Normal Human Breast Epithelium Research Articles

Modular DNA Tetrahedron Nanomachine-Guided Dual-Responsive Hybridization Chain Reactions for Discernible Bivariate Assay and Cell Imaging.

Engineering of multivariate biosensing and imaging platforms involved in disease plays a vital role in effectively discerning cancer cells from normal cells and facilitating reliable targeted therapy. Multiple biomarkers such as mucin 1 (MUC1) and nucleolin are typically overexpressed in breast cancer cells compared to normal human breast epithelium cells. Motivated by this knowledge, a dual-responsive DNA tetrahedron nanomachine (drDT-NM) is constructed through immobilizing two recognition modules, MUC1 aptamer (MA) and a hairpin H1* encoding nucleolin-specific G-rich AS1411 aptamer, in two separate vertexes of a functional DT architecture tethering two localized pendants (PM and PN). When drDT-NM identifiably binds bivariate MUC1 and nucleolin, two independent hybridization chain reactions (HCRM and HCRN) as amplification modules are initiated with two sets of four functional hairpin reactants. Among them, one hairpin for HCRM is dually ended by fluorescein and quencher BHQ1 to sense MUC1. The responsiveness of nucleolin is executed by operating HCRN utilizing another two hairpins programmed with two pairs of AS1411 splits. In the shared HCRN duplex products, the parent AS1411 aptamers are cooperatively merged and folded into G-quadruplex concatemers to embed Zn-protoporphyrin IX (ZnPPIX/G4) for fluorescence signaling readout, thereby achieving a highly sensitive intracellular assay and discernible cell imaging. The tandem ZnPPIX/G4 unities also act as imaging agents and therapeutic cargos for efficient photodynamic therapy of cancer cells. Based on drDT-NM to guide bispecific HCR amplifiers for adaptive bivariate detection, we present a paradigm of exquisitely integrating modular DNA nanostructures with nonenzymatic nucleic acid amplification, thus creating a versatile biosensing platform as a promising candidate for accurate assay, discernible cell imaging, and targeted therapy.

Breast-Specific Epigenetic Regulation of DeltaNp73 and Its Role in DNA-Damage-Response of BRCA1-Mutated Human Mammary Epithelial Cells.

The function of BRCA1/2 proteins is essential for maintaining genomic integrity in all cell types. However, why women who carry deleterious germline mutations in BRCA face an extremely high risk of developing breast and ovarian cancers specifically has remained an enigma. We propose that breast-specific epigenetic modifications, which regulate tissue differentiation, could team up with BRCA deficiency and affect tissue susceptibility to cancer. In earlier work, we compared genome-wide methylation profiles of various normal epithelial tissues and identified breast-specific methylated gene promoter regions. Here, we focused on deltaNp73, the truncated isoform of p73, which possesses antiapoptotic and pro-oncogenic functions. We showed that the promoter of deltaNp73 is unmethylated in normal human breast epithelium and methylated in various other normal epithelial tissues and cell types. Accordingly, deltaNp73 was markedly induced by DNA damage in human mammary epithelial cells (HMECs) but not in other epithelial cell types. Moreover, the induction of deltaNp73 protected HMECs from DNA damage-induced cell death, and this effect was more substantial in HMECs from BRCA1 mutation carriers. Notably, when BRCA1 was knocked down in MCF10A, a non-malignant breast epithelial cell line, both deltaNp73 induction and its protective effect from cell death were augmented upon DNA damage. Interestingly, deltaNp73 induction also resulted in inhibition of BRCA1 and BRCA2 expression following DNA damage. In conclusion, breast-specific induction of deltaNp73 promotes survival of BRCA1-deficient mammary epithelial cells upon DNA damage. This might result in the accumulation of genomic alterations and allow the outgrowth of breast cancers. These findings indicate deltaNp73 as a potential modifier of breast cancer susceptibility in BRCA1 mutation carriers and may stimulate novel strategies of prevention and treatment for these high-risk women.

Analysis of clonal expansions through the normal and premalignant human breast epithelium reveals the presence of luminal stem cells.

