An oestrogen-dependent model of breast cancer created by transformation of normal human mammary epithelial cells
© Duss et al.; licensee BioMed Central Ltd. 2007
Received: 26 January 2007
Accepted: 15 June 2007
Published: 15 June 2007
About 70% of breast cancers express oestrogen receptor α (ESR1/ERα) and are oestrogen-dependent for growth. In contrast with the highly proliferative nature of ERα-positive tumour cells, ERα-positive cells in normal breast tissue rarely proliferate. Because ERα expression is rapidly lost when normal human mammary epithelial cells (HMECs) are grown in vitro, breast cancer models derived from HMECs are ERα-negative. Currently only tumour cell lines are available to model ERα-positive disease. To create an ERα-positive breast cancer model, we have forced normal HMECs derived from reduction mammoplasty tissue to express ERα in combination with other relevant breast cancer genes.
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.
Microarray analysis of ERα-positive tumours reveals that they commonly overexpress the Polycomb-group gene BMI1. Lentiviral transduction with ERα, 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 ERα-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.
We have created a genetically defined model of ERα-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.
Classic epidemiological studies on the increase in cancer incidence with age predicted that from three to six independent events would be required to convert a normal cell into a tumour cell . Experimental studies have now proven that it is indeed possible to transform a cell in culture by modifying the activity of only a few critical genes. Cell lines quantitatively transformed by expressing oncogenes or inactivating tumour suppressor genes have been produced from normal fibroblasts, embryonic kidney cells and human mammary epithelial cells (HMECs) [2, 3]. The genes initially chosen for these studies were those encoding simian virus 40 large T and small t antigen, activated Ras and telomerase. Subsequently it was shown that viral oncogenes can be replaced by activated MYC and genes targeting the retinoblastoma pathway . This combination will transform HMECs, but the resulting tumours do not express oestrogen receptor α (ERα); this is an important weakness of current models, because about 70% of human breast tumours are ERα-positive.
ERα behaves quite differently in ERα-positive cell lines derived from human breast cancer and in normal human mammary epithelium in vivo. Oestradiol is a direct mitogen for ERα-positive cancer cell lines, but in normal human breast tissue the ERα-positive cells do not themselves divide in response to oestrogen . Instead, they relay a proliferative signal to neighbouring ERα-negative cells. The barrier to proliferation of ERα-positive normal cells may explain why HMECs rapidly lose ERα expression in culture and why the transformation studies performed so far have produced ERα-negative tumour cell lines.
The target cell of oncogenic mutations in the breast is probably a stem cell or bipotent progenitor cell [6, 7]. It is possible to enrich for these cells by growing primary HMECs in the non-adherent conditions previously developed for culture of neural stem cells, leading to the formation of so-called floating mammospheres . The Polycomb-group gene BMI1 can suppress activation of the p53 and Rb pathways by silencing the expression of p14ARF and p16CDKN2A  and it has been shown to increase the rate of self-renewal of mammospheres in response to Wnt, Hedgehog and Notch signals . Since Polycomb-group genes are overexpressed in breast cancer [11, 12], BMI1 is a relevant candidate to test in HMEC transformation assays. BMI1 was originally identified as an oncogene that cooperates with MYC to induce lymphomas in mice , and MYC is commonly amplified in breast cancer, so it is reasonable to use MYC in a transformation protocol that includes BMI1. We show here that lentiviral transduction of HMECs with ERα, BMI1, MYC and TERT leads to the formation of ERα-positive tumours whose growth is dependent on oestrogen.
Materials and methods
Approval for culture of reduction mammoplasty tissue was granted by the Lausanne University Hospital ethics committee, and patients gave informed consent. The patients were healthy women with no previous history of breast cancer. All samples were confirmed by histopathological examination to be free of malignancy. Primary HMECs and human mammary fibroblasts (HMFs) were prepared by standard techniques [8, 14]. HMECs were cultured in human mammosphere medium (HMM): Hepes-buffered DMEM/F12 without phenol red (Gibco, Basel, Switzerland) supplemented with 20 ng/ml EGF (Invitrogen, Basel, Switzerland), 1 × B-27 (Gibco) and 1 nM 17-β-oestradiol (Sigma, Buchs, Switzerland). B27 is a serum-free medium supplement containing antioxidants, vitamins, growth factors and hormones including progesterone . HMM was used for suspension and adherent culture of HMECs; tissue culture plastic was not coated with collagen for adherent culture. HMFs and MCF7 cells were grown in DMEM containing 10% fetal calf serum. For the proliferation assays, 20,000 cells were seeded per well in six-well plates in duplicate and stained with crystal violet (Sigma) after 10 days of culture in HMM containing 1 nM 17-β-oestradiol or 1 μM fulvestrant (ICI 182,780; Torcis Pharmaceuticals, Bristol, UK). The area covered with cells was quantified with ImageJ software (NIH, Bethesda, MD, USA). For the growth curves, 20,000 cells per well were seeded in 12-well plates in duplicate in HMM containing 1 nM 17-β-oestradiol or 1 μM fulvestrant. A separate plate was used for each time point and cells were counted in a Neubauer chamber. For colony formation and SNP assays from tumours, explanted cells were grown briefly in puromycin to eliminate murine cells.
