumours, the most common malignant neoplasm in bitches, often display myoepithelial (ME) cell proliferation. The aim of this study was to isolate, purify, culture and characterise ME cells from normal and neoplastic canine mammary glands. Monodispersed cells from three normal canine mammary glands and five canine mammary tumours were incubated with an anti-Thy1 antibody and isolated by magnetic-activated cell sorting (MACS). Cells isolated from two normal glands (cell lines CmME-N1 and CmME-N2) and four tumours (cell lines CmME-K1 from a complex carcinoma, CmME-K2 from a simple tubulopapillary carcinoma, and CmME-K3 and CmME-K4 from two carcinomas within benign tumours) were cultured in supplemented DMEM/F12 media for 40days. Cell purity was >90%. Tumour-derived ME cell lines exhibited heterogeneous morphology, growth patterns and immunocytochemical expression of cytokeratins, whereas cell lines from normal glands retained their morphology and levels of cytokeratin expression during culture. Cell lines from normal glands and carcinomas within benign tumours grew more slowly than those from simple and complex carcinomas. This methodology has the potential to be used for in vitro analysis of the role of ME cells in the growth and progression of canine mammary tumours. Copyright © 2013 Elsevier Ltd. All rights reserved.

Quantification of regenerative potential in primary human mammary epithelial cells.

PubMed

Linnemann, Jelena R; Miura, Haruko; Meixner, Lisa K; Irmler, Martin; Kloos, Uwe J; Hirschi, Benjamin; Bartsch, Harald S; Sass, Steffen; Beckers, Johannes; Theis, Fabian J; Gabka, Christian; Sotlar, Karl; Scheel, Christina H

2015-09-15

We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49f(hi)/EpCAM(-) population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. © 2015. Published by The Company of Biologists Ltd.

Quantification of regenerative potential in primary human mammary epithelial cells

PubMed Central

Linnemann, Jelena R.; Miura, Haruko; Meixner, Lisa K.; Irmler, Martin; Kloos, Uwe J.; Hirschi, Benjamin; Bartsch, Harald S.; Sass, Steffen; Beckers, Johannes; Theis, Fabian J.; Gabka, Christian; Sotlar, Karl; Scheel, Christina H.

2015-01-01

We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49fhi/EpCAM− population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. PMID:26071498

The Ets transcription factor Elf5 specifies mammary alveolar cell fate

PubMed Central

Oakes, Samantha R.; Naylor, Matthew J.; Asselin-Labat, Marie-Liesse; Blazek, Katrina D.; Gardiner-Garden, Margaret; Hilton, Heidi N.; Kazlauskas, Michael; Pritchard, Melanie A.; Chodosh, Lewis A.; Pfeffer, Peter L.; Lindeman, Geoffrey J.; Visvader, Jane E.; Ormandy, Christopher J.

2008-01-01

Hormonal cues regulate mammary development, but the consequent transcriptional changes and cell fate decisions are largely undefined. We show that knockout of the prolactin-regulated Ets transcription factor Elf5 prevented formation of the secretory epithelium during pregnancy. Conversely, overexpression of Elf5 in an inducible transgenic model caused alveolar differentiation and milk secretion in virgin mice, disrupting ductal morphogenesis. CD61+ luminal progenitor cells accumulated in Elf5-deficient mammary glands and were diminished in glands with Elf5 overexpression. Thus Elf5 specifies the differentiation of CD61+ progenitors to establish the secretory alveolar lineage during pregnancy, providing a link between prolactin, transcriptional events, and alveolar development. PMID:18316476

Repression of mammary adipogenesis by genistein limits mammosphere formation of human MCF-7 cells

USDA-ARS?s Scientific Manuscript database

Mammary adipose tissue may contribute to breast cancer development and progression by altering neighboring epithelial cell behavior and phenotype through paracrine signaling. Dietary exposure to soy foods is associated with lower mammary tumor risk and reduced body weight and adiposity in humans and...

Peripheral Serotonin Regulates Maternal Calcium Trafficking in Mammary Epithelial Cells during Lactation in Mice

PubMed Central

Laporta, Jimena; Keil, Kimberly P.; Vezina, Chad M.; Hernandez, Laura L.

2014-01-01

Lactation is characterized by massive transcellular flux of calcium, from the basolateral side of the mammary alveolar epithelium (blood) into the ductal lumen (milk). Regulation of calcium transport during lactation is critical for maternal and neonatal health. The monoamine serotonin (5-HT) is synthesized by the mammary gland and functions as a homeostatic regulation of lactation. Genetic ablation of tryptophan hydroxylase 1 (Tph1), which encodes the rate-limiting enzyme in non-neuronal serotonin synthesis, causes a deficiency in circulating serotonin. As a consequence maternal calcium concentrations decrease, mammary epithelial cell morphology is altered, and cell proliferation is decreased during lactation. Here we demonstrate that serotonin deficiency decreases the expression and disrupts the normal localization of calcium transporters located in the apical (PMCA2) and basolateral (CaSR, ORAI-1) membranes of the lactating mammary gland. In addition, serotonin deficiency decreases the mRNA expression of calcium transporters located in intracellular compartments (SERCA2, SPCA1 and 2). Mammary expression of serotonin receptor isoform 2b and its downstream pathways (PLCβ3, PKC and MAP-ERK1/2) are also decreased by serotonin deficiency, which might explain the numerous phenotypic alterations described above. In most cases, addition of exogenous 5-hydroxy-L-tryptophan to the Tph1 deficient mice rescued the phenotype. Our data supports the hypothesis that serotonin is necessary for proper mammary gland structure and function, to regulate blood and mammary epithelial cell transport of calcium during lactation. These findings can be applicable to the treatment of lactation-induced hypocalcemia in dairy cows and can have profound implications in humans, given the wide-spread use of selective serotonin reuptake inhibitors as antidepressants during pregnancy and lactation. PMID:25299122

Genotoxic Exposure during Juvenile Growth of Mammary Gland Depletes Stem Cell Activity and Inhibits Wnt Signaling

PubMed Central

Klos, Kristine S.; Kim, Soyoung; Alexander, Caroline M.

2012-01-01

Various types of somatic stem cell have been tested for their response to genotoxic exposure, since these cells are likely to be important to regeneration, aging and cancer. In this study, we evaluated the response of mammary stem cells to genotoxic exposure during ductal growth in juveniles. Exposure to the polycyclic aromatic hydrocarbon (DMBA; 7,12 dimethylbenz[a]anthracene) had no gross effect on outgrowth and morphogenesis of the ductal tree, or upon lobuloalveolar growth during pregnancy. However, by fat pad assay, we found that mammary stem cell activity was reduced by 80% in glands from adults that were exposed to genotoxins as juveniles. The associated basal cell lineage was depleted. Both basal and luminal cells showed a robust response to genotoxic exposure (including γH2AX phosphorylation, pS15p53 and pT68Chk2), with durable hyperproliferation, but little cytotoxicity. Since the phenotype of these glands (low basal cell fraction, low stem cell activity) phenocopies mammary glands with loss of function for Wnt signaling, we measured Wnt signaling in genotoxin-exposed glands, and found a durable reduction in the activation of the canonical signaling Wnt receptors, Lrp5/6. Furthermore, when mammary epithelial cells were treated with Wnt3a, DMBA exposure reduced the basal cell population and Lrp activation was ablated. We conclude that during active ductal growth, Wnt-dependent mammary stem cells are sensitized to cell death by genotoxin exposure. Our conclusion may be important for other tissues, since all solid tumor stem cell activities have been shown to be Wnt-dependent to date. PMID:23185480

Molecular Analysis of Motility in Metastatic Mammary Adenocarcinoma Cells

DTIC Science & Technology

1996-09-01

elements of epidermoid carcinoma (A43 1) cells. J. Cell. Biol. 103: 87-94 Winkler, M. (1988). Translational regulation in sea urchin eggs: a complex...and Methods. Error bars show SEM . Figure 2. Rhodamine-actin polymerizes preferentially at the tips of lamellipods in EGF- stimulated cells. MTLn3...lamellipods. B) rhodamine-actin intensity at the cell center. Data for each time point is the average and SEM of 15 different cells. Images A and B

A cyclized peptide derived from alpha fetoprotein inhibits the proliferation of ER-positive canine mammary cancer cells.

PubMed

Torres, Cristian Gabriel; Pino, Ana María; Sierralta, Walter Daniel

2009-06-01

The effects of estradiol (E2) and of an AFP-derived cyclized peptide (cP) on the proliferation of primary cultures of cancer cells isolated from spontaneous canine mammary tumors were studied. The cellular response to E2 and cP was related to the expression of estradiol receptor (isoforms alpha and beta). In ER-positive cells, 2 nM estradiol increased cell proliferation and the phosphorylation of ERK1/2; 2 microg/ml cP inhibited all these effects. Estradiol also increased HER2 immunoreactivity in ER-positive cells, an effect that was reverted to its basal values by cP. Estradiol stimulated in these cells the release of MMP2 and MMP9 and the shedding of HB-EGF, effects that the cP did not affect. ER-negative cells were refractory to estradiol or cP. All canine mammary tumor cells in culture responded to treatments analogously to human mammary cancer cells. Our results support the proposal of cP as a new, potentially effective therapeutic agent for the management of mammary cancer.

Cytomorphological features of ALK-positive lung adenocarcinomas: psammoma bodies and signet ring cells.

PubMed

Pareja, Fresia; Crapanzano, John P; Mansukhani, Mahesh M; Bulman, William A; Saqi, Anjali

2015-03-01

Correlation between histology and genotype has been described in lung adenocarcinomas. For example, studies have demonstrated that adenocarcinomas with an anaplastic lymphoma kinase (ALK) gene rearrangement may have mucinous features. The objective of the current study was to determine whether a similar association can be identified in cytological specimens. A retrospective search for ALK-rearranged cytopathology (CP) and surgical pathology (SP) lung carcinomas was conducted. Additional ALK-negative (-) lung adenocarcinomas served as controls. For CP and SP cases, the clinical data (i.e., age, sex, and smoking history), architecture, nuclear features, presence of mucin-containing cells (including signet ring cells), and any additional salient characteristics were evaluated. The search yielded 20 ALK-positive (+) adenocarcinomas. Compared with patients with ALK(-) lung adenocarcinomas (33 patients; 12 with epidermal growth factor receptor [EGFR]-mutation, 11 with Kristen rat sarcoma [KRAS]-mutation, and 10 wild-type adenocarcinomas), patients with ALK(+) adenocarcinoma presented at a younger age; and there was no correlation noted with sex or smoking status. The most common histological pattern in SP was papillary/micropapillary. Mucinous features were associated with ALK rearrangement in SP specimens. Signet ring cells and psammoma bodies were evident in and significantly associated with ALK(+) SP and CP specimens. However, psammoma bodies were observed in rare adenocarcinomas with an EGFR mutation. Both the ALK(+) and ALK(-) groups had mostly high nuclear grade. Salient features, including signet ring cells and psammoma bodies, were found to be significantly associated with ALK(+) lung adenocarcinomas and are identifiable on CP specimens. Recognizing these may be especially helpful in the molecular triage of scant CP samples. © 2014 American Cancer Society.

Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland.

PubMed

Lilja, Anna M; Rodilla, Veronica; Huyghe, Mathilde; Hannezo, Edouard; Landragin, Camille; Renaud, Olivier; Leroy, Olivier; Rulands, Steffen; Simons, Benjamin D; Fre, Silvia

2018-06-01

Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.5 and no statistically discernable bipotency after E15.5. To gain insights into the mechanisms governing the switch from multipotency to unipotency, we used gain-of-function Notch1 mice and demonstrated that Notch activation cell autonomously dictates luminal cell fate specification to both embryonic and basally committed mammary cells. These functional studies have important implications for understanding the signals underlying cell plasticity and serve to clarify how reactivation of embryonic programs in adult cells can lead to cancer.

Expansion of stem cells counteracts age-related mammary regression in compound Timp1/Timp3 null mice.

PubMed

Jackson, Hartland W; Waterhouse, Paul; Sinha, Ankit; Kislinger, Thomas; Berman, Hal K; Khokha, Rama

2015-03-01

Age is the primary risk factor for breast cancer in women. Bipotent basal stem cells actively maintain the adult mammary ductal tree, but with age tissues atrophy. We show that cell-extrinsic factors maintain the adult stem cell pool during ageing and dictate tissue stoichiometry. Mammary stem cells spontaneously expand more than 11-fold in virgin adult female mice lacking specific genes for TIMPs, the natural metalloproteinase inhibitors. Compound Timp1/Timp3 null glands exhibit Notch activation and accelerated gestational differentiation. Proteomics of mutant basal cells uncover altered cytoskeletal and extracellular protein repertoires, and we identify aberrant mitotic spindle orientation in these glands, a process that instructs asymmetric cell division and fate. We find that progenitor activity normally declines with age, but enriched stem/progenitor pools prevent tissue regression in Timp mutant mammary glands without affecting carcinogen-induced cancer susceptibility. Thus, improved stem cell content can extend mouse mammary tissue lifespan without altering cancer risk in this mouse model.

Cell structure and function and response to chemotherapy in tumors heterotransplanted into the subrenal capsule of mice and rats.

PubMed

Stenbäck, F; Kangas, L; Wasenius, V M

1985-12-01

Specimens from 16 freshly biopsied human tumors, two mammary adenocarcinomas, ten ovarian adenocarcinomas, two squamous cell carcinomas, one malignant histiocytoma and one chondrosarcoma of the bone, two human ovarian adenocarcinomas established by transplantation into nude mice and two adenocarcinomas induced in rat mammary gland were transplanted under the renal capsule of 510 normal immunocompetent mice and 180 rats and the effects of chemotherapy were evaluated. The results showed successful transplantation of all types of tumors in both animal species. Morphological analysis revealed preserved glandular structures with surface microvilli, mucin and CEA production and partially preserved basement membranes. Treatment with cyclophosphamide, vinblastine, adriamycin and cisplatin caused cell shrinkage, degradation and partial or total disappearance of the tumor cells. Vascularization was distinct in all specimens. A cellular infiltrate was found frequently but not consistently. A common end stage was a fibrotic scar with no cellular activity, occasionally giving a misleading impression of a growing tumor on gross observation. The results were obtained rapidly and suggest that the subrenal capsule assay would be useful for evaluating the sensitivity of human tumors to therapeutic manipulation, but needs supplementary histological examination.

Radiogenic transformation of human mammary epithelial cells in vitro

NASA Technical Reports Server (NTRS)

Yang, T. C.; Georgy, K. A.; Tavakoli, A.; Craise, L. M.; Durante, M.

1996-01-01

Cancer induction by space radiations is a major concern for manned space exploration. Accurate assessment of radiation risk at low doses requires basic understanding of mechanism(s) of radiation carcinogenesis. For determining the oncogenic effects of ionizing radiation in human epithelial cells, we transformed a mammary epithelial cell line (185B5), which was immortalized by benzo(a)pyrene, with energetic heavy ions and obtained several transformed clones. These transformed cells showed growth properties on Matrigel similar to human mammary tumor cells. To better understand the mechanisms of radiogenic transformation of human cells, we systematically examined the alterations in chromosomes and cancer genes. Among 16 autosomes examined for translocations, by using fluorescence in situ hybridization (FISH) technique, chromosomes 3, 12, 13, 15, 16, and 18 appeared to be normal in transformed cells. Chromosomes 1, 4, 6, 8, and 17 in transformed cells, however, showed patterns different from those in nontransformed cells. Southern blot analyses indicated no detectable alterations in myc, ras, Rb, or p53 genes. Further studies of chromosome 17 by using in situ hybridization with unique sequence p53 gene probe and a centromere probe showed no loss of p53 gene in transformed cells. Experimental results from cell fusion studies indicated that the transforming gene(s) is recessive. The role of genomic instability and tumor suppressor gene(s) in radiogenic transformation of human breast cells remains to be identified.

Self-organization is a dynamic and lineage-intrinsic property of mammary epithelial cells

PubMed Central

Chanson, Lea; Brownfield, Douglas; Garbe, James C.; Kuhn, Irene; Stampfer, Martha R.; Bissell, Mina J.; LaBarge, Mark A.

2011-01-01

Loss of organization is a principle feature of cancers; therefore it is important to understand how normal adult multilineage tissues, such as bilayered secretory epithelia, establish and maintain their architectures. The self-organization process that drives heterogeneous mixtures of cells to form organized tissues is well studied in embryology and with mammalian cell lines that were abnormal or engineered. Here we used a micropatterning approach that confined cells to a cylindrical geometry combined with an algorithm to quantify changes of cellular distribution over time to measure the ability of different cell types to self-organize relative to each other. Using normal human mammary epithelial cells enriched into pools of the two principal lineages, luminal and myoepithelial cells, we demonstrated that bilayered organization in mammary epithelium was driven mainly by lineage-specific differential E-cadherin expression, but that P-cadherin contributed specifically to organization of the myoepithelial layer. Disruption of the actomyosin network or of adherens junction proteins resulted in either prevention of bilayer formation or loss of preformed bilayers, consistent with continual sampling of the local microenvironment by cadherins. Together these data show that self-organization is an innate and reversible property of communities of normal adult human mammary epithelial cells. PMID:21300877

Self-organization is a dynamic and lineage-intrinsic property of mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chanson, L.; Brownfield, D.; Garbe, J. C.

Loss of organization is a principle feature of cancers; therefore it is important to understand how normal adult multilineage tissues, such as bilayered secretory epithelia, establish and maintain their architectures. The self-organization process that drives heterogeneous mixtures of cells to form organized tissues is well studied in embryology and with mammalian cell lines that were abnormal or engineered. Here we used a micropatterning approach that confined cells to a cylindrical geometry combined with an algorithm to quantify changes of cellular distribution over time to measure the ability of different cell types to self-organize relative to each other. Using normal humanmore » mammary epithelial cells enriched into pools of the two principal lineages, luminal and myoepithelial cells, we demonstrated that bilayered organization in mammary epithelium was driven mainly by lineage-specific differential E-cadherin expression, but that P-cadherin contributed specifically to organization of the myoepithelial layer. Disruption of the actomyosin network or of adherens junction proteins resulted in either prevention of bilayer formation or loss of preformed bilayers, consistent with continual sampling of the local microenvironment by cadherins. Together these data show that self-organization is an innate and reversible property of communities of normal adult human mammary epithelial cells.« less

MUCI Facilitation of Growth in Chemically Induced Mammary Gland Tumors in Muc-1 Mutant and MUCI Transgenic Mice.

DTIC Science & Technology

1998-08-01

present grant proposed to initiate tumor development using chemical carcinogenesis. Pazos et al. (1991) demonstrated chemical induction of murine...latency of 154 ±19 days. Tumors were mammary adenocarcinomas of the B type of Dunn’s classification ( Pazos , 1991). My hypothesis for these studies was...in rats. Murine response to NMU is only briefly documented in the literature ( Pazos et al., 1991). Following the protocol for NMU induction of mammary

Gallic Acid Induces Apoptosis in Human Gastric Adenocarcinoma Cells.

PubMed

Tsai, Chung-Lin; Chiu, Ying-Ming; Ho, Tin-Yun; Hsieh, Chin-Tung; Shieh, Dong-Chen; Lee, Yi-Ju; Tsay, Gregory J; Wu, Yi-Ying

2018-04-01

Gastric cancer is one of the most common malignant cancers with a poor prognosis and high mortality rate worldwide. Current treatment of gastric cancer includes surgery and chemotherapy as the main modalities, but the potentially severe side-effects of chemotherapy present a considerable challenge. Gallic acid is a trihydroxybenzoic acid found to exert an anticancer effect against a variety of cancer cells. The purpose of this study was to determine the anti-cancer activity of Galla chinensis and its main component gallic acid on human gastric adenocarcinoma cells. MTT assay and cell death ELISA were used to determine the apoptotic effect of Gallic Chinensis and gallic acid on human gastric adenocarcinoma cells. To determine the pathway and relevant components by which gallic acid-induced apoptosis is mediated through, cells were transfected with siRNA (Fas, FasL, DR5, p53) using Lipofectamine 2000. Reults: Gallic Chinensis and gallic acid induced apoptosis of human gastric adenocarcinoma cells. Gallic acid induced up-regulation of Fas, FasL, and DR5 expression in AGS cells. Transfection of cells with Fas, FasL, or DR5 siRNA reduced gallic acid-induced cell death. In addition, p53 was shown to be involved in gallic acid-mediated Fas, FasL, and DR5 expression as well as cell apoptosis in AGS cells. These results suggest that gallic acid has a potential role in the treatment of gastric cancer. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Cooperative Interactions During Human Mammary Epithelial Cell Immortalization

DTIC Science & Technology

2005-07-01

papilloma virus 16 E6 or E7. Proc. Nat. Acad. Sci. USA, 92: 3687-3691, 1995. 6. Huschtscha, L. I., Neumann, A. A., Noble, J. R., and Reddel, R. R. Effects...Oncology, In press. 5. Wazer, D. E., Liu, X.-L., Chu, Q., Gao, Q., and Band, V. Immortalization of distinct human mammary epithelial cell types by human

Canonical Wnt Signaling as a Specific Marker for Normal and Tumorigenic Mammary Stem Cells

DTIC Science & Technology

2012-02-01

These cells were identified by  flow   cytometry  to detect cells that were positive for CD24 and CD49f.   2. We have established that activation of Wnt...09-1-0072 TITLE: Canonical Wnt Signaling as a Specific marker for Normal and Tumorigenic Mammary Stem Cells PRINCIPAL...activation of canonical Wnt signaling may be a very specific marker for mammary stem cells and be a target for transformation that results in the

Radiation induced esophageal adenocarcinoma in a woman previously treated for breast cancer and renal cell carcinoma.

PubMed

Raissouni, Soundouss; Raissouni, Ferdaous; Rais, Ghizlane; Aitelhaj, Meryem; Lkhoyaali, Siham; Latib, Rachida; Mohtaram, Amina; Rais, Fadoua; Mrabti, Hind; Kabbaj, Nawal; Amrani, Naima; Errihani, Hassan

2012-08-09

Secondary radiation-induced cancers are rare but well-documented as long-term side effects of radiation in large populations of breast cancer survivors. Multiple neoplasms are rare. We report a case of esophageal adenocarcinoma in a patient treated previously for breast cancer and clear cell carcinoma of the kidney. A 56 year-old non smoking woman, with no alcohol intake and no familial history of cancer; followed in the National Institute of Oncology of Rabat Morocco since 1999 for breast carcinoma, presented on consultation on January 2011 with dysphagia. Breast cancer was treated with modified radical mastectomy, 6 courses of chemotherapy based on CMF regimen and radiotherapy to breast, inner mammary chain and to pelvis as castration. Less than a year later, a renal right mass was discovered incidentally. Enlarged nephrectomy realized and showed renal cell carcinoma. A local and metastatic breast cancer recurrence occurred in 2007. Patient had 2 lines of chemotherapy and 2 lines of hormonotherapy with Letrozole and Tamoxifen assuring a stable disease. On January 2011, the patient presented dysphagia. Oesogastric endoscopy showed middle esophagus stenosing mass. Biopsy revealed adenocarcinoma. No evidence of metastasis was noticed on computed tomography and breast disease was controlled. Palliative brachytherapy to esophagus was delivered. Patient presented dysphagia due to progressive disease 4 months later. Jejunostomy was proposed but the patient refused any treatment. She died on July 2011. We present here a multiple neoplasm in a patient with no known family history of cancers. Esophageal carcinoma is most likely induced by radiation. However the presence of a third malignancy suggests the presence of genetic disorders.

Rac3 Regulates Cell Invasion, Migration and EMT in Lung Adenocarcinoma through p38 MAPK Pathway

PubMed Central

Zhang, Chenlei; Liu, Tieqin; Wang, Gebang; Wang, Huan; Che, Xiaofang; Gao, Xinghua; Liu, Hongxu

2017-01-01

Background: The role of Rac3 in cell proliferation in lung adenocarcinoma has been tackled in our previous study. However, the role of Rac3 in cell invasion and migration of lung adenocarcinoma is still not clear. Methods: The expression of Rac3 in lung adenocarcinoma specimens and paired noncancerous normal tissues were evaluated by immunohistochemistry. Lentivirus-mediated RNA interference (RNAi) was employed to silence Rac3 in lung adenocarcinoma cell lines A549 and H1299. A p38 MAPK inhibitor (LY2228820) was employed to inhibit activity of p38 MAPK pathway. Cell invasion and migration in vitro were examined by invasion and migration assays, respectively. PathScan® intracellular signaling array kit and western blot were employed in mechanism investigation. Results: Rac3 expression was frequently higher in lung adenocarcinoma than paired noncancerous normal tissues. Rac3 expression was an independent risk factor for lymphonode metastasis, and was associated with worse survival outcome. Silencing of Rac3 inhibited cell invasion and cell migration in lung adenocarcinoma cell lines. Knockdown of Rac3 decreased activity of p38 MAPK pathway. LY2228820, which was an important p38 MAPK inhibitor, inhibited Rac3-induced cell invasion and migration of lung adenocarcinoma. E-cadherin expression was increased and vimentin expression was decreased after silencing of Rac3 or following the treatment of LY2228820. Conclusions: Our findings suggest that Rac3 regulates cell invasion, migration and EMT via p38 MAPK pathway. Rac3 may be a potential biomarker of invasion and metastasis for lung adenocarcinoma, and knockdown of Rac3 may potentially serve as a promising therapeutic target for lung adenocarcinoma. PMID:28900489

Mammary Gland Development

PubMed Central

Macias, Hector

2012-01-01

The mammary gland develops through several distinct stages. The first transpires in the embryo as the ectoderm forms a mammary line that resolves into placodes. Regulated by epithelial/mesenchymal interactions, the placodes descend into the underlying mesenchyme and produce the rudimentary ductal structure of the gland present at birth. Subsequent stages of development – pubertal growth, pregnancy, lactation and involution – occur postnatally under the regulation of hormones. Puberty initiates branching morphogenesis, which requires growth hormone and estrogen, as well as IGF1, to create a ductal tree that fills the fat pad. Upon pregnancy the combined actions of progesterone and prolactin generate alveoli, which secrete milk during lactation. Lack of demand for milk at weaning initiates the process of involution whereby the gland is remodeled back to its pre-pregnancy state. These processes require numerous signaling pathways that have distinct regulatory functions at different stages of gland development. Signaling pathways also regulate a specialized subpopulation of mammary stem cells that fuel the dramatic changes in the gland occurring with each pregnancy. Our knowledge of mammary gland development and mammary stem cell biology has significantly contributed to our understanding of breast cancer and has advanced the discovery of therapies to treat this disease. PMID:22844349

Mechanisms of rolipram-induced increase in the incidence of mammary adenocarcinoma: histopathological study of a 104-week oral carcinogenicity study in female Sprague-Dawley rats.

PubMed

Nishiyama, Shoji; Okudaira, Masahiko; Saito, Noboru

2006-02-01

The present study was carried out to elucidate the mechanisms behind an increase in the incidence of malignant or multiple mammary tumors as a result of oral administration of rolipram in a 104-week carcinogenicity study. The organs and tissues of Sprague-Dawley (SD) rats of both sexes, which had been subjected to a 104-week oral carcinogenicity study at doses of 0.2, 0.6 and 2.0 mg/kg, were examined. No treatment-related effects were seen in males; however, in females, there was a significant increase in the number of malignant or multiple mammary tumor bearers at a dose of 2.0 mg/kg. No other target organs were identified and the incidence of other tumor types were within the female control range. To clarify the mechanisms behind a rolipram-induced increase in the incidence of mammary adenocarcinoma at time points earlier than 104 weeks, the hormonal changes associated with pituitary adenoma were identified, and estrous cycling in the ovary, uterus, and vagina were examined in female rats treated with rolipram for 52 weeks. The plasma prolactin (PRL) concentration in all female groups exceeded the control value at Week 52, and all these differences were statistically significant. There was also a dose-dependent relationship with PRL-producing pituitary adenomas. Changes in estrous cycling in the uterus and vagina and a decrease in the size and number of corpora lutea in the ovaries of female rats treated with rolipram at 2.0 mg/kg for 52 weeks indicated that an increase in the estrus phase of the cycle corresponded to a marked decrease in the diestrus phase, which might result from the increased plasma estrogen concentration. Together, all of the above mentioned data suggest that rolipram not only stimulates an increase in the number and size of PRL adenomas in the pituitary gland but also in the estrus phase of the estrous cycle. These events might cause progression of the mammary gland tissues from hyperplasia to carcinoma.

Proliferative Activity of Mammary Carcinoma Cells by AgNOR Count in C3H mice Receiving Ethanol Extract of Sponge Haliclona sp

NASA Astrophysics Data System (ADS)

Sijabat, Lanceria; Susilaningsih, Neni; Trianto, Agus; Murwani, Retno

2018-02-01

Quantification of argyrophilic nucleolar organizer region (AgNORs) was considered as one of markers of proliferative activity of cancer cells. Sponge Haliclona sp extract contains anticancer bioactive compounds and our previous study showed that the extract was able to improve histological grade of induced mammary adenocarcinoma in mice. The following research was conducted to study the extract administration on the proliferative activity of the carcinoma cells represented by AgNOR count in mice. This experimental study applied post test only control group design. Twenty C3H mice were divided into four groups namely C (control), H1, H2 and H3. Each group was given 0, 0.15, 1.5, and 15 mg Haliclona sp extract respectively. After three weeks of extract administration, mice were inoculated with breast cancer cells from donor mice. The extract administration were continued for another three weeks. AgNOR count was performed on tumor sections and expressed as mean of AgNOR (mAgNOR) and percentage of AgNOR (pAgNOR). Means of mAgNOR in C, H1, H2 and H3 were 4.070, 3.195, 3.450, and 3.190 respectively. Means of pAgNOR in C, H1, H2 and H3 were 34,40, 25,40, 38,40 and 19,80 respectively. The lowest means of mAgNOR and pAgNOR which is an indication of lower proliferative activity of the cancer cells was found in H3. However no significant difference can be found among treatment groups (p>0.05). Using AgNOR count, the ethanol extract of Haliclona sp could not show significant reduction in proliferation of mammary carcinoma cells of C3H mice. This finding support the view that AgNOR alone could not be used to determine pathology of cancer cells.

Xanthosine administration does not affect the proportion of epithelial stem cells in bovine mammary tissue, but has a latent negative effect on cell proliferation.

PubMed

Rauner, Gat; Barash, Itamar

2014-10-15

The challenge in manipulating the proportion of somatic stem cells lies in having to override tissue homeostasis. Xanthosine infusion via the teat canal has been reported to augment the number of label-retaining cells in the mammary gland of 3-month-old bovine calves. To further delineate xanthosine׳s effect on defined stem cells in the mammary gland of heifers-which are candidates for increased prospective milk production following such manipulation-bovine mammary parenchymal tissue was transplanted and integrated into the cleared mammary fat pad of immunodeficient mice. Xanthosine administration for 14 days did not affect the number of label-retaining cells after 10- and 11-week chases. No change in stem cell proportion, analyzed according to CD49f and CD24 expression, was noted. Clone formation and propagation rate of cultured cells, as well as expression of stem cell markers, were also unaffected. In contrast, a latent 50% decrease in bovine mammary cell proliferation rate was observed 11 weeks after xanthosine administration. Tumor development in mice was also limited by xanthosine administration. These effects may have resulted from an initial decrease in expression of the rate-limiting enzyme in guanine synthesis, IMPDH. The data indicate that caution should be exerted when considering xanthosine for stem cell manipulation. Copyright © 2014 Elsevier Inc. All rights reserved.

miR-133 involves in lung adenocarcinoma cell metastasis by targeting FLOT2.

PubMed

Wei, Guangxia; Xu, Yahuan; Peng, Tao; Yan, Jie

2018-03-01

Dysregulated microRNAs (miRNAs) reported to involve into the oncogenesis and progression in various human cancers. However, the roles and mechanism of miR-133 in lung adenocarcinoma remain largely unclear. In this study, qPCR assay and western blot were used to detect the expression levels of miR-133, Akt and FLOT2. Luciferase reporter assay was used to identify the target role of miR-133 on FLOT2. The cell invasion and the migration capability were performed using the transwell invasion assay and wound healing assay. We found that miR-133 expression levels were downregulated in human lung adenocarcinoma specimens and cell lines compared with the adjacent normal tissues and normal human bronchial epithelial cell. miR-133 significantly suppressed metastasis of lung adenocarcinoma cells in vitro. Furthermore, FLOT2 (flotillin-2) identified as a direct target of miR-133, and FLOT2 expression levels were inversely correlated with miR-133 expression levels in human lung adenocarcinoma specimens. And the restoration studies suggested FGF2 as a downstream effector of miR-133 which acted through Akt signalling pathway. Our study revealed the mechanism that miR-133 suppresses lung adenocarcinoma metastasis by targeting FLOT2 via Akt signalling pathway, implicating a potential prognostic biomarker and therapeutic target for lung adenocarcinoma treatment.