It is widely accepted that the cell of origin of breast cancer is the adult mammary epithelial stem cell; however, demonstrating the presence and location of tissue stem cells in the human breast has proved difficult. Furthermore, we do not know the clonal architecture of the normal and premalignant mammary epithelium or its cellular hierarchy. Here, we use deficiency in the mitochondrial enzyme cytochrome c oxidase (CCO), typically caused by somatic mutations in the mitochondrial genome, as a means to perform lineage tracing in the human mammary epithelium. PCR sequencing of laser‐capture microdissected cells in combination with immunohistochemistry for markers of lineage differentiation was performed to determine the clonal nature of the mammary epithelium. We have shown that in the normal human breast, clonal expansions (defined here by areas of CCO deficiency) are typically uncommon and of limited size, but can occur at any site within the adult mammary epithelium. The presence of a stem cell population was shown by demonstrating multi‐lineage differentiation within CCO‐deficient areas. Interestingly, we observed infrequent CCO deficiency that was restricted to luminal cells, suggesting that niche succession, and by inference stem cell location, is located within the luminal layer. CCO‐deficient areas appeared large within areas of ductal carcinoma in situ, suggesting that the rate of clonal expansion was altered in the premalignant lesion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Abstract 4915: Alcohol-induced reprogramming of tumor plasticity is mediated via suppression of CCN5 signaling in breast cancer cells

Abstract Background and Objective: The epidemiological and experimental studies suggest that alcohol consumption is associated with increased risk for breast cancer development and metastasis. However, the mechanisms of alcohol-induced breast cancer progression and metastasis remain unknown. In this study, we examined the roles of CCN5, a tumor suppressor gene in alcohol induced breast cancer cell migration/invasion, epithelial to mesenchymal transition (EMT) and stemness. Methods: Estrogen receptor-positive (ER+) human breast cancer cell line MCF-7 and immortalized normal human epithelial breast cell line MCF-12A were treated with ethanol at various concentrations in the presence and absence of human recombinant human CCN5 protein (hrCCN5) . Invasive/migratory ability of treated and untreated cells was measured by Boyden chamber assays. The mRNA and protein expression level of CCN5 was determined by real-time qRT-PCR and Western blot. EMT and stemness markers were evaluated by Western blot. Single-cell suspensions from pre-treated cells were re-suspended at a density of 500 cells/ml mammocult media in ultralow attachment dishes. Number as well as the size of the mammosphere in specified experimental set-up was monitored and recorded alternate day for 8-10 days. Results: The studies demonstrated that alcohol promotes significantly the invasive/migratory ability and EMT phenotypes of breast cancer cells through the suppression of CCN5. Interestingly, hrCCN5 protein treatment suppresses the effect of alcohol and reprograms MET (mesenchymal to epithelial transition). In addition, alcohol-induced mammosphere formation efficiency is also suppressed by hrCCN5 protein-treatment. Conclusions: Our findings suggest that the CCN5 signaling plays a preventive role in alcohol-induced reprogramming of tumor plasticity in breast cancer cells. Citation Format: Inamul Haque, Arvind Subramanian, Vijayalaxmi Gupta, Sandipto Sarkar, Snigdha Banerjee, Sushanta K. Banerjee. Alcohol-induced reprogramming of tumor plasticity is mediated via suppression of CCN5 signaling in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4915. doi:10.1158/1538-7445.AM2017-4915

Abstract 5486: Oxidative stress and selective protein carbonylation in human breast cancer tissue

Abstract Protein carbonylation is an irreversible modification to the side chain of amino acid residues induced by severe oxidative stress. Reactive oxygen species (ROS) are constantly produced under normal as well as stress-induced conditions, and play a role in both cancer progression and cancer suppression. Tumor tissue are known to have higher ROS levels compared to surrounding healthy tissue but ROS-induced specific protein carbonylation and its unique role in cancer progression/suppression are poorly understood. In this study we compared the relative total and specific protein carbonylation in flash frozen human breast cancer and matched adjacent normal tissue using ELISA, two-color western blot, mass spectrometry, and immunoprecipitation approaches. To understand antioxidant capacity in tumor tissue, we analyzed superoxide dismutase (SOD) for its antioxidant activity and protein level. Our results indicate that tumor tissue has greater total protein carbonylation, lower SOD activity, lower SOD protein levels, and elevated levels of autophagy compared to matched adjacent healthy tissue. We also identified three specific proteins that showed higher level of carbonylation selectively in tumor tissue compared to adjacent normal tissue. Our findings were further confirmed using the immortalized MDA-MB-231 breast cancer cell line and MCF-12A noncancerous normal human epithelial breast cell. Identification of selectively carbonylated proteins in cancer tissue, and understanding their specific role in cancer progression may promote the development of targeted therapeutic approaches to mitigate or enhance oxidative damage of such proteins selectively in tumor tissue. Citation Format: Baikuntha P. Aryal, Ashutosh Rao. Oxidative stress and selective protein carbonylation in human breast cancer tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5486. doi:10.1158/1538-7445.AM2017-5486

Long-term exposure of MCF-12A normal human breast epithelial cells to ethanol induces epithelial mesenchymal transition and oncogenic features.