The pSD-69 plasmid contains the human phosphoglycerate kinase (PGK) promoter, a Gateway attR cassette (Invitrogen), the mouse PGK promoter and the puromycin acetyltransferase gene cloned into pRRLhPGK.GFP.SIN18 . Preliminary studies showed that the human PGK promoter is active in mammospheres and differentiated HMECs. Gateway BMI1 and ESR1 clones were obtained from Flexgene (Boston, MA, USA). The MYC (c-Myc) and TERT (hTERT) clones were provided by J Lingner and A Trumpp, respectively, and cloned into pENTR1A (Invitrogen). The BMI1, ESR1, MYC and TERT cDNAs were transferred to pSD-69 by LR recombination (Invitrogen) to give pSD-84, 82, 94 and 83. The MYC clone was wild-type in sequence. The β-glucuronidase gene (gusA) was transferred from pENTR-GUS (Invitrogen) into pSD-69 by LR recombination to give the control vector pSD-86. The cyan fluorescent protein gene (CFP) was cloned into pRRLhPGK.GFP.SIN18 from pECFP (Clontech, Saint-Germain-en-Laye, France) by standard techniques to give pSD-25. Lentivirus was produced by calcium phosphate transfection of 293T cells . To transduce HMECs with multiple genes, infections were performed simultaneously with different viruses: cells were infected in suspension with the ERα and BMI1 vectors 24 hours after harvest from the patient (that is, after digestion with collagenase and dissociation of organoids to single cells), grown in suspension for 6 days in ultra-low-attachment dishes (Corning, New York, NY, USA), then dissociated, plated and infected with the MYC, TERT and CFP vectors. HMFs were infected with the CFP-expressing lentivirus in the initial experiment to facilitate identification in mice but were not transduced with TERT or transforming genes. The titre of each lentiviral batch was determined on primary HMECs. All infections were performed at a multiplicity of infection of 50 viral particles per cell.
The following antibodies were used: antibodies against p14 (FL-132), p16 (M-156), MYC (9E10) (Santa Cruz Biotechnologies, Santa Cruz, USA); keratin 14 (RB-9020), keratin 18 (MS-142), progesterone receptor (PGR; Ab1), ERα (SP1), high molecular weight keratins (AB-3; Neomarkers, Stehelin, Basel, Switzerland); BMI1 (F6; Upstate, Lucerna-Chem AG, Lucerne, Switzerland); β-tubulin (Sigma); GFP (A11122) (Molecular Probes, Invitrogen); Ki67 (Novocastra, Newcastle, UK) and hTERT (R484) . For Western blotting, goat anti-mouse or goat anti-rabbit antibodies coupled to horseradish peroxidase (Jackson ImmunoResearch, Newmarket, UK) were used, followed by chemiluminescent detection (Amersham, Little Chalfont, Bucks., UK). For immunofluorescent staining of tissue culture cells, samples were fixed with cold methanol. For staining of tissue, samples were fixed for 2 hours at 4°C in 4% paraformaldehyde and embedded in paraffin. Antigens were retrieved by boiling sections for 20 minutes in trisodium citrate buffer pH 6. Goat anti-mouse or goat anti-rabbit antibodies coupled to Alexa 488 or Alexa 568 (Jackson ImmunoResearch) were used for detection and the slides were mounted with 1,4-diazabicyclo [2.2.2]octane (DABCO; Sigma).
RNA was extracted with an RNeasy kit (Qiagen, Hombrechtikon, Switzerland), amplified as described previously  and hybridised to U133Plus 2.0 gene chips (Affymetrix, CA, USA). CEL files were normalised with RMA . The CEL files have been deposited in the GEO database under accession number GSE6548. DNA was extracted with a DNeasy kit (Qiagen), and 250 ng per chip was processed and hybridised to 50K HindIII SNP chips in accordance with the manufacturer's instructions (Affymetrix). The CEL files were analysed with CNAG 2.0 .
Cells were grown for 8 hours in HMM plus 100 ng/ml colcemid (Sigma). Metaphase spreads were prepared and stained with Giemsa (Sigma) essentially as described . Fifty-five metaphase spreads of HMEC strains established from different donors were photographed and the chromosomes were counted.
RNA was extracted from HMECs before injection into mice and from tumours in the fat pad with the use of an RNeasy kit (Qiagen). p53 status was determined by yeast functional assay with total RNA .
