Paracrine signaling through receptor activator of NF-κB (RANK) pathway mediates the expansion of mammary epithelia that occurs during pregnancy, and activation of RANK pathway promotes mammary tumorigenesis in mice. In this study we extend these previous data to human cells and show that the RANK pathway promotes the development of mammary stem cells and breast cancer. Overexpression of RANK (FL-RANK) in a panel of tumoral and normal human mammary cells induces the expression of breast cancer stem and basal/stem cell markers. High levels of RANK in untransformed MCF10A cells induce changes associated with both stemness and transformation, including mammary gland reconstitution, epithelial–mesenchymal transition (EMT), increased migration, and anchorage-independent growth. In addition, spheroids of RANK overexpressing MCF10A cells display disrupted acinar formation, impair growth arrest and polarization, and luminal filling. RANK overexpression in tumor cells with nonfunctional BRCA1 enhances invasiveness in acinar cultures and increases tumorigenesis and metastasis in immunodeficient mice. High levels of RANK were found in human primary breast adenocarcinomas that lack expression of the hormone receptors, estrogen and progesterone, and in tumors with high pathologic grade and proliferation index; high RANK/RANKL expression was significantly associated with metastatic tumors. Together, our findings show that RANK promotes tumor initiation, progression, and metastasis in human mammary epithelial cells by increasing the population of CD44+CD24 cells, inducing stemness and EMT. These results suggest that RANK expression in primary breast cancer associates with poor prognosis. Cancer Res; 72(11); 2879–88. ©2012 AACR.

Receptor activator of NF-κB (RANK) ligand (RANKL) and its receptor RANK, members of the TNF ligand and receptor super family, respectively, are key regulators of bone remodeling and metastasis (1), and mammary gland development (2). Impaired mammary gland development of the RANK and RANKL null mice is due to defective proliferation and increased apoptosis of mammary epithelium (2). Conversely, overexpression of RANK or RANKL in the mammary gland leads to increased proliferation of the mammary epithelia (3, 4). It has been postulated that paracrine signaling through RANK/RANKL is responsible for the expansion of mammary stem cells observed during pregnancy and luteal cycles (5, 6).

We and others have recently shown that activation of RANK signaling promotes mammary tumorigenesis in mice (4, 7, 8). MMTV-RANK transgenic mice are prone to mammary tumors (4, 7). Reciprocally, pharmacologic inhibition of RANKL or genetic ablation of RANK attenuates mammary tumor development (7, 8).

Epithelial to mesenchymal transition (EMT) involves the loss of E-cadherin–mediated cell–cell adhesion and apical–basal polarity, concomitantly with the acquisition of a motile behavior contributing to invasion and metastasis (9). Induction of EMT in immortalized human mammary epithelial cells also results in the expression of stem cell markers (10), suggesting that these 2 processes may be functionally linked. Because rodent studies have shown that activation of RANK signaling promotes tumor initiation, progression, and metastasis involving mechanisms including increased proliferation, survival (7, 8), and enhanced regenerative capacity of cancer stem cells (8), then we analyzed the contribution of the RANK pathway to human breast stem cells and EMT using a panel of normal and tumoral human mammary epithelial cells. Our results highlight the relevance of the RANK pathway in human mammary stem cells and breast cancer.

Culture of human mammary epithelial cells

All cell lines were purchased from the American Type Culture Collection (ATCC), except UACC3199, which was obtained from the Arizona Cancer Center (Tucson, AZ). ATCC provides molecular authentication in support of their collection through their genomics, immunology, and proteomic cores, as described, by using DNA barcoding and species identification, quantitative gene expression, and transcriptomic analyses (ATCC Bulletin, 2010). UACC3199 cells harbor a methylated BRCA1 promoter suppressing gene transcription (11). UACC3199 was authenticated by its ability to reexpress BRCA1 after DNA demethylation treatment with 5-aza-2′-deoxycytidine. All lines were expanded and frozen within 2 weeks of purchase and used for a maximum of 2 months after resuscitation of frozen aliquots. MCF10A and HMECs immortalized with telomerase were cultured as described in Debnath and colleagues (12). Other cell culture conditions are described in Supplementary Methods.

Lentiviral infection

Lentiviral infection using pLV409-RANK, pLV417 control that contain a luciferase reporter, or pLenti6/V5-DEST-RANK, pLenti6/V5-DEST-tubGFP was done following the manufacturer's indications (Invitrogen) and Supplementary Methods.