Alcoholism is associated with breast cancer incidence and progression, and moderate chronic consumption of ethanol is a risk factor. The mechanisms involved in alcohol's oncogenic effects are unknown, but it has been speculated that they may be mediated by acetaldehyde. We used the immortalized normal human epithelial breast cell line MCF-12A to determine whether short- or long-term exposure to ethanol or to acetaldehyde, using in vivo compatible ethanol concentrations, induces their oncogenic transformation and/or the acquisition of epithelial mesenchymal transition (EMT). Cultures of MCF-12A cells were incubated with 25 mM ethanol or 2.5 mM acetaldehyde for 1 week, or with lower concentrations (1.0–2.5 mM for ethanol, 1.0 mM for acetaldehyde) for 4 weeks. In the 4-week incubation, cells were also tested for anchorage-independence, including isolation of soft agar selected cells (SASC) from the 2.5 mM ethanol incubations. Cells were analyzed by immunocytofluorescence, flow cytometry, western blotting, DNA microarrays, RT/PCR, and assays for miRs. We found that short-term exposure to ethanol, but not, in general, to acetaldehyde, was associated with transcriptional upregulation of the metallothionein family genes, alcohol metabolism genes, and genes suggesting the initiation of EMT, but without related phenotypic changes. Long-term exposure to the lower concentrations of ethanol or acetaldehyde induced frank EMT changes in the monolayer cultures and in SASC as demonstrated by changes in cellular phenotype, mRNA expression, and microRNA expression. This suggests that low concentrations of ethanol, with little or no mediation by acetaldehyde, induce EMT and some traits of oncogenic transformation such as anchorage-independence in normal breast epithelial cells.

LASP-1: a nuclear hub for the UHRF1-DNMT1-G9a-Snail1 complex.

Nuclear LASP-1 has a direct correlation with overall survival of breast cancer patients. In this study, immunohistochemical analysis of a human breast TMA showed that LASP-1 is absent in normal human breast epithelium but the expression increases with malignancy and is highly nuclear in aggressive breast cancer. We investigated whether the chemokines and growth factors present in the tumor microenvironment could trigger nuclear translocation of LASP-1.Treatment of human breast cancer cells with CXCL12, EGF and Heregulin and HMEC-CXCR2 cells with CXCL8 facilitated nuclear shuttling of LASP-1. Data from the biochemical analysis of the nuclear and cytosolic fractions further confirmed the nuclear translocation of LASP-1 upon chemokine and growth factor treatment. CXCL12-dependent nuclear import of LASP-1 could be blocked by CXCR4 antagonist, AMD-3100. Knock down of LASP-1 resulted in alterations in gene expression leading to an increased level of cell junction and extracellular matrix proteins and an altered cytokine secretory profile. Three dimensional cultures of human breast cancer cells on Matrigel revealed an altered colony growth, morphology and arborization pattern in LASP-1 knock down cells. Functional analysis of the LASP-1 knock down cells revealed increased adhesion to collagen IV and decreased invasion through the Matrigel. Proteomics analysis of immunoprecipitates of LASP-1 and subsequent validation approaches revealed that LASP-1associated with the epigenetic machinery especially UHRF1, DNMT1, G9a and the transcription factor Snail1. Interestingly, LASP-1 associated with UHRF1, G9a, Snail1 and di- and tri-methylated histoneH3 in a CXCL12-dependent manner based on immunoprecipitation and proximity ligation assays. LASP-1 also directly bound to Snail1 which may stabilize Snail1. Thus, nuclear LASP-1 appears to functionally serve as a hub for the epigenetic machinery.