Animal experiments were authorised by the Veterinary Office of the Canton de Vaud, Switzerland. One million HMECs and 200,000 normal human mammary fibroblasts from separate cultures were mixed with 12.5% Matrigel (BD Biosciences) and injected into the fourth mammary gland of 8-week-old female NOD/SCID mice (NOD.CB17-Prkdc scid /J; Jackson Laboratory, Bar Harbor, ME, USA). Either the HMECs or the HMFs expressed CFP but never both: only one cell type in a single experiment was ever CFP-positive. The total time in tissue culture ex vivo before the epithelial cells were injected into the mammary fat pad was 28 days. Silicon pellets containing 1.5 mg of oestradiol  were inserted subcutaneously into the neck region of the experimental animals at the time that the cells were injected. For fulvestrant treatment, 5 mg of Faslodex (AstraZeneca AG, Zug, Switzerland) was injected subcutaneously at weekly intervals. The oestrogen pellets were not removed from fulvestrant-treated animals.
BMI1 allows expansion of HMECs that express ERα
The medium used for both suspension and adherent culture was HMM . Because it is based on neurosphere medium and the microarray showed the expression of some neural genes, such as NEFL, we sought to confirm the epithelial nature of the cells by staining for keratins. Control cells plated after one round of mammosphere culture formed three types of colony: pure keratin 18 (K18)-positive luminal colonies, pure keratin 14 (K14)-positive myoepithelial colonies, and mixed colonies containing both luminal and myoepithelial cells (Figure 1g, top panels). This is the same pattern as that reported by Dontu and colleagues  after mammosphere culture; similar observations have been made by other groups using different HMEC culture conditions [28–30]. At later passages, luminal cells were lost from the control cultures, resulting in the formation of increasingly pure K14-positive myoepithelial cell cultures (Figure 1g, middle panels). This is the expected result when HMECs are put into culture (reviewed in ). In contrast with the single-positive staining pattern of the controls, cells transduced with ERα and BMI1 were double-positive, staining for both K14 and K18 (Figure 1g, bottom panels). We conclude that the transduced cells are HMECs, the ERα and BMI1 proteins are correctly expressed and biologically active, and that BMI1 expression permits the outgrowth of ERα-positive colonies.
Growth of ERα-transduced cells is dependent on oestrogen
Creation of an ERα-positive tumour model
Tumour formation in NOD/SCID mice.
E2 + fulvestrant
The physiological level of oestradiol is higher in women than in mice, and it is frequently necessary to administer exogenous oestradiol to study human oestrogen-dependent phenotypes in mice . The xenografts were therefore repeated in mice given slow-release oestradiol pellets. In these experiments, the HMECs were tagged with CFP. All except one of the 32 injected mammary glands developed tumours; the tumorigenicity of the transgene-expressing cells was confirmed by using HMECs from three different patients (Table 1, 'E2'). The process of engraftment and tumour formation was followed by killing mice at different time points (Figure 4d–f, day 5; then Figure 4g–i, days 14, 21 and 35, respectively). Five days after injection there was a large necrotic mass of tumour cells at the site of injection, accompanied by a vigorous vascular response (Figure 4e). On the surface of the mass, patches of brightly fluorescent CFP-positive epithelial cells were visible (Figure 4d). Histological examination confirmed that the main mass was necrotic (Figure 4f, the asterisked region bounded by the dotted line), presumably because it was insufficiently vascularised, but the brightly staining patches on the surface contained viable cells that were forming invasive tumour even at this early time point (Figure 4f, open arrowheads; closed arrowheads show mouse ducts). The absence of any lag suggests that the cells are quantitatively transformed. Every subsequent time point showed the presence of invasive tumour cells with a similar histological pattern: dense islands of squamous carcinoma adjacent to diffuse regions of invasive ductal carcinoma. We conclude that HMECs transduced with ERα, BMI1, MYC and TERT readily form oestrogen-dependent tumours in mice.
ERα is active in the tumours
ERα-transduced cells are genetically stable
Response to anti-oestrogen therapy
Metastasis of tumour cells
We have developed a model for ERα-positive breast cancer by transformation of normal HMECs with ERα, BMI1, MYC and TERT. Metastasis occurred in 38% of the mice after 90 days. Previous attempts to make an ERα-positive model probably failed because ERα induces growth arrest and differentiation. We have not addressed the mechanism in this study, but transforming growth factor-β is known to restrain the proliferation of ERα-positive murine mammary epithelial cells . Expression of BMI1 prevents differentiation and relieves the growth arrest, allowing the expansion of oestrogen-dependent HMECs in culture.
There are several differences between our culture system and those used previously. We grew the cells in floating mammosphere conditions before the first passage for several reasons. At a practical level, the final step after tissue digestion is a single-cell straining step; this facilitates efficient infection of the cells with lentiviral vectors less than 24 hours after the cells are removed from the patient. A second practical advantage is that fibroblasts do not survive in suspension, so floating mammosphere culture is an efficient way to eliminate fibroblasts. More importantly, it is based on techniques developed initially for propagation of neural stem cells  and later adapted for culture of HMECs . The mammosphere approach enriches for bipotent progenitor cells that are capable of differentiating to myoepithelial and luminal cells, with production of milk proteins by the latter after treatment with prolactin in three-dimensional Matrigel culture . The main difference between our approach and that of Dontu and colleagues  is that we used the same medium for suspension and adherent cell culture, and we omitted basic fibroblast growth factor. The medium is based on B27 , a serum-free medium supplement that is known to preserve the phenotype of human tumour cells in culture better than serum-containing media . In our study, the relative importance of the medium versus the suspension culture is unclear but we have preliminary evidence that suspension culture may not be strictly necessary.