Six1 expands the mouse mammary epithelial stem/progenitor cell pool and induces mammary tumors that undergo epithelial-mesenchymal transition

PubMed Central

McCoy, Erica L.; Iwanaga, Ritsuko; Jedlicka, Paul; Abbey, Nee-Shamo; Chodosh, Lewis A.; Heichman, Karen A.; Welm, Alana L.; Ford, Heide L.

2009-01-01

Six1 is a developmentally regulated homeoprotein with limited expression in most normal adult tissues and frequent misexpression in a variety of malignancies. Here we demonstrate, using a bitransgenic mouse model, that misexpression of human Six1 in adult mouse mammary gland epithelium induces tumors of multiple histological subtypes in a dose-dependent manner. The neoplastic lesions induced by Six1 had an in situ origin, showed diverse differentiation, and exhibited progression to aggressive malignant neoplasms, as is often observed in human carcinoma of the breast. Strikingly, the vast majority of Six1-induced tumors underwent an epithelial-mesenchymal transition (EMT) and expressed multiple targets of activated Wnt signaling, including cyclin D1. Interestingly, Six1 and cyclin D1 coexpression was found to frequently occur in human breast cancers and was strongly predictive of poor prognosis. We further show that Six1 promoted a stem/progenitor cell phenotype in the mouse mammary gland and in Six1-driven mammary tumors. Our data thus provide genetic evidence for a potent oncogenic role for Six1 in mammary epithelial neoplasia, including promotion of EMT and stem cell–like features. PMID:19726883

Neem leaf extract inhibits mammary carcinogenesis by altering cell proliferation, apoptosis, and angiogenesis

PubMed Central

Arumugam, Arunkumar; Agullo, Pamela; Boopalan, Thiyagarajan; Nandy, Sushmita; Lopez, Rebecca; Gutierrez, Christina; Narayan, Mahesh; Rajkumar, Lakshmanaswamy

2014-01-01

Plant-based medicines are useful in the treatment of cancer. Many breast cancer patients use complementary and alternative medicine in parallel with conventional treatments. Neem is historically well known in Asia and Africa as a versatile medicinal plant with a wide spectrum of biological activities. The experiments reported herein determined whether the administration of an ethanolic fraction of Neem leaf (EFNL) inhibits progression of chemical carcinogen-induced mammary tumorigenesis in rat models. Seven-week-old female Sprague Dawley rats were given a single intraperitoneal injection of N-methyl-N-nitrosourea (MNU). Upon the appearance of palpable mammary tumors, the rats were divided into vehicle-treated control groups and EFNL-treated groups. Treatment with EFNL inhibited MNU-induced mammary tumor progression. EFNL treatment was also highly effective in reducing mammary tumor burden and in suppressing mammary tumor progression even after the cessation of treatment. Further, we found that EFNL treatment effectively upregulated proapoptotic genes and proteins such as p53, B cell lymphoma-2 protein (Bcl-2)-associated X protein (Bax), Bcl-2-associated death promoter protein (Bad) caspases, phosphatase and tensin homolog gene (PTEN), and c-Jun N-terminal kinase (JNK). In contrast, EFNL treatment caused downregulation of anti-apoptotic (Bcl-2), angiogenic proteins (angiopoietin and vascular endothelial growth factor A [VEGF-A]), cell cycle regulatory proteins (cyclin D1, cyclin-dependent kinase 2 [Cdk2], and Cdk4), and pro-survival signals such as NFκB, mitogen-activated protein kinase 1 (MAPK1). The data obtained in this study demonstrate that EFNL exert a potent anticancer effect against mammary tumorigenesis by altering key signaling pathways. PMID:24146019

Pim-1 kinase expression during murine mammary development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gapter, Leslie A.; School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4234; Magnuson, Nancy S.

2006-07-07

Pim-1 kinase phosphorylates substrates whose activities are linked to proliferation, survival, differentiation, and apoptosis. Although pim-1 is induced by hormones and cytokines, the hormonal control and contribution of Pim-1 to mammary gland development have not been evaluated. We examined Pim-1 expression in mammary cell lines, investigated whether Pim-1 levels could be altered in breast epithelia by mammogenic hormones, and evaluated Pim-1 expression during mammary development. We found that Pim-1 was elevated in most mammary carcinoma cell lines and progesterone increased Pim-1 protein to some extent in non-tumorigenic mammary epithelia. Pim-1 expression in situ was consistent with the documented profile ofmore » progesterone activity in mouse mammary glands. Pim-1 nuclear localization correlated with cytoplasmic distribution for its substrate, p21{sup CIP/Waf1}, and we found that Pim-1 and p21 associate in vitro. Our results suggest that Pim-1 expression may be regulated by progesterone during mammary development and Pim-1 associates with p21 in mammary epithelial cells.« less

Oxidative DNA damage and mammary cell proliferation by alcohol-derived salsolinol.

PubMed

Murata, Mariko; Midorikawa, Kaoru; Kawanishi, Shosuke

2013-10-21

Drinking alcohol is a risk factor for breast cancer. Salsolinol (SAL) is endogenously formed by a condensation reaction of dopamine with acetaldehyde, a major ethanol metabolite, and SAL is detected in blood and urine after alcohol intake. We investigated the possibility that SAL can participate in tumor initiation and promotion by causing DNA damage and cell proliferation, leading to alcohol-associated mammary carcinogenesis. SAL caused oxidative DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), in the presence of transition metal ions, such as Cu(II) and Fe(III)EDTA. Inhibitory effects of scavengers on SAL-induced DNA damage and the electron spin resonance study indicated the involvement of H₂O₂, which is generated via the SAL radical. Experiments on scavengers and site specificity of DNA damage suggested ·OH generation via a Fenton reaction and copper-peroxide complexes in the presence of Fe(III)EDTA and Cu(II), respectively. SAL significantly increased 8-oxodG formation in normal mammary epithelial MCF-10A cells. In addition, SAL induced cell proliferation in estrogen receptor (ER)-negative MCF-10A cells, and the proliferation was inhibited by an antioxidant N-acetylcysteine and an epidermal growth factor receptor (EGFR) inhibitor AG1478, suggesting that reactive oxygen species may participate in the proliferation of MCF-10A cells via EGFR activation. Furthermore, SAL induced proliferation in estrogen-sensitive breast cancer MCF-7 cells, and a surface plasmon resonance sensor revealed that SAL significantly increased the binding activity of ERα to the estrogen response element but not ERβ. In conclusion, SAL-induced DNA damage and cell proliferation may play a role in tumor initiation and promotion of multistage mammary carcinogenesis in relation to drinking alcohol.

Bifunctional role of ephrin A1-Eph system in stimulating cell proliferation and protecting cells from cell death through the attenuation of ER stress and inflammatory responses in bovine mammary epithelial cells.

PubMed

Kang, Minkyung; Jeong, Wooyoung; Bae, Hyocheol; Lim, Whasun; Bazer, Fuller W; Song, Gwonhwa

2018-03-01

Structural and functional development of the mammary gland is constant in the mammary gland life cycle. Eph receptors and their ligands, ephrins, control events through cell-to-cell interactions during embryonic development, and adult tissue homeostasis; however, little information on participation of ephrin A1, a representative ligand of the Eph receptor, in the development and function of normal mammary glands is known. In this study, we demonstrated functional effects of the ephrin A1-Eph system and mechanisms of its action on bovine mammary epithelial (MAC-T) cells. The in vitro cultured MAC-T cells expressed the ephrin A1 ligand and EphA1, A2, A4, A7, and A8 among the eight members of the Eph A family. Our results revealed that ephrin A1 induced MAC-T cell cycle progression and stimulated cell proliferation with abundant expression of nucleic PCNA and cyclin D1 proteins. Additionally, ephrin A1 induced activation of intracellular signaling molecules involved in PI3 K/AKT and MAPK signaling, and the proliferation-stimulating effect of ephrin A1 was mediated by activation of these pathways. Furthermore, ephrin A1 influenced expression and activation of various ER stress-related proteins and protected MAC-T cells from stress-induced cell death. Finally, ephrin A1 alleviated LPS-induced cell death through down-regulation of inflammatory cytokines. In conclusion, the results of this study suggest that the Eph A-ephrin A1 system is a positive factor in the increase and maintenance of epithelial cells in mammary glands of cows; the signaling system contributes to development, remodeling, and functionality of normal mammary glands and could overcome mastitis in cows and other mammals. © 2017 Wiley Periodicals, Inc.

IL-12 Can Target Human Lung Adenocarcinoma Cells and Normal Bronchial Epithelial Cells Surrounding Tumor Lesions

PubMed Central

Airoldi, Irma; Di Carlo, Emma; Cocco, Claudia; Caci, Emanuela; Cilli, Michele; Sorrentino, Carlo; Sozzi, Gabriella; Ferrini, Silvano; Rosini, Sandra; Bertolini, Giulia; Truini, Mauro; Grossi, Francesco; Galietta, Luis Juan Vicente; Ribatti, Domenico; Pistoia, Vito

2009-01-01

Background Non small cell lung cancer (NSCLC) is a leading cause of cancer death. We have shown previously that IL-12rb2 KO mice develop spontaneously lung adenocarcinomas or bronchioalveolar carcinomas. Aim of the study was to investigate i) IL-12Rβ2 expression in human primary lung adenocarcinomas and in their counterparts, i.e. normal bronchial epithelial cells (NBEC), ii) the direct anti-tumor activity of IL-12 on lung adenocarcinoma cells in vitro and vivo, and the mechanisms involved, and iii) IL-12 activity on NBEC. Methodology/Principal Findings Stage I lung adenocarcinomas showed significantly (P = 0.012) higher frequency of IL-12Rβ2 expressing samples than stage II/III tumors. IL-12 treatment of IL-12R+ neoplastic cells isolated from primary adenocarcinoma (n = 6) inhibited angiogenesis in vitro through down-regulation of different pro-angiogenic genes (e.g. IL-6, VEGF-C, VEGF-D, and laminin-5), as assessed by chorioallantoic membrane (CAM) assay and PCR array. In order to perform in vivo studies, the Calu6 NSCLC cell line was transfected with the IL-12RB2 containing plasmid (Calu6/β2). Similar to that observed in primary tumors, IL-12 treatment of Calu6/β2+ cells inhibited angiogenesis in vitro. Tumors formed by Calu6/β2 cells in SCID/NOD mice, inoculated subcutaneously or orthotopically, were significantly smaller following IL-12 vs PBS treatment due to inhibition of angiogenesis, and of IL-6 and VEGF-C production. Explanted tumors were studied by histology, immuno-histochemistry and PCR array. NBEC cells were isolated and cultured from lung specimens of non neoplastic origin. NBEC expressed IL-12R and released constitutively tumor promoting cytokines (e.g. IL-6 and CCL2). Treatment of NBEC with IL-12 down-regulated production of these cytokines. Conclusions This study demonstrates that IL-12 inhibits directly the growth of human lung adenocarcinoma and targets the adjacent NBEC. These novel anti-tumor activities of IL-12 add to the well

Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium

PubMed Central

Ewald, Andrew J.; Huebner, Robert J.; Palsdottir, Hildur; Lee, Jessie K.; Perez, Melissa J.; Jorgens, Danielle M.; Tauscher, Andrew N.; Cheung, Kevin J.; Werb, Zena; Auer, Manfred

2012-01-01

Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organizations. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayered epithelium. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in three-dimensional culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, whereas PAR3 displayed a cytoplasmic localization, suggesting that the apico-basal polarity was incomplete. Despite membrane localization of E-cadherin and β-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex interdigitating membrane protrusions. Single-cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes re-formed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression might reflect partial recapitulation of a normal developmental program. PMID:22344263

Gas6 Induces Growth, β-Catenin Stabilization, and T-Cell Factor Transcriptional Activation in Contact-Inhibited C57 Mammary Cells

PubMed Central

Goruppi, Sandro; Chiaruttini, Cristina; Ruaro, Maria Elisabetta; Varnum, Brian; Schneider, Claudio

2001-01-01

Gas6 is a growth factor related to protein S that was identified as the ligand for the Axl receptor tyrosine kinase (RTK) family. In this study, we show that Gas6 induces a growth response in a cultured mammalian mammary cell line, C57MG. The presence of Gas6 in the medium induces growth after confluence and similarly causes cell cycle reentry of density-inhibited C57MG cells. We show that Axl RTK but not Rse is efficiently activated by Gas6 in density-inhibited C57MG cells. We have analyzed the signaling required for the Gas6 proliferative effect and found a requirement for PI3K-, S6K-, and Ras-activated pathways. We also demonstrate that Gas6 activates Akt and concomitantly inhibits GSK3 activity in a wortmannin-dependent manner. Interestingly, Gas6 induces up-regulation of cytosolic β-catenin, while membrane-associated β-catenin remains unaffected. Stabilization of β-catenin in C57MG cells is correlated with activation of a T-cell factor (TCF)-responsive transcriptional element. We thus provide evidence that Gas6 is mitogenic and induces β-catenin proto-oncogene stabilization and subsequent TCF/Lef transcriptional activation in a mammary system. These results suggest that Gas6-Axl interaction, through stabilization of β-catenin, may have a role in mammary development and/or be involved in the progression of mammary tumors. PMID:11154277

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells.

PubMed

Yu, Qing Cissy; Song, Wenqian; Lai, Dengwen; Zeng, Yi Arial

2017-08-03

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr + VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

VHL deletion impairs mammary alveologenesis but is not sufficient for mammary tumorigenesis.

PubMed

Seagroves, Tiffany N; Peacock, Danielle L; Liao, Debbie; Schwab, Luciana P; Krueger, Robin; Handorf, Charles R; Haase, Volker H; Johnson, Randall S

2010-05-01

Overexpression of hypoxia inducible factor-1 (HIF-1)alpha, which is common in most solid tumors, correlates with poor prognosis and high metastatic risk in breast cancer patients. Because HIF-1alpha protein stability is tightly controlled by the tumor suppressor von Hippel-Lindau (VHL), deletion of VHL results in constitutive HIF-1alpha expression. To determine whether VHL plays a role in normal mammary gland development, and if HIF-1alpha overexpression is sufficient to initiate breast cancer, Vhl was conditionally deleted in the mammary epithelium using the Cre/loxP system. During first pregnancy, loss of Vhl resulted in decreased mammary epithelial cell proliferation and impaired alveolar differentiation; despite these phenotypes, lactation was sufficient to support pup growth. In contrast, in multiparous dams, Vhl(-/-) mammary glands exhibited a progressive loss of alveolar epithelium, culminating in lactation failure. Deletion of Vhl in the epithelium also impacted the mammary stroma, as there was increased microvessel density accompanied by hemorrhage and increased immune cell infiltration. However, deletion of Vhl was not sufficient to induce mammary tumorigenesis in dams bred continuously for up to 24 months of age. Moreover, co-deletion of Hif1a could not rescue the Vhl(-/-)-dependent phenotype as dams were unable to successfully lactate during the first lactation. These results suggest that additional VHL-regulated genes besides HIF1A function to maintain the proliferative and regenerative potential of the breast epithelium.

Megalin-Mediated Endocytosis of Vitamin D Binding Protein Correlates with 25-Hydroxycholecalciferol Actions in Human Mammary Cells1

PubMed Central

Rowling, Matthew J.; Kemmis, Carly M.; Taffany, David A.; Welsh, JoEllen

2007-01-01

The major circulating form of vitamin D is 25-hydroxycholecalciferol [25(OH)D3], which is delivered to target tissues in complex with the serum vitamin D binding protein (DBP). We recently observed that mammary cells can metabolize 25(OH)D3 to 1,25-dihydroxycholecalciferol [1,25(OH)2D3], the vitamin D receptor (VDR) ligand, and the objective of our study was to elucidate the mechanisms by which the 25(OH)D3-DBP complex is internalized by mammary cells prior to metabolism. Using fluorescent microscopy and temperature-shift techniques, we found that T-47D breast cancer cells rapidly internalize DBP via endocytosis, which is blunted by receptor-associated protein, a specific inhibitor of megalin-mediated endocytosis. Endocytosis of DBP was associated with activation of VDR by 25(OH)D3 but not 1,25(OH)2D3 (as measured by induction of the VDR target gene, CYP24). We also found that megalin and its endocytic partner, cubilin, are coexpressed in normal murine mammary tissue, in nontransformed human mammary epithelial cell lines, and in some established human breast cancer cell lines. To our knowledge, our studies are the first to demonstrate that mammary-derived cells express megalin and cubilin, which contribute to the endocytic uptake of 25(OH)D3-DBP and activation of the VDR pathway. PMID:17056796

The spectrum of STAT functions in mammary gland development

PubMed Central

Hughes, Katherine; Watson, Christine J.

2012-01-01

The signal transducer and activator of transcription (STAT) family of transcription factors have a spectrum of functions in mammary gland development. In some cases these roles parallel those of STATs in other organ systems, while in other instances the function of individual STATs in the mammary gland is specific to this tissue. In the immune system, STAT6 is associated with differentiation of T helper cells, while in the mammary gland, it has a fundamental role in the commitment of luminal epithelial cells to the alveolar lineage. STAT5A is required for the production of luminal progenitor cells from mammary stem cells and is essential for the differentiation of milk producing alveolar cells during pregnancy. By contrast, the initiation of regression following weaning heralds a dramatic and specific activation of STAT3, reflecting its pivotal role in the regulation of cell death and tissue remodeling during mammary involution. Although it has been demonstrated that STAT1 is regulated during a mammary developmental cycle, it is not yet determined whether it has a specific, non-redundant function. Thus, the mammary gland constitutes an unusual example of an adult organ in which different STATs are sequentially activated to orchestrate the processes of functional differentiation, cell death and tissue remodeling. PMID:24058764

Enhanced degradation of p53 protein in HPV-6 and BPV-1 E6-immortalized human mammary epithelial cells.

PubMed Central

Band, V; Dalal, S; Delmolino, L; Androphy, E J

1993-01-01

Normal mammary epithelial cells are efficiently immortalized by the E6 gene of human papillomavirus (HPV)-16, a virus commonly associated with cervical cancers. Surprisingly, introduction of the E6 gene from HPV-6, which is rarely found in cervical cancer, or bovine papillomavirus (BPV)-1, into normal mammary cells resulted in the generation of immortal cell lines. The establishment of HPV-6 and BPV-1 E6-immortalized cells was less efficient and required a longer period in comparison to HPV-16 E6. These HPV-6- and BPV-1 E6-immortalized cells demonstrated dramatically reduced levels of p53 protein by immunoprecipitation. While the half-life of p53 protein in normal mammary epithelial cells was approximately 3 h, it was reduced to approximately 15 min in all the E6-immortalized cells. These results demonstrate that the E6 genes of both high-risk and low-risk papilloma viruses immortalize human mammary epithelial cells and induce a marked degradation of p53 protein in vivo. Images PMID:8387914

Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro.

PubMed

Mießler, Katharina S; Markov, Alexander G; Amasheh, Salah

2018-01-01

During lactation, accumulation of milk in mammary glands (MG) causes hydrostatic pressure (HP) and concentration of bioactive compounds. Previously, a changed expression of tight junction (TJ) proteins was observed in mice MGs by accumulation of milk, in vivo. The TJ primarily determines the integrity of the MG epithelium. The present study questioned whether HP alone can affect the TJ in a mammary epithelial cell model, in vitro. Therefore, monolayers of HC11, a mammary epithelial cell line, were mounted into modified Ussing chambers and incubated with 10 kPa bilateral HP for 4 h. Short circuit current and transepithelial resistance were recorded and compared to controls, and TJ proteins were analyzed by Western blotting and immunofluorescent staining. In our first approach HC11 cells could withstand the pressure incubation and a downregulation of occludin was observed. In a second approach, using prolactin- and dexamethasone-induced cells, a decrease of short circuit current was observed, beginning after 2 h of incubation. With the addition of 1 mM barium chloride to the bathing solution the decrease could be blocked temporarily. On molecular level an upregulation of ZO-1 could be observed in hormone-induced cells, which was downregulated after the incubation with barium chloride. In conclusion, bilateral HP incubation affects mammary epithelial monolayers, in vitro. Both, the reduction of short circuit current and the change in TJ proteins may be interpreted as physiological requirements for lactation. Copyright © 2017 Elsevier Inc. All rights reserved.

Unsaturated fatty acids promote proliferation via ERK1/2 and Akt pathway in bovine mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yonezawa, Tomo; Haga, Satoshi; Kobayashi, Yosuke

2008-03-21

GPR40 has recently been identified as a G protein-coupled cell-surface receptor for long-chain fatty acids (LCFAs). The mRNA of the bovine ortholog of GPR40 (bGPR40) was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and in the bovine mammary gland at various stages of lactation. Oleate and linoleate caused an increase in intracellular Ca{sup 2+} concentrations in these cells, and significantly reduced forskolin-induced cAMP concentrations. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase, which regulates cell proliferation and survival, was rapidly increased by oleate. Incubation with oleate and linoleate for 24 h significantly promoted cell proliferation.more » Moreover, in serum-free medium, oleate significantly stimulated cell proliferation during a 7-day culture. These results suggest that bGPR40 mediates LCFA signaling in mammary epithelial cells and thereby plays an important role in cell proliferation and survival.« less

The mammary cellular hierarchy and breast cancer.

PubMed

Oakes, Samantha R; Gallego-Ortega, David; Ormandy, Christopher J

2014-11-01

Advances in the study of hematopoietic cell maturation have paved the way to a deeper understanding the stem and progenitor cellular hierarchy in the mammary gland. The mammary epithelium, unlike the hematopoietic cellular hierarchy, sits in a complex niche where communication between epithelial cells and signals from the systemic hormonal milieu, as well as from extra-cellular matrix, influence cell fate decisions and contribute to tissue homeostasis. We review the discovery, definition and regulation of the mammary cellular hierarchy and we describe the development of the concepts that have guided our investigations. We outline recent advances in in vivo lineage tracing that is now challenging many of our assumptions regarding the behavior of mammary stem cells, and we show how understanding these cellular lineages has altered our view of breast cancer.

Microenvironmental Regulation of Mammary Carcinogenesis

DTIC Science & Technology

2008-06-01

cells. These models share many of the hallmarks of multistage human breast cancer development including histological disease progression and immune cell... developed by Muller and colleagues20, represents a reasonable recapitulation of late-stage human breast cancer as determined by histological progression ...Annual Progress Report d. Develop a profile of proteolytic activities in normal and neoplastic mammary tissues from mouse models of mammary

Different effects of H2O2 treatment on cervical squamous carcinoma cells and adenocarcinoma cells

PubMed Central

Zhang, Peihai; Yin, Haiqin; Wang, Sie; Wei, Yuping; Peng, Nan

2015-01-01

Introduction This study aims to compare the antioxidant abilities of cervical squamous carcinoma cells and cervical adenocarcinoma cells and to study the related mechanisms. Material and methods Cervical squamous carcinoma and adenocarcinoma cells were treated with H2O2. Cell proliferation was determined with the MTT assay. The reactive oxygen species (ROS) level was detected by the 2’,7’-dichlorofluorescein-diacetate (DCFH-DA) method. The 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) method was performed to measure intracellular concentrations of reduced glutathione (GSH) and oxidized glutathione (GSSG). The nitrite formation method, the molybdate colorimetric method, and the DTNB colorimetric method were used to determine activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), respectively. Results Compared with untreated control cells, cell proliferation of cervical squamous carcinoma cells and cervical adenocarcinoma cells was significantly inhibited by H2O2 treatment (p < 0.05). Reactive oxygen species levels and GSSG levels were significantly increased (p < 0.01), whereas GSH levels were significantly decreased (p < 0.05 or 0.01) in both cells after H2O2 treatment. Thus the ratio of GSH/GSSG was significantly decreased by H2O2 treatment in both cells (p < 0.01). In addition, H2O2 treatment significantly increased activities of SOD, CAT, and GPx in both cells (p < 0.05 or 0.01). Furthermore, the above-mentioned changes induced by H2O2 treatment were more dramatic in cervical squamous carcinoma cells. Conclusions The antioxidant ability of cervical squamous carcinoma cells is lower than that of cervical adenocarcinoma cells, which may be related to the increased ROS levels in cervical squamous carcinoma cells induced by H2O2 treatments. PMID:26788095

Proliferation of Estrogen Receptor alpha Positive Mammary Epithelial Cells is Restrained by TGFbeta1 in Adult Mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ewan, Kenneth B.R.; Oketch-Rabah, Hellen A.; Ravani, Shraddha A.

2005-03-03

Transforming growth factor {beta}1 (TGF{beta}1) is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor {alpha} (ER{alpha}) cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF{beta}1 is necessary for the quiescence of ER{alpha}-positive population, we examined mouse mammary epithelial gland at estrus. Approximately 35% of cells showed TGF{beta}1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF{beta} signaling is autocrine. Furthermore, nuclear Smad co-localized with nuclear ER{alpha}. To test whether TGF{beta} was functional, we examined genetically engineered mice with different levels of TGF{beta}1. ER{alpha}more » co-localization with markers of proliferation (i.e. Ki-67 or BrdU) at estrus was significantly increased in the mammary glands of Tgf{beta}1 C57/bl/129SV heterozygote mice. This relationship was maintained following pregnancy, but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF{beta}1 via the MMTV promoter suppressed proliferation of ER{alpha} positive cells. Thus, TGF{beta}1 activation functionally restrains ER{alpha} positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF{beta}1 dysregulation may promote proliferation of ER{alpha} positive cells associated with breast cancer risk in humans.« less

Effects of xanthosine in isoform switch and splice variants of expressed genes in mammary epithelial cells

USDA-ARS?s Scientific Manuscript database

Although intramammary xanthosine (XS) treatment was reported to increase the mammary stem cell population and milk yield in bovine and caprine, underlying molecular mechanisms remain unclear. The goal of this study was to evaluate effects of XS treatment on the mammary transcriptome in early-lactati...

Host-pathogen interactions in bovine mammary epithelial cells and HeLa cells by Staphylococcus aureus isolated from subclinical bovine mastitis.

PubMed

Castilho, Ivana G; Dantas, Stéfani Thais Alves; Langoni, Hélio; Araújo, João P; Fernandes, Ary; Alvarenga, Fernanda C L; Maia, Leandro; Cagnini, Didier Q; Rall, Vera L M

2017-08-01

Staphylococcus aureus is a common pathogen that causes subclinical bovine mastitis due to several virulence factors. In this study, we analyzed S. aureus isolates collected from the milk of cows with subclinical mastitis that had 8 possible combinations of bap, icaA, and icaD genes, to determine their capacity to produce biofilm on biotic (bovine primary mammary epithelial cells and HeLa cells) and abiotic (polystyrene microplates) surfaces, and their ability to adhere to and invade these cells. We also characterized isolates for microbial surface components recognizing adhesive matrix molecules (MSCRAMM) and agr genes, and for their susceptibility to cefquinome sulfate in the presence of biofilm. All isolates adhered to and invaded both cell types, but invasion indexes were higher in bovine primary mammary epithelial cells. Using tryptic soy broth + 1% glucose on abiotic surfaces, 5 out of 8 isolates were biofilm producers, but only the bap + icaA + icaD + isolate was positive in Dulbecco's Modified Eagle's medium. The production of biofilm on biotic surfaces occurred only with this isolate and only on HeLa cells, because the invasion index for bovine primary mammary epithelial cells was too high, making it impossible to use these cells in this assay. Of the 5 biofilm producers in tryptic soy broth + 1% glucose, 4 presented with the bap/fnbA/clfA/clfB/eno/fib/ebpS combination, and all were protected from cefquinome sulfate. We found no predominance of any agr group. The high invasive potential of S. aureus made it impossible to observe biofilm in bovine primary mammary epithelial cells, and we concluded that cells with lower invasion rates, such as HeLa cells, were more appropriate for this assay. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

CXCR4 expression in feline mammary carcinoma cells: evidence of a proliferative role for the SDF-1/CXCR4 axis.

PubMed

Ferrari, Angelo; Petterino, Claudio; Ratto, Alessandra; Campanella, Chiara; Wurth, Roberto; Thellung, Stefano; Vito, Guendalina; Barbieri, Federica; Florio, Tullio

2012-03-14

Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation. A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4 immunoreactivity. CXCR4 score

Ligand activation of peroxisome proliferator-activated receptor-β/δ (PPAR β/δ) inhibits cell growth in a mouse mammary gland cancer cell line

PubMed Central

Foreman, Jennifer E.; Sharma, Arun K.; Amin, Shantu; Gonzalez, Frank J.; Peters, Jeffrey M.

2009-01-01

The effects of ligand activation of PPARβ/δ were examined in the mouse mammary tumor cell line (C20). Expression of PPARβ/δ was markedly lower in C20 cells as compared to the human non-tumorigenic mammary gland derived cell line (MCF10A) and mouse keratinocytes. Ligand activation of PPARβ/δ in C20 cells caused upregulation of the PPARβ/δ target gene angiopoietin-like 4 (Angptl4). Inhibition of C20 cell proliferation and clonogenicity was observed following treatment with GW0742 or GW501516, two highly specific PPARβ/δ ligands. In addition, an increase in apoptosis was observed in C20 cells cultured with 10 µM GW501516 that preceded the observed inhibition of cell proliferation. Results from this study show that proliferation of the C20 mouse mammary gland cancer cell line is inhibited by ligand activation of PPARβ/δ due in part to increased apoptosis. PMID:19660859

Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System.

PubMed

Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

2013-01-01

Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1) on bovine mammary epithelial cells in monolayer and three-dimensional cultures. Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 µL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells.

Contralateral breast metastasis from pulmonary adenocarcinoma: two cases report and literature review

PubMed Central

Ji, Fang-Fang; Gao, Peng; Wang, Ji-Gang; Zhao, Jie

2012-01-01

Carcinoma metastatic to breast from extra-mammary malignancy is rare and only accounts for 0.4-1.3% of all breast cancer. Two rare cases of single breast metastasis from pulmonary adenocarcinoma were reported here with a brief review of the pertinent literature. The only complaint of the these two female patients was painless breast mass found recently. Most breast metastasis previously reported are present in the upper outer quadrant, however, in our study, one case was found to be located in the lower inner quadrant and the other in the upper inner quadrant. Tumor cells from breast biopsy were immune-positive for thyroid transcription factor-1. The two patients survived 5 and 8 months, respectively, following the diagnosis of both the primary lung tumor and the breast metastasis. Breast metastasis from lung adenocarcinoma is rare but does exist. The awareness of this possibility may help to differentiate the tumor from primary breast carcinoma. Clinical history and immunohistochemical studies are essential to reach the final diagnosis. PMID:22934141

Folic Acid Supplementation Promotes Mammary Tumor Progression in a Rat Model

PubMed Central

Deghan Manshadi, Shaidah; Ishiguro, Lisa; Sohn, Kyoung-Jin; Medline, Alan; Renlund, Richard; Croxford, Ruth; Kim, Young-In

2014-01-01

Folic acid supplementation may prevent the development of cancer in normal tissues but may promote the progression of established (pre)neoplastic lesions. However, whether or not folic acid supplementation can promote the progression of established (pre)neoplastic mammary lesions is unknown. This is a critically important issue because breast cancer patients and survivors in North America are likely exposed to high levels of folic acid owing to folic acid fortification and widespread supplemental use after cancer diagnosis. We investigated whether folic acid supplementation can promote the progression of established mammary tumors. Female Sprague-Dawley rats were placed on a control diet and mammary tumors were initiated with 7,12-dimethylbenza[a]anthracene at puberty. When the sentinel tumor reached a predefined size, rats were randomized to receive a diet containing the control, 2.5x, 4x, or 5x supplemental levels of folic acid for up to 12 weeks. The sentinel mammary tumor growth was monitored weekly. At necropsy, the sentinel and all other mammary tumors were analyzed histologically. The effect of folic acid supplementation on the expression of proteins involved in proliferation, apoptosis, and mammary tumorigenesis was determined in representative sentinel adenocarcinomas. Although no clear dose-response relationship was observed, folic acid supplementation significantly promoted the progression of the sentinel mammary tumors and was associated with significantly higher sentinel mammary tumor weight and volume compared with the control diet. Furthermore, folic acid supplementation was associated with significantly higher weight and volume of all mammary tumors. The most significant and consistent mammary tumor-promoting effect was observed with the 2.5x supplemental level of folic acid. Folic acid supplementation was also associated with an increased expression of BAX, PARP, and HER2. Our data suggest that folic acid supplementation may promote the progression

A Novel Nectin-mediated Cell Adhesion Apparatus That Is Implicated in Prolactin Receptor Signaling for Mammary Gland Development*

PubMed Central

Kitayama, Midori; Mizutani, Kiyohito; Maruoka, Masahiro; Mandai, Kenji; Sakakibara, Shotaro; Ueda, Yuki; Komori, Takahide; Shimono, Yohei; Takai, Yoshimi

2016-01-01

Mammary gland development is induced by the actions of various hormones to form a structure consisting of collecting ducts and milk-secreting alveoli, which comprise two types of epithelial cells known as luminal and basal cells. These cells adhere to each other by cell adhesion apparatuses whose roles in hormone-dependent mammary gland development remain largely unknown. Here we identified a novel cell adhesion apparatus at the boundary between the luminal and basal cells in addition to desmosomes. This apparatus was formed by the trans-interaction between the cell adhesion molecules nectin-4 and nectin-1, which were expressed in the luminal and basal cells, respectively. Nectin-4 of this apparatus further cis-interacted with the prolactin receptor in the luminal cells to enhance the prolactin-induced prolactin receptor signaling for alveolar development with lactogenic differentiation. Thus, a novel nectin-mediated cell adhesion apparatus regulates the prolactin receptor signaling for mammary gland development. PMID:26757815

[NUCLEAR STRUCTURE IN THE SECRETORY CELLS OF MAMMARY GLANDS IN LACTATING AND NON-LACTATING RATS].