Abstract 225: Overexpression of truncated Ron augments benzyl isothiocyanate-induced apoptosis in human breast cancer cells

Abstract Phytochemicals from edible plants remain attractive for prevention of breast cancer, which is a leading cause of cancer-related mortality in women worldwide. We have shown previously that benzyl isothiocyanate (BITC), a small-molecule found in edible cruciferous vegetables such as garden cress, confers significant protection against mammary cancer development in MMTV-neu transgenic mice. BITC-mediated prevention of mammary carcinogenesis in MMTV-neu mice in vivo is associated with tumor cell apoptosis induction and elimination of breast cancer stem cells (bCSC). In cultured human breast cancer cells, BITC-induced apoptosis is accompanied by activation of multidomain proapoptotic protein Bax whereas inhibition of bCSC self-renewal is significantly attenuated by overexpression of truncated Ron (recepteur d’origine nantais) tyrosine kinase. Ron expression is very low in normal human breast epithelium but its overexpression has been reported in about 50% of primary breast cancers. In addition, mammary-specific overexpression of wild-type Ron as well as its constitutively active form induces highly metastatic breast tumors in mice. The present study was undertaken to study the role of truncated Ron (sfRon) in BITC-induced apoptosis using MCF-7 and MDA-MB-361 human breast cancer cells. Overexpression of sfRon resulted in statistically significant augmentation of BITC-induced apoptotic cell death in both cell lines as judged by flow cytometry after staining with Annexin V and propidium iodide, analysis of histone associated DNA fragment release into the cytosol, and/or cell proliferation assay. On the other hand, sfRon overexpressing MCF-7 cells were resistant to BITC-mediated G2/M phase cell cycle arrest when compared with parental cells. Increased apoptosis following BITC treatment in sfRon overexpressing cells was due to enhanced activation of Bax and/or Bak. Taken together, these findings indicate that overexpression of sfRon sensitizes human breast cancer cells to BITC-induced apoptosis. This investigation was supported by the NCI grant CA129347-06. Citation Format: Anuradha Sehrawat, Shivendra V. Singh. Overexpression of truncated Ron augments benzyl isothiocyanate-induced apoptosis in human breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 225. doi:10.1158/1538-7445.AM2014-225

Aldehyde dehydrogenase and estrogen receptor define a hierarchy of cellular differentiation in the normal human mammary epithelium.

IntroductionAlthough estrogen and progesterone play a key role in normal mammary development and in breast cancer, the potential for proliferation and lineage differentiation as well as origin of cells that express the estrogen receptor (ER) in normal breast epithelium are not known. Some evidence suggests that normal human mammary stem/progenitor cells are ER–, but the identity of these cells and the cellular hierarchy of breast epithelium are still subjects of controversy. It is likely that elucidation of these aspects will bring insight into the cellular origin of breast cancer subtypes.MethodsWe used fluorescence-activated cell sorting of primary human mammary epithelial cells along with in vitro and in vivo functional assays to examine the hierarchic relation between cells with aldehyde dehydrogenase enzymatic activity (ALDH+ cells) and ER+ cells in the normal human breast epithelium. We assessed the proliferation and lineage differentiation potential of these cells in vitro and in vivo. A gene reporter assay was used to separate live ER+ and ER– mammary epithelial cells. With shRNA-mediated knockdown, we investigated the role of ALDH isoforms in the functionality of mammary epithelial progenitor cells.ResultsWe describe a cellular hierarchy in the normal human mammary gland in which ER–/ALDH+ cells with functional properties of stem/progenitor cells generate ER+ progenitor cells, which in turn give rise to cells of luminal lineage. We show that the ALDH1A1 isoform, through its function in the retinoic acid metabolism, affects the proliferation and/or early differentiation of stem/progenitor cells and is important for branching morphogenesis.ConclusionsThis study presents direct evidence that ER+ cells are generated by ER–/ALDH+ stem/progenitor cells. We also show that ER+ cells are able to generate cell progeny of luminal lineage in vitro and in vivo. Loss of ALDH1A1 function impairs this process, as well as branching morphogenesis and clonogenicity in suspension culture. This latter effect is reversed by treatment with retinoic acid.

Tissue specific DNA methylation in normal human breast epithelium and in breast cancer.