In the absence of a stem cell assay for HMECs it is not possible to state definitively whether mammospheres contain true human mammary epithelial stem cells (MaSCs). It is possible that the mammosphere approach enriches for mammary colony-forming cells (Ma-CFCs) rather than mammary repopulating units (MRUs) [37, 38]. The nature of the cell initially infected with lentiviruses in our protocol is unknown because the infections were performed on the mixed population of cells present in reduction mammoplasty tissue. An intriguing question is whether cells expressing the recently identified murine MaSC markers would be more sensitive to transformation. Given the uncertainty surrounding the identity of human MaSCs, our main aim was to reduce the duration of growth in vitro to limit the potential for selection of adaptations to culture in vitro. The present study used 106 cells per fat pad injection, for which we needed to expand the cultures in vitro for a total of 28 days. We have preliminary results indicating that 5,000 cells are sufficient to form tumours, so it should be possible to greatly reduce the duration of culture in vitro.
Polycomb-group genes such as BMI1, EZH2 and SUZ12 have repeatedly been identified as adverse prognostic factors in breast cancer [11, 12]. BMI1 is required for proliferation and renewal of stem cells in the brain and hematopoietic system. In mammospheres, BMI1 is thought to act as a point of convergence of the Wnt, Notch and Hedgehog signals that promote stem cell renewal . BMI1 probably has at least a dual role, allowing cell proliferation by suppressing p14ARF and p16CDKN2A expression, and preventing differentiation through a more complex mechanism. Both processes are clearly visible in the microarray data reported here (Figure 2). ERα-positive tumours typically contain wild-type p53 and have fewer genomic changes than ERα-negative tumours . The ERα-positive tumour model we have produced matches the human disease in this respect. The most likely explanation for the tumours to have retained wild-type p53 is that BMI1 suppresses p14ARF expression . Previous quantitative transformation models included genes such as those encoding simian virus 40 T antigen and p53DD to inactivate p53 [3, 4]. The MCF10A and MCF15 HMEC-derived cell lines show large differences in their DNA damage response despite both retaining wild-type p53 , and ERα-positive human breast tumours respond poorly to chemotherapy despite having wild-type p53. It is therefore important to note that although we have shown that the p53 cDNA is wild type, we have not shown that the p53 pathway is functional in our cells. The genes suppressed by BMI1 in ERα-expressing cells include many associated with neural and squamous differentiation. Suppression of these genes presumably favours proliferation by avoiding entry into a terminal differentiation program. We found that BMI1 itself was one of the genes suppressed by exogenous BMI1 expression. Bracken and colleagues showed by chromatin immunoprecipitation (ChIP) that the Polycomb-repressive complex 1 (PRC1) component CDX8 and the PRC2 component SUZ12 were present at the BMI1 promoter . Suppression of PRC function by RNA-mediated interference (RNAi) led to derepression of genes with PRC proteins at the promoter . On the basis of the ChIP data and the transcriptional response to RNAi against BMI1, EED, SUZ12 and EZH2, Bracken and colleagues suggested that PcG proteins autoregulate their own synthesis . Autoregulation of BMI1 itself would by definition not have been detectable in their RNAi experiment, but when taken together with our results it is plausible that BMI1 suppresses its own expression through binding to its own promoter.
Wild-type ERα is not normally considered to be an oncogene, but behaves like one in our protocol. It is well known that ERα expression is rapidly lost from HMECs in culture. This is not solely a consequence of growth inhibition by ERα, because expression is still lost when cells are forced to express exogenous BMI1. In comparison with previous studies, the combination of genes we used to transform the cells seems rather gentle. In particular, we see no need to activate Ras signalling [2–4, 41]. TERT was essential for successful transformation of HMECs in previous studies , but BMI1 and MYC can both activate TERT expression [42, 43], so it is possible that TERT may not be required in our protocol. We included MYC in the protocol because BMI1 was originally identified as an oncogene that cooperates with MYC in lymphoma production in mice , MYC is commonly amplified in human breast cancer, and several groups have reported that MYC is required for HMEC transformation. Indeed, when Elenbaas and colleagues  used an HMEC transformation protocol lacking MYC, the cells spontaneously amplified MYC during culture in vitro. We used a wild-type MYC (c-myc) clone for our studies, rather than the activated form of MYC (T58A) used by Kendall and colleagues . Despite the strong evidence that MYC is important, the requirements may differ when the selection conditions are changed, and we have preliminary evidence that MYC may not be required in ERα/BMI/TERT-transduced HMECs, at least for the initial stages of tumour formation.