PubMed

Tyutina, K V; Skopichev, V G; Bogolyubov, D S; Bogolyubova, I O

2016-01-01

The features of structural and functional organization of the main nuclear compartments and distribution of their key molecular components (chromatin-remodeling protein ATRX, RNA polymerase I and II, and the splicing factor SC35) has been studied in the nuclei of mammary gland cells at different functional states. No significant differences between the nuclei of the cells in the lactating and non-lactating mammary glands have been revealed at the ultrastructural level. At the same time, photometric analysis has revealed higher intensity of nucleoplasmic immunofluorescent staining of mammary glands in the lactating animals when antibodies against the proteins ATRX and SC35 were used. Apparently, this observation reflects the changes of the structural and functional status of chromatin as well as the redistribution of splicing factors between the sites of their deposition and transcription.

GAS6 is an estrogen-inducible gene in mammary epithelial cells

PubMed Central

Mo, Rigen; Zhu, Yiwei Tony; Zhang, Zhongyi; Rao, Sambasiva M.; Zhu, Yi-Jun

2007-01-01

To identify estrogen responsive genes in mammary glands, microarray assays were performed. Twenty genes were found to be up-regulated while 16 genes were repressed in the 9h estrogen treated glands. The induction of GAS6, one of the genes up-regulated by estrogen, was confirmed by RNase protection assay. Furthermore, GAS6 was also demonstrated to be induced by estrogen in ER positive breast cancer cells. Analysis of GAS6 promoter revealed that GAS6 promoter was regulated by estrogen. An estrogen response element (ERE) was identified in the GAS6 promoter. Electrophoretic mobility shift assay revealed that ERα interacted with the ERE in the GAS6 promoter. Chromatin immunoprecipitation demonstrated that ERα was recruited to the GAS6 promoter upon estrogen stimulation. These results suggested that GAS6 is an estrogen target gene in mammary epithelial cells. PMID:17174935

Oncogene-Induced Changes in Mammary Cell Fate and EMT in Breast Tumorigenesis

DTIC Science & Technology

2015-04-01

have worked on the project? Name: Lauren Walheim Project Role: Chemistry pre-medicine undergraduate student Researcher Identifier (e.g. ORCID ID...Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author( s ) and should not be construed as an...Examine if IGF1R alters mammary cell fate in vivo and the effect this has on mammary tumorigenesis (months 13-30) ...…………………………………………….......11 2a

R-spondin3 is associated with basal-progenitor behavior in normal and tumor mammary cells.

PubMed

Tocci, Johanna Melisa; Felcher, Carla María; García Solá, Martín E; Goddio, María Victoria; Zimberlin, María Noel; Rubinstein, Natalia; Srebrow, Anabella; Coso, Omar Adrián; Abba, Martín C; Meiss, Roberto P; Kordon, Edith C

2018-05-10

R-spondin3 (RSPO3) is a member of a family of secreted proteins that enhance Wnt signaling pathways in diverse processes including cancer. However, the role of RSPO3 in mammary gland and breast cancer development remains unclear. In this study, we show that RSPO3 is expressed in the basal stem cell-enriched compartment of normal mouse mammary glands but is absent from committed mature luminal cells in which exogenous RSPO3 impairs lactogenic differentiation. RSPO3 knockdown in basal-like mouse mammary tumor cells reduced canonical Wnt signaling, epithelial-to-mesenchymal transition-like features, migration capacity, and tumor formation in vivo. Conversely, RSPO3 overexpression, which was associated with some LGR and RUNX factors, highly correlated with the basal-like subtype among breast cancer patients. Thus we identified RSPO3 as a novel key modulator of breast cancer development and a potential target for treatment of basal-like breast cancers. Copyright ©2018, American Association for Cancer Research.

A redefinition of the representation of mammary cells and enzyme activities in a lactating dairy cow model.

PubMed

Hanigan, M D; Rius, A G; Kolver, E S; Palliser, C C

2007-08-01

The Molly model predicts various aspects of digestion and metabolism in the cow, including nutrient partitioning between milk and body stores. It has been observed previously that the model underpredicts milk component yield responses to nutrition and consequently overpredicts body energy store responses. In Molly, mammary enzyme activity is represented as an aggregate of mammary cell numbers and activity per cell with minimal endocrine regulation. Work by others suggests that mammary cells can cycle between active and quiescent states in response to various stimuli. Simple models of milk production have demonstrated the utility of this representation when using the model to simulate variable milking and nutrient restriction. It was hypothesized that replacing the current representation of mammary cells and enzyme activity in Molly with a representation of active and quiescent cells and improving the representation of endocrine control of cell activity would improve predictions of milk component yield. The static representation of cell numbers was replaced with a representation of cell growth during gestation and early lactation periods and first-order cell death. Enzyme capacity for fat and protein synthesis was assumed to be proportional to cell numbers. Enzyme capacity for lactose synthesis was represented with the same equation form as for cell numbers. Data used for parameter estimation were collected as part of an extended lactation trial. Cows with North American or New Zealand genotypes were fed 0, 3, or 6 kg of concentrate dry matter daily during a 600-d lactation. The original model had root mean square prediction errors of 17.7, 22.3, and 19.8% for lactose, protein, and fat yield, respectively, as compared with values of 8.3, 9.4, and 11.7% for the revised model, respectively. The original model predicted body weight with an error of 19.7% vs. 5.7% for the revised model. Based on these observations, it was concluded that representing mammary synthetic

Runx2 contributes to the regenerative potential of the mammary epithelium.

PubMed

Ferrari, Nicola; Riggio, Alessandra I; Mason, Susan; McDonald, Laura; King, Ayala; Higgins, Theresa; Rosewell, Ian; Neil, James C; Smalley, Matthew J; Sansom, Owen J; Morris, Joanna; Cameron, Ewan R; Blyth, Karen

2015-10-22

Although best known for its role in bone development and associated structures the transcription factor RUNX2 is expressed in a wide range of lineages, including those of the mammary gland. Previous studies have indicated that Runx2 can regulate aspects of mammary cell function and influence the properties of cancer cells. In this study we investigate the role of Runx2 in the mammary stem/progenitor population and its relationship with WNT signalling. Results show that RUNX2 protein is differentially expressed throughout embryonic and adult development of the murine mammary gland with high levels of expression in mammary stem-cell enriched cultures. Importantly, functional analysis reveals a role for Runx2 in mammary stem/progenitor cell function in in vitro and in vivo regenerative assays. Furthermore, RUNX2 appears to be associated with WNT signalling in the mammary epithelium and is specifically upregulated in mouse models of WNT-driven breast cancer. Overall our studies reveal a novel function for Runx2 in regulating mammary epithelial cell regenerative potential, possibly acting as a downstream target of WNT signalling.

Runx2 contributes to the regenerative potential of the mammary epithelium

PubMed Central

Ferrari, Nicola; Riggio, Alessandra I.; Mason, Susan; McDonald, Laura; King, Ayala; Higgins, Theresa; Rosewell, Ian; Neil, James C.; Smalley, Matthew J.; Sansom, Owen J.; Morris, Joanna; Cameron, Ewan R.; Blyth, Karen

2015-01-01

Although best known for its role in bone development and associated structures the transcription factor RUNX2 is expressed in a wide range of lineages, including those of the mammary gland. Previous studies have indicated that Runx2 can regulate aspects of mammary cell function and influence the properties of cancer cells. In this study we investigate the role of Runx2 in the mammary stem/progenitor population and its relationship with WNT signalling. Results show that RUNX2 protein is differentially expressed throughout embryonic and adult development of the murine mammary gland with high levels of expression in mammary stem-cell enriched cultures. Importantly, functional analysis reveals a role for Runx2 in mammary stem/progenitor cell function in in vitro and in vivo regenerative assays. Furthermore, RUNX2 appears to be associated with WNT signalling in the mammary epithelium and is specifically upregulated in mouse models of WNT-driven breast cancer. Overall our studies reveal a novel function for Runx2 in regulating mammary epithelial cell regenerative potential, possibly acting as a downstream target of WNT signalling. PMID:26489514

[Mammary gland tumor induction in rats by N-nitroso-N-methylurea and N-methyl-N1-nitro-N-nitrosoguanidine].

PubMed

Eliseev, V V; Vlasov, N N

1980-01-01

Cancer of the mammary gland was induced in female non-inbred rats under the local effect of N-nitroso-N-methylurea (NMU) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG). During 10 weeks 2.5 mg of the substance in 0.2 ml of saline was injected in the region of the third mammary gland once a week. Under NMU exposure a primary tumor arose 3 months following the initiation of the experiment, the average latent period being 5.8 months, the incidence rate--76.7%. All tumors of this series were adenocarcinomas, in 5 cases there were noted sites of fibroadenomatosis with malignification along the tumor node margins. MNNG produced a primary tumor at the 7th month of the experiment, an average latent period--8.3 months, the incidence rate--56.7%. Tumors were mostly adenocarcinomas.

Canine mammary cancer cells direct macrophages toward an intermediate activation state between M1/M2.

PubMed

Beirão, Breno C B; Raposo, Teresa; Pang, Lisa Y; Argyle, David J

2015-07-15

Canine mammary carcinoma is the most common cancer in female dogs and is often fatal due to the development of distance metastasis. The microenvironment of a tumour often contains abundant infiltrates of macrophages called tumour-associated macrophages (TAMs). TAMs express an activated phenotype, termed M2, which sustains proliferation of cancer cells, and has been correlated with poor clinical outcomes in human cancer patients. Cancer cells themselves have been implicated in stimulating the conversion of macrophages to a TAM with an M2 phenotype. This process has yet to be fully elucidated. Here we investigate the interplay between cancer cells and macrophages in the context of canine mammary carcinoma. We show that cancer cells inhibit lipopolysaccharide (LPS)-induced macrophage activation. Further, we show that macrophage associated proteins, colony-stimulating factor (CSF)-1 and C-C motif ligand (CCL)-2, stimulate macrophages and are responsible for the effects of cancer cells on macrophages. We suggest the existence of a feedback loop between macrophages and cancer cells; while cancer cells influence the phenotype of the TAMs through CSF-1 and CCL2, the macrophages induce canine mammary cancer cells to upregulate their own expression of the receptors for CSF-1 and CCL2 and increase the cancer cellular metabolic activity. However, these cytokines in isolation induce a phenotypic state in macrophages that is between M1 and M2 phenotypes. Overall, our results demonstrate the extent to which canine mammary carcinoma cells influence the macrophage phenotype and the relevance of a feedback loop between these cells, involving CSF-1 and CCL2 as important mediators.

Sequencing the transcriptome of milk production: milk trumps mammary tissue

PubMed Central

2013-01-01

Background Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells. Results We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat, whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from milk-producing mammary epithelial cells. Conclusions RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation. PMID:24330573

Sequencing the transcriptome of milk production: milk trumps mammary tissue.

PubMed

Lemay, Danielle G; Hovey, Russell C; Hartono, Stella R; Hinde, Katie; Smilowitz, Jennifer T; Ventimiglia, Frank; Schmidt, Kimberli A; Lee, Joyce W S; Islas-Trejo, Alma; Silva, Pedro Ivo; Korf, Ian; Medrano, Juan F; Barry, Peter A; German, J Bruce

2013-12-12

Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells. We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat, whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from milk-producing mammary epithelial cells. RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation.

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

PubMed Central

Tenan, Mirna; Ferrari, Paolo; Sappino, André‐Pascal

2016-01-01

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. PMID:27541736

Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System

PubMed Central

Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

2013-01-01

Purpose: Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1) on bovine mammary epithelial cells in monolayer and three-dimensional cultures. Methods: Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 µL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. Results: Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. Conclusion: According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells. PMID:24312827

CXCR4 expression in feline mammary carcinoma cells: evidence of a proliferative role for the SDF-1/CXCR4 axis

PubMed Central

2012-01-01

Background Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation. Results A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4

Ethanolic and aqueous extracts derived from Australian fungi inhibit cancer cell growth in vitro.

PubMed

Beattie, Karren D; Ulrich, Rahel; Grice, I Darren; Uddin, Shaikh J; Blake, Tony B; Wood, Kyle A; Steele, Jules; Iu, Fontaine; May, Tom W; Tiralongo, Evelin

2011-01-01

Fifteen Australian macrofungi were investigated for cytotoxic activity. Ethanol, cold and hot water extracts of each species were screened for cytotoxic activity against normal mouse fibroblast cells (NIH/3T3), healthy human epithelial kidney cells (HEK-293), four cancer cell lines, gastric adenocarcinoma cells (AGS), two mammary gland adenocarcinoma cells (MDA-MB-231, MCF7) and colorectal adenocarcinoma cells (HT-29) with a validated MTT assay. Most extracts derived from Omphalotus nidiformis, Cordyceps cranstounii and Cordyceps gunnii demonstrated significant cytotoxic activity toward a variety of cancer cell lines. In contrast only some extracts from Coprinus comatus, Cordyceps hawkesii, Hypholoma fasciculare, Lepista nuda, Leratiomyces ceres and Ophiocordyceps robertsii displayed significant cytotoxic activity, which was usually selective for only one or two cancer cell lines tested. The least cytotoxic species evaluated in this study were Agaricus bitorquis, Coprinopsis atrametaria, Psathyrella asperospora, Russula clelandii, Tricholoma sp. AU2 and Xerula mundroola.

The Type 7 Serotonin Receptor, 5-HT7, Is Essential in the Mammary Gland for Regulation of Mammary Epithelial Structure and Function

PubMed Central

Pai, Vaibhav P.; Hernandez, Laura L.; Stull, Malinda A.; Horseman, Nelson D.

2015-01-01

Autocrine-paracrine activity of serotonin (5-hydroxytryptamine, 5-HT) is a crucial homeostatic parameter in mammary gland development during lactation and involution. Published studies suggested that the 5-HT7 receptor type was important for mediating several effects of 5-HT in the mammary epithelium. Here, using 5-HT7 receptor-null (HT7KO) mice we attempt to understand the role of this receptor in mediating 5-HT actions within the mammary gland. We demonstrate for the first time that HT7KO dams are inefficient at sustaining their pups. Histologically, the HT7KO mammary epithelium shows a significant deviation from the normal secretory epithelium in morphological architecture, reduced secretory vesicles, and numerous multinucleated epithelial cells with atypically displaced nuclei, during lactation. Mammary epithelial cells in HT7KO dams also display an inability to transition from lactation to involution as normally seen by transition from a columnar to a squamous cell configuration, along with alveolar cell apoptosis and cell shedding. Our results show that 5-HT7 is required for multiple actions of 5-HT in the mammary glands including core functions that contribute to changes in cell shape and cell turnover, as well as specialized secretory functions. Understanding these actions may provide new interventions to improve lactation performance and treat diseases such as mastitis and breast cancer. PMID:25664318

The prognostic value of natural killer cell infiltration in resected pulmonary adenocarcinoma.

PubMed

Takanami, I; Takeuchi, K; Giga, M

2001-06-01

Natural cytotoxicity caused by mediated natural killer cells is believed to play an important role in host-cancer defense mechanisms. Immunohistochemically, we have detected natural killer cells in tissue specimens from patients with pulmonary adenocarcinoma and have assessed their clinical characteristics. Using the monoclonal antibody for CD57 specific marker for natural killer cells, we quantified natural killer cell infiltration in 150 patients with pulmonary adenocarcinoma who underwent curative tumor resection to investigate the relationship between natural killer cell counts and clinicopathologic factors and prognosis. The natural killer cell count was significantly related to the regulation of tumor progression, involving T classification, N classification, and stage (P =.01 for T classification or stage; P =.02 for N classification). A significant difference in the rate of patient survival was detected between those patients whose tumors had either high or low natural killer cell counts in both the overall and stage I groups (P =.0002 for the overall group; P =.049 for the stage I group). These data indicate that natural killer infiltration may contribute to the regulation of tumor progression and that the natural killer cell count can serve as a useful prognostic marker in overall and stage I pulmonary adenocarcinoma.

Active Plasma Kallikrein Localizes to Mast Cells and Regulates Epithelial Cell Apoptosis, Adipocyte Differentiation, and Stromal Remodeling during Mammary Gland Involution*

PubMed Central

Lilla, Jennifer N.; Joshi, Ravi V.; Craik, Charles S.; Werb, Zena

2009-01-01

The plasminogen cascade of serine proteases directs both development and tumorigenesis in the mammary gland. Plasminogen can be activated to plasmin by urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasma kallikrein (PKal). The dominant plasminogen activator for mammary involution is PKal, a serine protease that participates in the contact activation system of blood coagulation. We observed that the prekallikrein gene (Klkb1) is expressed highly in the mammary gland during stromal remodeling periods including puberty and postlactational involution. We used a variant of ecotin (ecotin-PKal), a macromolecular inhibitor of serine proteases engineered to be highly specific for active PKal, to demonstrate that inhibition of PKal with ecotin-PKal delays alveolar apoptosis, adipocyte replenishment, and stromal remodeling in the involuting mammary gland, producing a phenotype resembling that resulting from plasminogen deficiency. Using biotinylated ecotin-PKal, we localized active PKal to connective tissue-type mast cells in the mammary gland. Taken together, these results implicate PKal as an effector of the plasminogen cascade during mammary development. PMID:19297327

Over-expression of angiotensin II type 2 receptor gene induces cell death in lung adenocarcinoma cells.

PubMed

Pickel, Lara; Matsuzuka, Takaya; Doi, Chiyo; Ayuzawa, Rie; Maurya, Dharmendra Kumar; Xie, Sheng-Xue; Berkland, Cory; Tamura, Masaaki

2010-02-01

The endogenous angiotensin II (Ang II) type 2 receptor (AT 2) has been shown to mediate apoptosis in cardiovascular tissues. Thus, the aim of this study was to explore the anti-cancer effect of AT 2 over-expression on lung adenocarcinoma cells in vitro using adenoviral (Ad), FuGENE, and nanoparticle vectors. All three gene transfection methods efficiently transfected AT 2 cDNA into lung cancer cells but caused minimal gene transfection in normal lung epithelial cells. Ad-AT 2 significantly attenuated multiple human lung cancer cell growth (A549 and H358) as compared to the control viral vector, Ad-LacZ, when cell viability was examined by direct cell count. Examination of annexin V by flow cytometry revealed the activation of the apoptotic pathway via AT 2 over-expression. Western Blot analysis confirmed the activation of caspase-3. Similarly, poly (lactide-co-glycolic acid) (PLGA) biodegradable nanoparticles encapsulated AT 2 plasmid DNA were shown to be effectively taken up into the lung cancer cell. Nanoparticle-based AT 2 gene transfection markedly increased AT 2 expression and resultant cell death in A549 cells. These results indicate that AT 2 over-expression effectively attenuates growth of lung adenocarcinoma cells through intrinsic apoptosis. Our results also suggest that PLGA nanoparticles can be used as an efficient gene delivery vector for lung adenocarcinoma targeted therapy.

Cathelicidin production and release by mammary epithelial cells during infectious mastitis.

PubMed

Cubeddu, Tiziana; Cacciotto, Carla; Pisanu, Salvatore; Tedde, Vittorio; Alberti, Alberto; Pittau, Marco; Dore, Simone; Cannas, Agnese; Uzzau, Sergio; Rocca, Stefano; Addis, Maria Filippa

2017-07-01

Cathelicidins are well-characterized antimicrobial peptides (AMPs) that are present in significant amounts in mastitic milk. Neutrophils are believed to be the main producers of these AMPs, while the role of mammary epithelial cells (MECs) in their production and release is still unclear. In this work, cathelicidin production patterns were investigated in mammary tissues of ewes infected by Staphylococcus aureus, Streptococcus uberis, or Mycoplasma agalactiae, with a combined approach including immunohistochemistry, immune-colocalization, and fluorescent in situ hybridization. Our results confirm that MECs produce and release cathelicidins in response to different mastitis pathogens. As opposed to neutrophils, however, MECs do not seem to store the preformed protein precursor in their cytoplasm, but appear to synthesize and release it only upon exposure to the microorganisms. Cathelicidin production by MECs appears to occur before leukocyte influx in the milk, suggesting a role for these cells in the initial response of the mammary epithelium to microbial infection. Once in the milk, infiltrating neutrophils release massive amounts of cathelicidin by degranulation and production of neutrophil extracellular traps, acting as the main contributor for cathelicidin abundance in mastitic milk. Taken together, our results support the active contribution of MECs to cathelicidin production and release, and reinforce the value of cathelicidins as sensitive and pathogen-independent mastitis markers. Copyright © 2017 Elsevier B.V. All rights reserved.

Mucinous colorectal adenocarcinoma with signet-ring cells: immunohistochemical and ultrastructural study.

PubMed

Seretis, E; Konstantinidou, A; Arnogiannakis, N; Xinopoulos, D; Voloudakis-Baltatzis, I E

2010-12-01

A primary mucinous colorectal adenocarcinoma tissue with signet-ring cells, as revealed after histological evaluation, was examined ultrastructurally. The authors also analyzed the immunohistochemical data of the tissue for serotonin, vasoactive intestinal polypeptide (VIP), bombesin, somatostatin, and glucagon, using the peroxidase anti-peroxidase (PAP) method and the immunogold labeling method for light and electron microscope, respectively. Electron microscopically mucinous adenocarcinoma was characterized by the formation of small lumen. Adenocarcinoma cells were full of mucous granules of varying electron density, providing a good environment for the tumor cells to grow. They also exhibited a significant loss of microvilli and intracytoplasmic junctions, which could allow the cells to disseminate. Signet-ring cells were located in the basal site of the ducts or in the lamina propria and appeared neoplastic, with mucin accumulation intracellularly and an eccentric crescent-shaped nucleus. The cytoplasmic organelles were decreased and at the periphery of the cell. The PAP method demonstrated that these cells were strongly positive for bombesin and also positive for vasointestinal polypeptide (VIP). The immunogold method detected bombesin immunoreactivity in the vacuoles as well as in other cytoplasmic membranes, whereas VIP was localized mainly in the plasma membrane. The location of signet-ring cells combined with the immunoreactivity for bombesin and VIP indicated that signet-ring cells were of neuroendocrine origin and probably dedifferentiated enterochromaffin-like endocrine cells. These findings have implications for understanding the biological behavior of these composite malignant tumors and could help in the knowledge of the origin of signet-ring cells.

Luminal Progenitors Restrict Their Lineage Potential during Mammary Gland Development

PubMed Central

Rodilla, Veronica; Dasti, Alessandro; Huyghe, Mathilde; Lafkas, Daniel; Laurent, Cécile; Reyal, Fabien; Fre, Silvia

2015-01-01

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes. PMID:25688859

Luminal progenitors restrict their lineage potential during mammary gland development.

PubMed

Rodilla, Veronica; Dasti, Alessandro; Huyghe, Mathilde; Lafkas, Daniel; Laurent, Cécile; Reyal, Fabien; Fre, Silvia

2015-02-01

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes.

The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle.

DTIC Science & Technology

1996-08-01

J-4030 TITLE: The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle PRINCIPAL...The In Vivo DNA Binding Properties of 5. FUNDING NUMBERS Wild-Type and Mutant p53 Proteins in Mammary Cell Lines DAMD17-94-J-4030 During the Course of...ABSTRACT (Maximum 200 Using a pair of murine cell lines, one lacking p53 and a derivative cell line containing temperature sensitive p53 val 135

Feline mammary carcinoma stem cells are tumorigenic, radioresistant, chemoresistant and defective in activation of the ATM/p53 DNA damage pathway

PubMed Central

Pang, L.Y.; Blacking, T.M.; Else, R.W.; Sherman, A.; Sang, H.M.; Whitelaw, B.A.; Hupp, T.R.; Argyle, D.J.

2013-01-01

Cancer stem cells were identified in a feline mammary carcinoma cell line by demonstrating expression of CD133 and utilising the tumour sphere assay. A population of cells was identified that had an invasive, mesenchymal phenotype, expressed markers of pluripotency and enhanced tumour formation in the NOD-SCID mouse and chick embryo models. This population of feline mammary carcinoma stem cells was resistant to chemotherapy and radiation, possibly due to aberrant activation of the ATM/p53 DNA damage pathway. Epithelial–mesenchymal transition was a feature of the invasive phenotype. These data demonstrate that cancer stem cells are a feature of mammary cancer in cats. PMID:23219486

Lineage-Restricted Mammary Stem Cells Sustain the Development, Homeostasis, and Regeneration of the Estrogen Receptor Positive Lineage.

PubMed

Van Keymeulen, Alexandra; Fioramonti, Marco; Centonze, Alessia; Bouvencourt, Gaëlle; Achouri, Younes; Blanpain, Cédric

2017-08-15

The mammary gland (MG) is composed of different cell lineages, including the basal and the luminal cells (LCs) that are maintained by distinct stem cell (SC) populations. LCs can be subdivided into estrogen receptor (ER) + and ER - cells. LCs act as the cancer cell of origin in different types of mammary tumors. It remains unclear whether the heterogeneity found in luminal-derived mammary tumors arises from a pre-existing heterogeneity within LCs. To investigate LC heterogeneity, we used lineage tracing to assess whether the ER + lineage is maintained by multipotent SCs or by lineage-restricted SCs. To this end, we generated doxycycline-inducible ER-rtTA mice that allowed us to perform genetic lineage tracing of ER + LCs and study their fate and long-term maintenance. Our results show that ER + cells are maintained by lineage-restricted SCs that exclusively contribute to the expansion of the ER + lineage during puberty and their maintenance during adult life. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Long-term primary culture of mouse mammary tumor cells: production of virus.

PubMed

Young, L J; Cardiff, R D; Ashley, R L

1975-05-01

Long-term primary cultures of mouse mammary tumor cells proved an excellent source of mouse mammary tumor virus (MMTV). Virus purified from these primary cultures had the same morphologic biochemical, immunologic, and biologic characteristics as MMTV. Quantitation of MMTV-protein equivalents released into the medium was measured by the radioimmunoassay for MMTV. Peak production levels were 20-40 mug MMTV protien equivalents/75-cm-2 flask/24 hours. These cultures produced MMTV for as long as 90 days. MMTV cultivation depended on the initial cell-plating density and hormones. Maximal MMTV release was obtained at a plating density of 1 times 10-6 cells/cm-2 in the presence of insulin and hydrocortisone. Insulin alone gave basal levels of MMTV, and hydrocortisone alone increased MMTV release only three-fold, but insulin and hydrocortisone together effected an eightfold increase in MMTV release. This suggested that hydrocortisone had a primary effect on MMTV release and insulin acted synergistically with hydrocortisone to maximize MMTV release.

Evaluation of STAT5A Gene Expression in Aflatoxin B1 Treated Bovine Mammary Epithelial Cells

PubMed Central

Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

2013-01-01

Purpose: Aflatoxin B1 (AFB1) is a potent mycotoxin which has been produced by fungi such as Aspergillus flavus and Aspergillus parasiticus as secondary metabolites due to their growth on food stuffs and induces hepatocellular carcinoma in many animal species, including humans. In the present study, the effect of AFB1 on STAT5A gene expression was investigated in bovine mammary epithelial cells using real time RT-PCR. Methods: Bovine mammary epithelial cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, cells were treated with AFB1 and incubated for 8 h. For real time PCR reaction, total RNA from the cultured and treated cells was extracted and used for complementary DNA synthesis. Results: The expression of STAT5A gene was significantly down regulated by AFB1 in dose- dependent manner and led to the reduction of proliferation and differentiation of epithelial cells, which has direct effect in milk protein quantity and quality. Conclusion: According to the results, it seems that down regulation of STAT5A gene in AFB1-treated cells maybe due to DNA damage induced by AFB1 in bovine mammary epithelial cells. PMID:24312879

Establishment of primary mixed cell cultures from spontaneous canine mammary tumors: Characterization of classic and new cancer-associated molecules

PubMed Central

Gentile, Luciana B.; Nagamine, Marcia K.; Biondi, Luiz R.; Sanches, Daniel S.; Toyota, Fábio; Giovani, Tatiane M.; de Jesus, Isis P.; da Fonseca, Ivone I. M.; Queiroz-Hazarbassanov, Nicolle; Diaz, Bruno L.; Salles Gomes, Cristina de O. Massoco

2017-01-01

There are many factors which make canine cancer like cancer in humans. The occurrence of spontaneous mammary tumors in pet dogs, tumor genetics, molecular targets and exposure to the same environmental risk factors are among these factors. Therefore, the study of canine cancer can provide useful information to the oncology field. This study aimed to establish and characterize a panel of primary mixed cell cultures obtained from spontaneous canine mammary tumors. Eight established cell cultures obtained from one normal mammary gland, one complex adenoma, one mixed adenoma, two complex carcinomas and two mixed carcinomas were analyzed. The gene expression levels of classic molecular cancer players such as fibroblast growth factor receptor (FGFR) 2, breast cancer (BRCA) 1, BRCA2 and estrogen receptor (ESR) 1 were evaluated. For the first time, three orphan nuclear receptors, estrogen-related receptors (ERRs) α, β and γ were studied in canine mammary cancer. The highest expression level of ERRα was observed in complex carcinoma-derived cell culture, while the highest levels of ERRβ and γ were observed in cells derived from a mixed carcinoma. Meanwhile, complex carcinomas presented the highest levels of expression of ESR1, BRCA1 and FGFR2 among all samples. BRCA2 was found exclusively in complex adenoma. The transcription factor GATA3 had its highest levels in mixed carcinoma samples and its lowest levels in complex adenoma. Proliferation assays were also performed to evaluate the mixed cell cultures response to ER ligands, genistein and DES, both in normoxia and hypoxic conditions. Our results demonstrate that morphological and functional studies of primary mixed cell cultures derived from spontaneous canine mammary tumors are possible and provide valuable tool for the study of various stages of mammary cancer development. PMID:28945747

PD-1 and PD-L1 Up-regulation Promotes T-cell Apoptosis in Gastric Adenocarcinoma.

PubMed

Chiu, Ying-Ming; Tsai, Chung-Lin; Kao, Jung-Ta; Hsieh, Chin-Tung; Shieh, Dong-Chen; Lee, Yi-Ju; Tsay, Gregory J; Cheng, Ken-Sheng; Wu, Yi-Ying

2018-04-01

The programmed death 1 (PD-1) receptor and its ligand (PD-L1) play pivotal roles in regulating host immune responses. However, the inhibitory effects of this pathway on the function of tumor infiltrating T lymphocytes in gastric adenocarcinoma patients are not well-defined. We characterized the expression of PD-1 and PD-L1 in peripheral blood and tumor infiltrating cells and analyzed the association between PD-1/PD-L1 expression and disease progression in a cohort of 60 patients with Helicobacter pylori infection, including 18 with gastric adenocarcinoma, 23 with gastritis, and 19 asymptomatic controls. Relative to controls, the expression of PD-1 on peripheral blood and tumor infiltrating T cells increased with disease progression. In vitro, T cells induced PD-L1 expression on primary gastric adenocarcinoma epithelial cells in an IFN-γ-dependent manner, which in turn promoted T cells apoptosis. Blocking of PD-L1 reversed this effect. This study provides evidence for a new therapeutic target in gastric adenocarcinoma patients. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Mammary cell-activating factor regulates the hormone-independent transcription of the early lactation protein (ELP) gene in a marsupial.