Cancer is a heterogeneous and tissue-specific disease. Thus, the tissue of origin reflects on the natural history of the disease and dictates the therapeutic approach. It is suggested that tissue differentiation, mediated mostly by epigenetic modifications, could guide tissue-specific susceptibility and protective mechanisms against cancer. Here we studied breast specific methylation in purified normal epithelium and its reflection in breast cancers. We established genome wide methylation profiles of various normal epithelial tissues and identified 110 genes that were differentially methylated in normal breast epithelium. A number of these genes also showed methylation alterations in breast cancers. We elaborated on one of them, TRIM29 (ATDC), and showed that its promoter was hypo-methylated in normal breast epithelium and heavily methylated in other normal epithelial tissues. Moreover, in breast carcinomas methylation increased and expression decreased whereas the reverse was noted for multiple other carcinomas. Interestingly, TRIM29 regulation in breast tumors clustered according to the PAM50 classification. Thus, it was repressed in the estrogen receptor positive tumors, particularly in the more proliferative luminal B subtype. This goes in line with previous reports indicating tumor suppressive activity of TRIM29 in estrogen receptor positive luminal breast cells in contrast to oncogenic function in pancreatic and lung cancers. Overall, these findings emphasize the linkage between breast specific epigenetic regulation and tissue specificity of cancer.

Abstract P4-11-03: Effect of tamoxifen and raloxifene on proliferative activity of breast epithelium in premenopausal women

Abstract Background: Both Tamoxifen and raloxifene have been demonstrated to be effective to reduce the incidence of breast cancer among women. Objective: To compare the effects of raloxifene (60 mg/day) and tamoxifen (20 mg/day) on the proliferative activity of normal human breast epithelium, during the luteal phase of the menstrual cycle, by means of the immunoexpression of Ki-67/MIB-1. Patients and Methods: Forty-eight women presenting benign breast nodules with an indication for lesion excision took part in this study. These women were randomized to use tamoxifen or raloxifene for 22 days. They underwent surgery after using the medication, and a one-centimeter fragment of normal breast tissue was removed to study the immunoexpression of Ki-67. For this, the frequency of cells immunolabeled for Ki-67/MIB-1 per thousand epithelial cells was quantified. Results: The mean percentage ratios between immunolabeled and non-labeled cells were 2.02 (±1.09) and 3.13 (±3.23) for the tamoxifen and raloxifene groups, respectively. There was no statistically significant difference between the two groups, tamoxifen (n = 16) and raloxifene (n = 14), in relation to the immunoexpression of Ki-67 (p = 0.205). The mean of progesterone level was 19.62 (±17.67) for the tamoxifen group and 14.59 (±8.98) for the raloxefene group (p = 0.32). The plasma levels of estradiol, the values found were: 355.72 (± 337.98) and 171.02 (± 119.83) for groups of tamoxifen e raloxifene groups, respectively (p = 0.055). Conclusion: In this study, we observed that there was no difference in the potential for reducing the proliferative activity of normal breast epithelium between tamoxifen and raloxifene in premenopausal women. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-11-03.

Abstract 5751: Detection of HMTV in breast and endometrium carcinomas

Abstract Retroviral sequences homologous to the betaretrovirus mouse mammary tumor virus (MMTV), the etiological agent of mammary tumor in mice, are present in 40% of American women's breast cancers. A 660 bp sequence homologous to MMTV env gene with no significant homology to any other viral or human sequence reported in the Gen Bank is found in breast cancer, but not in adjacent normal breast tissues, indicating its exogenous origin. A complete provirus structure with 95% homology to MMTV has been isolated from two human breast tumors and named human mammary tumor virus (HMTV). Betaretroviral particles from primary cultures of metastatic breast cancer cells (MSSM) have been isolated and characterized. HMTV virion RNA is more than 90% homologous to MMTV RNA and to the HMTV proviral DNA. HMTV is able to infect a variety of cells bringing about striking molecular changes, as seen by co-culture experiments between MSSM cells and normal human epithelial breast cells, B and T human lymphocytes and human dendritic cells. Protein expression was only observed in 10-15% of the infected cells by FACS analysis, suggesting the presence of innate resistance in human cells after HMTV infection, as well as in breast cancer cells. The human retroviral restriction factors APOBEC F and G and the TRIM proteins are all highly expressed in HMTV infected cells as well as in breast cancer cells as measured by quantitative RT-PCR. Disruption of the cytoskeleton, evidence for epithelial mesenchymal transition (EMT) and increase in invasiveness are additional molecular changes seen in HMTV infected cells. Analysis of the sequencing data showed that HMTV also contains hormone response elements (HRE) homologous to MMTV HRE. This result encouraged us to investigate if HMTV were present in hormonal responsive tissues other than breast. Endometrium is a classical example of such tissue. Endometrial cancers and normal endometrium were analyzed for HMTV env sequences. HMTV env sequences were present in 0% of 29 normal tissues and 23.2% of 56 tumors analyzed. Johal et al. recently reported that MMTV-like sequence was found in 10% of the endometrial cancers (J. Med. Virology; 82: 1044-50). We describe here the detection of HMTV proteins by immunohistochemistry (IHC) in formalin fixed paraffin embedded (FFPE) slides obtained from breast and endometrium carcinomas but not in their respective normal tissues. These results are compared with the results obtained by PCR. PCR is a very sensitive technique able to detect a single DNA copy number but potential contaminations can result in false positives. In addition, IHC is accessible to pathology laboratories since no hybridization step with radioactive material is necessary as in the HMTV PCR. In conclusion, we have developed a contamination resistant method like IHC using monoclonal antibodies against HMTV that correlates with the results we have obtained by western blot and by PCR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5751. doi:1538-7445.AM2012-5751

