An oestrogen-dependent model of breast cancer created by transformation of normal human mammary epithelial cells

INTRODUCTION: About 70% of breast cancers express oestrogen receptor alpha (ESR1/ERalpha) and are oestrogen-dependent for growth. In contrast with the highly proliferative nature of ERalpha-positive tumour cells, ERalpha-positive cells in normal breast tissue rarely proliferate. Because ERalpha expression is rapidly lost when normal human mammary epithelial cells (HMECs) are grown in vitro, breast cancer models derived from HMECs are ERalpha-negative. Currently only tumour cell lines are available to model ERalpha-positive disease. To create an ERalpha-positive breast cancer model, we have forced normal HMECs derived from reduction mammoplasty tissue to express ERalpha in combination with other relevant breast cancer genes. METHODS: Candidate genes were selected based on breast cancer microarray data and cloned into lentiviral vectors. Primary HMECs prepared from reduction mammoplasty tissue were infected with lentiviral particles. Infected HMECs were characterised by Western blotting, immunofluorescence microscopy, microarray analysis, growth curves, karyotyping and SNP chip analysis. The tumorigenicity of the modified HMECs was tested after orthotopic injection into the inguinal mammary glands of NOD/SCID mice. Cells were marked with a fluorescent protein to allow visualisation in the fat pad. The growth of the graft was analysed by fluorescence microscopy of the mammary glands and pathological analysis of stained tissue sections. Oestrogen dependence of tumour growth was assessed by treatment with the oestrogen antagonist fulvestrant. RESULTS: Microarray analysis of ERalpha-positive tumours reveals that they commonly overexpress the Polycomb-group gene BMI1. Lentiviral transduction with ERalpha, BMI1, TERT and MYC allows primary HMECs to be expanded in vitro in an oestrogen-dependent manner. Orthotopic xenografting of these cells into the mammary glands of NOD/SCID mice results in the formation of ERalpha-positive tumours that metastasise to multiple organs. The cells remain wild type for TP53, diploid and genetically stable. In vivo tumour growth and in vitro proliferation of cells explanted from tumours are dependent on oestrogen. CONCLUSION: We have created a genetically defined model of ERalpha-positive human breast cancer based on normal HMECs that has the potential to model human oestrogen-dependent breast cancer in a mouse and enables the study of mechanisms involved in tumorigenesis and metastasi

An oestrogen-dependent model of breast cancer created by transformation of normal human mammary epithelial cells

Introduction About 70% of breast cancers express oestrogen receptor a ( ESR1/ ER alpha) and are oestrogen-dependent for growth. In contrast with the highly proliferative nature of ER alpha-positive tumour cells, ER alpha-positive cells in normal breast tissue rarely proliferate. Because ER alpha expression is rapidly lost when normal human mammary epithelial cells ( HMECs) are grown in vitro, breast cancer models derived from HMECs are ER alpha-negative. Currently only tumour cell lines are available to model ER alpha-positive disease. To create an ER alpha-positive breast cancer model, we have forced normal HMECs derived from reduction mammoplasty tissue to express in combination with other relevant breast cancer genes. Methods Candidate genes were selected based on breast cancer microarray data and cloned into lentiviral vectors. Primary HMECs prepared from reduction mammoplasty tissue were infected with lentiviral particles. Infected HMECs were characterised by Western blotting, immunofluorescence microscopy, microarray analysis, growth curves, karyotyping and SNP chip analysis. The tumorigenicity of the modified HMECs was tested after orthotopic injection into the inguinal mammary glands of NOD/ SCID mice. Cells were marked with a fluorescent protein to allow visualisation in the fat pad. The growth of the graft was analysed by fluorescence microscopy of the mammary glands and pathological analysis of stained tissue sections. Oestrogen dependence of tumour growth was assessed by treatment with the oestrogen antagonist fulvestrant. Results Microarray analysis of ER alpha-positive tumours reveals that they commonly overexpress the Polycomb-group gene BMI1. Lentiviral transduction with ER a, BMI1, TERT and MYC allows primary HMECs to be expanded in vitro in an oestrogen-dependent manner. Orthotopic xenografting of these cells into the mammary glands of NOD/ SCID mice results in the formation of ER alpha-positive tumours that metastasise to multiple organs. The cells remain wild type for TP53, diploid and genetically stable. In vivo tumour growth and in vitro proliferation of cells explanted from tumours are dependent on oestrogen. Conclusion We have created a genetically defined model of ERa-positive human breast cancer based on normal HMECs that has the potential to model human oestrogen-dependent breast cancer in a mouse and enables the study of mechanisms involved in tumorigenesis and metastasis.</p