PubMed

Pharo, Elizabeth A; Renfree, Marilyn B; Cane, Kylie N

2016-11-15

The regulation of the tammar wallaby (Macropus eugenii) early lactation protein (ELP) gene is complex. ELP is responsive to the lactogenic hormones; insulin (I), hydrocortisone (HC) and prolactin (PRL) in mammary gland explants but could not be induced with lactogenic hormones in tammar primary mammary gland cells, nor in KIM-2 conditionally immortalised murine mammary epithelial cells. Similarly, ELP promoter constructs transiently-transfected into human embryonic kidney (HEK293T) cells constitutively expressing the prolactin receptor (PRLR) and Signal Transducer and Activator of Transcription (STAT)5A were unresponsive to prolactin, unlike the rat and mouse β-casein (CSN2) promoter constructs. Identification of the minimal promoter required for the hormone-independent transcription of tammar ELP in HEK293Ts and comparative analysis of the proximal promoters of marsupial ELP and the orthologous eutherian colostrum trypsin inhibitor (CTI) gene suggests that mammary cell-activating factor (MAF), an E26 transformation-specific (ETS) factor, may bind to an AGGAAG motif and activate tammar ELP. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Transforming growth factor-beta regulates mammary carcinoma cell survival and interaction with the adjacent microenvironment.

PubMed

Bierie, Brian; Stover, Daniel G; Abel, Ty W; Chytil, Anna; Gorska, Agnieszka E; Aakre, Mary; Forrester, Elizabeth; Yang, Li; Wagner, Kay-Uwe; Moses, Harold L

2008-03-15

Transforming growth factor (TGF)-beta signaling has been associated with early tumor suppression and late tumor progression; however, many of the mechanisms that mediate these processes are not known. Using Cre/LoxP technology, with the whey acidic protein promoter driving transgenic expression of Cre recombinase (WAP-Cre), we have now ablated the type II TGF-beta receptor (T beta RII) expression specifically within mouse mammary alveolar progenitors. Transgenic expression of the polyoma virus middle T antigen, under control of the mouse mammary tumor virus enhancer/promoter, was used to produce mammary tumors in the absence or presence of Cre (T beta RII((fl/fl);PY) and T beta RII((fl/fl);PY;WC), respectively). The loss of TGF-beta signaling significantly decreased tumor latency and increased the rate of pulmonary metastasis. The loss of TGF-beta signaling was significantly correlated with increased tumor size and enhanced carcinoma cell survival. In addition, we observed significant differences in stromal fibrovascular abundance and composition accompanied by increased recruitment of F4/80(+) cell populations in T beta RII((fl/fl);PY;WC) mice when compared with T beta RII((fl/fl);PY) controls. The recruitment of F4/80(+) cells correlated with increased expression of known inflammatory genes including Cxcl1, Cxcl5, and Ptgs2 (cyclooxygenase-2). Notably, we also identified an enriched K5(+) dNp63(+) cell population in primary T beta RII((fl/fl);PY;WC) tumors and corresponding pulmonary metastases, suggesting that loss of TGF-beta signaling in this subset of carcinoma cells can contribute to metastasis. Together, our current results indicate that loss of TGF-beta signaling in mammary alveolar progenitors may affect tumor initiation, progression, and metastasis through regulation of both intrinsic cell signaling and adjacent stromal-epithelial interactions in vivo.

The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells.

PubMed

Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

2015-01-01

The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival.

Integrin β4 Signaling Promotes Mammary Tumor Cell Adhesion to Brain Microvascular Endothelium by Inducing ErbB2-mediated Secretion of VEGF

PubMed Central

Fan, Jie; Cai, Bin; Zeng, Min; Hao, Yanyan

2015-01-01

Prior studies have indicated that the β4 integrin promotes mammary tumor invasion and metastasis by combining with ErbB2 and amplifying its signaling capacity. However, the effector pathways and cellular functions by which the β4 integrin exerts these effects are incompletely understood. To examine if β4 signaling plays a role during mammary tumor cell adhesion to microvascular endothelium, we have examined ErbB2-transformed mammary tumor cells expressing either a wild-type (WT) or a signaling-defective form of β4 (1355T). We report that WT cells adhere to brain microvascular endothelium in vitro to a significantly larger extent as compared to 1355T cells. Interestingly, integrin β4 signaling does not exert a direct effect on adhesion to the endothelium or the underlying basement membrane. Rather, it enhances ErbB2-dependent expression of VEGF by tumor cells. VEGF in turn disrupts the tight and adherens junctions of endothelial monolayers, enabling the exposure of underlying basement membrane and increasing the adhesion of tumor cells to the intercellular junctions of endothelium. Inhibition of ErbB2 on tumor cells or the VEGFR-2 on endothelial cells suppresses mammary tumor cell adhesion to microvascular endothelium. Our results indicate that β4 signaling regulates VEGF expression by the mammary tumor cells thereby enhancing their adhesion to microvascular endothelium. PMID:21556948

Hormesis effect of trace metals on cultured normal and immortal human mammary cells.

PubMed

Schmidt, Craig M; Cheng, Chun N; Marino, Angelo; Konsoula, Roula; Barile, Frank A

2004-06-01

An in vitro study was conducted to determine the effects of variable concentrations of trace metals on human cultured mammary cells. Monolayers of human mortal (MCF-12A) and immortal (MDA-MB231) mammary epithelial cells were incubated in the absence or presence of increasing concentrations of arsenic (As), mercury (Hg) and copper (Cu) for 24-h, 72-h, 4-d, and 7-d. The MTT assay was used to assess viability for all time periods and cell proliferation was monitored for 4-d and 7-d studies. Monolayers were also labeled with rhodamine-110 (R-6501), Sytox green, and Celltiter blue fluorescent dyes as indicators for intracellular esterase activity, nucleic acid staining, and cell reduction/viability, respectively. Total incubation time with chemical plus dyes was 24 h. For 24-h and 72-h studies, cells were seeded in 96-well plates, after which confluent monolayers were exposed to increasing concentrations of chemicals. For 4-d and 7-d studies, cells were seeded in 12-well plates at 1/3 confluent density (day 0) and exposed to increasing concentrations of metals on day 1. All cells were counted on days 4 and 7. In addition, test medium was removed from select groups of cultures on day 4, replaced with fresh medium in the absence of chemical (recovery studies), and assays were performed on day 7 as above. The data suggest that there is a consistent protective and/or stimulating effect of metals at the lowest concentrations in MCF-12A cells that is not observed in immortal MDA-MB231 cells. In fact, cell viability of MCF-12A cells is stimulated by otherwise equivalent inhibitory concentrations of As, Cu, and Hg on MDA-MB231 cells at 24-h. Whereas As and Hg suppress proliferation and viability in both cell lines after 4-d and 7-d of exposure, Cu enhances cell proliferation and viability of MCF-12A cells. MDA-MB231, however, recover better after 4-days of toxic insult. In addition, nutritional manipulation of media between the cell lines, or pretreatment with penicillamine

CDB-4124, a progesterone receptor modulator, inhibits mammary carcinogenesis by suppressing cell proliferation and inducing apoptosis.

PubMed

Wiehle, Ronald; Lantvit, Daniel; Yamada, Tohru; Christov, Konstantin

2011-03-01

CDB-4124 (Proellex or telapristone acetate) is a modulator of progesterone receptor (PR) signaling, which is currently employed in preclinical studies for prevention and treatment of breast cancer and has been used in clinical studies for treatment of uterine fibroids and endometriosis. Here we provide evidence for its action on steroid hormone-signaling, cell cycle-regulated genes and in vivo on mammary carcinogenesis. When CDB-4124 is given to rats at 200 mg/kg for 24 months, it prevents the development of spontaneous mammary hyperplastic and premalignant lesions. Also, CDB-4124 given as subcutaneous pellets at two different doses suppressed, dose dependently, N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis. The high dose (30 mg, over 84 days) increased tumor latency from 66 ± 24 days to 87 ± 20 days (P < 0.02), decreased incidence from 85% to 35% (P < 0.001), and reduced multiplicity from 3.0 to 1.1 tumors/animal (P < 0.001). Tumor burden decreased from 2.6 g/animal to 0.26 g/animal (P < 0.01). CDB-4124 inhibited cell proliferation and induced apoptosis in MNU-induced mammary tumors, which correlated with a decreased proportion of PR(+) tumor cells and with decreased serum progesterone. CDB-4124 did not affect serum estradiol. In a mechanistic study employing T47D cells we found that CDB-4124 suppressed G(1)/G(0)-S transition by inhibiting CDK2 and CDK4 expressions, which correlated with inhibition of estrogen receptor (ER) expression. Taken together, these data indicate that CDB-4124 can suppress the development of precancerous lesions and carcinogen-induced ER(+) mammary tumors in rats, and may have implications for prevention and treatment of human breast cancer.

Lysine-specific demethylase 2A expression is associated with cell growth and cyclin D1 expression in colorectal adenocarcinoma.

PubMed

Cao, Lin-Lin; Du, Changzheng; Liu, Hangqi; Pei, Lin; Qin, Li; Jia, Mei; Wang, Hui

2018-04-01

Lysine-specific demethylase 2A (KDM2A), a specific H3K36me1/2 demethylase, has been reported to be closely associated with several types of cancer. In this study, we aimed to investigate the expression and function of KDM2A in colorectal adenocarcinoma. A total of 215 colorectal adenocarcinoma specimens were collected, and then subjected to immunohistochemistry assay to evaluate the expression levels of KDM2A, cyclin D1 and other proteins in colorectal adenocarcinoma tissues. Real-time polymerase chain reaction, Western blot, and other molecular biology methods were used to explore the role of KDM2A in colorectal adenocarcinoma cells. In this study, we report that the expression level of KDM2A is high in colorectal adenocarcinoma tissues, and this high expression promotes the proliferation and colony formation of colorectal adenocarcinoma cells, as demonstrated by KDM2A knockdown experiments. In addition, the expression of KDM2A is closely associated with cyclin D1 expression in colorectal adenocarcinoma tissues and cell lines. Our study reveals a novel role for high-expressed KDM2A in colorectal adenocarcinoma cell growth, and that the expression of KDM2A is associated with that of cyclin D1 in colorectal adenocarcinoma.

Staphylococcus aureus Phenol-Soluble Modulins Impair Interleukin Expression in Bovine Mammary Epithelial Cells

PubMed Central

Deplanche, Martine; Alekseeva, Ludmila; Semenovskaya, Ksenia; Fu, Chih-Lung; Dessauge, Frederic; Finot, Laurence; Petzl, Wolfram; Zerbe, Holm; Le Loir, Yves; Rainard, Pascal; Smith, David G. E.; Germon, Pierre; Otto, Michael

2016-01-01

The role of the recently described interleukin-32 (IL-32) in Staphylococcus aureus-induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6, and IL-8 in S. aureus- and Escherichia coli-infected bovine mammary gland epithelial cells. Using live bacteria, we found that in S. aureus-infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli-infected cells. Notably, IL-32 expression was decreased in S. aureus-infected cells, while it was increased in E. coli-infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6, and IL-8 expression by epithelial cells exposed to psm mutant strains was significantly increased compared to that in cells exposed to the isogenic S. aureus wild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S. aureus-related mastitis. PMID:27001539

Human Wharton's jelly stem cell conditioned medium and cell-free lysate inhibit human osteosarcoma and mammary carcinoma cell growth in vitro and in xenograft mice.

PubMed

Gauthaman, Kalamegam; Fong, Chui-Yee; Arularasu, Suganya; Subramanian, Arjunan; Biswas, Arijit; Choolani, Mahesh; Bongso, Ariff

2013-02-01

Human Wharton's jelly stem cells (hWJSCs) were shown to inhibit the growth of human mammary carcinomas. It is not known whether cell-free secretions or lysates of hWJSCs do the same on different cancers. They may be less controversial than cells to regulatory bodies for clinical application. We examined the influence of hWJSC conditioned medium (hWJSC-CM) and cell-free lysate (hWJSC-CL) on two osteosarcoma cell lines (MG-63, SKES-1) in vitro and on human mammary carcinomas in immunodeficient mice. When exposed to hWJSC-CL, increased vacuolations in MG-63 and increased membrane fragmentation in SKES-1 cells were observed, with greater cell death in SKES-1. Exposure of SKES-1 and MG-63 cells to hWJSC-CL showed significant decreases in cell proliferation of 46.48 ± 6.66% and 24.32 ± 5.67% respectively compared to controls. MG-63 and SKES-1 cells were annexin V-FITC positive and SKES-1 TUNEL positive following treatment with hWJSC-CM and hWJSC-CL. MG-63 cells were positive and SKES-1 cells negative for anti-BECLIN-1 and anti-LC3B following treatment with hWJSC-CM and hWJSC-CL. RT-PCR showed that the pro-apoptotic BAX gene and the autophagy-related ATG-5 and BECLIN-1 genes were up-regulated while the anti-apoptotic BCL2 and SURVIVIN genes were down-regulated in MG-63 and SKES-1 cells treated with hWJSC-CM and hWJSC-CL. Injections of hWJSCs and hWJSC-CM into mammary carcinomas in immunodeficient mice resulted in decreased tumor sizes and weights of 24.86 ± 6.05% to 37.03 ± 5.91% and 47.14 ± 7.36% to 55.09 ± 5.87% respectively at 6 weeks compared to controls. hWJSC-CM and hWJSC-CL inhibit mammary carcinoma and osteosarcoma cells via apoptosis and autophagy. Copyright © 2012 Wiley Periodicals, Inc.

Characteristics and EGFP expression of goat mammary gland epithelial cells.

PubMed

Zheng, Y-M; He, X-Y; Zhang, Y

2010-12-01

The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing. © 2010 Blackwell Verlag GmbH.

Mammary gland involution is associated with rapid down regulation of major mammary Ca**2+-ATPases

USDA-ARS?s Scientific Manuscript database

Sixty percent of calcium in milk is transported across the mammary cells apical membrane by the plasma membrane Ca**2+-ATPase 2 (PMCA2). The effect of abrupt cessation of milk production on the Ca**2+-ATPases and mammary calcium transport is unknown. We found that 24 hours after stopping milk prod...

Expression Pattern of the Pro-apoptotic Gene PAR-4 During the Morphogenesis of MCF-10A Human Mammary Epithelial Cells.

PubMed

de Bessa Garcia, Simone A; Pereira, Michelly C; Nagai, Maria A

2010-12-21

The histological organization of the mammary gland involves a spatial interaction of epithelial and myoepithelial cells with the specialized basement membrane (BM), composed of extra-cellular matrix (ECM) proteins, which is disrupted during the tumorigenic process. The interactions between mammary epithelial cells and ECM components play a major role in mammary gland branching morphogenesis. Critical signals for mammary epithelial cell proliferation, differentiation, and survival are provided by the ECM proteins. Three-dimensional (3D) cell culture was developed to establish a system that simulates several features of the breast epithelium in vivo; 3D cell culture of the spontaneously immortalized cell line, MCF10A, is a well-established model system to study breast epithelial cell biology and morphogenesis. Mammary epithelial cells grown in 3D form spheroids, acquire apicobasal polarization, and form lumens that resemble acini structures, processes that involve cell death. Using this system, we evaluated the expression of the pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) by immunofluorescence and quantitative real time PCR (qPCR). A time-dependent increase in PAR-4 mRNA expression was found during the process of MCF10A acinar morphogenesis. Confocal microscopy analysis also showed that PAR-4 protein was highly expressed in the MCF10A cells inside the acini structure. During the morphogenesis of MCF10A cells in 3D cell culture, the cells within the lumen showed caspase-3 activation, indicating apoptotic activity. PAR-4 was only partially co-expressed with activated caspase-3 on these cells. Our results provide evidence, for the first time, that PAR-4 is differentially expressed during the process of MCF10A acinar morphogenesis.

Aging is associated with an expansion of CD49fhi mammary stem cells that show a decline in function and increased transformation potential

PubMed Central

Dong, Qiaoxiang; Gao, Hui; Shi, Yuanshuo; Zhang, Fuchuang; Gu, Xiang; Wu, Anqi; Wang, Danhan; Chen, Yuanhong; Bandyopadhyay, Abhik; Yeh, I-Tien; Daniel, Benjamin J.; Chen, Yidong; Zou, Yi; Rebel, Vivienne L.; Walter, Christi A.; Lu, Jianxin; Huang, Changjiang; Sun, Lu-Zhe

2016-01-01

Breast cancer incidence increases during aging, yet the mechanism of age-associated mammary tumorigenesis is unclear. Mammary stem cells are believed to play an important role in breast tumorigenesis, but how their function changes with age is unknown. We compared mammary epithelial cells isolated from young and old mammary glands of different cohorts of C57BL6/J and BALB/c mice, and our findings revealed that old mammary glands were characterized by increased basal cell pool comprised of mostly CD49fhi cells, altered luminal-to-basal cell ratio, and irregular ductal morphology. More interestingly, basal stem cells in old mice were increased in frequency, but showed a functional decline of differentiation and increased neoplastic transformation potential. Gene signature enrichment analysis revealed a significant enrichment of a luminal cell gene expression signature in the basal stem cell-enriched population from old mice, suggesting some luminal cells were expressing basal markers. Immunofluorescence staining confirmed the presence of luminal cells with high CD49f expression in hyperplastic lesions implicating these cells as undergoing luminal to basal phenotypic changes during aging. Whole transcriptome analysis showed elevated immune and inflammatory responses in old basal stem cells and stromal cells, which may be the underlying cause for increased CD49fhi basal-like cells in aged glands. PMID:27852980

Aging is associated with an expansion of CD49fhi mammary stem cells that show a decline in function and increased transformation potential.

PubMed

Dong, Qiaoxiang; Gao, Hui; Shi, Yuanshuo; Zhang, Fuchuang; Gu, Xiang; Wu, Anqi; Wang, Danhan; Chen, Yuanhong; Bandyopadhyay, Abhik; Yeh, I-Tien; Daniel, Benjamin J; Chen, Yidong; Zou, Yi; Rebel, Vivienne L; Walter, Christi A; Lu, Jianxin; Huang, Changjiang; Sun, Lu-Zhe

2016-11-15

Breast cancer incidence increases during aging, yet the mechanism of age-associated mammary tumorigenesis is unclear. Mammary stem cells are believed to play an important role in breast tumorigenesis, but how their function changes with age is unknown. We compared mammary epithelial cells isolated from young and old mammary glands of different cohorts of C57BL6/J and BALB/c mice, and our findings revealed that old mammary glands were characterized by increased basal cell pool comprised of mostly CD49f hi cells, altered luminal-to-basal cell ratio, and irregular ductal morphology. More interestingly, basal stem cells in old mice were increased in frequency, but showed a functional decline of differentiation and increased neoplastic transformation potential. Gene signature enrichment analysis revealed a significant enrichment of a luminal cell gene expression signature in the basal stem cell-enriched population from old mice, suggesting some luminal cells were expressing basal markers. Immunofluorescence staining confirmed the presence of luminal cells with high CD49f expression in hyperplastic lesions implicating these cells as undergoing luminal to basal phenotypic changes during aging. Whole transcriptome analysis showed elevated immune and inflammatory responses in old basal stem cells and stromal cells, which may be the underlying cause for increased CD49f hi basal-like cells in aged glands.

Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

PubMed Central

Young, Christian D.; Lewis, Andrew S.; Rudolph, Michael C.; Ruehle, Marisa D.; Jackman, Matthew R.; Yun, Ui J.; Ilkun, Olesya; Pereira, Renata; Abel, E. Dale; Anderson, Steven M.

2011-01-01

Tumor cells exhibit an altered metabolism characterized by elevated aerobic glycolysis and lactate secretion which is supported by an increase in glucose transport and consumption. We hypothesized that reducing or eliminating the expression of the most prominently expressed glucose transporter(s) would decrease the amount of glucose available to breast cancer cells thereby decreasing their metabolic capacity and proliferative potential. Of the 12 GLUT family glucose transporters expressed in mice, GLUT1 was the most abundantly expressed at the RNA level in the mouse mammary tumors from MMTV-c-ErbB2 mice and cell lines examined. Reducing GLUT1 expression in mouse mammary tumor cell lines using shRNA or Cre/Lox technology reduced glucose transport, glucose consumption, lactate secretion and lipid synthesis in vitro without altering the concentration of ATP, as well as reduced growth on plastic and in soft agar. The growth of tumor cells with reduced GLUT1 expression was impaired when transplanted into the mammary fat pad of athymic nude mice in vivo. Overexpression of GLUT1 in a cell line with low levels of endogenous GLUT1 increased glucose transport in vitro and enhanced growth in nude mice in vivo as compared to the control cells with very low levels of GLUT1. These studies demonstrate that GLUT1 is the major glucose transporter in mouse mammary carcinoma models overexpressing ErbB2 or PyVMT and that modulation of the level of GLUT1 has an effect upon the growth of mouse mammary tumor cell lines in vivo. PMID:21826239

Effects of xanthosine on gene expression of mammary epithelial cells using RNA sequencing of goat milk fat globules

USDA-ARS?s Scientific Manuscript database

Although intramammary xanthosine (XS) treatment was reported to increase the mammary stem cell population and milk yield in bovine and caprine, underlying molecular mechanisms remain unclear. The goal of this study was to evaluate effects of XS treatment on the mammary transcriptome in early lactati...

Number of circulating endothelial progenitor cells and intratumoral microvessel density in non-small cell lung cancer patients: differences in angiogenic status between adenocarcinoma histologic subtypes.

PubMed

Maeda, Ryo; Ishii, Genichiro; Ito, Masami; Hishida, Tomoyuki; Yoshida, Junji; Nishimura, Mitsuyo; Haga, Hironori; Nagai, Kanji; Ochiai, Atsushi

2012-03-01

Angiogenesis plays a significant role in tumor progression. This study examined the association between the number of circulating endothelial progenitor cells (EPCs), intratumoral microvessel density (MVD) (both of which may be markers for neovascularization), and lung cancer histological types, particularly adenocarcinoma histological subtypes. A total of 83 stage I non-small cell lung cancer (NSCLC) patients underwent complete tumor resection between November 2009 and July 2010. The number of EPCs from the pulmonary artery of the resected lungs was measured by assaying CD34/vascular endothelial growth factor receptor 2 positive cells, and the MVD was assessed immunohistochemically in tumor specimens by staining for CD34. A statistically significant correlation between the number of EPCs from pulmonary artery and intratumoral MVD was found (p < 0.001). No statistically significant differences in the number of EPCs and the MVD were observed between the adenocarcinomas and the squamous cell carcinomas. Among the adenocarcinoma histological subtypes, a higher number of EPCs and MVD were found significantly more frequently in solid adenocarcinomas than in nonsolid adenocarcinomas (p < 0.001 and p = 0.011, respectively). In addition, solid adenocarcinomas showed higher levels of vascular endothelial growth factor using quantitative real-time polymerase chain reaction in the tumor tissue samples than in the nonsolid adenocarcinomas (p = 0.005). The higher number of circulating EPCs and the MVD of solid adenocarcinoma may indicate the presence of differences in the tumor angiogenic status between early-stage adenocarcinoma histological subtypes. Among adenocarcinoma patients, patients with solid adenocarcinoma may be the best candidates for antiangiogenic therapies.

Embryonic mammary signature subsets are activated in Brca1-/- and basal-like breast cancers

PubMed Central

2013-01-01

Introduction Cancer is often suggested to result from development gone awry. Links between normal embryonic development and cancer biology have been postulated, but no defined genetic basis has been established. We recently published the first transcriptomic analysis of embryonic mammary cell populations. Embryonic mammary epithelial cells are an immature progenitor cell population, lacking differentiation markers, which is reflected in their very distinct genetic profiles when compared with those of their postnatal descendents. Methods We defined an embryonic mammary epithelial signature that incorporates the most highly expressed genes from embryonic mammary epithelium when compared with the postnatal mammary epithelial cells. We looked for activation of the embryonic mammary epithelial signature in mouse mammary tumors that formed in mice in which Brca1 had been conditionally deleted from the mammary epithelium and in human breast cancers to determine whether any genetic links exist between embryonic mammary cells and breast cancers. Results Small subsets of the embryonic mammary epithelial signature were consistently activated in mouse Brca1-/- tumors and human basal-like breast cancers, which encoded predominantly transcriptional regulators, cell-cycle, and actin cytoskeleton components. Other embryonic gene subsets were found activated in non-basal-like tumor subtypes and repressed in basal-like tumors, including regulators of neuronal differentiation, transcription, and cell biosynthesis. Several embryonic genes showed significant upregulation in estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and/or grade 3 breast cancers. Among them, the transcription factor, SOX11, a progenitor cell and lineage regulator of nonmammary cell types, is found highly expressed in some Brca1-/- mammary tumors. By using RNA interference to silence SOX11 expression in breast cancer cells, we found evidence that SOX11 regulates breast cancer cell

Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging

PubMed Central

Sobolik, Tammy; Su, Ying-Jun; Ashby, Will; Schaffer, David K.; Wells, Sam; Wikswo, John P.; Zijlstra, Andries; Richmond, Ann

2016-01-01

ABSTRACT We developed mammary imaging windows (MIWs) to evaluate leukocyte infiltration and cancer cell dissemination in mouse mammary tumors imaged by confocal microscopy. Previous techniques relied on surgical resection of a skin flap to image the tumor microenvironment restricting imaging time to a few hours. Utilization of mammary imaging windows offers extension of intravital imaging of the tumor microenvironment. We have characterized strengths and identified some previously undescribed potential weaknesses of MIW techniques. Through iterative enhancements of a transdermal portal we defined conditions for improved quality and extended confocal imaging time for imaging key cell-cell interactions in the tumor microenvironment. PMID:28243517

Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging.

PubMed

Sobolik, Tammy; Su, Ying-Jun; Ashby, Will; Schaffer, David K; Wells, Sam; Wikswo, John P; Zijlstra, Andries; Richmond, Ann

2016-01-01

We developed mammary imaging windows (MIWs) to evaluate leukocyte infiltration and cancer cell dissemination in mouse mammary tumors imaged by confocal microscopy. Previous techniques relied on surgical resection of a skin flap to image the tumor microenvironment restricting imaging time to a few hours. Utilization of mammary imaging windows offers extension of intravital imaging of the tumor microenvironment. We have characterized strengths and identified some previously undescribed potential weaknesses of MIW techniques. Through iterative enhancements of a transdermal portal we defined conditions for improved quality and extended confocal imaging time for imaging key cell-cell interactions in the tumor microenvironment.

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland.

PubMed

Flint, D J; Boutinaud, M; Tonner, E; Wilde, C J; Hurley, W; Accorsi, P A; Kolb, A F; Whitelaw, C B A; Beattie, J; Allan, G J

2005-08-01

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue

Isolation of Endoplasmic Reticulum Fractions from Mammary Epithelial Tissue.

PubMed

Chanat, Eric; Le Parc, Annabelle; Lahouassa, Hichem; Badaoui, Bouabid

2016-06-01

In the mammary glands of lactating animals, the mammary epithelial cells that surround the lumen of the acini produce and secrete copious amounts of milk. Functional differentiation of these mammary epithelial cells depends on the development of high-efficiency secretory pathways, notably for protein and lipid secretion. Protein secretion is a fundamental process common to all animal cells that involves a subset of cellular organelles, including the endoplasmic reticulum and the Golgi apparatus. In contrast, en masse secretion of triglycerides and cholesterol esters in the form of milk fat globules is a unique feature of the mammary epithelial cell. Cytoplasmic lipid droplets, the intracellular precursors of milk fat globules, originate from the endoplasmic reticulum, as do most milk-specific proteins. This organelle is therefore pivotal in the biogenesis of milk components. Fractionation of the cell into its subcellular parts is an approach that has proven very powerful for understanding organelle function and for studying the specific role of an organelle in a given cell activity. Here we describe a method for the purification of both smooth and rough microsomes, the membrane-bound endoplasmic reticulum fragments that form from endoplasmic reticulum domains when cells are broken up, from mammary gland tissue at lactation.

Notch pathway activity identifies cells with cancer stem cell-like properties and correlates with worse survival in lung adenocarcinoma

PubMed Central

Hassan, Khaled A.; Wang, Luo; Korkaya, Hasan; Chen, Guoan; Maillard, Ivan; Beer, David G.; Kalemkerian, Gregory P.; Wicha, Max S.

2013-01-01

Purpose The cancer stem cell theory postulates that tumors contain a subset of cells with stem cell properties of self-renewal, differentiation and tumor-initiation. The purpose of this study is to determine the role of Notch activity in identifying lung cancer stem cells. Experimental Design We investigated the role of Notch activity in lung adenocarcinoma utilizing a Notch GFP-reporter construct and a gamma-secretase inhibitor (GSI), which inhibits Notch pathway activity. Results Transduction of lung cancer cells with Notch GFP-reporter construct identified a subset of cells with high Notch activity (GFP-bright). GFP-bright cells had the ability to form more tumor spheres in serum-free media, and were able to generate both GFP-bright and GFP-dim (lower Notch activity) cell populations. GFP-bright cells were resistant to chemotherapy and were tumorigenic in serial xenotransplantation assays. Tumor xenografts of mice treated with GSI had decreased expression of downstream effectors of Notch pathway and failed to regenerate tumors upon reimplantation in NOD/SCID mice. Using multivariate analysis, we detected a statistically significant correlation between poor clinical outcome and Notch activity (reflected in increased Notch ligand expression or decreased expression of the negative modulators), in a group of 441 lung adenocarcinoma patients. This correlation was further confirmed in an independent group of 89 adenocarcinoma patients where Hes-1 overexpression correlated with poor overall survival. Conclusions Notch activity can identify lung cancer stem cell-like population and its inhibition may be an appropriate target for treating lung adenocarcinoma. PMID:23444212

Characterization of mammary epithelial stem/progenitor cells and their changes with aging in common marmosets.

PubMed

Wu, Anqi; Dong, Qiaoxiang; Gao, Hui; Shi, Yuanshuo; Chen, Yuanhong; Zhang, Fuchuang; Bandyopadhyay, Abhik; Wang, Danhan; Gorena, Karla M; Huang, Changjiang; Tardif, Suzette; Nathanielsz, Peter W; Sun, Lu-Zhe

2016-08-25

Age is the number one risk factor for breast cancer, yet the underlying mechanisms are unexplored. Age-associated mammary stem cell (MaSC) dysfunction is thought to play an important role in breast cancer carcinogenesis. Non-human primates with their close phylogenetic relationship to humans provide a powerful model system to study the effects of aging on human MaSC. In particular, the common marmoset monkey (Callithrix jacchus) with a relatively short life span is an ideal model for aging research. In the present study, we characterized for the first time the mammary epithelial stem/progenitor cells in the common marmoset. The MaSC-enriched cells formed four major types of morphologically distinct colonies when cultured on plates pre-seeded with irradiated NIH3T3 fibroblasts, and were also capable of forming mammospheres in suspension culture and subsequent formation of 3D organoids in Matrigel culture. Most importantly, these 3D organoids were found to contain stem/progenitor cells that can undergo self-renewal and multi-lineage differentiation both in vitro and in vivo. We also observed a significant decrease of luminal-restricted progenitors with age. Our findings demonstrate that common marmoset mammary stem/progenitor cells can be isolated and quantified with established in vitro and in vivo assays used for mouse and human studies.

The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells

PubMed Central

Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

2015-01-01

The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival. PMID:25616580

Clear cell adenocarcinoma of the ovary associated with in utero diethylstilbestrol exposure: case report and clinical overview.

PubMed

Dasanu, Constantin A; Herzog, Thomas J

2009-01-01

Clear cell adenocarcinoma of the vagina and cervix were previously shown to be tumors occurring in female offspring exposed prenatally to diethylstilbestrol. This report describes the first clinical case of clear cell adenocarcinoma of the ovary linked to early diethylstilbestrol exposure in utero. A 45-year-old woman presented with a self-discovered lump in the lower abdominal quadrant. She underwent surgery and staging that revealed clear cell adenocarcinoma confined to the left ovary. Foci of high-grade squamous neoplastic proliferation, inflammation, and a paratubal cyst were also present on the pathology specimen. Medical records established unequivocally that the patient's mother received diethylstilbestrol therapy throughout the pregnancy. Our case is consistent with clear cell adenocarcinoma, probably related to diethylstilbestrol exposure in utero. It reinforces the need for continued vigilance in individuals prenatally exposed to this drug.

Tumours and dysplasias of the mammary gland

PubMed Central

Hampe, J. F.; Misdorp, W.