Identification of specific nuclear structural protein alterations in human breast cancer

Breast cancer is the most commonly diagnosed type of cancer and a major cause of death in women. Reliable biomarkers are urgently needed to improve early detection or to provide evidence of the prognosis for each individual patient through expression levels in tumor tissue or body fluids. This proteomic analysis focused on the nuclear structure of human breast cancer tissue, which has been shown to be a promising tool for cancer biomarker development. The nuclear matrix composition of human breast cancer (n = 14), benign controls (n = 2), and healthy controls (n = 2) was analyzed by high-resolution two-dimensional gel electrophoresis and mass spectrometry. Validation studies were performed in an individual sample set consisting of additional breast cancer tissues (n = 3) and additional healthy control tissues (n = 2) by one-dimensional immunoblot. In this setting, we identified five proteins that were upregulated in human breast cancer tissue, but absent in the healthy and benign controls (P < 0.001). These spots were also present in the investigated human breast cancer cell lines, but absent in the MCF10a cell line, which represents normal human epithelial breast cells. Two of the breast cancer-specific proteins have been confirmed to be calponin h2 and calmodulin-like protein 5 by one-dimensional immunoblot. This is the first study demonstrating the expression of both proteins in human breast cancer tissue. Further studies are required to investigate the potential role of these proteins as biomarkers for early diagnosis or prognosis in human breast cancer.

Use of in vivo and mechanistic evidence for the development of structural alerts for the prediction of non-genotoxic carcinogenicity

Receptor (PR) from the steroid hormone receptors superfamily. AR binding has been associated with i) the transient stimulation of cell proliferation and consequent tumour formation in the prostate of rats and humans [3], and also in organs such as the liver [4] and ii) strong tumour promoter activity to enhance carcinogenicity of endogenous species such as oestrogen reactive metabolites or reactive oxygen species [3]. PR binding has been correlated i) with cell proliferation in normal human epithelial breast cell via an indirect production of paracrine growth factors [5] as well as in rodent and/or human mammary tumour cells with medroxyprogesterone acetate [6] and ii) tumour promoter activity [7]. Use of in vivo and mechanistic evidence for the development of structural alerts for the prediction of non-genotoxic carcinogenicity Laurence Coquin, Laura Gibson, Mukesh L. Patel and Susanne A. Stalford. Lhasa Limited, 22-23 Blenheim Terrace, Woodhouse Lane, Leeds LS2 9HD, UK