It is intriguing that the transformed HMECs in our model can form polarised epithelial structures in vivo that express the correct luminal and basal keratins (Figure 6). This indicates that the double-positive keratin staining pattern in vitro is more likely to reflect a specific progenitor state than a loss of control of lineage-specific gene expression. Unlike the primary tumours in the mammary gland, the metastases were K18-negative. This suggests that the cells differentiate in response to signals from their local environment and that the mammary fat pad supplies specific signals that promote luminal keratin expression. Although the tumours contained regions of invasive adenocarcinoma, the predominant pathology was squamous carcinoma. Squamous differentiation is uncommon in human breast tumours. The squamous differentiation we observed in the NOD/SCID mice may reflect a general property of the mouse mammary fat pad model, a specific property of the target cell of the in vitro transformation protocol, or a defect in the transactivation of critical ERα target genes in the transformed cells. In comparison with the human breast, the mouse mammary gland contains much less fibrous connective tissue . To promote engraftment of HMECs in the mouse mammary gland, human fibroblasts are commonly injected either at the same time as the HMECs or a few days earlier, to 'humanise' the stroma . Human cancer-associated fibroblasts (CAFs) are similarly used to promote engraftment of human tumour cells in mice . It is possible that the HMFs we injected together with the HMECs may have contributed to the squamous phenotype, but injection of our HMECs without HMFs led to the formation of tumours with similar kinetics and histology (data not shown). As mentioned above, we do not know the identity of the target cell of the transformation protocol. The mammosphere protocol enriches for mammary epithelial progenitor cells, but it is possible that expression of BMI1 promotes the survival of more differentiated cells or, conversely, forces progenitors to adopt a more stem-cell-like phenotype. HMEC protocols commonly give rise to squamous tumours in mice [3, 46, 47], so we consider it unlikely that the mammosphere protocol has led to the expansion of cells that are unrelated to mammary epithelium. Another possibility is that, despite expressing ERα, the cells are unable to respond appropriately to it. The gene expression profile of breast tumours is dominated by genes that are tightly associated with ERα status . Many of these genes are direct targets of ERα but others are thought to represent markers of cell type. Some classic ERα target genes, such as the progesterone and prolactin receptor genes, were induced by oestradiol in the ERα/BMI1 HMECs, but others, such as TFF1 and XBP1, were not (Figure 2; the full data set is available in the GEO database under accession number GSE6548). The uninduced group includes many of the genes shown by RNA interference to require co-activation by FOXA1 . Because FOXA1 is expressed only weakly in the transformed cells, transduction with a FOXA1 vector might lead to the activation of a broader range of ERα target genes and suppression of the squamous phenotype. More generally, there may be regulators of the differentiation programme of mammary epithelial cells, such as GATA3 or TP73L, that are not correctly expressed in the xenografts. We are currently testing these models to develop a transformation protocol that more faithfully reproduces the histology of human breast tumours.
It has long been known that ERα-positive breast tumours metastasise early, leading to distant relapse many years after excision of the primary tumour. In comparison with ERα-negative tumours, they have a better initial prognosis but this is followed by a relentless increase in breast cancer-specific mortality that continues even 15 years after treatment of the primary tumour . It is tempting to speculate that the correct paradigm for these tumours is a low-grade lymphoma: a systemic disease characterised by few genetic changes and poor response to therapy. Intriguingly, there is a class of ERα-positive breast tumours that have only a single change on genomic profiling: an unbalanced translocation leading to gain of chromosome 1q and loss of chromosome 16q . For this model to be correct, metastasis would have to occur early. Metastasis has not previously been reported in studies with HMECs transformed with defined oncogenes . At early time points the transformed HMECs reported here invaded the fat pad rapidly, forming a prominent duct-like structure that arose either from cells deposited in the needle track or by the migration of cells out from the main mass (Figure 4g–i). We have not tested the invasive properties of the cells in vitro, but the specific combination of genes used to transform the cells certainly triggers an invasive and metastatic program in vivo. Because the key difference between this model and previous HMEC models is ERα expression, it is tempting to speculate that ERα itself has a critical role in the early metastasis of ERα-positive human breast tumours.
We have created a new model for ERα-positive breast cancer by transduction of normal HMECs with lentiviruses expressing ERα, BMI1, MYC and TERT. The transformed cells are oestrogen-dependent for growth, wild-type for p53, diploid, and genetically normal as judged by hybridisation of tumour-cell DNA to SNP chips. The lack of secondary genetic changes and the high efficiency of tumour formation suggest that the cells are quantitatively transformed by the transgenes. The cells form disseminated peritoneal and liver metastases, a feature not previously seen with genetically defined, ERα-negative breast cancer models.
cyan fluorescent protein
Dulbecco's modified Eagle's medium
oestrogen receptor alpha
green fluorescent protein
human mammary epithelial cell
human mammary fibroblast
human mammosphere medium
mammary epithelial stem cell
single nucleotide polymorphism.