1974-01-01

As mammary tumours occur frequently in the dog and cat but rarely in other domestic animals, only the tumours of these two species are classified. The epithelial tumours are termed “complex” when they consist of cells resembling both secretory and myoepithelial cells: these tumours are biologically less malignant than tumours of the “simple” type in which only one of these kinds of cell is present. The carcinomas are subdivided into adenocarcinoma, solid carcinoma, spindle cell carcinoma, anaplastic carcinoma, squamous cell carcinoma, and mucinous carcinoma. The term “carcinosarcoma or malignant mixed tumour” was used only when there were cells morphologically resembling not only one or both of the epithelial components but also connective tissue cells with their products of differentiation. The benign tumours are classed as adenoma, papilloma, fibroadenoma, or benign soft tissue tumour. The dysplasias are described under the following headings: cyst, adenosis, regular typical epithelial proliferation in ducts and lobules (epitheliosis), duct ectasia, fibrosclerosis, and lobular hyperplasia. ImagesFig. 41Fig. 42Fig. 43Fig. 44Fig. 5Fig. 6Fig. 7Fig. 8Fig. 13Fig. 14Fig. 15Fig. 16Fig. 45Fig. 46Fig. 47Fig. 48Fig. 17Fig. 18Fig. 19Fig. 20Fig. 25Fig. 26Fig. 27Fig. 28Fig. 29Fig. 30Fig. 31Fig. 32Fig. 21Fig. 22Fig. 23Fig. 24Fig. 37Fig. 38Fig. 39Fig. 40Fig. 9Fig. 10Fig. 11Fig. 12Fig. 1Fig. 2Fig. 3Fig. 4Fig. 33Fig. 34Fig. 35Fig. 36 PMID:4371737

Identification of cancer initiating cells in K-Ras driven lung adenocarcinoma.

PubMed

Mainardi, Sara; Mijimolle, Nieves; Francoz, Sarah; Vicente-Dueñas, Carolina; Sánchez-García, Isidro; Barbacid, Mariano

2014-01-07

Ubiquitous expression of a resident K-Ras(G12V) oncogene in adult mice revealed that most tissues are resistant to K-Ras oncogenic signals. Indeed, K-Ras(G12V) expression only induced overt tumors in lungs. To identify these transformation-permissive cells, we induced K-Ras(G12V) expression in a very limited number of adult lung cells (0.2%) and monitored their fate by X-Gal staining, a surrogate marker coexpressed with the K-Ras(G12V) oncoprotein. Four weeks later, 30% of these cells had proliferated to form small clusters. However, only SPC(+) alveolar type II (ATII) cells were able to form hyperplastic lesions, some of which progressed to adenomas and adenocarcinomas. In contrast, induction of K-Ras(G12V) expression in lung cells by intratracheal infection with adenoviral-Cre particles generated hyperplasias in all regions except the proximal airways. Bronchiolar and bronchioalveolar duct junction hyperplasias were primarily made of CC10(+) Clara cells. Some of them progressed to form benign adenomas. However, only alveolar hyperplasias, exclusively made up of SPC(+) ATII cells, progressed to yield malignant adenocarcinomas. Adenoviral infection induced inflammatory infiltrates primarily made of T and B cells. This inflammatory response was essential for the development of K-Ras(G12V)-driven bronchiolar hyperplasias and adenomas, but not for the generation of SPC(+) ATII lesions. Finally, activation of K-Ras(G12V) during embryonic development under the control of a Sca1 promoter yielded CC10(+), but not SPC(+), hyperplasias, and adenomas. These results, taken together, illustrate that different types of lung cells can generate benign lesions in response to K-Ras oncogenic signals. However, in adult mice, only SPC(+) ATII cells were able to yield malignant adenocarcinomas.

An Organotypic 3D Assay for Primary Human Mammary Epithelial Cells that Recapitulates Branching Morphogenesis.

PubMed

Linnemann, Jelena R; Meixner, Lisa K; Miura, Haruko; Scheel, Christina H

2017-01-01

We have developed a three-dimensional organotypic culture system for primary human mammary epithelial cells (HMECs) in which the cells are cultured in free floating collagen type I gels. In this assay, luminal cells predominantly form multicellular spheres, while basal/myoepithelial cells form complex branched structures resembling terminal ductal lobular units (TDLUs), the functional units of the human mammary gland in situ. The TDLU-like organoids can be cultured for at least 3 weeks and can then be passaged multiple times. Subsequently, collagen gels can be stained with carmine or by immunofluorescence to allow for the analysis of morphology, protein expression and polarization, and to facilitate quantification of structures. In addition, structures can be isolated for gene expression analysis. In summary, this technique is suitable for studying branching morphogenesis, regeneration, and differentiation of HMECs as well as their dependence on the physical environment.

MUC4 immunohistochemistry is useful in distinguishing epithelioid mesothelioma from adenocarcinoma and squamous cell carcinoma of the lung.

PubMed

Mawas, Amany Sayed; Amatya, Vishwa Jeet; Kushitani, Kei; Kai, Yuichiro; Miyata, Yoshihiro; Okada, Morihito; Takeshima, Yukio

2018-01-09

The differential diagnosis of epithelioid mesothelioma from lung adenocarcinoma and squamous cell carcinoma requires the positive and negative immunohistochemical markers of mesothelioma. The IMIG guideline has suggested the use of Calretinin, D2-40, WT1, and CK5/6 as mesothelial markers, TTF-1, Napsin-A, Claudin 4, CEA as lung adenocarcinoma markers p40, p63, CK5/6, MOC-31 as squamous cell markers. However, use of other immunohistochemical markers is still necessary. We evaluated 65 epithelioid mesotheliomas, 60 adenocarcinomas, and 57 squamous cell carcinomas of the lung for MUC4 expression by immunohistochemistry and compared with the previously known immunohistochemical markers. MUC4 expression was not found in any of 65 cases of epithelioid mesothelioma. In contrast, MUC4 expression was observed in 50/60(83.3%) cases of lung adenocarcinoma and 50/56(89.3%) cases of lung squamous cell carcinoma. The negative MUC4 expression showed 100% sensitivity, 86.2% specificity and accuracy rate of 91.2% to differentiate epithelioid mesothelioma from lung carcinoma. The sensitivity, specificity, and accuracy of MUC4 are comparable to that of previously known markers of lung adenocarcinoma and squamous cell carcinoma, namely CEA, Claudin 4 and better than that of MOC-31. In conclusion, MUC4 immunohistochemistry is useful for differentiation of epithelioid mesothelioma from lung carcinoma, either adenocarcinoma or squamous cell carcinoma.

Staphylococcus aureus Phenol-Soluble Modulins Impair Interleukin Expression in Bovine Mammary Epithelial Cells.

PubMed

Deplanche, Martine; Alekseeva, Ludmila; Semenovskaya, Ksenia; Fu, Chih-Lung; Dessauge, Frederic; Finot, Laurence; Petzl, Wolfram; Zerbe, Holm; Le Loir, Yves; Rainard, Pascal; Smith, David G E; Germon, Pierre; Otto, Michael; Berkova, Nadia

2016-06-01

The role of the recently described interleukin-32 (IL-32) in Staphylococcus aureus-induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6, and IL-8 in S. aureus- and Escherichia coli-infected bovine mammary gland epithelial cells. Using live bacteria, we found that in S. aureus-infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli-infected cells. Notably, IL-32 expression was decreased in S. aureus-infected cells, while it was increased in E. coli-infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6, and IL-8 expression by epithelial cells exposed to psm mutant strains was significantly increased compared to that in cells exposed to the isogenic S. aureus wild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S. aureus-related mastitis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Ethanol potentiates the genotoxicity of the food-derived mammary carcinogen PhIP in human estrogen receptor-positive mammary cells: mechanistic support for lifestyle factors (cooked red meat and ethanol) associated with mammary cancer.

PubMed

Malik, Durr-E-Shahwar; David, Rhiannon M; Gooderham, Nigel J

2018-04-01

Consumption of cooked/processed meat and ethanol are lifestyle risk factors in the aetiology of breast cancer. Cooking meat generates heterocyclic amines such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Epidemiology, mechanistic and animal studies indicate that PhIP is a mammary carcinogen that could be causally linked to breast cancer incidence; PhIP is DNA damaging, mutagenic and oestrogenic. PhIP toxicity involves cytochrome P450 (CYP1 family)-mediated metabolic activation to DNA-damaging species, and transcriptional responses through Aryl hydrocarbon receptor (AhR) and estrogen-receptor-α (ER-α). Ethanol consumption is a modifiable lifestyle factor strongly associated with breast cancer risk. Ethanol toxicity involves alcohol dehydrogenase metabolism to reactive acetaldehyde, and is also a substrate for CYP2E1, which when uncoupled generates reactive oxygen species (ROS) and DNA damage. Here, using human mammary cells that differ in estrogen-receptor status, we explore genotoxicity of PhIP and ethanol and mechanisms behind this toxicity. Treatment with PhIP (10 -7 -10 -4 M) significantly induced genotoxicity (micronuclei formation) preferentially in ER-α positive human mammary cell lines (MCF-7, ER-α+) compared to MDA-MB-231 (ER-α-) cells. PhIP-induced CYP1A2 in both cell lines but CYP1B1 was selectively induced in ER-α(+) cells. ER-α inhibition in MCF-7 cells attenuated PhIP-mediated micronuclei formation and CYP1B1 induction. PhIP-induced CYP2E1 and ROS via ER-α-STAT-3 pathway, but only in ER-α (+) MCF-7 cells. Importantly, simultaneous treatments of physiological concentrations ethanol (10 -3 -10 -1 M) with PhIP (10 -7 -10 -4 M) increased oxidative stress and genotoxicity in MCF-7 cells, compared to the individual chemicals. Collectively, these data offer a mechanistic basis for the increased risk of breast cancer associated with dietary cooked meat and ethanol lifestyle choices.

Pomegranate exerts chemoprevention of experimentally induced mammary tumorigenesis by suppression of cell proliferation and induction of apoptosis.

PubMed

Bishayee, Anupam; Mandal, Animesh; Bhattacharyya, Piyali; Bhatia, Deepak

2016-01-01

Breast cancer is the second leading cause of cancer-related death in women in the United States and discovery and development of safe chemopreventive drugs is urgently needed. The fruit pomegranate (Punica granatum) is gaining importance because of its various health benefits. This study was initiated to investigate chemopreventive potential of a pomegranate emulsion (PE) against 7,12-dimethylbenz(a)anthracene (DMBA) rat mammary carcinogenesis. The animals were orally administered with PE (0.2-5.0 g/kg), starting 2 wk before and 16 wk following DMBA treatment. PE exhibited a striking reduction of DMBA-induced mammary tumor incidence, total tumor burden, and reversed histopathological changes. PE dose-dependently suppressed cell proliferation and induced apoptosis in mammary tumors. Immunohistochemical studies showed that PE increased intratumor Bax, decreased Bcl2 and manifested a proapoptotic shift in Bax/Bcl2 ratio. In addition, our gene expression study showed PE-mediated upregulation of Bad, caspase-3, caspase-7, caspase-9, poly (ADP ribose) polymerase and cytochrome c in mammary tumors. Thus, PE exerts chemoprevention of mammary carcinogenesis by suppressing cell proliferation and inducing apoptosis mediated through upregulation of Bax and downregulation of Bcl2 in concert with caspase cascades. Pomegranate bioactive phytoconstituents could be developed as a chemopreventive drug to reduce the risk of breast cancer.

Telomere maintenance in laser capture microdissection-purified Barrett's adenocarcinoma cells and effect of telomerase inhibition in vivo.

PubMed

Shammas, Masood A; Qazi, Aamer; Batchu, Ramesh B; Bertheau, Robert C; Wong, Jason Y Y; Rao, Manjula Y; Prasad, Madhu; Chanda, Diptiman; Ponnazhagan, Selvarangan; Anderson, Kenneth C; Steffes, Christopher P; Munshi, Nikhil C; De Vivo, Immaculata; Beer, David G; Gryaznov, Sergei; Weaver, Donald W; Goyal, Raj K

2008-08-01

The aims of this study were to investigate telomere function in normal and Barrett's esophageal adenocarcinoma (BEAC) cells purified by laser capture microdissection and to evaluate the effect of telomerase inhibition in cancer cells in vitro and in vivo. Epithelial cells were purified from surgically resected esophagi. Telomerase activity was measured by modified telomeric repeat amplification protocol and telomere length was determined by real-time PCR assay. To evaluate the effect of telomerase inhibition, adenocarcinoma cell lines were continuously treated with a specific telomerase inhibitor (GRN163L) and live cell number was determined weekly. Apoptosis was evaluated by Annexin labeling and senescence by beta-galactosidase staining. For in vivo studies, severe combined immunodeficient mice were s.c. inoculated with adenocarcinoma cells and following appearance of palpable tumors, injected i.p. with saline or GRN163L. Telomerase activity was significantly elevated whereas telomeres were shorter in BEAC cells relative to normal esophageal epithelial cells. The treatment of adenocarcinoma cells with telomerase inhibitor, GRN163L, led to loss of telomerase activity, reduction in telomere length, and growth arrest through induction of both the senescence and apoptosis. GRN163L-induced cell death could also be expedited by addition of the chemotherapeutic agents doxorubicin and ritonavir. Finally, the treatment with GRN163L led to a significant reduction in tumor volume in a subcutaneous tumor model. We show that telomerase activity is significantly elevated whereas telomeres are shorter in BEAC and suppression of telomerase inhibits proliferation of adenocarcinoma cells both in vitro and in vivo.

A hybrid of coumarin and phenylsulfonylfuroxan induces caspase-dependent apoptosis and cytoprotective autophagy in lung adenocarcinoma cells.

PubMed

Wang, Qian; Guo, Yalan; Jiang, Shanshan; Dong, Mengxue; Kuerban, Kudelaidi; Li, Jiyang; Feng, Meiqing; Chen, Ying; Ye, Li

2018-01-15

Lung adenocarcinoma is the most primary histologic subtype of non-small cell lung cancer (NSCLC). Compound 8b, a novel coumarin derivative with phenylsulfonylfuroxan group, shows significant antiproliferation activity against lung adenocarcinoma cell with low toxicity. This study aims to uncover the potential of compound 8b in relation to apoptosis as well as autophagy induction in lung adenocarcinoma cells. The cytotoxicity and apoptosis of A549 and H1299 cells induced by compound 8b were detected by MTT, microscope and western blot analysis. Autophagy was determined by TEM, confocal microscopy and western blot analysis. Akt/mTOR and Erk signaling pathway were also examined by western blot analysis. First, significant growth inhibition and caspase-dependent apoptosis were observed in compound 8b-treated A549 and H1299 cells. Then, we confirmed compound 8b-induced autophagy by autophagosomes formation, upregulated expression of autophagy-related protein LC3-II and autophagic flux. Importantly, abolishing autophagy using inhibitors and ATG5 siRNA enhanced the cytotoxicity of compound 8b, indicating the cytoprotective role of autophagy in lung adenocarcinoma. Further mechanistic investigations suggested that Akt/mTOR and Erk signaling pathways contributed to autophagy induction by compound 8b. This results demonstrate that compound 8b induces caspase-dependent apoptosis as well as cytoprotective autophagy in lung adenocarcinoma cells, which may provide scientific evidence for developing this furoxan-based NO-releasing coumarin derivative as a potential anti-lung adenocarcinoma therapeutic agents. Copyright © 2017 Elsevier GmbH. All rights reserved.

Th-POK regulates mammary gland lactation through mTOR-SREBP pathway.

PubMed

Zhang, Rui; Ma, Huimin; Gao, Yuan; Wu, Yanjun; Qiao, Yuemei; Geng, Ajun; Cai, Cheguo; Han, Yingying; Zeng, Yi Arial; Liu, Xiaolong; Ge, Gaoxiang

2018-02-01

The Th-inducing POK (Th-POK, also known as ZBTB7B or cKrox) transcription factor is a key regulator of lineage commitment of immature T cell precursors. It is yet unclear the physiological functions of Th-POK besides helper T cell differentiation. Here we show that Th-POK is restrictedly expressed in the luminal epithelial cells in the mammary glands that is upregulated at late pregnancy and lactation. Lineage restrictedly expressed Th-POK exerts distinct biological functions in the mammary epithelial cells and T cells in a tissue-specific manner. Th-POK is not required for mammary epithelial cell fate determination. Mammary gland morphogenesis in puberty and alveologenesis in pregnancy are phenotypically normal in the Th-POK-deficient mice. However, Th-POK-deficient mice are defective in triggering the onset of lactation upon parturition with large cellular lipid droplets retained within alveolar epithelial cells. As a result, Th-POK knockout mice are unable to efficiently secret milk lipid and to nurse the offspring. Such defect is mainly attributed to the malfunctioned mammary epithelial cells, but not the tissue microenvironment in the Th-POK deficient mice. Th-POK directly regulates expression of insulin receptor substrate-1 (IRS-1) and insulin-induced Akt-mTOR-SREBP signaling. Th-POK deficiency compromises IRS-1 expression and Akt-mTOR-SREBP signaling in the lactating mammary glands. Conversely, insulin induces Th-POK expression. Thus, Th-POK functions as an important feed-forward regulator of insulin signaling in mammary gland lactation.

Th-POK regulates mammary gland lactation through mTOR-SREBP pathway

PubMed Central

Wu, Yanjun; Qiao, Yuemei; Geng, Ajun; Cai, Cheguo; Han, Yingying; Zeng, Yi Arial

2018-01-01

The Th-inducing POK (Th-POK, also known as ZBTB7B or cKrox) transcription factor is a key regulator of lineage commitment of immature T cell precursors. It is yet unclear the physiological functions of Th-POK besides helper T cell differentiation. Here we show that Th-POK is restrictedly expressed in the luminal epithelial cells in the mammary glands that is upregulated at late pregnancy and lactation. Lineage restrictedly expressed Th-POK exerts distinct biological functions in the mammary epithelial cells and T cells in a tissue-specific manner. Th-POK is not required for mammary epithelial cell fate determination. Mammary gland morphogenesis in puberty and alveologenesis in pregnancy are phenotypically normal in the Th-POK-deficient mice. However, Th-POK-deficient mice are defective in triggering the onset of lactation upon parturition with large cellular lipid droplets retained within alveolar epithelial cells. As a result, Th-POK knockout mice are unable to efficiently secret milk lipid and to nurse the offspring. Such defect is mainly attributed to the malfunctioned mammary epithelial cells, but not the tissue microenvironment in the Th-POK deficient mice. Th-POK directly regulates expression of insulin receptor substrate-1 (IRS-1) and insulin-induced Akt-mTOR-SREBP signaling. Th-POK deficiency compromises IRS-1 expression and Akt-mTOR-SREBP signaling in the lactating mammary glands. Conversely, insulin induces Th-POK expression. Thus, Th-POK functions as an important feed-forward regulator of insulin signaling in mammary gland lactation. PMID:29420538

Mammary Cancer and Activation of Transposable Elements

DTIC Science & Technology

2015-03-01

EGFP). No other cell type in the mammary fat pad was observed to express EGFP. Wholemount and FACS analyses of mammary fat pads after involution from...were sacrificed for PI-MEC isolation in groups of up to 4 control or cancer-prone uniparous or triparous females. Both 4 th mammary fat pads were...to unknown reason (n=46), and smaller numbers of animals with various conditions (malocclusion, head tilt , dystocia, respiratory complaints, identity

Lachrymal Gland Basal Cell Adenocarcinoma in a Ferret (Mustela putorius furo).

PubMed

Chambers, J K; Nakamori, T; Kishimoto, T E; Nakata, M; Miwa, Y; Nakayama, H; Uchida, K

2016-01-01

A 1 cm diameter mass was detected in the caudal superotemporal area of the left eye of a 6-year-old neutered male ferret (Mustela putorius furo). The mass and the left eye were removed surgically. Microscopical examination revealed a tumour of the adnexal gland of the eye that had invaded the surrounding ocular muscle. The tumour was composed of basal-type epithelial cells arranged in a solid, or occasionally tubular, pattern. Immunohistochemically, the tumour cells expressed cytokeratin and p63, but not smooth muscle actin. Based on these findings, the tumour was diagnosed as a basal cell adenocarcinoma of the lachrymal gland. In addition to the tumour, the retina of the left eye was detached and folded at the centre of the globe. This is the first report of a non-human case of basal cell adenocarcinoma of the lachrymal gland. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stromal and Epithelial Caveolin-1 Both Confer a Protective Effect Against Mammary Hyperplasia and Tumorigenesis

PubMed Central

Williams, Terence M.; Sotgia, Federica; Lee, Hyangkyu; Hassan, Ghada; Di Vizio, Dolores; Bonuccelli, Gloria; Capozza, Franco; Mercier, Isabelle; Rui, Hallgeir; Pestell, Richard G.; Lisanti, Michael P.

2006-01-01

Here, we investigate the role of caveolin-1 (Cav-1) in breast cancer onset and progression, with a focus on epithelial-stromal interactions, ie, the tumor microenvironment. Cav-1 is highly expressed in adipocytes and is abundant in mammary fat pads (stroma), but it remains unknown whether loss of Cav-1 within mammary stromal cells affects the differentiated state of mammary epithelia via paracrine signaling. To address this issue, we characterized the development of the mammary ductal system in Cav-1−/− mice and performed a series of mammary transplant studies, using both wild-type and Cav-1−/− mammary fat pads. Cav-1−/− mammary epithelia were hyperproliferative in vivo, with dramatic increases in terminal end bud area and mammary ductal thickness as well as increases in bromodeoxyuridine incorporation, extracellular signal-regulated kinase-1/2 hyperactivation, and up-regulation of STAT5a and cyclin D1. Consistent with these findings, loss of Cav-1 dramatically exacerbated mammary lobulo-alveolar hyperplasia in cyclin D1 Tg mice, whereas overexpression of Cav-1 caused reversion of this phenotype. Most importantly, Cav-1−/− mammary stromal cells (fat pads) promoted the growth of both normal mammary ductal epithelia and mammary tumor cells. Thus, Cav-1 expression in both epithelial and stromal cells provides a protective effect against mammary hyperplasia as well as mammary tumorigenesis. PMID:17071600

Adenocarcinoma of the urinary bladder

PubMed Central

Dadhania, Vipulkumar; Czerniak, Bogdan; Guo, Charles C

2015-01-01

Adenocarcinoma is an uncommon malignancy in the urinary bladder which may arise primarily in the bladder as well as secondarily from a number of other organs. Our aim is to provide updated information on primary and secondary bladder adenocarcinomas, with focus on pathologic features, differential diagnosis, and clinical relevance. Primary bladder adenocarcinoma exhibits several different growth patterns, including enteric, mucinous, signet-ring cell, not otherwise specified, and mixed patterns. Urachal adenocarcinoma demonstrates similar histologic features but it can be distinguished from bladder adenocarcinoma on careful pathologic examination. Secondary bladder adenocarcinomas may arise from the colorectum, prostate, endometrium, cervix and other sites. Immunohistochemical study is valuable in identifying the origin of secondary adenocarcinomas. Noninvasive neoplastic glandular lesions, adenocarcinoma in situ and villous adenoma, are frequently associated with bladder adenocarcinoma. It is also important to differentiate bladder adenocarcinoma from a number of nonneoplastic lesions in the bladder. Primary bladder adenocarcinoma has a poor prognosis largely because it is usually diagnosed at an advanced stage. Urachal adenocarcinoma shares similar histologic features with bladder adenocarcinoma, but it has a more favorable prognosis than bladder adenocarcinoma, partly due to the relative young age of patients with urachal adenocarcinoma. PMID:26309895

Prognosis of oesophageal adenocarcinoma and squamous cell carcinoma following surgery and no surgery in a nationwide Swedish cohort study

PubMed Central

Mattsson, Fredrik

2018-01-01

Objectives To assess the recent prognostic trends in oesophageal adenocarcinoma and oesophageal squamous cell carcinoma undergoing resectional surgery and no such surgery. Additionally, risk factors for death were assessed in each of these patient groups. Design Cohort study. Setting A population-based, nationwide study in Sweden. Participants All patients diagnosed with oesophageal adenocarcinoma and oesophageal squamous cell carcinoma in Sweden from 1 January 1990 to 31 December 2013, with follow-up until 14 May 2017. Outcome measures Observed and relative (to the background population) 1-year, 3-year and 5-year survivals were analysed using life table method. Multivariable Cox regression provided HR with 95% CI for risk factors of death. Results Among 3794 patients with oesophageal adenocarcinoma and 4631 with oesophageal squamous cell carcinoma, 82% and 63% were men, respectively. From 1990–1994 to 2010–2013, the relative 5-year survival increased from 12% to 15% for oesophageal adenocarcinoma and from 9% to 12% for oesophageal squamous cell carcinoma. The corresponding survival following surgery increased from 27% to 45% in oesophageal adenocarcinoma and from 24% to 43% in oesophageal squamous cell carcinoma. In patients not undergoing surgery, the survival increased from 3% to 4% for oesophageal adenocarcinoma and from 3% to 6% for oesophageal squamous cell carcinoma. Women with oesophageal squamous cell carcinoma had better prognosis than men both following surgery (HR 0.71, 95% CI 0.61 to 0.83) and no surgery (HR 0.86, 95% CI 0.81 to 0.93). Conclusions The prognosis has improved over calendar time both in oesophageal adenocarcinoma and oesophageal squamous cell carcinoma in Sweden that did and did not undergo surgery. Women appear to have better prognosis in oesophageal squamous cell carcinoma than men, independent of treatment. PMID:29748347

The RhoGEF Net1 Is Required for Normal Mammary Gland Development

PubMed Central

Zuo, Yan; Berdeaux, Rebecca

2014-01-01

Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily-specific guanine nucleotide exchange factor that is overexpressed in human breast cancer and is required for breast cancer cell migration and invasion. However, the role of Net1 in normal mammary gland development or function has never been assessed. To understand the role of Net1 in the mammary gland, we have created a conditional Net1 knockout mouse model. Whole-body deletion of Net1 results in delayed mammary gland development during puberty characterized by slowed of ductal extension and reduced ductal branching. Epithelial cells within the developing ducts show reduced proliferation that is accompanied by diminished estrogen receptor-α expression and activity. Net1-deficient mammary glands also exhibit reduced phosphorylation of regulatory subunits of myosin light chain and myosin light-chain phosphatase, indicating that RhoA-dependent actomyosin contraction is compromised. Net1 deficiency also leads to disorganization of myoepithelial and ductal epithelial cells and increased periductal collagen deposition. Mammary epithelial cell transplantation experiments indicate that reduced ductal branching and disorganization are cell autonomous. These data identify for the first time a role for NET1 in vivo and indicate that NET1 expression is essential for the proliferation and differentiation of mammary epithelial cells in the developing mammary gland. PMID:25321414

Notch ligand Dll1 mediates cross-talk between mammary stem cells and the macrophageal niche.

PubMed

Chakrabarti, Rumela; Celià-Terrassa, Toni; Kumar, Sushil; Hang, Xiang; Wei, Yong; Choudhury, Abrar; Hwang, Julie; Peng, Jia; Nixon, Briana; Grady, John J; DeCoste, Christina; Gao, Jie; van Es, Johan H; Li, Ming O; Aifantis, Iannis; Clevers, Hans; Kang, Yibin

2018-05-17

The stem cell niche is a specialized environment that dictates stem cell function during development and homeostasis. Here, we show that Dll1, a Notch pathway ligand, is enriched in mammary gland stem cells (MaSCs) and mediates critical interactions with stromal macrophages in the surrounding niche. Conditional deletion of Dll1 reduced the number of MaSCs and impaired ductal morphogenesis in the mammary gland. Moreover, MaSC-expressed Dll1 activates Notch signaling in stromal macrophages, increasing their expression of Wnt family ligands such as Wnt3, Wnt10A, and Wnt16, thereby initiating a feed back loop that promotes the function of Dll1 + MaSCs. Together, these findings reveal functionally important cross-talk between MaSCs and their macrophageal niche through Dll1/Notch-mediated signaling. Copyright © 2018, American Association for the Advancement of Science.

The effect of neighboring cells on the stiffness of cancerous and non-cancerous human mammary epithelial cells

NASA Astrophysics Data System (ADS)

Guo, Xinyi; Bonin, Keith; Scarpinato, Karin; Guthold, Martin

2014-10-01

Using an Atomic Force Microscope (AFM) with a 5.3 μm diameter spherical probe, we determined mechanical properties of individual human mammary epithelial cells. The cells were derived from a pair of cell lines that mimic cell progression through four phases of neoplastic transformation: normal (non-transformed), immortal, tumorigenic, and metastatic. Measurements on cells in all four phases were taken over both the cytoplasmic and nuclear regions. Moreover, the measurements were made for cells in different microenvironments as related to cell-cell contacts: isolated cells; cells residing on the periphery of a contiguous cell monolayer; and cells on the inside of a contiguous cell monolayer. By fitting the AFM force versus indentation curves to a Hertz model, we determined the pseudo-elastic Young’s modulus, E. Combining all data for the cellular subregions (over nucleus and cytoplasm) and the different cell microenvironments, we obtained stiffness values for normal, immortal, tumorigenic, and metastatic cells of 870 Pa, 870 Pa, 490 Pa, and 580 Pa, respectively. That is, cells become softer as they advance to the tumorigenic phase and then stiffen somewhat in the final step to metastatic cells. We also found a distinct contrast in the influence of a cell’s microenvironment on cell stiffness. Normal mammary epithelial cells inside a monolayer are stiffer than peripheral cells, which are stiffer than isolated cells. However, the microenvironment had a slight, opposite effect on tumorigenic and little effect on immortal and metastatic cell stiffness. Thus, the stiffness of cancer cells is less sensitive to the microenvironment than normal cells. Our results show that the mechanical properties of a cell can depend on cancer progression and microenvironment (cell-cell interactions).

Metastatic, papillary cystadenocarcinoma of the mammary gland in a black-footed ferret

USGS Publications Warehouse

Carpenter, J.W.; Davidson, J.P.; Novilla, M.N.; Huang, J.C.M.

1980-01-01

A simple, papillary cystic adenocarcinoma of the mammary gland with metastases to the internal iliac and mesenteric lymph nodes, liver, and spleen was observed in a 12 to 13 year old female black-footed ferret (Mustela nigripes). Histologically, the tumor was aggressive, and lymphatic invasion was found. Attempts at virus isolation were negative. Other findings were bilateral infarcts in the kidneys, apparently resulting in acute renal shutdown and death, multiple thrombi in the right atrium, aortic arteriosclerosis, and focal interstitial pneumonia.

Metastatic, papillary cystadenocarcinoma of the mammary gland in a black-footed ferret.

PubMed

Carpenter, J W; Davidson, J P; Novilla, M N; Huang, J C

1980-10-01

A simple, papillary cystic adenocarcinoma of the mammary gland with metastases to the internal iliac and mesenteric lymph nodes, liver, and spleen was observed in a 12 to 13 year old female black-footed ferret (Mustela nigripes). Histologically, the tumor was aggressive, and lymphatic invasion was found. Attempts at virus isolation were negative. Other findings were bilateral infarcts in the kidneys, apparently resulting in acute renal shutdown and death, multiple thrombi in the right atrium, aortic arteriosclerosis, and focal interstitial pneumonia.

Genistein-mediated inhibition of mammary stromal adipocyte differentiation limits expansion of mammary stem/progenitor cells by paracrine signaling

USDA-ARS?s Scientific Manuscript database

Mammary adiposity may contribute to breast cancer development and progression by releasing cytokines and other inflammatory mediators that promote mammary epithelial proliferation. We evaluated the effects of soy isoflavone genistein (GEN) on the adipogenic differentiation of a SV40-immortalized mou...

Mouse Mammary Tumor Virus c-rel Transgenic Mice Develop Mammary Tumors

PubMed Central

Romieu-Mourez, Raphaëlle; Kim, Dong W.; Min Shin, Sang; Demicco, Elizabeth G.; Landesman-Bollag, Esther; Seldin, David C.; Cardiff, Robert D.; Sonenshein, Gail E.