Abstract 2723: Host response to HMTV

Abstract Work from our laboratory demonstrated the presence of retroviral sequences homologous to the mouse mammary tumor virus (MMTV) in a large proportion of human breast cancers. These observations have been confirmed by others. A 660 bp sequence of the MMTV env gene, with no significant homology to any viral or human sequence reported in the GenBank, has been found in 40% of the U.S. human breast cancers studied. This sequence is expressed in most of the env positive tumors, and is not detected in the normal breast tissue of patients with env positive carcinomas, indicating its exogenous origin. A complete provirus structure with 95% homology to MMTV has been described and designated as human mammary tumor virus (HMTV). Betaretroviral particles from primary cultures of breast cancer cells (MSSM) have been isolated and characterized. Virion RNA is more than 90% homologous to MMTV RNA and to the HMTV proviral DNA previously identified. To find out if these particles were infectious, co-culture experiments between MSSM cells and normal human epithelial breast cells, B and T human lymphocytes and human dendritic cells were performed. The results demonstrated that the recipient human cells became infected with HMTV. However, protein expression was only observed in 10-15% of the infected cells by FACS analysis suggesting the presence of an innate resistance to HMTV in human cells. We have then investigated whether human APOBEC and TRIM proteins are implicated in resistance to HMTV expression. The results indicated that APOBEC G and F, TRIM 5, 21 and 25 are all highly expressed in HMTV cells as measured by quantitative RT-PCR. Additional molecular changes seen in infected cells are disruption of the cytoskeleton and evidence for epithelial-mesenchymal transition (EMT). Since expression of an endogenous retrovirus (HERV-K) env gene has been reported to be upregulated in human breast cancers we have also investigated its expression in HMTV-infected cells, primary MSSM cells and breast tumors by quantitative RT PCR. The results indicated that in all instances where HMTV was present, expression of HERV-K (HML-2) was less than the control counterpart. In conclusion, HMTV is able to infect a variety of cells bringing about striking molecular changes. Whether these changes play a role in tumorigenesis remains to be shown. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2723. doi:10.1158/1538-7445.AM2011-2723

Mammary gland stem cells: current status and future challenges

Distinct subsets of cells, including cells with stem cell-like properties, have been proposed to exist in normal human breast epithelium and breast carcinomas. The cellular origins of epithelial cells contributing to gland development, tissue homeostasis and cancer are, however, still poorly understood. The mouse is a widely used model of mammary gland development, both directly by studying the mouse mammary epithelial cells themselves and indirectly, by studying development, morphogenesis, differentiation and carcinogenesis of xenotransplanted human breast epithelium in vivo. While in early studies, human or mouse epithelium was implanted as fragments into the mouse gland, more recent technical progress has allowed the self-renewal capacity and differentiation potential of distinct cell populations or even individual cells to be interrogated. Here, we review and discuss similarities and differences between mouse and human gland development with particular emphasis on the identity and localization of stem cells, and the influence of the surrounding microenvironment. It is concluded that while recent advances in the field have contributed immense insight into how the normal mammary gland develops and is maintained, significant discrepancies exist between the mouse and human gland which should be taken into consideration in current and future models of mammary stem cell biology.

Differential Contextual Responses of Normal Human Breast Epithelium to Ionizing Radiation in a Mouse Xenograft Model

Radiotherapy is a key treatment option for breast cancer, yet the molecular responses of normal human breast epithelial cells to ionizing radiation are unclear. A murine subcutaneous xenograft model was developed in which nonneoplastic human breast tissue was maintained with the preservation of normal tissue architecture, allowing us to study for the first time the radiation response of normal human breast tissue in situ. Ionizing radiation induced dose-dependent p53 stabilization and p53 phosphorylation, together with the induction of p21(CDKN1A) and apoptosis of normal breast epithelium. Although p53 was stabilized in both luminal and basal cells, induction of Ser392-phosphorylated p53 and p21 was higher in basal cells and varied along the length of the ductal system. Basal breast epithelial cells expressed ΔNp63, which was unchanged on irradiation. Although stromal responses themselves were minimal, the response of normal breast epithelium to ionizing radiation differed according to the stromal setting. We also demonstrated a dose-dependent induction of γ-H2AX foci in epithelial cells that was similarly dependent on the stromal environment and differed between basal and luminal epithelial cells. The intrinsic differences between human mammary cell types in response to in vivo irradiation are consistent with clinical observation that therapeutic ionizing radiation is associated with the development of basal-type breast carcinomas. Furthermore, there may be clinically important stromal-epithelial interactions that influence DNA damage responses in the normal breast. These findings demonstrate highly complex responses of normal human breast epithelium following ionizing radiation exposure and emphasize the importance of studying whole-tissue effects rather than single-cell systems.