We thank V Sechet, A Cottilard and W Raffoul for providing reduction mammoplasty samples, S Mallepell and RD Rajaram for breeding mice, C Pythoud, C Morel and N Mueller for expert technical assistance, X Schmidt for analysing p53 responses, J Huelsken, D Trono, L Naldini, J Lingner and A Trumpp for providing reagents, F Paulin and A Thompson for help with microarrays, CS Herrington and V Becette for advice on pathology, and P Gonczy for critical reading of the manuscript. We acknowledge financial support from Oncosuisse, the Swiss National Science Foundation NCCR Molecular Oncology program, Breast Cancer Research Scotland, EU Active p53 project and ISREC.
- Vogelstein B, Kinzler KW: The multistep nature of cancer. Trends Genet. 1993, 9: 138-141. 10.1016/0168-9525(93)90209-Z.PubMedView ArticleGoogle Scholar
- Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg RA: Creation of human tumour cells with defined genetic elements. Nature. 1999, 400: 464-468. 10.1038/22780.PubMedView ArticleGoogle Scholar
- Elenbaas B, Spirio L, Koerner F, Fleming MD, Zimonjic DB, Donaher JL, Popescu NC, Hahn WC, Weinberg RA: Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells. Genes Dev. 2001, 15: 50-65. 10.1101/gad.828901.PubMedPubMed CentralView ArticleGoogle Scholar
- Kendall SD, Linardic CM, Adam SJ, Counter CM: A network of genetic events sufficient to convert normal human cells to a tumorigenic state. Cancer Res. 2005, 65: 9824-9828. 10.1158/0008-5472.CAN-05-1543.PubMedView ArticleGoogle Scholar
- Clarke RB, Howell A, Potten CS, Anderson E: Dissociation between steroid receptor expression and cell proliferation in the human breast. Cancer Res. 1997, 57: 4987-4991.PubMedGoogle Scholar
- Li Y, Rosen JM: Stem/progenitor cells in mouse mammary gland development and breast cancer. J Mammary Gland Biol Neoplasia. 2005, 10: 17-24. 10.1007/s10911-005-2537-2.PubMedView ArticleGoogle Scholar
- Smalley M, Ashworth A: Stem cells and breast cancer: a field in transit. Nat Rev Cancer. 2003, 3: 832-844. 10.1038/nrc1212.PubMedView ArticleGoogle Scholar
- Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS: In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 2003, 17: 1253-1270. 10.1101/gad.1061803.PubMedPubMed CentralView ArticleGoogle Scholar
- Jacobs JJL, Kieboom K, Marino S, DePinho RA, van Lohuizen M: The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature. 1999, 397: 164-168. 10.1038/16476.PubMedView ArticleGoogle Scholar
- Liu S, Dontu G, Wicha MS: Mammary stem cells, self-renewal pathways, and carcinogenesis. Breast Cancer Res. 2005, 7: 86-95. 10.1186/bcr1021.PubMedPubMed CentralView ArticleGoogle Scholar
- Sparmann A, van Lohuizen M: Polycomb silencers control cell fate, development and cancer. Nat Rev Cancer. 2006, 6: 846-856. 10.1038/nrc1991.PubMedView ArticleGoogle Scholar
- Kim JH, Yoon SY, Jeong SH, Kim SY, Moon SK, Joo JH, Lee Y, Choe IS, Kim JW: Overexpression of Bmi-1 oncoprotein correlates with axillary lymph node metastases in invasive ductal breast cancer. Breast. 2004, 13: 383-388. 10.1016/j.breast.2004.02.010.PubMedView ArticleGoogle Scholar
- van Lohuizen M, Verbeek S, Scheijen B, Wientjens E, van der Gulden H, Berns A: Identification of cooperating oncogenes in E mu-myc transgenic mice by provirus tagging. Cell. 1991, 65: 737-752. 10.1016/0092-8674(91)90382-9.PubMedView ArticleGoogle Scholar
- Stingl J, Emerman JT, Eaves CJ: Enzymatic dissociation and culture of normal human mammary tissue to detect progenitor activity. Methods Mol Biol. 2005, 290: 249-263.PubMedGoogle Scholar
- Brewer GJ, Torricelli JR, Evege EK, Price PJ: Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res. 1993, 35: 567-576. 10.1002/jnr.490350513.PubMedView ArticleGoogle Scholar
- Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L: A third-generation lentivirus vector with a conditional packaging system. J Virol. 1998, 72: 8463-8471.PubMedPubMed CentralGoogle Scholar
- Wenz C, Enenkel B, Amacker M, Kelleher C, Damm K, Lingner J: Human telomerase contains two cooperating telomerase RNA molecules. EMBO J. 2001, 20: 3526-3534. 10.1093/emboj/20.13.3526.PubMedPubMed CentralView ArticleGoogle Scholar