2003-01-01

Amplification, overexpression, or rearrangement of the c-rel gene, encoding the c-Rel NF-κB subunit, has been reported in solid and hematopoietic malignancies. For example, many primary human breast cancer tissue samples express high levels of nuclear c-Rel. While the Rev-T oncogene v-rel causes tumors in birds, the ability of c-Rel to transform in vivo has not been demonstrated. To directly test the role of c-Rel in breast tumorigenesis, mice were generated in which overexpression of mouse c-rel cDNA was driven by the hormone-responsive mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, and four founder lines identified. In the first cycle of pregnancy, the expression of transgenic c-rel mRNA was observed, and levels of c-Rel protein were increased in the mammary gland. Importantly, 31.6% of mice developed one or more mammary tumors at an average age of 19.9 months. Mammary tumors were of diverse histology and expressed increased levels of nuclear NF-κB. Analysis of the composition of NF-κB complexes in the tumors revealed aberrant nuclear expression of multiple subunits, including c-Rel, p50, p52, RelA, RelB, and the Bcl-3 protein, as observed previously in human primary breast cancers. Expression of the cancer-related NF-κB target genes cyclin D1, c-myc, and bcl-xl was significantly increased in grossly normal transgenic mammary glands starting the first cycle of pregnancy and increased further in mammary carcinomas compared to mammary glands from wild-type mice or virgin transgenic mice. In transient transfection analysis in untransformed breast epithelial cells, c-Rel-p52 or -p50 heterodimers either potently or modestly induced cyclin D1 promoter activity, respectively. Lastly, stable overexpression of c-Rel resulted in increased cyclin D1 and NF-κB p52 and p50 subunit protein levels. These results indicate for the first time that dysregulated expression of c-Rel, as observed in breast cancers, is capable of contributing to mammary

Citrus-derived oils inhibit Satphylococcus aureus growth and alter its interaction with bovine mammary cells

USDA-ARS?s Scientific Manuscript database

This experiment examined the effects of cold-pressed, terpeneless citrus oil (CDO) on growth of Staphylococcus aureus, which a major cause of contagious bovine mastitis, and invasion of epithelial cells as modeled with bovine mammary cells (MAC-T). The broth dilution method (Muthaiyan et al., 2012)...

Overexpression of RBM5 induces autophagy in human lung adenocarcinoma cells.

PubMed

Su, Zhenzhong; Wang, Ke; Li, Ranwei; Yin, Jinzhi; Hao, Yuqiu; Lv, Xuejiao; Li, Junyao; Zhao, Lijing; Du, Yanwei; Li, Ping; Zhang, Jie

2016-02-29

Dysfunctions in autophagy and apoptosis are closely interacted and play an important role in cancer development. RNA binding motif 5 (RBM5) is a tumor suppressor gene, which inhibits tumor cells' growth and enhances chemosensitivity through inducing apoptosis in our previous studies. In this study, we investigated the relationship between RBM5 overexpression and autophagy in human lung adenocarcinoma cells. Human lung adenocarcinoma cancer (A549) cells were cultured in vitro and were transiently transfected with a RBM5 expressing plasmid (GV287-RBM5) or plasmid with scrambled control sequence. RBM5 expression was determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Intracellular LC-3 I/II, Beclin-1, lysosome associated membrane protein-1 (LAMP1), Bcl-2, and NF-κB/p65 protein levels were detected by Western blot. Chemical staining with monodansylcadaverine (MDC) and acridine orange (AO) was applied to detect acidic vesicular organelles (AVOs). The ultrastructure changes were observed under transmission electron microscope (TEM). Then, transplanted tumor models of A549 cells on BALB/c nude mice were established and treated with the recombinant plasmids carried by attenuated Salmonella to induce RBM5 overexpression in tumor tissues. RBM5, LC-3, LAMP1, and Beclin1 expression was determined by immunohistochemistry staining in plasmids-treated A549 xenografts. Our study demonstrated that overexpression of RBM5 caused an increase in the autophagy-related proteins including LC3-I, LC3-II, LC3-II/LC3-I ratio, Beclin1, and LAMP1 in A549 cells. A large number of autophagosomes with double-membrane structure and AVOs were detected in the cytoplasm of A549 cells transfected with GV287-RBM5 at 24 h. We observed that the protein level of NF-κB/P65 was increased and the protein level of Bcl-2 decreased by RBM5 overexpression. Furthermore, treatment with an autophagy inhibitor, 3-MA, enhanced RBM5-induced cell death and

Using 3D Culture of Primary Mammary Epithelial Cells to Define Molecular Entities Required for Acinus Formation: Analyzing MAP Kinase Phosphatases.

PubMed

Gajewska, Malgorzata; McNally, Sara

2017-01-01

Three-dimensional (3D) cell cultures on reconstituted basement membrane (rBM) enable the study of complex interactions between extracellular matrix (ECM) components and epithelial cells, which are crucial for the establishment of cell polarity and functional development of epithelia. 3D cultures of mammary epithelial cells (MECs) on Matrigel (a laminin-rich ECM derived from the Engelbreth-Holm-Swarm (EHS) murine tumor) promote interactions of MECs with the matrix via integrins, leading to formation of spherical monolayers of polarized cells surrounding a hollow lumen (acini). Acini closely resemble mammary alveoli found in the mammary gland. Thus, it is possible to study ECM-cell interactions and signalling pathways that regulate formation and maintenance of tissue-specific shape and functional differentiation of MECs in 3D under in vitro conditions. Here we present experimental protocols used to investigate the role of mitogen-activated protein kinase phosphatases (MKPs) during development of the alveoli-like structures by primary mouse mammary epithelial cells (PMMEC) cultured on Matrigel. We present detailed protocols for PMMEC isolation, and establishment of 3D cultures using an "on top" method, use of specific kinase and phosphatases inhibitors (PD98059 and pervanadate, respectively) administered at different stages of acinus development, and give examples of analyses carried out post-culture (Western blot, immunofluorescence staining, and confocal imaging).

Breast metastasis of gastric signet-ring cell carcinoma: a case report and literature review.

PubMed

He, Chun-Lan; Chen, Ping; Xia, Bing-Lan; Xiao, Qin; Cai, Feng-Lin

2015-03-26

Cases of primary gastric adenocarcinoma with metastasis to the breast are extremely rare. Till now, only 38 cases have been reported in PubMed since 1908. We herein reported a race case of gastric adenocarcinoma with metastasis to the right breast. Breast biopsy showed invasive signet-ring cell breast carcinoma in the right breast. She was given a TEC regimen (docetaxel 75 mg/m(2), epirubicin 75 mg/m(2), and cyclophosphamide 600 mg/m(2) every 3 weeks) for one cycle but showed no objective response. Upper gastrointestinal endoscopy demonstrated an ulcerative mass in the gastric body. Biopsy demonstrated low-grade gastric adenocarcinoma with signet-ring features. In immunohistochemistry, mammary malignant cells were positive for cytokeratin 7, cytokeratin 20, villin, and ErbB2/HER2, but negative for gross cystic disease fluid protein-15, estrogen receptor, and progesterone receptor. The diagnosis of metastatic poorly differentiated signet-ring cell adenocarcinoma of the right breast identical to gastric primary was confirmed finally. Gastric cancer with metastasis to the breast can be diagnosed by clinical history, histological findings, and immunohistochemical markers.

Keeping abreast of the mammary epithelial hierarchy and breast tumorigenesis.

PubMed

Visvader, Jane E

2009-11-15

The epithelium of the mammary gland exists in a highly dynamic state, undergoing dramatic morphogenetic changes during puberty, pregnancy, lactation, and regression. The recent identification of stem and progenitor populations in mouse and human mammary tissue has provided evidence that the mammary epithelium is organized in a hierarchical manner. Characterization of these normal epithelial subtypes is an important step toward understanding which cells are predisposed to oncogenesis. This review summarizes progress in the field toward defining constituent cells and key molecular regulators of the mammary epithelial hierarchy. Potential relationships between normal epithelial populations and breast tumor subtypes are discussed, with implications for understanding the cellular etiology underpinning breast tumor heterogeneity.

Epigenetic regulation of normal human mammary cell type-specific miRNAs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.

2011-08-26

Epigenetic mechanisms are important regulators of cell type–specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type–specific pattern of expression that was linkedmore » to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type–specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type–specific miRNA expression.« less

Radiation-induced chromosomal instability in human mammary epithelial cells

NASA Technical Reports Server (NTRS)

Durante, M.; Grossi, G. F.; Yang, T. C.

1996-01-01

Karyotypes of human cells surviving X- and alpha-irradiation have been studied. Human mammary epithelial cells of the immortal, non-tumorigenic cell line H184B5 F5-1 M/10 were irradiated and surviving clones isolated and expanded in culture. Cytogenetic analysis was performed using dedicated software with an image analyzer. We have found that both high- and low-LET radiation induced chromosomal instability in long-term cultures, but with different characteristics. Complex chromosomal rearrangements were observed after X-rays, while chromosome loss predominated after alpha-particles. Deletions were observed in both cases. In clones derived from cells exposed to alpha-particles, some cells showed extensive chromosome breaking and double minutes. Genomic instability was correlated to delayed reproductive death and neoplastic transformation. These results indicate that chromosomal instability is a radiation-quality-dependent effect which could determine late genetic effects, and should therefore be carefully considered in the evaluation of risk for space missions.

Radiation-induced chromosomal instability in human mammary epithelial cells

NASA Astrophysics Data System (ADS)

Durante, M.; Grossi, G. F.; Yang, T. C.

Karyotypes of human cells surviving X- and alpha-irradiation have been studied. Human mammary epithelial cells of the immortal, non-tumorigenic cell line H184B5 F5-1 M/10 were irradiated and surviving clones isolated and expanded in culture. Cytogenetic analysis was performed using dedicated software with an image analyzer. We have found that both high- and low-LET radiation induced chromosomal instability in long-term cultures, but with different characteristics. Complex chromosomal rearrangements were observed after X-rays, while chromosome loss predominated after alpha-particles. Deletions were observed in both cases. In clones derived from cells exposed to alpha-particles, some cells showed extensive chromosome breaking and double minutes. Genomic instability was correlated to delayed reproductive death and neoplastic transformation. These results indicate that chromosomal instability is a radiation-quality-dependent effect which could determine late genetic effects, and should therefore be carefully considered in the evaluation of risk for space missions.

hEcd, A Novel Regulator of Mammary Epithelial Cell Survival

DTIC Science & Technology

2009-09-01

theYeast Two hybrid analysis with human papilloma virus oncogene E6 (the most efficient oncogene to immortalize hMECs in vitro) as a bait and mammary...transformation. We have identified a novel protein us ing the Yeast Two hybrid analysis with human papilloma virus oncogene E6 (the most efficient...epithelial cell cDNA library, we identified hEcd ( human orthologue of Drosophila Ecdysoneless) as a novel E6 binding partner. To study the cellular

Lrp5 Has a Wnt-Independent Role in Glucose Uptake and Growth for Mammary Epithelial Cells

PubMed Central

Chin, Emily N.; Martin, Joshua A.; Kim, Soyoung; Fakhraldeen, Saja A.

2015-01-01

Lrp5 is typically described as a Wnt signaling receptor, albeit a less effective Wnt signaling receptor than the better-studied sister isoform, Lrp6. Here we show that Lrp5 is only a minor player in the response to Wnt3a-type ligands in mammary epithelial cells; instead, Lrp5 is required for glucose uptake, and glucose uptake regulates the growth rate of mammary epithelial cells in culture. Thus, a loss of Lrp5 leads to profound growth suppression, whether growth is induced by serum or by specific growth factors, and this inhibition is not due to a loss of Wnt signaling. Depletion of Lrp5 decreases glucose uptake, lactate secretion, and oxygen consumption rates; inhibition of glucose consumption phenocopies the loss of Lrp5 function. Both Lrp5 knockdown and low external glucose induce mitochondrial stress, as revealed by the accumulation of reactive oxygen species (ROS) and the activation of the ROS-sensitive checkpoint, p38α. In contrast, loss of function of Lrp6 reduces Wnt responsiveness but has little impact on growth. This highlights the distinct functions of these two Lrp receptors and an important Wnt ligand-independent role of Lrp5 in glucose uptake in mammary epithelial cells. PMID:26711269

Mammaglobin expression in gynecologic adenocarcinomas.

PubMed

Hagemann, Ian S; Pfeifer, John D; Cao, Dengfeng

2013-04-01

Mammaglobin (MGB) has been proposed as a sensitive and specific immunohistochemical marker for adenocarcinoma of the breast. The differential diagnosis of breast adenocarcinoma versus a gynecologic primary frequently arises. We performed a semiquantitative survey of MGB immunoreactivity in 26 benign gynecologic tissues (6 ectocervices, 9 endocervices, 11 endometria), 86 ovarian adenocarcinomas, 70 endometrial adenocarcinomas, and 10 endocervical adenocarcinomas. Among ovarian tumors, MGB was present in 40% of endometrioid carcinomas; 36%, serous carcinomas; 21%, clear cell carcinomas; and 6%, mucinous carcinomas. Among endometrial cancers, MGB reactivity was present in 57% of endometrioid carcinomas, but only 30% of serous carcinomas and 6% of clear cell carcinomas. MGB was absent in endocervical adenocarcinomas. Across all tumor types with positive staining, MGB was focal or patchy (ie, less than diffuse) in 50 of 57 cases. Using a scale of 0 to 3+, the only 3 tumors with 3+ MGB reactivity were all serous carcinomas (1 ovarian and 2 endometrial). There were no cases with diffuse 3+ MGB expression. On the other hand, diffuse 2+ MGB was seen in 4 cases: 1 endometrioid carcinoma of ovary, 1 serous carcinoma of ovary, and 2 clear cell carcinomas of ovary. In conclusion, a diagnostically significant proportion of gynecologic carcinomas are immunoreactive for MGB. Gynecologic primaries should be considered in the differential diagnosis of MGB-positive malignancies of unknown origin. Copyright © 2013 Elsevier Inc. All rights reserved.

Enhanced growth medium and method for culturing human mammary epithelial cells

DOEpatents

Stampfer, Martha R.; Smith, Helene S.; Hackett, Adeline J.

1983-01-01

Methods are disclosed for isolating and culturing human mammary epithelial cells of both normal and malignant origin. Tissue samples are digested with a mixture including the enzymes collagenase and hyaluronidase to produce clumps of cells substantially free from stroma and other undesired cellular material. Growing the clumps of cells in mass culture in an enriched medium containing particular growth factors allows for active cell proliferation and subculture. Clonal culture having plating efficiencies of up to 40% or greater may be obtained using individual cells derived from the mass culture by plating the cells on appropriate substrates in the enriched media. The clonal growth of cells so obtained is suitable for a quantitative assessment of the cytotoxicity of particular treatment. An exemplary assay for assessing the cytotoxicity of the drug adriamycin is presented.

CSF-1R as an inhibitor of apoptosis and promoter of proliferation, migration and invasion of canine mammary cancer cells

PubMed Central

2013-01-01

Background Tumor-associated macrophages (TAMs) have high impact on the cancer development because they can facilitate matrix invasion, angiogenesis, and tumor cell motility. It gives cancer cells the capacity to invade normal tissues and metastasize. The signaling of colony-stimulating factor-1 receptor (CSF-1R) which is an important regulator of proliferation and differentiation of monocytes and macrophages regulates most of the tissue macrophages. However, CSF-1R is expressed also in breast epithelial tissue during some physiological stages i.g.: pregnancy and lactation. Its expression has been also detected in various cancers. Our previous study has showed the expression of CSF-1R in all examined canine mammary tumors. Moreover, it strongly correlated with grade of malignancy and ability to metastasis. This study was therefore designed to characterize the role of CSF-1R in canine mammary cancer cells proliferation, apoptosis, migration, and invasion. As far as we know, the study presented hereby is a pioneering experiment in this field of veterinary medicine. Results We showed that csf-1r silencing significantly increased apoptosis (Annexin V test), decreased proliferation (measured as Ki67 expression) and decreased migration (“wound healing” assay) of canine mammary cancer cells. Treatment of these cells with CSF-1 caused opposite effect. Moreover, csf-1r knock-down changed growth characteristics of highly invasive cell lines on Matrigel matrix, and significantly decreased the ability of these cells to invade matrix. CSF-1 treatment increased invasion of cancer cells. Conclusion The evidence of the expression and functional role of the CSF-1R in canine mammary cancer cells indicate that CSF-1R targeting may be a good therapeutic approach. PMID:23561040

A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes

PubMed Central

Zhao, Xiangshan; Malhotra, Gautam K.; Band, Hamid; Band, Vimla

2011-01-01

Introduction: Emerging evidence suggests a direct role of cancer stem cells (CSCs) in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs) immortalized with human telomerase reverse transcriptase (hTERT) possess properties of mammary stem / progenitor cells. Materials and Methods: In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with γ-radiation), share with hTERT, the ability to maintain mammary stem / progenitor cells. Results: The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC. Conclusions: Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers. PMID:22279424

A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes.

PubMed

Zhao, Xiangshan; Malhotra, Gautam K; Band, Hamid; Band, Vimla

2011-01-01

Emerging evidence suggests a direct role of cancer stem cells (CSCs) in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs) immortalized with human telomerase reverse transcriptase (hTERT) possess properties of mammary stem / progenitor cells. In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with γ-radiation), share with hTERT, the ability to maintain mammary stem / progenitor cells. The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC. Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers.

Lactation stage-dependent expression of transporters in rat whole mammary gland and primary mammary epithelial organoids.

PubMed

Gilchrist, Samuel E; Alcorn, Jane

2010-04-01

Since solute carrier (SLC) and ATP-binding cassette (ABC) transporters play pivotal roles in the transport of both nutrients and drugs into breast milk, drug-nutrient transport interactions at the lactating mammary gland are possible. Our purpose was to characterize lactation stage-dependent changes in transporter expression in rat mammary gland and isolated mammary epithelial organoids (MEO) to provide additional insight for the safe use of maternal medications during breastfeeding. We used quantitative reverse transcription-polymerase chain reaction to assess the temporal expression patterns of SLC and ABC transporters in rat mammary gland and isolated MEO at different stages of lactation. In whole mammary gland five distinct patterns of expression emerged relative to late gestation: (i) decreasing throughout lactation (Mdr1a, Mdr1b, Mrp1, Octn2, Ent2, Ent3, Ncbt2, Mtx1); (ii) prominent increase in early lactation, which may remain elevated or decline with advancing lactation (Octn1, Cnt2, Cnt3, Ent1, Pept1, Pept2); (iii) constant but decreasing later in lactation (Octn3, Dmt1); (iv) increasing until mid-to-late lactation (Oct1, Cnt1); and (v) prominent increase late in lactation (Ncbt1). In isolated MEO (an enriched source of mammary epithelial cells) major differences in expression patterns were noted for Octn3, Ncbt1, and Mtx1, but otherwise were reasonably similar with the whole mammary gland. In conclusion our study augments existing data on transporter expression in the lactating mammary gland. These data should facilitate investigations into lactation-stage dependent changes in drug or nutrient milk-to-serum concentration ratios, the potential for drug- or disease-transporter interactions, and mechanistic studies of transporter function in the lactating mammary gland.

Canonical Wnt Signaling as a Specific Marker of Normal and Tumorigenic Mammary Stem Cells

DTIC Science & Technology

2013-02-01

get enough sorted mammary cells for the transplantation experiments. We are currently working with our Flow Cytometry Core to sort Lin-/CD24+/CD49...activity our flow cytometry data suggests t here is a 2-fold increase in the number of FOG+ MEC’s in BATgal animals compared to contro ls which...this populat ion of cells is enriched for stem cell activity. Flow cytometry will determine the percentage of FOG+ cells within pre-neoplastic BATgai

Combined Allogeneic Tumor Cell Vaccination and Systemic Interleukin 12 Prevents Mammary Carcinogenesis in HER-2/neu Transgenic Mice

PubMed Central

Nanni, Patrizia; Nicoletti, Giordano; De Giovanni, Carla; Landuzzi, Lorena; Di Carlo, Emma; Cavallo, Federica; Pupa, Serenella M.; Rossi, Ilaria; Colombo, Mario P.; Ricci, Cinzia; Astolfi, Annalisa; Musiani, Piero; Forni, Guido; Lollini, Pier-Luigi

2001-01-01

Transgenic Balb/c mice expressing the transforming rat HER-2/neu oncogene develop early and multifocal mammary carcinomas. Within the first 5 months of life the tissue-specific expression of HER-2/neu causes a progression in all their 10 mammary glands from atypical hyperplasia to invasive carcinoma. It was previously observed that chronic administration of interleukin (IL)-12 increased tumor latency, but every mouse eventually succumbed to multiple carcinomas. A significant improvement in tumor prevention was sought by administering allogeneic mammary carcinoma cells expressing HER-2/neu combined with systemic IL-12. This treatment reduced tumor incidence by 90% and more than doubled mouse lifetime. For the maximum prevention p185neu antigen must be expressed by allogeneic cells. IL-12 treatment strongly increased the cell vaccine efficacy. The mammary glands of mice receiving the combined treatment displayed a markedly reduced epithelial cell proliferation, angiogenesis, and HER-2/neu expression, while the few hyperplastic foci were heavily infiltrated by granulocytes, macrophages, and CD8+ lymphocytes. Specific anti–HER-2/neu antibodies were produced and a nonpolarized activation of CD4+ and CD8+ cells secreting IL-4 and interferon (IFN)-γ were evident. A central role for IFN-γ in the preventive effect was proven by the lack of efficacy of vaccination in IFN-γ gene knockout HER-2/neu transgenic Balb/c mice. A possible requirement for IFN-γ is related to its effect on antibody production, in particular on IgG2a and IgG2b subclasses, that were not induced in IFN-γ knockout HER-2/neu mice. In conclusion, our data show that an allogeneic HER-2/neu–expressing cell vaccine combined with IL-12 systemic treatment can prevent the onset of genetically determined tumors. PMID:11696586

Bee Venom Decreases LPS-Induced Inflammatory Responses in Bovine Mammary Epithelial Cells.

PubMed

Jeong, Chang Hee; Cheng, Wei Nee; Bae, Hyojin; Lee, Kyung Woo; Han, Sang Mi; Petriello, Michael C; Lee, Hong Gu; Seo, Han Geuk; Han, Sung Gu

2017-10-28

The world dairy industry has long been challenged by bovine mastitis, an inflammatory disease, which causes economic loss due to decreased milk production and quality. Attempts have been made to prevent or treat this disease with multiple approaches, primarily through increased abuse of antibiotics, but effective natural solutions remain elusive. Bee venom (BV) contains a variety of peptides ( e.g. , melittin) and shows multiple bioactivities, including prevention of inflammation. Thus, in the current study, it was hypothesized that BV can reduce inflammation in bovine mammary epithelial cells (MAC-T). To examine the hypothesis, cells were treated with LPS (1 μg/ml) to induce an inflammatory response and the anti-inflammatory effects of BV (2.5 and 5 μg/ml) were investigated. The cellular mechanisms of BV against LPS-induced inflammation were also investigated. Results showed that BV can attenuate expression of an inflammatory protein, COX2, and pro-inflammatory cytokines such as IL-6 and TNF-α. Activation of NF-κB, an inflammatory transcription factor, was significantly downregulated by BV in cells treated with LPS, through dephosphorylation of ERK1/2. Moreover, pretreatment of cells with BV attenuated LPS-induced production of intracellular reactive oxygen species ( e.g. , superoxide anion). These results support our hypothesis that BV can decrease LPS-induced inflammatory responses in bovine mammary epithelial cells through inhibition of oxidative stress, NF-κB, ERK1/2, and COX-2 signaling.

Basal cell adenocarcinoma of minor salivary and seromucous glands of the head and neck region.

PubMed

Fonseca, I; Soares, J

1996-05-01

Basal cell adenocarcinoma of salivary glands is an uncommon and recently described entity occurring almost exclusively at the major salivary glands. This report provides an overview of the clinicopathologic profile of this neoplasm by including the personal experience on the clinical features, microscopic and ultrastructural characteristics, proliferation activity, and DNA tumor patterns of 12 lesions occurring at the minor salivary glands of the head and neck region, where basal cell adenocarcinoma is probably an underecognized entity, previously reported under different designations. Basal cell adenocarcinoma predominates at the seventh decade without sex preference. The tumors affecting the minor salivary glands occur most frequently at the oral cavity (jugal mucosa, palate) and the upper respiratory tract. The prevalent histologic tumor pattern is represented by solid neoplastic aggregates with a peripheral cell palisading arrangement frequently delineated by basement membrane-like material. The neoplastic clusters are formed by two cell populations: the small dark cell type (that predominates) and a large cell type. Necrosis, either of the comedo or the apoptotic type, is a frequent finding. Perineural growth occurs in 50% of the cases and vascular permeation in 25%. Immunohistochemistry identifies a dual differentiation with a reactivity pattern indicative of ductal epithelial and myoepithelial differentiation, which can be confirmed by electron microscopy. The differential diagnosis of the neoplasm includes its benign counterpart, the basal cell adenoma, solid variant of adenoid cystic carcinoma, undifferentiated carcinoma, and basaloid squamous carcinoma. The tumors recur more frequently than lesions originating in major salivary glands. Mortality is associated with the anatomic site of the lesion, advanced stage, residual neoplasia at surgery, and tumor recurrence. The importance of recognizing basal cell adenocarcinoma outside major salivary glands is

Elevated circulating IGF-I promotes mammary gland development and proliferation.

PubMed

Cannata, Dara; Lann, Danielle; Wu, Yingjie; Elis, Sebastien; Sun, Hui; Yakar, Shoshana; Lazzarino, Deborah A; Wood, Teresa L; Leroith, Derek

2010-12-01

Animal studies have shown that IGF-I is essential for mammary gland development. Previous studies have suggested that local IGF-I rather than circulating IGF-I is the major mediator of mammary gland development. In the present study we used the hepatic IGF-I transgenic (HIT) and IGF-I knockout/HIT (KO-HIT) mouse models to examine the effects of enhanced circulating IGF-I on mammary development in the presence and absence of local IGF-I. HIT mice express the rat IGF-I transgene under the transthyretin promoter in the liver and have elevated circulating IGF-I and normal tissue IGF-I levels. The KO-HIT mice have no tissue IGF-I and increased circulating IGF-I. Analysis of mammary gland development reveals a greater degree of complexity in HIT mice as compared to control and KO-HIT mice, which demonstrate similar degrees of mammary gland complexity. Immunohistochemical evaluation of glands of HIT mice also suggests an enhanced degree of proliferation of the mammary gland, whereas KO-HIT mice exhibit mammary gland proliferation similar to control mice. In addition, HIT mice have a higher percentage of proliferating myoepithelial and luminal cells than control mice, whereas KO-HIT mice have an equivalent percentage of proliferating myoepithelial and luminal cells as control mice. Thus, our findings show that elevated circulating IGF-I levels are sufficient to promote normal pubertal mammary epithelial development. However, HIT mice demonstrate more pronounced mammary gland development when compared to control and KO-HIT mice. This suggests that both local and endocrine IGF-I play roles in mammary gland development and that elevated circulating IGF-I accelerates mammary epithelial proliferation.

Low-Dose Alkylphenol Exposure Promotes Mammary Epithelium Alterations and Transgenerational Developmental Defects, But Does Not Enhance Tumorigenic Behavior of Breast Cancer Cells

PubMed Central

Chamard-Jovenin, Clémence; Thiebaut, Charlène; Chesnel, Amand; Bresso, Emmanuel; Morel, Chloé; Smail-Tabbone, Malika; Devignes, Marie-Dominique; Boukhobza, Taha; Dumond, Hélène

2017-01-01

Fetal and neonatal exposure to long-chain alkylphenols has been suspected to promote breast developmental disorders and consequently to increase breast cancer risk. However, disease predisposition from developmental exposures remains unclear. In this work, human MCF-10A mammary epithelial cells were exposed in vitro to a low dose of a realistic (4-nonylphenol + 4-tert-octylphenol) mixture. Transcriptome and cell-phenotype analyses combined to functional and signaling network modeling indicated that long-chain alkylphenols triggered enhanced proliferation, migration ability, and apoptosis resistance and shed light on the underlying molecular mechanisms which involved the human estrogen receptor alpha 36 (ERα36) variant. A male mouse-inherited transgenerational model of exposure to three environmentally relevant doses of the alkylphenol mix was set up in order to determine whether and how it would impact on mammary gland architecture. Mammary glands from F3 progeny obtained after intrabuccal chronic exposure of C57BL/6J P0 pregnant mice followed by F1–F3 male inheritance displayed an altered histology which correlated with the phenotypes observed in vitro in human mammary epithelial cells. Since cellular phenotypes are similar in vivo and in vitro and involve the unique ERα36 human variant, such consequences of alkylphenol exposure could be extrapolated from mouse model to human. However, transient alkylphenol treatments combined to ERα36 overexpression in mammary epithelial cells were not sufficient to trigger tumorigenesis in xenografted Nude mice. Therefore, it remains to be determined if low-dose alkylphenol transgenerational exposure and subsequent abnormal mammary gland development could account for an increased breast cancer susceptibility. PMID:29109696

Role of the Stem Cell Niche in Hormone-induced Tumorigenesis in Fetal Mouse Mammary Epithelium

DTIC Science & Technology

2006-08-01

Develop an immunohistochemical method for identifying stem cells and stem cell niches, and to use this to determine if in utero estrogenic...overstimulation causes changes in the number of stem cells or their niches. To extend the power of ex vivo stem cell isolation and enumeration by providing a...marginal success due primarily to 1) most antibodies previously reputed to be stem cell specific turned out to be present in differentiated mammary

Analysis of papillary renal adenocarcinoma.

PubMed

Mydlo, J H; Bard, R H

1987-12-01

A retrospective review was conducted comparing the angiographic findings, tumor volumes, staging, and survival of patients with papillary renal adenocarcinoma as compared with the more common clear and granular cell renal adenocarcinoma. The data suggest that the papillary histopathologic organization confers an improved prognosis, which concurs with previous findings. We speculate on why this tumor behaves differently from clear cell carcinoma.

Keratinocyte growth factor is a growth factor for mammary epithelium in vivo. The mammary epithelium of lactating rats is resistant to the proliferative action of keratinocyte growth factor.

PubMed Central

Ulich, T. R.; Yi, E. S.; Cardiff, R.; Yin, S.; Bikhazi, N.; Biltz, R.; Morris, C. F.; Pierce, G. F.

1994-01-01

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. KGF is secreted by stromal cells and affects epithelial but not mesenchymal cell proliferation. KGF injected intravenously was found to cause dramatic proliferation of mammary epithelium in the mammary glands of rats. KGF causes ductal neogenesis and intraductal epithelial hyperplasia but not lobular differentiation in nulliparous female rats. KGF causes ductal and lobular epithelial hyperplasia in male rats. KGF causes proliferation of ductal and acinar cells in the mammary glands of pregnant rats. On the other hand, the ductal epithelium of lactating postpartum rats is resistant to the proliferative action of KGF. The mammary glands of lactating rats did not express less KGF receptor mRNA than the glands of pregnant rats, suggesting that the resistance of the ductal epithelium to KGF during lactation is not related to KGF receptor mRNA down-regulation. The mammary glands of both pregnant and postpartum lactating rats express KGF mRNA with more KGF present in the glands of lactating rats. In conclusion, the KGF and KGF receptor genes are expressed in rat mammary glands and recombinant KGF is a potent growth factor for mammary epithelium. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8178937

Prognostic Value of Occult Isolated Tumour Cells within Regional Lymph Nodes of Dogs with Malignant Mammary Tumours.

PubMed

Coleto, A F; Wilson, T M; Soares, N P; Gundim, L F; Castro, I P; Guimarães, E C; Bandarra, M B; Medeiros-Ronchi, A A

2018-01-01

Canine mammary tumours (CMTs) are the most common type of neoplasm in bitches. As in women, the presence of metastasis in regional lymph nodes is an important prognostic factor in bitches with mammary carcinomas, but the clinical significance of occult isolated tumour cells (ITCs) within lymph nodes is still undefined in this species. The effectiveness of immunohistochemistry (IHC) in identifying occult ITCs and micrometastasis (MIC) was compared with that of the conventional haematoxylin and eosin staining technique. The relationship between tumour size, histological type, histological grade and the presence of metastasis was evaluated. The overall survival (OS) of female dogs with occult mammary carcinomas and ITCs within lymph nodes was analysed. Fragments of mammary carcinoma and regional lymph nodes of 59 female dogs were also evaluated. Histological sections of mammary carcinoma and lymph node samples were studied for tumour diagnosis and lymph node samples were tested by IHC using a pan-cytokeratin antibody. It was found that 35.2% of occult ITCs and 2.8% of hidden MIC were detected when IHC was used. There was a good correlation between the size of the tumour and metastasis to the lymph nodes (P = 0.77). ITCs were observed more frequently in the medullary region (60.7%) and metastases in the cortical region (44.4%). There was no significant difference in the OS between female dogs with occult ITCs and lymph nodes without ITCs. IHC can detect occult tumour cells in lymph nodes that are negative by histopathological examination. Female dogs with nodal ITCs do not have lower survival. Copyright © 2017 Elsevier Ltd. All rights reserved.