Cadmium and Breast Cancer: Exposure Associated with Basal-Like Phenotype

Cadmium has been linked with several human diseases including chronic kidney disease and cancer. As a carcinogen, cadmium targets several sites that are considered endocrine-sensitive, and some data suggest the breast may be among them. Although cadmium has been hypothesized to act as a metalloestrogen—a metal that triggers an estrogen-like reaction—research to date has not confirmed this as a mechanism linking cadmium and breast cancer. Additionally, although many breast cancers are estrogen-dependent, some of the most difficult-to-treat cases are not. A new study finds that cadmium can induce malignant transformation in breast cells in vitro regardless of the absence of estrogen receptors, strengthening evidence that cadmium exposure may be a factor in breast cancer, a leading cause of cancer deaths among women [EHP 117:1847–1852; Benbrahim-Tallaa et al.]. MCF-10A cells, which are derived from normal human breast epithelium, were grown with either no cadmium exposure or continuous cadmium exposure (2.5 μM) for up to 40 weeks. Positive controls included MCF-7 human breast cancer cells (which express the estrogen receptors ER-α and ER-β) and SKBR3 breast cancer cells (which express HER2, a receptor that can be overexpressed in certain malignant breast cancer cells). In contrast, MCF-10A cells do not express ER-α, ER-β, or HER2 proteins, although expression can be acquired in carcinogenesis. Chronic cadmium exposure of the MCF-10A cells yielded increased expression of matrix metalloproteinase-9, an enzyme that facilitates tumor cell invasion. These cells also formed cell mounds, indicating a loss of contact inhibition (the natural process of cell growth stopping once a certain density of cells is reached). When these transformed cells were implanted in mice, they formed highly aggressive tumors that demonstrated metastatic potential. Transformed MCF-10A cells remained negative for ER-α and ER-β and also lacked HER2 protein. However, metallothionein, typically overexpressed in ER-negative breast cancers, was elevated as were several other breast cancer markers. These characteristics collectively suggest that cadmium could be a risk factor for a basal-like breast cancer phenotype, which is clinically associated with a higher risk of relapse after treatment and lower survival rates. The precise mechanism by which cadmium may transform breast cells is unknown, but the results of this study suggest it is unlikely to be a metalloestrogenic effect via estrogen receptors. Although additional research is needed to define the mechanism, the current study provides strong evidence that cadmium may play a role in human breast cancer.

Progestins induce catalase activities in breast cancer cells through PRB isoform: Correlation with cell growth inhibition

Reactive oxygen species (ROS) have been suggested to participate in tumor emergence due to their mitogenic and apoptotic signaling, and as contributors to DNA structural damage. Here we report that progesterone and various synthetic steroids with progestin potencies (norethisterone acetate, MPA, and Tibolone) counteract cell growth induced by hydrogen peroxide (H 2O 2), through a potent induction of catalase activities, in breast cancer cells and normal human epithelial breast cells. At physiological concentrations, progesterone and the pure progestin, Org2058, displayed the most potent H 2O 2 detoxification ability suggesting its effect was characteristic of its progestin potency. We also report on the enhancement of catalase activities by progesterone receptor isoform B (PRB), as determined from experiments using antiprogestins and MDA-MB-231, cells engineered for the selective expression of progesterone receptor isoform A or B. The potent action of progesterone on catalase activities indicates its contribution to a beneficial role in breast cell homeostasis.

Comparison of the Effects of Phenethyl Isothiocyanate and Sulforaphane on Gene Expression in Breast Cancer and Normal Mammary Epithelial Cells

Phenethyl isothiocyanate (PEITC) and sulforaphane (SF) exhibit tumor preventive activity in lung, prostate, breast and colon cancers. Our objective was to examine the effect of these two isothiocyanates on estrogen receptor-related genes, and genes related to apoptosis and cell cycle in the estrogen-dependent breast cancer cell line MCF7 and in normal human epithelial breast (HME) cells. We treated cells with 0.3 microM or 3.0 microM concentrations of PEITC or SF. In HME cells, gene expression was significantly altered for 23 genes by PEITC at a concentration of 0.3 microM and 4 genes at 3.0 microM. SF altered the expression of 16 genes at a concentration of 0.3 microM and 2 genes at 3.0 microM. In HME cells, genes altered by both PEITC and SF exhibited changes in gene expression that were similar in extent as well as direction of change. In MCF-7 cells, PEITC did not produce any significant changes in the gene expression at both treatment levels. SF produced significant changes in 7 genes, but only at the higher treatment level of 3.0 microM. Normal mammary cells exhibited more changes in the expression of estrogen receptor related genes than did breast cancer cells, and significantly these changes occurred predominantly at the low concentration of 0.3 microM, a concentration achievable by dietary input of isothiocyanates. Novel findings were the upregulation of the pro-apoptotic gene BAD and estrogen receptor beta gene in normal human mammary cells. These gene alterations observed, along with upregulation of tumor suppressors p21 and p27, may provide a protective effect to mammary cells against breast cancer.