Expression of oestrogen receptor alpha (ERα) and BMI1 in human mammary epithelial cells

<p><b>Copyright information:</b></p><p>Taken from "An oestrogen-dependent model of breast cancer created by transformation of normal human mammary epithelial cells"</p><p>http://breast-cancer-research.com/content/9/3/R38</p><p>Breast Cancer Research 2007;9(3):R38-R38.</p><p>Published online 15 Jun 2007</p><p>PMCID:PMC1929103.</p><p></p> Plot of microarray data [18] showing BMI1 expression in ERα-positive and ERα-negative breast tumours. BMI1 is significantly overexpressed in ERα-positive tumours (< 0.001). Colony formation assay. Human mammary epithelial cells (HMECs) transduced with either glucuronidase (; a negative control gene) alone, alone or and were fixed after growth for 10 days in the presence of oestrogen and then stained with crystal violet. The surface area covered with cells in the fixed plates was used to estimate growth. Western blot for ERα and β-tubulin in MCF7 control cells or in passage 3 (P3, left) and passage 4 (P4, right) HMECs transduced with alone or with and together. Endogenous ERα expression is progressively lost with passage. Western blot for ERα, BMI1 and β-tubulin in passage 6 (P6) HMECs transduced with alone or with and together. Before being harvested, cells were treated for 24 hours with 1 nM oestrogen (E) or 1 μM fulvestrant (F). Endogenous ERα expression is no longer detectable. Exogenous ERα is destabilised by fulvestrant. Immunofluorescent staining of HMECs infected with and viruses for ERα (lower left panel) and BMI1 (upper left panel). 4',6-Diamidino-2-phenylindole (DAPI; right panels) was used to counterstain nuclei. ERα and BMI1 are both nuclear. Western blot for TERT, ERα, BMI1, MYC, p14, p16and β-tubulin in HMECs infected with control virus (, lane 1) or , , and lentiviruses. HMECs from three different patients are shown in lanes 2 to 4. The transgenes are expressed, and BMI1 suppresses p14and p16expression. Immunofluorescent staining of HMECs for keratin 14 (K14, green) and keratin 18 (K18, red). DAPI (blue) was used to counterstain nuclei. At passage 1, HMECs infected with control virus and plated at clonal density formed mixed colonies with central K18-positive luminal cells and peripheral K14-positive myoepithelial cells (GUS P1). Passaging of these cultures led to progressive loss of luminal cells (GUS P4). HMECs infected with and viruses maintained K18 expression at passage 4, but individual cells were positive for both K14 and K18 (ERα BMI1 P4)

Immunofluorescent staining of tumours harvested 35 days after injection of -transduced HMECs

<p><b>Copyright information:</b></p><p>Taken from "An oestrogen-dependent model of breast cancer created by transformation of normal human mammary epithelial cells"</p><p>http://breast-cancer-research.com/content/9/3/R38</p><p>Breast Cancer Research 2007;9(3):R38-R38.</p><p>Published online 15 Jun 2007</p><p>PMCID:PMC1929103.</p><p></p> Matched sections from one region of a tumour; matched sections from a different region. The haematoxylin/eosin staining (H&E) in (a) shows the formation of tumours with dense squamous islands and diffuse infiltrating regions. The antibodies used for immunofluorescence in (b-l) are indicated in the lower right corner of each panel. The cyan fluorescent protein (CFP) staining in (b) and keratin 18 (K18) staining in (i) show that the tumour cells are derived from the injected human mammary epithelial cells (HMECs; the anti-K18 antibody is human-specific). BMI1 staining in (c) and oestrogen receptor alpha (ERα) staining in (d), (f) and (j) show that the HMECs retain nuclear expression of the transgenes. In (d-f), (j) and (k) it can be seen that some cells expressing ERα are also Ki-67-positive; (g) and (l) show expression of the target gene progesterone receptor (PGR) in the tumour cells. There is a tendency, seen in (j) and (l), for cells with higher ERα expression to have lower PGR expression. In (h) and (i) it can be seen that the tumour cells are positive for keratins. The arrows in (a), (h) and (i) show a group of K18-positive glandular cells within a squamous island

Identification of molecular apocrine breast tumours by microarray analysis

Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor alpha gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a 'molecular apocrine' gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov-Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8-14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER- AR-) and molecular apocrine (ER- AR+).SCOPUS: ar.jinfo:eu-repo/semantics/publishe