CD24 cell surface expression in Mvt1 mammary cancer cells serves as a biomarker for sensitivity to anti-IGF1R therapy.

PubMed

Rostoker, Ran; Ben-Shmuel, Sarit; Rashed, Rola; Shen Orr, Zila; LeRoith, Derek

2016-05-14

The pro-tumorigenic effects of the insulin-like growth factor receptor (IGF1R) are well described. IGF1R promotes cancer cell survival and proliferation and prevents apoptosis, and, additionally it was shown that IGF1R levels are significantly elevated in most common human malignancies including breast cancer. However, results from phase 3 clinical trials in unselected patients demonstrated lack of efficacy for anti-IGF1R therapy. These findings suggest that predictive biomarkers are greatly warranted in order to identify patients that will benefit from anti-IGF1R therapeutic strategies. Using the delivery of shRNA vectors into the Mvt1 cell line, we tested the role of the IGF1R in the development of mammary tumors. Based on CD24 cell surface expression, control and IGF1R-knockdown (IGF1R-KD) cells were FACS sorted into CD24(-) and CD24(+) subsets and further characterized in vitro. The tumorigenic capacity of each was determined following orthotopic inoculation into the mammary fat pad of female mice. Tumor cells were FACS characterized upon sacrifice to determine IGF1R effect on the plasticity of this cell's phenotype. Metastatic capacity of the cells was assessed using the tail vein assay. In this study we demonstrate that downregulation of the IGF1R specifically in cancer cells expressing CD24 on the cell surface membrane affect both their morphology (from mesenchymal-like into epithelial-like morphology) and phenotype in vitro. Moreover, we demonstrate that IGF1R-KD abolished both CD24(+) cells capacity to form mammary tumors and lung metastatic lesions. We found in both cells and tumors a marked upregulation in CTFG and a significant reduction of SLP1 expression in the CD24(+)/IGF1R-KD; tumor-suppressor and tumor-promoting genes respectively. Moreover, we demonstrate here that the IGF1R is essential for the maintenance of stem/progenitor-like cancer cells and we further demonstrate that IGF1R-KD induces in vivo differentiation of the CD24(+) cells toward the

Regulation of Mammary Stem Cell Quiescence via Post-Translational Modification of DeltaNp63alpha

DTIC Science & Technology

2012-12-01

This document is the Annual Summary Report on the training grant awarded to Andrew DeCastro entitled Regulation of Mammary Stem Cell Quiescence via...screen) mediated phosphorylation of deltaNPdelta3 on stem cell behavior and mitotic activity. Task 1 aims to determine the effects of wild-type, phospho...ablative and phospho-mimetic alleles of deltaNP63delta phosphorylation on stem cell behavior in vitro. Thus far, we demonstrate that stem cell enriched

Inhibition of the mammary carcinoma angiogenic switch in C3(1)/SV40 transgenic mice by a mutated form of human endostatin.

PubMed

Calvo, Alfonso; Yokoyama, Yumi; Smith, Lois E; Ali, Iqbal; Shih, Shu-Ching; Feldman, Andrew L; Libutti, Steven K; Sundaram, Ramakrishnan; Green, Jeffrey E

2002-09-20

Cancer therapies based on the inhibition of angiogenesis by endostatin have recently been developed. We demonstrate that a mutated form of human endostatin (P125A) can inhibit the angiogenic switch in the C3(1)/Tag mammary cancer model. P125A has a stronger growth-inhibitory effect on endothelial cell proliferation than wild-type endostatin. We characterize the angiogenic switch, which occurs during the transition from preinvasive lesions to invasive carcinoma in this model, and which is accompanied by a significant increase in total protein levels of vascular endothelial growth factor (VEGF) and an invasion of blood vessels. Expression of the VEGF(188) mRNA isoform, however, is suppressed in invasive carcinomas. The VEGF receptors fetal liver kinase-1 (Flk-1) and Fms-like tyrosine kinase-1 (Flt-1) become highly expressed in epithelial tumor and endothelial cells in the mammary carcinomas, suggesting a potential autocrine effect for VEGF on tumor cell growth. Angiopoietin-2 mRNA levels are also increased during tumor progression. CD-31 (platelet-endothelial cell adhesion molecule [PECAM]) staining revealed that blood vessels developed in tumors larger than 1 mm The administration of P125A human endostatin in C3(1)/Tag females resulted in a significant delay in tumor onset, decreased tumor multiplicity and tumor burden and prolonged survival of the animals. Endostatin treatment did not reduce the number of preinvasive lesions, proliferation rates or apoptotic index, compared with controls. However, mRNA levels of a variety of proangiogenic factors (VEGF, VEGF receptors Flk-1 and Flt-1, angiopoietin-2, Tie-1, cadherin-5 and PECAM) were significantly decreased in the endostatin-treated group compared with controls. These results demonstrate that P125A endostatin inhibits the angiogenic switch during mammary gland adenocarcinoma tumor progression in the C3(1)/Tag transgenic model. Copyright 2002 Wiley-Liss, Inc.

[Increased expressions of peripheral PD-1+ lymphocytes and CD4+CD25+FOXP3+ T cells in gastric adenocarcinoma patients].

PubMed

Li, Hao; Li, Songyan; Hu, Shidong; Zou, Guijun; Hu, Zilong; Wei, Huahua; Wang, Yufeng; Du, Xiaohui

2017-01-01

Objective To detect the frequencies of peripheral programmed death-1 + (PD-1 + ) lymphocytes and CD4 + CD25 + FOXP3 + regulatory T cells in patients with gastric adenocarcinoma. Methods The study enrolled 29 patients with gastric adenocarcinoma and 29 age- and sex-matched healthy controls. Frequencies of PD-1 + lymphocytes and CD4 + CD25 + FOXP3 + regulatory T cells were detected using flow cytometry. Results The number of PD-1 + lymphocytes and CD4 + CD25 + FOXP3 + regulatory T cells in peripheral blood was higher in patients with gastric adenocarcinoma than that in the control group. Moreover, linear correlation analysis indicated a positive correlation between PD-1 expression and frequency of CD4 + CD25 + FOXP3 + regulatory T cells in peripheral blood of the patients. Conclusion Gastric adenocarcinoma patients present with increased PD-1 + lymphocytes and CD4 + CD25 + FOXP3 + regulatory T cells in the peripheral blood.

Analysis of Normal Human Mammary Epigenomes Reveals Cell-Specific Active Enhancer States and Associated Transcription Factor Networks.

PubMed

Pellacani, Davide; Bilenky, Misha; Kannan, Nagarajan; Heravi-Moussavi, Alireza; Knapp, David J H F; Gakkhar, Sitanshu; Moksa, Michelle; Carles, Annaick; Moore, Richard; Mungall, Andrew J; Marra, Marco A; Jones, Steven J M; Aparicio, Samuel; Hirst, Martin; Eaves, Connie J

2016-11-15

The normal adult human mammary gland is a continuous bilayered epithelial system. Bipotent and myoepithelial progenitors are prominent and unique components of the outer (basal) layer. The inner (luminal) layer includes both luminal-restricted progenitors and a phenotypically separable fraction that lacks progenitor activity. We now report an epigenomic comparison of these three subsets with one another, with their associated stromal cells, and with three immortalized, non-tumorigenic human mammary cell lines. Each genome-wide analysis contains profiles for six histone marks, methylated DNA, and RNA transcripts. Analysis of these datasets shows that each cell type has unique features, primarily within genomic regulatory regions, and that the cell lines group together. Analyses of the promoter and enhancer profiles place the luminal progenitors in between the basal cells and the non-progenitor luminal subset. Integrative analysis reveals networks of subset-specific transcription factors. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Prevention of carcinogenesis of mouse mammary epithelial cells RIII/MG by epigallocatechin gallate.

PubMed

Yanaga, Hiroshi; Fujii, Teruhiko; Koga, Toshihiro; Araki, Ruriko; Shirouzu, Kazuo

2002-09-01

The chemopreventive effect of the polyphenols abundant in green tea on carcinogenesis has been attracting attention in recent years. Among tea polyphenols, epigallocatechin gallate (EGCG) has been studied as a preventive substance for carcinogenesis. We investigated the chemopreventive effect in a group treated with EGCG in vitro and in vivo using mouse mammary epithelial cells RIII/MG. In the in vitro experiment, crude catechin (catechin) containing 50% or more EGCG significantly inhibited the growth of RIII/MG cells, which were precancerous cultured cells. Many cells died, and a DNA ladder was observed. In the in vivo experiment, RIII/MG cells formed a tumor after 13 weeks in a group without catechin treatment, and the tumor formation rate in the 20th week was 40%. In a group treated with 0.1% catechin, a tumor began to grow in the 13th week, and the tumor formation rate in the 20th week was 20%. In a group treated with 1% catechin, no tumor was detected even in the 20th week. There was no significant difference in the change in body weight between the catechin treatment groups and the non-treatment group during the observation period. Tissue samples were stained by the nick-end-labeling method and apoptosis was observed in many cells. Based on the above findings, EGCG inhibited growth in the mouse viral mammary epithelial carcinogenesis model RIII/MG, and induced apoptosis, suggesting a clinical usefulness of EGCG as a chemopreventive substance.

Genotoxic stress induces Sca-1 expressing metastatic mammary cancer cells.

PubMed

Gong, Jianlin; Lang, Benjamin J; Weng, Desheng; Eguchi, Takanori; Murshid, Ayesha; Borges, Thiago J; Doshi, Sachin; Song, Baizheng; Stevenson, Mary Ann; Calderwood, Stuart K

2018-05-08

We describe a cell damage-induced phenotype in mammary carcinoma cells involving acquisition of enhanced migratory and metastatic properties. Induction of this state by radiation required increased activity of the Ptgs2 gene product cyclooxygenase 2 (Cox2), secretion of its bioactive lipid product prostaglandin E2 (PGE2) and the activity of the PGE2 receptor EP4. Although largely transient, decaying to low levels in a few days to a week, this phenotype was cumulative with damage and levels of cell markers Sca-1 and ALDH1 increased with treatment dose. The Sca-1 + , metastatic phenotype was inhibited by both Cox2 inhibitors and PGE2 receptor antagonists suggesting novel approaches to radiosensitization. Molecular Oncology (2018) © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Stromal matrix metalloproteinase-11 is involved in the mammary gland postnatal development.

PubMed

Tan, J; Buache, E; Alpy, F; Daguenet, E; Tomasetto, C-L; Ren, G-S; Rio, M-C

2014-07-31

MMP-11 is a bad prognosis paracrine factor in invasive breast cancers. However, its mammary physiological function remains largely unknown. In the present study we have investigated MMP-11 function during postnatal mammary gland development and function using MMP-11-deficient (MMP-11-/-) mice. Histological and immunohistochemical analyses as well as whole-mount mammary gland staining show alteration of the mammary gland in the absence of MMP-11, where ductal tree, alveolar structures and milk production are reduced. Moreover, a series of transplantation experiments allowed us to demonstrate that MMP-11 exerts an essential local paracrine function that favors mammary gland branching and epithelial cell outgrowth and invasion through adjacent connective tissues. Indeed, MMP-11-/- cleared fat pads are not permissive for wild-type epithelium development, whereas MMP-11-/- epithelium transplants grow normally when implanted in wild-type cleared fat pads. In addition, using primary mammary epithelial organoids, we show in vitro that this MMP-11 pro-branching effect is not direct, suggesting that MMP-11 acts via production/release of stroma-associated soluble factor(s). Finally, the lack of MMP-11 leads to decreased periductal collagen content, suggesting that MMP-11 has a role in collagen homeostasis. Thus, local stromal MMP-11 might also regulate mammary epithelial cell behavior mechanically by promoting extracellular matrix stiffness. Collectively, the present data indicate that MMP-11 is a paracrine factor involved during postnatal mammary gland morphogenesis, and support the concept that the stroma strongly impact epithelial cell behavior. Interestingly, stromal MMP-11 has previously been reported to favor malignant epithelial cell survival and promote cancer aggressiveness. Thus, MMP-11 has a paracrine function during mammary gland development that might be harnessed to promote tumor progression, exposing a new link between development and malignancy.

Retinoids Modulate Expression of the Endocytic Partners Megalin, Cubilin, and Disabled-2 and Uptake of Vitamin D-Binding Protein in Human Mammary Cells12

PubMed Central

Chlon, Timothy M.; Taffany, David A.; Welsh, JoEllen; Rowling, Matthew J.

2008-01-01

The major circulating form of vitamin D, 25-hydroxycholecalciferol (25D3), circulates bound to vitamin D-binding protein (DBP). Prior to activation to 1,25-dihydroxycholecalciferol in the kidney, the 25D3-DBP complex is internalized via receptor-mediated endocytosis, which is absolutely dependent on the membrane receptors megalin and cubilin and the adaptor protein disabled-2 (Dab2). We recently reported that mammary epithelial cells (T-47D) expressing megalin, cubilin, and Dab2 rapidly internalize DBP via endocytosis, whereas cells that do not express all 3 proteins (MCF-7) do not. The objectives of this study were to characterize megalin, cubilin, and Dab2 expression and transport of DBP in human mammary epithelial cells. Using immunoblotting and real-time PCR, we found that megalin, cubilin, and Dab2 were expressed and dose dependently induced by all-trans-retinoic acid (RA) in T-47D human breast cancer cells and that RA-treated T-47D cells exhibited enhanced DBP internalization. These are the first studies to our knowledge to demonstrate that mammary epithelial cells express megalin, cubilin, and Dab2, which are enhanced during differentiation and may explain, at least in part, our finding that receptor-mediated endocytosis of DBP is upregulated in differentiated mammary epithelial cells. PMID:18567755

Culture models of human mammary epithelial cell transformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stampfer, Martha R.; Yaswen, Paul

2000-11-10

Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the know in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcomemore » stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TFGB. We propose th at overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.« less

Serotonin suppresses β-casein expression via PTP1B activation in human mammary epithelial cells.

PubMed

Chiba, Takeshi; Maeda, Tomoji; Sanbe, Atsushi; Kudo, Kenzo

2016-04-22

Serotonin (5-hydroxytriptamine, 5-HT) has an important role in milk volume homeostasis within the mammary gland during lactation. We have previously shown that the expression of β-casein, a differentiation marker in mammary epithelial cells, is suppressed via 5-HT-mediated inhibition of signal transduction and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial MCF-12A cell line. In addition, the reduction of β-casein in turn was associated with 5-HT7 receptor expression in the cells. The objective of this study was to determine the mechanisms underlying the 5-HT-mediated suppression of β-casein and STAT5 phosphorylation. The β-casein level and phosphorylated STAT5 (pSTAT5)/STAT5 ratio in the cells co-treated with 5-HT and a protein kinase A (PKA) inhibitor (KT5720) were significantly higher than those of cells treated with 5-HT alone. Exposure to 100 μM db-cAMP for 6 h significantly decreased the protein levels of β-casein and pSTAT5 and the pSTAT5/STAT5 ratio, and significantly increased PTP1B protein levels. In the cells co-treated with 5-HT and an extracellular signal-regulated kinase1/2 (ERK) inhibitor (FR180294) or Akt inhibitor (124005), the β-casein level and pSTAT5/STAT5 ratio were equal to those of cells treated with 5-HT alone. Treatment with 5-HT significantly induced PTP1B protein levels, whereas its increase was inhibited by KT5720. In addition, the PTP1B inhibitor sc-222227 increased the expression levels of β-casein and the pSTAT5/STAT5 ratio. Our observations indicate that PTP1B directly regulates STAT5 phosphorylation and that its activation via the cAMP/PKA pathway downstream of the 5-HT7 receptor is involved in the suppression of β-casein expression in MCF-12A cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Enhancement of NAD+-dependent SIRT1 deacetylase activity by methylselenocysteine resets the circadian clock in carcinogen-treated mammary epithelial cells

PubMed Central

Fang, Mingzhu; Guo, Wei-Ren; Park, Youngil; Kang, Hwan-Goo; Zarbl, Helmut

2015-01-01

We previously reported that dietary methylselenocysteine (MSC) inhibits N-methyl-N-nitrosourea (NMU)-induced mammary tumorigenesis by resetting circadian gene expression disrupted by the carcinogen at the early stage of tumorigenesis. To investigate the underlying mechanism, we developed a circadian reporter system comprised of human mammary epithelial cells with a luciferase reporter driven by the promoter of human PERIOD 2 (PER2), a core circadian gene. In this in vitro model, NMU disrupted cellular circadian rhythm in a pattern similar to that observed with SIRT1-specific inhibitors; in contrast, MSC restored the circadian rhythms disrupted by NMU and protected against SIRT1 inhibitors. Moreover, NMU inhibited intracellular NAD+/NADH ratio and reduced NAD+-dependent SIRT1 activity in a dose-dependent manner, while MSC restored NAD+/NADH and SIRT1 activity in the NMU-treated cells, indicating that the NAD+-SIRT1 pathway was targeted by NMU and MSC. In rat mammary tissue, a carcinogenic dose of NMU also disrupted NAD+/NADH oscillations and decreased SIRT1 activity; dietary MSC restored NAD+/NADH oscillations and increased SIRT1 activity in the mammary glands of NMU-treated rats. MSC-induced SIRT1 activity was correlated with decreased acetylation of BMAL1 and increased acetylation of histone 3 lysine 9 at the Per2 promoter E-Box in mammary tissue. Changes in SIRT1 activity were temporally correlated with loss or restoration of rhythmic Per2 mRNA expression in NMU-treated or MSC-rescued rat mammary glands, respectively. Together with our previous findings, these results suggest that enhancement of NAD+-dependent SIRT1 activity contributes to the chemopreventive efficacy of MSC by restoring epigenetic regulation of circadian gene expression at early stages of mammary tumorigenesis. PMID:26544624

Normal and cancer mammary stem cells evade interferon-induced constraint through the miR-199a-LCOR Axis

PubMed Central

Celià-Terrassa, Toni; Liu, Daniel; Choudhury, Abrar; Hang, Xiang; Wei, Yong; Zamalloa, Jose; Alfaro-Aco, Raymundo; Chakrabarti, Rumela; Jiang, Yi-Zhou; Koh, Bong Ihn; Smith, Heath; DeCoste, Christina; Li, Jun-Jing; Shao, Zhi-Ming; Kang, Yibin

2017-01-01

Tumor-initiating cells (TICs), or cancer stem cells (CSC), possess stem cell-like properties observed in normal adult tissue stem cells. Normal and cancerous stem cells may therefore share regulatory mechanisms for maintaining self-renewing capacity and resisting differentiation elicited by cell-intrinsic or microenvironmental cues. Here, we show that miR-199a promotes stem cell properties in mammary stem cells (MaSCs) and breast CSCs by directly repressing nuclear receptor corepressor LCOR, which primes interferon (IFN) responses. Elevated miR-199a expression in stem cell-enriched populations protects normal and malignant stem-like cells from differentiation and senescence induced by IFNs that are produced by epithelial and immune cells in the mammary gland. Importantly, the miR-199a-LCOR-IFN axis is activated in poorly differentiated ER− breast tumors, functionally promotes tumor initiation and metastasis, and is associated with poor clinical outcome. Our study therefore reveals a common mechanism shared by normal and malignant stem cells to protect them from suppressive immune cytokine signaling. PMID:28530657

Insulin receptor substrate-2 regulates aerobic glycolysis in mouse mammary tumor cells via glucose transporter 1.

PubMed

Pankratz, Shannon L; Tan, Ernest Y; Fine, Yumiko; Mercurio, Arthur M; Shaw, Leslie M

2009-01-23

The insulin receptor substrate (IRS) proteins are cytoplasmic adaptor molecules that function as signaling intermediates downstream of activated cell surface receptors. Based on data implicating IRS-2 but not IRS-1 in breast cancer invasion, survival, and metastasis, we assessed the contribution of IRS-1 and IRS-2 to aerobic glycolysis, which is known to impact tumor growth and progression. For this purpose, we used tumor cell lines derived from transgenic mice that express the polyoma virus middle T antigen (PyV-MT) in the mammary gland and that are wild-type (WT) or null for either Irs-1 (Irs-1-/-) or Irs-2 (Irs-2-/-). Aerobic glycolysis, as assessed by the rate of lactic acid production and glucose consumption, was diminished significantly in Irs-2-/- cells when compared with WT and Irs-1-/- cells. Expression of exogenous Irs-2 in Irs-2-/- cells restored the rate of glycolysis to that observed in WT cells. The transcription factor FoxO1 does not appear to be involved in Irs-2-mediated glycolysis. However, Irs-2 does regulate the surface expression of glucose transporter 1 (Glut1) as assessed by flow cytometry using a Glut1-specific ligand. Suppression of Glut1 expression inhibits Irs-2-dependent invasion, which links glycolysis to mammary tumor progression. Irs-2 was shown to be important for mammalian target of rapamycin (mTor) activation, and Irs-2-dependent regulation of Glut1 surface expression is rapamycin-sensitive. Collectively, our data indicate that Irs-2, but not Irs-1, promotes invasion by sustaining the aerobic glycolysis of mouse mammary tumor cells and that it does so by regulating the mTor-dependent surface expression of Glut1.

Immune cell landscape in therapy-naïve squamous cell and adenocarcinomas of the lung.

PubMed

Brcic, Luka; Stanzer, Stefanie; Krenbek, Dagmar; Gruber-Moesenbacher, Ulrike; Absenger, Gudrun; Quehenberger, Franz; Valipour, Arschang; Lindenmann, Joerg; Stoeger, Herbert; Al Effah, Mohamed; Fediuk, Melanie; Balic, Marija; Popper, Helmut H

2018-04-01

Squamous cell and adenocarcinomas of the lung develop different mechanisms during carcinogenesis to evade attacks of the immune system. Besides the well-known check-point control programmed death 1 and its ligand, many more mechanisms, acting either tumoricidal or in favor of tumor progression, exist. Analysis of the immune cell profiles in resected tissues and bronchoalveolar lavage samples and correlation between them and with overall survival data was performed. In all tumor samples in this study, cells of the immune system expressed a tumor-cooperating phenotype. High numbers of regulatory T cells, or alternatively expression of Vista on lymphocytes was present. Tumoricidal dendritic cells were absent in tumor tissue, and barely present in bronchoalveolar lavage, whereas tumor-friendly monocytoid and plasmocytoid dendritic cells were seen in both. Alveolar macrophages were predominantly differentiated into tumor-cooperating M2 types, whereas tumoricidal M1 macrophages were absent or rare. The expression of PDL1 on tumor cells did not correlate with any other immune cells. Expression of PD1 on lymphocytes was frequently encountered. None of analyzed immune cells showed correlation with overall survival. Immune cells in bronchoalveolar lavage and tissue did not correlate. For the first time, a tissue-based analysis of different immune cells in squamous cell and adenocarcinomas of the lung is provided, trying to explain their potential role in tumor development and progression. Discordant numbers of cells with bronchoalveolar lavage are most probably due to the fact that bronchoalveolar lavage reflects the situation in the whole lung, where chronic obstructive lung disease and other conditions are present.

Isoform-specific function of calpains in cell adhesion disruption: studies in postlactational mammary gland and breast cancer.

PubMed

Rodríguez-Fernández, Lucía; Ferrer-Vicens, Iván; García, Concha; Oltra, Sara S; Zaragozá, Rosa; Viña, Juan R; García-Trevijano, Elena R

2016-09-15

Cleavage of adhesion proteins is the first step for physiological clearance of undesired cells during postlactational regression of the mammary gland, but also for cell migration in pathological states such as breast cancer. The intracellular Ca(2+)-dependent proteases, calpains (CAPNs), are known to cleave adhesion proteins. The isoform-specific function of CAPN1 and CAPN2 was explored and compared in two models of cell adhesion disruption: mice mammary gland during weaning-induced involution and breast cancer cell lines according to tumor subtype classification. In both models, E-cadherin, β-catenin, p-120, and talin-1 were cleaved as assessed by western blot analysis. Both CAPNs were able to cleave adhesion proteins from lactating mammary gland in vitro Nevertheless, CAPN2 was the only isoform found to co-localize with E-cadherin in cell junctions at the peak of lactation. CAPN2/E-cadherin in vivo interaction, analyzed by proximity ligation assay, was dramatically increased during involution. Calpain inhibitor administration prevented the cytosolic accumulation of truncated E-cadherin cleaved by CAPN2. Conversely, in breast cancer cells, CAPN2 was restricted to the nuclear compartment. The isoform-specific expression of CAPNs and CAPN activity was dependent on the breast cancer subtype. However, CAPN1 and CAPN2 knockdown cells showed that cleavage of adhesion proteins and cell migration was mediated by CAPN1, independently of the breast cancer cell line used. Data presented here suggest that the subcellular distribution of CAPN1 and CAPN2 is a major issue in target-substrate recognition; therefore, it determines the isoform-specific role of CAPNs during disruption of cell adhesion in either a physiological or a pathological context. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

The Role of Epithelial-Mesenchymal Transition in the Formation of Normal and Neoplastic Mammary Epithelial Stem Cells

DTIC Science & Technology

2011-09-01

separating stem cell and non- stem cell populations of normal and breast cancer cells and identified EMT transcription factors most likely involved in... stem cell biology. Preliminary results directly demonstrate that transient induction of EMT increases the number of mammary epithelial stem cells...EMT and entrance into a stem - cell state. The outcome of these experiments holds important implications for the mechanisms controlling the formation of

Sesamol induced apoptotic effect in lung adenocarcinoma cells through both intrinsic and extrinsic pathways.

PubMed

Siriwarin, Boondaree; Weerapreeyakul, Natthida

2016-07-25

Sesamol is a phenolic lignan found in sesame seeds (Sesamum indicum L.) and sesame oil. The anticancer effects and molecular mechanisms underlying its apoptosis-inducing effect were investigated in human lung adenocarcinoma (SK-LU-1) cells. Sesamol inhibited SK-LU-1 cell growth with an IC50 value of 2.7 mM and exhibited less toxicity toward normal Vero cells after 48 h of treatment (Selective index = 3). Apoptotic bodies-the hallmark of apoptosis-were observed in sesamol-treated SK-LU-1 cells, stained with DAPI. Sesamol increased the activity of caspase 8, 9, and 3/7, indicating that apoptotic cell death occurred through both extrinsic and intrinsic pathways. Sesamol caused the loss of mitochondrial transmembrane potential signifying intrinsic apoptosis induction. Decreasing Bid expression revealed crosstalk between the intrinsic and extrinsic apoptotic pathways; demonstrating clearly that sesamol induces apoptosis through both pathways in human lung adenocarcinoma (SK-LU-1) cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer.

PubMed

He, Y L; Wu, Y H; He, X N; Liu, F J; He, X Y; Zhang, Y

2009-06-01

Although mammary epithelial cell lines can provide a rapid and reliable indicator of gene expression efficiency of transgenic animals, their short lifespan greatly limits this application. To provide stable and long lifespan cells, goat mammary epithelial cells (GMECs) were transduced with pLNCX2-hTERT by retrovirus-mediated gene transfer. Transduced GMECs were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR), proliferation assays, karyotype analysis, telomerase activity assay, western blotting, soft agar assay, and injection into nude mice. Non-transduced GMECs were used as a control. The hTERT-GMECs had higher telomerase activity and extended proliferative lifespan compared to non-transfected GMECs; even after Passage 50, hTERT-GMECs had a near diploid complement of chromosomes. Furthermore, they did not gain the anchorage-independent growth property and were not associated with a malignant phenotype in vitro or in vivo.

Enhancement of Thermal Damage to Adenocarcinoma Cells by Iron Nanoparticles Modified with MUC1 Aptamer.

PubMed

Guo, Fangqin; Hu, Yan; Yu, Lianyuan; Deng, Xiaoyuan; Meng, Jie; Wang, Chen; Yang, Xian-Da

2016-03-01

Hyperthermia cancer treatment is an adjunctive therapy that aims at killing the tumor cells with excessive heat that is usually generated by metal contrasts exposed to alternating magnetic field. The efficacy of hyperthermia is often limited by the heat damage to normal tissue due to indiscriminate distribution of the metal contrasts within the body. Tumor-targeting metal contrasts may reduce the toxicity of hyperthermia and improve the efficacy of thermotherapy against cancer. MUC1 is a glycoprotein over expressed in most adenocarcinomas, and represents an attractive therapeutic target. In this study, a MUC1 aptamer is conjugated with iron nanoparticles to construct adenocarcinoma-targeting metal contrasts. DNA hybridization studies confirmed that the aptamers were conjugated to the iron nanoparticles. Importantly, more aptamer-modified nanoparticles attached to the MUC1-positive cancer cells compared with the unmodified nanoparticles. Moreover, aptamer-modified nanoparticles significantly enhanced the targeted hyperthermia damage to MUC1-positive cancer cells in vitro (p < 0.05). The results suggest that MUC1 aptamer-modified metal particles may have potential in development of targeted hyperthermia therapy against adenocarcinomas.

Microcalcifications in breast cancer: novel insights into the molecular mechanism and functional consequence of mammary mineralisation

PubMed Central

Cox, R F; Hernandez-Santana, A; Ramdass, S; McMahon, G; Harmey, J H; Morgan, M P

2012-01-01

Background: Mammographic microcalcifications represent one of the most reliable features of nonpalpable breast cancer yet remain largely unexplored and poorly understood. Methods: We report a novel model to investigate the in vitro mineralisation potential of a panel of mammary cell lines. Primary mammary tumours were produced by implanting tumourigenic cells into the mammary fat pads of female BALB/c mice. Results: Hydroxyapatite (HA) was deposited only by the tumourigenic cell lines, indicating mineralisation potential may be associated with cell phenotype in this in vitro model. We propose a mechanism for mammary mineralisation, which suggests that the balance between enhancers and inhibitors of physiological mineralisation are disrupted. Inhibition of alkaline phosphatase and phosphate transport prevented mineralisation, demonstrating that mineralisation is an active cell-mediated process. Hydroxyapatite was found to enhance in vitro tumour cell migration, while calcium oxalate had no effect, highlighting potential consequences of calcium deposition. In addition, HA was also deposited in primary mammary tumours produced by implanting the tumourigenic cells into the mammary fat pads of female BALB/c mice. Conclusion: This work indicates that formation of mammary HA is a cell-specific regulated process, which creates an osteomimetic niche potentially enhancing breast tumour progression. Our findings point to the cells mineralisation potential and the microenvironment regulating it, as a significant feature of breast tumour development. PMID:22233923

Synergistic effects of androgen and estrogen on the mouse uterus and mammary gland.

PubMed

Zhang, Jian; Sun, Yibin; Liu, Yunhai; Sun, Yi; Liao, Dezhong Joshua

2004-10-01

Many studies have suggested that elevated estrogens and androgens may be etiologically related to the development of breast cancer, endometrial cancer and uterine leiomyomas. We and other investigators have previously shown that estrogen and androgen are synergistic in the induction of mammary carcinogenesis in the Noble rat. However, the mechanisms behind the synergy is unknown, and it is unclear whether such synergy is unique for the Noble rat and for the mammary gland. In this study we treated female FVB mice with 17beta-estradiol (E2) and 5alpha-dihydrotestosterone-bezonate (DHT-B), alone and in combination, using silastic tubing for 2-7 months. The results showed that DHT-B alone induced proliferation of uterine endometrial epithelium and myometrial smooth muscle cells, whereas E2 alone induced much more pronounced growth of endometrial epithelium without affecting smooth muscle cells. Combined treatment with E2+DHT-B caused an even more severe hyperplasia of endometrial epithelium and myometrial muscle cells, compared with the treatment with each hormone alone. Uterine leiomyomas were observed in 2 of 6 mice at 7 months of combined treatment but not in any of 6 or 7 mice receiving each single hormone. DHT-B alone induced growth and secretion of mammary ductal cells, as well as growth of mammary stroma. E2 alone stimulated much more pronounced growth of both ductal cells and alveolar cells and secretion of alveolar cells, but had no effect on mammary stroma. Treatment with both E2 and DHT-B caused more severe hyperplasia of mammary ducts and alveoli, compared to the treatment with each hormone alone. Intraductal hyperplasia occurred early and frequently in the E2+DHT-B- treated mice, but no mammary tumors were observed. These results suggest that E2 and DHT-B have synergistic effects on the growth of uterine endometrial epithelium and myometrial muscle cells, as well as mammary epithelial ducts and alveoli.

Histochemical properties of bovine and ovine mammary glands during fetal development.