- Farmer P, Bonnefoi H, Becette V, Tubiana-Hulin M, Fumoleau P, Larsimont D, Macgrogan G, Bergh J, Cameron D, Goldstein D, et al: Identification of molecular apocrine breast tumours by microarray analysis. Oncogene. 2005, 24: 4660-4671. 10.1038/sj.onc.1208561.PubMedView ArticleGoogle Scholar
- Bioconductor. [http://www.bioconductor.org]
- Nannya Y, Sanada M, Nakazaki K, Hosoya N, Wang L, Hangaishi A, Kurokawa M, Chiba S, Bailey DK, Kennedy GC, et al: A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays. Cancer Res. 2005, 65: 6071-6079. 10.1158/0008-5472.CAN-05-0465.PubMedView ArticleGoogle Scholar
- Henegariu O, Heerema NA, Lowe Wright L, Bray-Ward P, Ward DC, Vance GH: Improvements in cytogenetic slide preparation: controlled chromosome spreading, chemical aging and gradual denaturing. Cytometry. 2001, 43: 101-109. 10.1002/1097-0320(20010201)43:2<101::AID-CYTO1024>3.0.CO;2-8.PubMedView ArticleGoogle Scholar
- Flaman JM, Frebourg T, Moreau V, Charbonnier F, Martin C, Chappuis P, Sappino AP, Limacher IM, Bron L, Benhattar J, et al: A simple p53 functional assay for screening cell lines, blood, and tumors. Proc Natl Acad Sci USA. 1995, 92: 3963-3967. 10.1073/pnas.92.9.3963.PubMedPubMed CentralView ArticleGoogle Scholar
- Laidlaw IJ, Clarke RB, Howell A, Owen AW, Potten CS, Anderson E: The proliferation of normal human breast tissue implanted into athymic nude mice is stimulated by estrogen but not progesterone. Endocrinology. 1995, 136: 164-171. 10.1210/en.136.1.164.PubMedGoogle Scholar
- Wang Y, Klijn JG, Zhang Y, Sieuwerts AM, Look MP, Yang F, Talantov D, Timmermans M, Meijer-van Gelder ME, Yu J, et al: Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet. 2005, 365: 671-679.PubMedView ArticleGoogle Scholar
- van de Vijver MJ, He YD, van't Veer LJ, Dai H, Hart AA, Voskuil DW, Schreiber GJ, Peterse JL, Roberts C, Marton MJ, et al: A gene-expression signature as a predictor of survival in breast cancer. N Engl J. 2002, 347: 1999-2009. 10.1056/NEJMoa021967.View ArticleGoogle Scholar
- Foster SA, Wong DJ, Barrett MT, Galloway DA: Inactivation of p16 in human mammary epithelial cells by CpG island methylation. Mol Cell Biol. 1998, 18: 1793-1801.PubMedPubMed CentralView ArticleGoogle Scholar
- Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K: Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev. 2006, 20: 1123-1136. 10.1101/gad.381706.PubMedPubMed CentralView ArticleGoogle Scholar
- Stingl J, Eaves CJ, Kuusk U, Emerman JT: Phenotypic and functional characterization in vitro of a multipotent epithelial cell present in the normal adult human breast. Differentiation. 1998, 63: 201-213. 10.1111/j.1432-0436.1998.00201.x.PubMedView ArticleGoogle Scholar
- Stingl J, Eaves CJ, Zandieh I, Emerman JT: Characterization of bipotent mammary epithelial progenitor cells in normal adult human breast tissue. Breast Cancer Res Treat. 2001, 67: 93-109. 10.1023/A:1010615124301.PubMedView ArticleGoogle Scholar
- Clayton H, Titley I, Vivanco M: Growth and differentiation of progenitor/stem cells derived from the human mammary gland. Exp Cell Res. 2004, 297: 444-460. 10.1016/j.yexcr.2004.03.029.PubMedView ArticleGoogle Scholar
- Villadsen R: In search of a stem cell hierarchy in the human breast and its relevance to breast cancer evolution. Apmis. 2005, 113: 903-921. 10.1111/j.1600-0463.2005.apm_344.x.PubMedView ArticleGoogle Scholar
- Kuperwasser C, Chavarria T, Wu M, Magrane G, Gray JW, Carey L, Richardson A, Weinberg RA: Reconstruction of functionally normal and malignant human breast tissues in mice. Proc Natl Acad Sci USA. 2004, 101: 4966-4971. 10.1073/pnas.0401064101.PubMedPubMed CentralView ArticleGoogle Scholar
- Metivier R, Penot G, Hubner MR, Reid G, Brand H, Kos M, Gannon F: Estrogen receptor-α directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell. 2003, 115: 751-763. 10.1016/S0092-8674(03)00934-6.PubMedView ArticleGoogle Scholar
- Ewan KB, Oketch-Rabah HA, Ravani SA, Shyamala G, Moses HL, Barcellos-Hoff MH: Proliferation of estrogen receptor-α-positive mammary epithelial cells is restrained by transforming growth factor-β1 in adult mice. Am J Pathol. 2005, 167: 409-417.PubMedPubMed CentralView ArticleGoogle Scholar
- Reynolds BA, Weiss S: Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol. 1996, 175: 1-13. 10.1006/dbio.1996.0090.PubMedView ArticleGoogle Scholar
- Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM, Pastorino S, Purow BW, Christopher N, Zhang W, et al: Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell. 2006, 9: 391-403. 10.1016/j.ccr.2006.03.030.PubMedView ArticleGoogle Scholar
- Shackleton M, Vaillant F, Simpson KJ, Stingl J, Smyth GK, Asselin-Labat ML, Wu L, Lindeman GJ, Visvader JE: Generation of a functional mammary gland from a single stem cell. Nature. 2006, 439: 84-88. 10.1038/nature04372.PubMedView ArticleGoogle Scholar
- Stingl J, Eirew P, Ricketson I, Shackleton M, Vaillant F, Choi D, Li HI, Eaves CJ: Purification and unique properties of mammary epithelial stem cells. Nature. 2006, 439: 993-997.PubMedGoogle Scholar
- Chin K, DeVries S, Fridlyand J, Spellman PT, Roydasgupta R, Kuo WL, Lapuk A, Neve RM, Qian Z, Ryder T, et al: Genomic and transcriptional aberrations linked to breast cancer pathophysiologies. Cancer Cell. 2006, 10: 529-541. 10.1016/j.ccr.2006.10.009.PubMedView ArticleGoogle Scholar
- Shen KC, Miller F, Tait L, Santner SJ, Pauley R, Raz A, Tainsky MA, Brooks SC, Wang YA: Isolation and characterization of a breast progenitor epithelial cell line with robust DNA damage responses. Breast Cancer Res Treat. 2006, 98: 357-364. 10.1007/s10549-006-9173-4.PubMedView ArticleGoogle Scholar
- Rangarajan A, Hong SJ, Gifford A, Weinberg RA: Species- and cell type-specific requirements for cellular transformation. Cancer Cell. 2004, 6: 171-183. 10.1016/j.ccr.2004.07.009.PubMedView ArticleGoogle Scholar
- Dimri GP, Martinez JL, Jacobs JJ, Keblusek P, Itahana K, Van Lohuizen M, Campisi J, Wazer DE, Band V: The Bmi-1 oncogene induces telomerase activity and immortalizes human mammary epithelial cells. Cancer Res. 2002, 62: 4736-4745.PubMedGoogle Scholar
- Wu KJ, Grandori C, Amacker M, Simon-Vermot N, Polack A, Lingner J, Dalla-Favera R: Direct activation of TERT transcription by c-MYC. Nat Genet. 1999, 21: 220-224. 10.1038/6010.PubMedView ArticleGoogle Scholar
- Parmar H, Cunha GR: Epithelial–stromal interactions in the mouse and human mammary gland in vivo. Endocr Relat Cancer. 2004, 11: 437-458. 10.1677/erc.1.00659.PubMedView ArticleGoogle Scholar
- Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, Naeem R, Carey VJ, Richardson AL, Weinberg RA: Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell. 2005, 121: 335-348. 10.1016/j.cell.2005.02.034.PubMedView ArticleGoogle Scholar
- Stampfer MR, Yaswen P: Culture systems for study of human mammary epithelial cell proliferation, differentiation and transformation. Cancer Surv. 1993, 18: 7-34.PubMedGoogle Scholar
- Santner SJ, Dawson PJ, Tait L, Soule HD, Eliason J, Mohamed AN, Wolman SR, Heppner GH, Miller FR: Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells. Breast Cancer Res Treat. 2001, 65: 101-110. 10.1023/A:1006461422273.PubMedView ArticleGoogle Scholar
- Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, et al: Molecular portraits of human breast tumours. Nature. 2000, 406: 747-752. 10.1038/35021093.PubMedView ArticleGoogle Scholar
- Carroll JS, Liu XS, Brodsky AS, Li W, Meyer CA, Szary AJ, Eeckhoute J, Shao W, Hestermann EV, Geistlinger TR, et al: Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead protein FoxA1. Cell. 2005, 122: 33-43. 10.1016/j.cell.2005.05.008.PubMedView ArticleGoogle Scholar
- EBCTCG: Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. The Lancet. 2005, 365: 1687-1717. 10.1016/S0140-6736(05)66544-0.View ArticleGoogle Scholar
- Fridlyand J, Snijders AM, Ylstra B, Li H, Olshen A, Segraves R, Dairkee S, Tokuyasu T, Ljung BM, Jain AN, et al: Breast tumor copy number aberration phenotypes and genomic instability. BMC Cancer. 2006, 6: 96-10.1186/1471-2407-6-96.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.