PubMed

Hara, Asuka; Abe, Tomoyuki; Hirao, Atsushi; Sanbe, Kazuhiro; Ayakawa, Hiromichi; Sarantonglaga, Borjigin; Yamaguchi, Mio; Sato, Akane; Khurchabilig, Atchalalt; Ogata, Kazuko; Fukumori, Rika; Sugita, Shoei; Nagao, Yoshikazu

2018-02-20

In order to obtain more information on the development of bovine and ovine fetal mammary glands, a series of mammary glands from fetuses of different ages were analyzed. A total of 16 bovine fetuses with curved crown rump lengths ranging from 12 cm (80 days) to 75 cm (240 days) and 15 ovine fetuses ranging from 55 days to 131 days were examined. We used hematoxylin and eosin stain and Oil-Red-O stain to analyze the developmental and morphogenetic processes of mammary glands. In addition, we used immunohistochemical staining to determine the pattern of expression of cytokeratin 18 (CK18) during luminal epithelial differentiation, α-smooth-muscle actin (α-SMA) for myoepithelial differentiation, Ki-67 for cell proliferation, and estrogen receptor α (ERα). Our analyzes showed: (a) The primary mammary duct begin to proliferate in a lengthwise within the teat at 90 days in bovine fetuses and 63 days in ovine fetus; (b) luminal epithelial cells and myoepithelial cells appeared from 90 days in bovine fetuses and 63 days in ovine fetus; (c) proliferation of epithelial cells appeared to coincide with the development of the primary and secondary ducts; and (d) ERα was not found in the fetal mammary gland, but adipocytes showed the presence of ERα. Overall, these results indicate that the sequence of events in the prenatal development of the mammary gland of sheep is similar to that of cattle.

Histochemical properties of bovine and ovine mammary glands during fetal development

PubMed Central

HARA, Asuka; ABE, Tomoyuki; HIRAO, Atsushi; SANBE, Kazuhiro; AYAKAWA, Hiromichi; SARANTONGLAGA, Borjigin; YAMAGUCHI, Mio; SATO, Akane; KHURCHABILIG, Atchalalt; OGATA, Kazuko; FUKUMORI, Rika; SUGITA, Shoei; NAGAO, Yoshikazu

2017-01-01

In order to obtain more information on the development of bovine and ovine fetal mammary glands, a series of mammary glands from fetuses of different ages were analyzed. A total of 16 bovine fetuses with curved crown rump lengths ranging from 12 cm (80 days) to 75 cm (240 days) and 15 ovine fetuses ranging from 55 days to 131 days were examined. We used hematoxylin and eosin stain and Oil-Red-O stain to analyze the developmental and morphogenetic processes of mammary glands. In addition, we used immunohistochemical staining to determine the pattern of expression of cytokeratin 18 (CK18) during luminal epithelial differentiation, α-smooth-muscle actin (α-SMA) for myoepithelial differentiation, Ki-67 for cell proliferation, and estrogen receptor α (ERα). Our analyzes showed: (a) The primary mammary duct begin to proliferate in a lengthwise within the teat at 90 days in bovine fetuses and 63 days in ovine fetus; (b) luminal epithelial cells and myoepithelial cells appeared from 90 days in bovine fetuses and 63 days in ovine fetus; (c) proliferation of epithelial cells appeared to coincide with the development of the primary and secondary ducts; and (d) ERα was not found in the fetal mammary gland, but adipocytes showed the presence of ERα. Overall, these results indicate that the sequence of events in the prenatal development of the mammary gland of sheep is similar to that of cattle. PMID:29249731

Uptake of choline by rat mammary-gland epithelial cells.

PubMed Central

Chao, C K; Pomfret, E A; Zeisel, S H

1988-01-01

The neonatal mammal requires especially large amounts of choline to sustain growth. Much of this choline is derived from the newborn's only source of food, milk. The concentration of choline in rat milk [182 +/- 24 microM (S.E.M.)] was much higher than that in maternal serum (11.6 +/- 0.9 microM), suggesting that a mechanism capable of concentrating choline into milk must exist. We characterized choline uptake by mammary epithelial cells (the site of milk production) of the lactating rat. We observed two uptake processes, one saturable and obeying Michaelis-Menten kinetics, and the other non-saturable and linear. At physiological blood choline concentrations, the saturable component of choline uptake predominated. The saturable component had Kapp. = 35 +/- 16 microM, and Vmax. = 1.24 +/- 0.19 nmol/h per mg of protein. Saturable uptake of choline was inhibited by hemicholinium-3. Ca2+ was required for uptake, but Mg2+ was not. Replacement Na+ with K+, Li+ or sucrose inhibited transport. Ouabain did not inhibit choline uptake. Choline concentration in epithelial cells was 67.7 +/- 1.9 nmol/g wet wt. at the start of incubation at 37 degrees C and rose to 80.9 +/- 6.5 nmol/g wet wt. over 30 min. Much of the choline accumulated by the mammary gland (in the presence of endogenous concentrations of choline) remained in the form of choline (50 +/- 1.2%), phosphatidylcholine (12 +/- 2.3%), lysophosphatidylcholine (0.1 +/- 0.03%), betaine (7 +/- 0.3% and phosphocholine (6 +/- 0.5%). In addition, we isolated 25 +/- 1.2% of choline-derived radiolabel in an unidentified compound. Images Fig. 1. Fig. 3. PMID:3178755

Flor-Essence? Herbal Tonic Promotes Mammary Tumor Development in Sprague Dawley Rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bennett, L; Montgomery, J; Steinberg, S

Background: Women who are diagnosed with breast cancer often self-administer complementary and alternative medicines to augment their conventional treatments, improve health, or prevent recurrence. Flor-Essence{reg_sign} Tonic is a complex mixture of herbal extracts used by cancer patients because of anecdotal evidence that it can treat or prevent disease. Methods: Female Sprague Dawley rats were given water or exposed to 3% or 6% Flor-Essence{reg_sign} beginning at one day of age. Mammary tumors were induced with a single oral 40 mg/kg/bw dose of dimethylbenz(a)anthracene at 50 days of age and sacrificed at 23 weeks. Rats were maintained on AIN-76A diet. Results: Controlmore » rats had palpable mammary tumor incidence of 51.0% at 19 weeks of age compared to 65.0% and 59.4% for the 3% and 6% Flor-Essence{reg_sign} groups respectively. Overall, no significant difference in time until first palpable tumor was detected among any of the groups. At necropsy, mammary tumor incidence was 82.5% for controls compared to 90.0% and 97.3% for rats consuming 3% and 6% Flor-Essence{reg_sign}, respectively. Mean mammary tumor multiplicity ({+-}SES) for the controls was 2.8 ({+-} 0.5) and statistically different from the 3% or 6% Flor- Essence{reg_sign} groups with 5.2 ({+-} 0.7), and 4.8 ({+-} 0.6), respectively (p{<=}0.01). As expected, the majority of isolated tumors were diagnosed as adenocarcinomas. Conclusions: Flor-Essence{reg_sign} can promote mammary tumor development in the Sprague Dawley rat model. This observation is contrary to widely available anecdotal evidence as well as the desire of the consumer that this commercially available herbal tonic will suppress and/or inhibit tumor growth.« less

Single-cell mRNA sequencing identifies subclonal heterogeneity in anti-cancer drug responses of lung adenocarcinoma cells.

PubMed

Kim, Kyu-Tae; Lee, Hye Won; Lee, Hae-Ock; Kim, Sang Cheol; Seo, Yun Jee; Chung, Woosung; Eum, Hye Hyeon; Nam, Do-Hyun; Kim, Junhyong; Joo, Kyeung Min; Park, Woong-Yang

2015-06-19

Intra-tumoral genetic and functional heterogeneity correlates with cancer clinical prognoses. However, the mechanisms by which intra-tumoral heterogeneity impacts therapeutic outcome remain poorly understood. RNA sequencing (RNA-seq) of single tumor cells can provide comprehensive information about gene expression and single-nucleotide variations in individual tumor cells, which may allow for the translation of heterogeneous tumor cell functional responses into customized anti-cancer treatments. We isolated 34 patient-derived xenograft (PDX) tumor cells from a lung adenocarcinoma patient tumor xenograft. Individual tumor cells were subjected to single cell RNA-seq for gene expression profiling and expressed mutation profiling. Fifty tumor-specific single-nucleotide variations, including KRAS(G12D), were observed to be heterogeneous in individual PDX cells. Semi-supervised clustering, based on KRAS(G12D) mutant expression and a risk score representing expression of 69 lung adenocarcinoma-prognostic genes, classified PDX cells into four groups. PDX cells that survived in vitro anti-cancer drug treatment displayed transcriptome signatures consistent with the group characterized by KRAS(G12D) and low risk score. Single-cell RNA-seq on viable PDX cells identified a candidate tumor cell subgroup associated with anti-cancer drug resistance. Thus, single-cell RNA-seq is a powerful approach for identifying unique tumor cell-specific gene expression profiles which could facilitate the development of optimized clinical anti-cancer strategies.

Isolation of stem-like cells from spontaneous feline mammary carcinomas: phenotypic characterization and tumorigenic potential.

PubMed

Barbieri, Federica; Wurth, Roberto; Ratto, Alessandra; Campanella, Chiara; Vito, Guendalina; Thellung, Stefano; Daga, Antonio; Cilli, Michele; Ferrari, Angelo; Florio, Tullio

2012-04-15

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-α and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs. Copyright © 2012 Elsevier Inc. All rights reserved.

Isolation of Estrogen Regulated Genes from MCF-7 Human Mammary Cancer Cells

DTIC Science & Technology

1990-07-13

Syrian hamster BHK cells. The expression of approximately five hundred clones increased at various points in Gl transit (Hirschhom er d., 1984). No...hyperplasia of ductal epithelium of the mammary gland (Lyons etal., 1958). Biochemical changes in uterine wet weight, protein content, and nucleic... transition from a resting to a growing state (Kaufman and Sharp, 1983). Similarly post- transcriptional processing appears to be important in the ceU

Tracing anti-cancer and cancer-promoting actions of all-trans retinoic acid in breast cancer to a RARα epigenetic mechanism of mammary epithelial cell fate.

PubMed

Rossetti, Stefano; Ren, MingQiang; Visconti, Nicolo; Corlazzoli, Francesca; Gagliostro, Vincenzo; Somenzi, Giulia; Yao, Jin; Sun, Yijun; Sacchi, Nicoletta

2016-12-27

A hallmark of cancer cells is the ability to evade the growth inhibitory/pro-apoptotic action of physiological all-trans retinoic acid (RA) signal, the bioactive derivative of Vitamin A. However, as we and others reported, RA can also promote cancer cell growth and invasion. Here we show that anticancer and cancer-promoting RA actions in breast cancer have roots in a mechanism of mammary epithelial cell morphogenesis that involves both transcriptional (epigenetic) and non-transcriptional RARα (RARA) functions. We found that the mammary epithelial cell-context specific degree of functionality of the RARA transcriptional (epigenetic) component of this mechanism, by tuning the effects of the non-transcriptional RARA component, determines different cell fate decisions during mammary morphogenesis. Indeed, factors that hamper the RARA epigenetic function make physiological RA drive aberrant morphogenesis via non-transcriptional RARA, thus leading to cell transformation. Remarkably, also the cell context-specific degree of functionality of the RARA epigenetic component retained by breast cancer cells is critical to determine cell fate decisions in response to physiological as well as supraphysiological RA variation. Overall this study supports the proof of principle that the epigenetic functional plasticity of the mammary epithelial cell RARA mechanism, which is essential for normal morphogenetic processes, is necessary to deter breast cancer onset/progression consequent to the insidious action of physiological RA.

The intranuclear PEX domain of MMP involves proliferation, migration, and metastasis of aggressive adenocarcinoma cells.

PubMed

Okusha, Yuka; Eguchi, Takanori; Sogawa, Chiharu; Okui, Tatsuo; Nakano, Keisuke; Okamoto, Kuniaki; Kozaki, Ken-Ichi

2018-05-15

Members of matrix metalloproteinase (MMP) family promote cancer cell migration, invasion, and metastasis through alteration of the tumor milieu, intracellular signaling pathways, and transcription. We examined gene expression signatures of colon adenocarcinoma cell lines with different metastatic potentials and found that rapidly metastatic cells powerfully expressed genes encoding MMP3 and MMP9. The non-proteolytic PEX isoform and proteolytic isoforms of MMPs were significantly expressed in the metastatic cells in vitro. Knockdown of MMP3 attenuated cancer cell migration and invasion in vitro and lung metastasis in vivo. Profound nuclear localization of MMP3/PEX was found in tumor-stroma marginal area. In contrast, MMP9 was localized in central area of subcutaneous tumors. Overexpression of the PEX isoform of MMP3 promoted proliferation and migration of the rapidly metastatic cells in vitro. Taken together, the non-proteolytic PEX isoform of MMPs locating in cell nuclei involves proliferation, migration, and subsequent metastasis of aggressive adenocarcinoma cells. © 2018 Wiley Periodicals, Inc.

Engineered chimeric antigen receptor-expressing T cells for the treatment of pancreatic ductal adenocarcinoma

PubMed Central

Beatty, Gregory L

2014-01-01

Adoptive cell therapy with chimeric antigen receptor (CAR)-engineered T cells is under investigation as an approach to restore productive T cell immunosurveillance in patients with pancreatic ductal adenocarcinoma. Early findings demonstrate safety of this cell-based therapy and the capacity of CAR-expressing T cells to mediate anti-tumor activity as well as induce endogeneous antitumoral immune responses. PMID:25050204

Morphological and immunohistochemical characterization of spontaneous mammary tumours in European hedgehogs (Erinaceus europaeus).

PubMed

Döpke, C; Fehr, M; Thiele, A; Pohlenz, J; Wohlsein, P

2007-07-01

Mammary tumour samples (11 surgical and five post-mortem) from 16 adult European hedgehogs submitted between 1980 and 2004 were examined. Histologically, the tumours were classified as simple tubulo-papillary carcinomas with local invasive growth. In six cases, tumour cell emboli were present in blood vessels or lymphatic vessels, or both. However, metastasis to regional lymph nodes was found only in one hedgehog. Malignant neoplastic epithelial cells were immunolabelled by antibodies specific for various cytokeratins (CKs), including CK1-8, 10, 13-16, 19 and 20. CK expression did not differ from that in normal mammary gland tissue. CK20 was expressed in the mammary tissue of hedgehogs, in contrast to that of dogs and cats; CK7 immunolabelling, however, which commonly occurs in mammary epithelial cells, was negative. CK20 expression, together with the lack of CK7 as determined by a protein-specific antibody, represented an important difference from the CK profile shown by mammary epithelial cells of other mammalian species, including the dog and cat.

Production and Release of Antimicrobial and Immune Defense Proteins by Mammary Epithelial Cells following Streptococcus uberis Infection of Sheep

PubMed Central

Pisanu, Salvatore; Marogna, Gavino; Cubeddu, Tiziana; Pagnozzi, Daniela; Cacciotto, Carla; Campesi, Franca; Schianchi, Giuseppe; Rocca, Stefano

2013-01-01

Investigating the innate immune response mediators released in milk has manifold implications, spanning from elucidation of the role played by mammary epithelial cells (MECs) in fighting microbial infections to the discovery of novel diagnostic markers for monitoring udder health in dairy animals. Here, we investigated the mammary gland response following a two-step experimental infection of lactating sheep with the mastitis-associated bacterium Streptococcus uberis. The establishment of infection was confirmed both clinically and by molecular methods, including PCR and fluorescent in situ hybridization of mammary tissues. Proteomic investigation of the milk fat globule (MFG), a complex vesicle released by lactating MECs, enabled detection of enrichment of several proteins involved in inflammation, chemotaxis of immune cells, and antimicrobial defense, including cathelicidins and calprotectin (S100A8/S100A9), in infected animals, suggesting the consistent involvement of MECs in the innate immune response to pathogens. The ability of MECs to produce and release antimicrobial and immune defense proteins was then demonstrated by immunohistochemistry and confocal immunomicroscopy of cathelicidin and the calprotectin subunit S100A9 on mammary tissues. The time course of their release in milk was also assessed by Western immunoblotting along the course of the experimental infection, revealing the rapid increase of these proteins in the MFG fraction in response to the presence of bacteria. Our results support an active role of MECs in the innate immune response of the mammary gland and provide new potential for the development of novel and more sensitive tools for monitoring mastitis in dairy animals. PMID:23774600

Production and release of antimicrobial and immune defense proteins by mammary epithelial cells following Streptococcus uberis infection of sheep.

PubMed

Addis, Maria Filippa; Pisanu, Salvatore; Marogna, Gavino; Cubeddu, Tiziana; Pagnozzi, Daniela; Cacciotto, Carla; Campesi, Franca; Schianchi, Giuseppe; Rocca, Stefano; Uzzau, Sergio

2013-09-01

Investigating the innate immune response mediators released in milk has manifold implications, spanning from elucidation of the role played by mammary epithelial cells (MECs) in fighting microbial infections to the discovery of novel diagnostic markers for monitoring udder health in dairy animals. Here, we investigated the mammary gland response following a two-step experimental infection of lactating sheep with the mastitis-associated bacterium Streptococcus uberis. The establishment of infection was confirmed both clinically and by molecular methods, including PCR and fluorescent in situ hybridization of mammary tissues. Proteomic investigation of the milk fat globule (MFG), a complex vesicle released by lactating MECs, enabled detection of enrichment of several proteins involved in inflammation, chemotaxis of immune cells, and antimicrobial defense, including cathelicidins and calprotectin (S100A8/S100A9), in infected animals, suggesting the consistent involvement of MECs in the innate immune response to pathogens. The ability of MECs to produce and release antimicrobial and immune defense proteins was then demonstrated by immunohistochemistry and confocal immunomicroscopy of cathelicidin and the calprotectin subunit S100A9 on mammary tissues. The time course of their release in milk was also assessed by Western immunoblotting along the course of the experimental infection, revealing the rapid increase of these proteins in the MFG fraction in response to the presence of bacteria. Our results support an active role of MECs in the innate immune response of the mammary gland and provide new potential for the development of novel and more sensitive tools for monitoring mastitis in dairy animals.

A Rare Case of an Adult with Untreated Bladder Exstrophy Presenting with Signet-Ring Cell Adenocarcinoma of Urinary Bladder.

PubMed

Savalia, Abhishek J; Kumar, Vikash; Kasat, Gaurav; Sawant, Ajit

2016-11-01

Untreated bladder exstrophy in an adult is rare, as the defect is obvious and primary reconstruction is usually done in infancy. There are less than 90 reported cases of primary adenocarcinoma in an untreated bladder exstrophy in literature and only two such case reports from India. Of these, only one case was of signet-ring cell type of mucinous adenocarcinoma. Here we report the second case of signet-ring cell adenocarcinoma in a 63 year old male with untreated bladder exstrophy (oldest patient in literature), to highlight the extreme rarity, yet distinct possibility and challenges faced in surgical management of such cases.

Effects of milk replacer formulation on measures of mammary growth and composition in Holstein heifers.

PubMed

Daniels, K M; Capuco, A V; McGilliard, M L; James, R E; Akers, R M

2009-12-01

Overfeeding prepubertal heifers may impair mammary parenchymal growth and reduce milk production, but evidence suggests that increased intake of a high-protein milk replacer before weaning may be beneficial. This study was designed to evaluate effects of milk replacer (MR) composition on mass and composition of mammary parenchyma and fat pad, growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis gene expression, and putative mammary epithelial stem cells. Specifically, we hypothesized that positive effects of faster rates of gain during the preweaning period alter the development, persistence, or activity of populations of putative mammary epithelial stem cells, possibly through involvement of GH/IGF-I axis molecules. Twenty-four newborn heifers were fed 1 of 4 MR diets (n = 6/diet): control [20% crude protein (CP), 21% fat MR fed at 441 g of dry matter (DM)/d], high protein, low fat (28% CP, 20% fat MR fed at 951 g of DM/d), high protein, high fat (27% CP, 28% fat MR fed at 951 g of DM/d), and high protein, high fat+ (27% CP, 28% fat MR fed at 1,431 g of DM/d). Water and starter (20% CP, 1.43% fat) were offered ad libitum. Animals were killed on d 65 and mammary tissue was subjected to biochemical, molecular, and histological examination. No differences in mammary parenchymal mass or composition, with or without adjusting for empty body weight, were detected. Mass was increased and composition of the mammary fat pad was altered by nutrient intake. No diet differences in putative mammary epithelial stem cell abundance or abundance of transcripts for genes of the GH/IGF-I axis were detected. In this study, growth of the mammary epithelium, size of the mammary epithelial stem cell population, and components of the GH/IGF-I axis did not depend on diet. However, an underlying positive correlation between telomerase, a marker of mammary stem cells, and growth of the mammary parenchyma was detected. Implications of diet-induced effects on mammary fat pad and

Effects of polyamine inhibitors on zinc uptake by COMMA-1D mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allen, J.C.; Haedrich, L.H.

1991-03-15

Zn uptake or transport is stimulated by glucocorticoids in many types of epithelial cells, including the COMMA-1D mouse mammary cell line. The current objective was to determine whether polyamines also mediate glucocorticoid stimulation of Zn-uptake. Initially, cells grown in lactogenic hormone supplemented-media had approximately 65% greater {sup 65}Zn-uptake over 24 h than cells in nonsupplemented growth media (GM). {sup 65}Zn-uptake from HM with 10{sup {minus}5}M methylglyoxal-bis(guanylhydrazone) (MGBG) (s-adenosyl-methionine decarboxylase inhibitor to block polyamine synthesis) added was less than from GM. Exogenous spermidine added to the MGBG-HM media increased {sup 65}Zn-uptake. However, up to 10mM difluoromethylornithine (DFMO), a more specific inhibitormore » of sperimidine synthesis, had no significant effect on 24-h {sup 65}Zn-uptake by cells in HM. In GM, DFMO caused a slight dose-dependent decrease in {sup 65}Zn-uptake over the range 10{sup {minus}6} to 5 {times} 10{sup 3}M. Also, with 8 h of incubation, DFMO tended to decrease {sup 65}Zn-uptake in HM-stimulated cells. These data cannot yet distinguish between the possibilities that DFMO is inactivated during the 24-h incubation or that the dramatic effects of MGBG on {sup 65}Zn-uptake in these mammary-derived cells is not related to its inhibition of polyamine synthesis. Because COMMA-1D cells alter Zn uptake in response to lactogenic hormones and MGBG, the model system is suitable for further studies of the mechanisms of zinc transport in epithelia.« less

Loss of p53 protein during radiation transformation of primary human mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wazer, D.E.; Chu, Qiuming; Liu, Xiao Long

1994-04-01

The causative factors leading to breast cancer are largely unknown. Increased incidence of breast cancer following diagnostic or therapeutic radiation suggests that radiation may contribute to mammary oncogenesis. This report describes the in vitro neoplastic transformation of a normal human mammary epithelial cell strain, 76N, by fractionated [gamma]-irradiation at a clinically used dose (30 Gy). The transformed cells (76R-30) were immortal, had reduced growth factor requirements, and produced tumors in nude mice. Remarkably, the 76R-30 cells completely lacked the p53 tumor suppressor protein. Loss of p53 was due to deletion of the gene on one allele and a 26-bp deletionmore » within the third intron on the second allele which resulted in abnormal splicing out of either the third or fourth exon from the mRNA. PCR with a mutation-specific primer showed that intron 3 mutation was present in irradiated cells before selection for immortal phenotype. 76R-30 cells did not exhibit G[sub 1] arrest in response to radiation, indicating a loss of p53-mediated function. Expression of the wild-type p53 gene in 76R-30 cells led to their growth inhibition. Thus, loss of p53 protein appears to have contributed to neoplastic transformation of these cells. This unique model should facilitate analyses of molecular mechanisms of radiation-induced breast cancer and allow identification of p53-regulated cellular genes in breast cells. 44 refs., 8 figs., 1 tab.« less

Specificity of gap junction communication among human mammary cells and connexin transfectants in culture

PubMed Central

1993-01-01

In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein. The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC

Beneficial bacteria stimulate host immune cells to counteract dietary and genetic predisposition to mammary cancer in mice.

PubMed

Lakritz, Jessica R; Poutahidis, Theofilos; Levkovich, Tatiana; Varian, Bernard J; Ibrahim, Yassin M; Chatzigiagkos, Antonis; Mirabal, Sheyla; Alm, Eric J; Erdman, Susan E

2014-08-01

Recent studies suggest health benefits including protection from cancer after eating fermented foods such as probiotic yogurt, though the mechanisms are not well understood. Here we tested mechanistic hypotheses using two different animal models: the first model studied development of mammary cancer when eating a Westernized diet, and the second studied animals with a genetic predilection to breast cancer. For the first model, outbred Swiss mice were fed a Westernized chow putting them at increased risk for development of mammary tumors. In this Westernized diet model, mammary carcinogenesis was inhibited by routine exposure to Lactobacillus reuteri ATCC-PTA-6475 in drinking water. The second model was FVB strain erbB2 (HER2) mutant mice, genetically susceptible to mammary tumors mimicking breast cancers in humans, being fed a regular (non-Westernized) chow diet. We found that oral supplement with these purified lactic acid bacteria alone was sufficient to inhibit features of mammary neoplasia in both models. The protective mechanism was determined to be microbially-triggered CD4+CD25+ lymphocytes. When isolated and transplanted into other subjects, these L. reuteri-stimulated lymphocytes were sufficient to convey transplantable anti-cancer protection in the cell recipient animals. These data demonstrate that host immune responses to environmental microbes significantly impact and inhibit cancer progression in distal tissues such as mammary glands, even in genetically susceptible mice. This leads us to conclude that consuming fermentative microbes such as L. reuteri may offer a tractable public health approach to help counteract the accumulated dietary and genetic carcinogenic events integral in the Westernized diet and lifestyle. © 2013 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.

Involvements of Estrogen Receptor, Proliferating Cell Nuclear Antigen and p53 in Endometrial Adenocarcinoma Development in Donryu Rats

PubMed Central

Yoshida, Midori; Katsuda, Shin-ichi; Maekawa, Akihiko

2012-01-01

Involvements of estrogen receptor (ER)α, proliferating cell nuclear antigen (PCNA) and p53 in the uterine carcinogenesis process in Donryu rats, a high yield strain of the uterine cancer were investigated immunohistochemically. ERα was expressed in atypical endometrial hyperplasia, accepted as a precancerous lesion of the uterine tumors, as well as well- and in moderately-differentiated endometrial adenocarcinomas, and the intensities of expression were similar to those in the luminal epithelial cells of the atrophic uterus at 15 months of age. The expression, however, was negative in the tumor cells of poorly differentiated type. Good growth of implanted grafts of the poorly-differentiated adenocarcinomas in both sexes with or without gonadectomy supported the estrogen independency of tumor progression to malignancy. PCNA labeling indices were increased with tumor development from atypical hyperplasia to adenocarcinoma. The tumor cells in poorly-differentiated adenocarcinomas were positive for p53 positive but negative for p21 expression, suggesting accumulation of mutated p53. These results indicate that the consistent ERα expression is involved in initiation and promotion steps of uterine carcinogenesis, but not progression. In addition, PCNA is related to tumor development and the expression of mutated p53 might be a late event during endometrial carcinogenesis. PMID:23345926

ApcMin, A Mutation in the Murine Apc Gene, Predisposes to Mammary Carcinomas and Focal Alveolar Hyperplasias

NASA Astrophysics Data System (ADS)

Moser, Amy Rapaich; Mattes, Ellen M.; Dove, William F.; Lindstrom, Mary J.; Haag, Jill D.; Gould, Michael N.

1993-10-01

ApcMin (Min, multiple intestinal neoplasia) is a point mutation in the murine homolog of the APC gene. Min/+ mice develop multiple intestinal adenomas, as do humans carrying germ-line mutations in APC. Female mice carrying Min are also prone to develop mammary tumors. Min/+ mammary glands are more sensitive to chemical carcinogenesis than are +/+ mammary glands. Transplantation of mammary cells from Min/+ or +/+ donors into +/+ hosts demonstrates that the propensity to develop mammary tumors is intrinsic to the Min/+ mammary cells. Long-term grafts of Min/+ mammary glands also gave rise to focal alveolar hyperplasias, indicating that the presence of the Min mutation also has a role in the development of these lesions.

Cytoplasmic Overexpression of CD95L in Esophageal Adenocarcinoma Cells Overcomes Resistance to CD95-Mediated Apoptosis1

PubMed Central

Watson, Gregory A; Naran, Sanjay; Zhang, Xinglu; Stang, Michael T; Queiroz de Oliveira, Pierre E; Hughes, Steven J

2011-01-01

Introduction The CD95/CD95L pathway plays a critical role in tissue homeostasis and immune system regulation; however, the function of this pathway in malignancy remains poorly understood. We hypothesized that CD95L expression in esophageal adenocarcinoma confers advantages to the neoplasm other than immune privilege. Methods CD95L expression was characterized in immortalized squamous esophagus (HET-1A) and Barrett esophagus (BAR-T) cells; adenocarcinoma cell lines FLO-1, SEG-1, and BIC-1, and MDA468 (- control); and KFL cells (+ control). Analyses included reverse transcription-polymerase chain reaction, immunoblots of whole cell and secretory vesicle lysates, FACScan analysis, laser scanning confocal microscopy of native proteins and fluorescent constructs, and assessment of apoptosis and ERK1/2 pathways. Results Cleaved, soluble CD95L is expressed at both the RNA and protein levels in these cell lines derived from esophageal adenocarcinoma and other human tissues. CD95L was neither trafficked to the cell membrane nor secreted into the media or within vesicles, rather the protein seems to be sequestered in the cytoplasm. CD95 and CD95L colocalize by immunofluorescence, but an interaction was not proven by immunoprecipitation. Overexpression of CD95L in the adenocarcinoma cell lines induced robust apoptosis and, under conditions of pan-caspase inhibition, resulted in activation of ERK signaling. Conclusions CD95L localization in EA cells is inconsistent with the conference of immune privilege and is more consistent with a function that promotes tumor growth through alternative CD95 signaling. Reduced cell surface expression of CD95 affects cell sensitivity to extracellular apoptotic signals more significantly than alterations in downstream modulators of apoptosis. PMID:21390183

Adipose and mammary epithelial tissue engineering.

PubMed

Zhu, Wenting; Nelson, Celeste M

2013-01-01

Breast reconstruction is a type of surgery for women who have had a mastectomy, and involves using autologous tissue or prosthetic material to construct a natural-looking breast. Adipose tissue is the major contributor to the volume of the breast, whereas epithelial cells comprise the functional unit of the mammary gland. Adipose-derived stem cells (ASCs) can differentiate into both adipocytes and epithelial cells and can be acquired from autologous sources. ASCs are therefore an attractive candidate for clinical applications to repair or regenerate the breast. Here we review the current state of adipose tissue engineering methods, including the biomaterials used for adipose tissue engineering and the application of these techniques for mammary epithelial tissue engineering. Adipose tissue engineering combined with microfabrication approaches to engineer the epithelium represents a promising avenue to replicate the native structure of the breast.

Adipose and mammary epithelial tissue engineering

PubMed Central

Zhu, Wenting; Nelson, Celeste M.

2013-01-01

Breast reconstruction is a type of surgery for women who have had a mastectomy, and involves using autologous tissue or prosthetic material to construct a natural-looking breast. Adipose tissue is the major contributor to the volume of the breast, whereas epithelial cells comprise the functional unit of the mammary gland. Adipose-derived stem cells (ASCs) can differentiate into both adipocytes and epithelial cells and can be acquired from autologous sources. ASCs are therefore an attractive candidate for clinical applications to repair or regenerate the breast. Here we review the current state of adipose tissue engineering methods, including the biomaterials used for adipose tissue engineering and the application of these techniques for mammary epithelial tissue engineering. Adipose tissue engineering combined with microfabrication approaches to engineer the epithelium represents a promising avenue to replicate the native structure of the breast. PMID:23628872

Hypoxia and prostaglandin E receptor 4 signalling pathways synergise to promote endometrial adenocarcinoma cell proliferation and tumour growth.

PubMed

Catalano, Rob D; Wilson, Martin R; Boddy, Sheila C; McKinlay, Andrew T M; Sales, Kurt J; Jabbour, Henry N

2011-05-12

The prostaglandin endoperoxide synthase (PTGS) pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG) E(2). PTGS2 expression and PGE(2) biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE(2) regulate endometrial tumour growth is unknown. Here we investigated (a) the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3) and PGE receptors (PTGER1-4) in endometrial adenocarcinomas compared with normal endometrium and (b) the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells) xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE(2) and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE(2) and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells.

Hypoxia and Prostaglandin E Receptor 4 Signalling Pathways Synergise to Promote Endometrial Adenocarcinoma Cell Proliferation and Tumour Growth