Protein isolation and Western blot analysis

To evaluate activation of RANK signaling, MDA-MB-436, HCC1937, and UACC3199 cells were starved (S) with 0% FBS for 48 hours, MCF10A cells were starved in 2% serum without EGF overnight and then stimulated with hRANKL (100 ng/mL; Amgen). Proteins were isolated and analyzed following standard procedures and antibodies (Supplementary Methods).

Three-dimensional cultures and immunofluorescence analysis from human cells

Acinar structures were cultured and stained as described (12) and Supplementary Methods. Confocal analysis was carried out using a Leica confocal microscopy system equipped with argon and HeNe lasers. Images were captured using LasAF software (Leica).

Flow cytometry

Cells were seeded at 50% of confluence in growth medium. After 24 hours 100 ng/mL hRANKL (Amgen) were added. Forty-eight hours later medium was removed, MCF10A cells were washed and stained as reported in Supplementary Methods, and analyzed with the fluorescence-activated cell sorting Canto BD Flow Cytometer.

Xenograft in immunodeficient mice

FL-RANK or parental MCF10A, MDA-MB-436, or UACC 3199 (3 × 106) cells mixed with Matrigel (50%) were injected into the fat pad or (106) in the tail vein of severe combined immunodeficient (SCID)/Beige (Charles River) or athymic nude (Harlan). After 110 to 160 days, MCF10A-injected mice were injected with 150 μg/g luciferin substrate and mammary glands and lungs were scored for bioluminescence and cell growth using (IVIS; Xenogen). Outgrowths were scored in mammary glands that were efficiently cleared. Other mice were observed for palpable tumors or health deterioration once a week. Lungs were collected at indicated times, sectioned every 100 μm, and scored for metastasis. All mice were bred and maintained in a specific pathogen-free AAALAC International accredited facility with controlled light/dark cycle, temperature, and humidity. Cages, bedding, food, and water were all autoclaved. Experimental procedures were approved by the Bellvitge Biomedical Research Institute (IDIBELL) ethics committee and were in accordance with Spanish and European regulations.

Human tumor samples

Samples from breast cancer patients for mRNA analysis were collected from the University Hospital of Bellvitge (details in Supplementary Methods), using protocols approved by the IDIBELL ethics committee and according to Declaration of Helsinki. Samples were collected immediately after surgery and subsequently frozen or fixed for RNA extraction or immunohistochemistry (IHC).

Samples from consented breast cancer patients for immunohistochemical analysis were ethically collected and procured through various human biospecimen providers (Asterand, Ardais, Bio-Options, Cytomyx Origene, and Zoion). Samples were obtained from providers through the Amgen Tissue Bank and immunohistochemical analysis done at Amgen.

Immunohistochemistry

Anti-human RANK or vimentin IHC was carried out on sections from formalin-fixed, paraffin-embedded specimens using anti-human RANK monoclonal antibodies (N-1H8 and N-2B10; Amgen) as described in Gonzalez-Suarez and colleagues (7) or anti-human vimentin.

Statistical analyses

To evaluate whether RANK/RANKL mRNA levels discriminate metastastatic tumors, we used Multivariate ANOVA (MANOVA) considering N0 = lymph node negative (n = 25); Ni+ = isolated tumor cells in nodes; isolated groups of epithelial cells less than 0.2 mm in diameter, only visible by IHC (n = 7); Nmi = lymph node metastases more than 0.2 mm but less than 2 mm in diameter (n = 5); N1 = lymph node positive or distant metastasis (n = 26). Analyses was adjusted by tumor subtype (S): ER+PR+Her2 (n = 39); ER+PR+Her2+ (n = 5); ERPRHer2+ (n = 8); ERPRHer2 (n = 10). Logarithmic transformation was required to avoid data heteroscedasticity.

Plasmids, antibodies, and other methods are included in Supplementary Methods.

RANK overexpression induces EMT in nontranformed human mammary epithelial cells

To evaluate the impact of RANK overexpression in human mammary epithelial cells, we obtained stable MCF10A cells, immortal but nontransformed cells (12), expressing high levels of the full-length hRANK receptor (FL-RANK) and a control vector (PARENTAL; Fig. 1A). FL-RANK expression levels were maintained during serial passages, whereas endogenous RANK in parental cells was low or undetectable by our methodology. RANKL expression was very low and similar in both genotypes (Fig. 1A, Supplementary Fig. S1A). Functionality of the protein was shown by phosphorylation of IkBα and the p65 subunit of NF-kB upon RANKL stimulation (Supplementary Fig. S1B). Increased basal levels of RANK dowstream targets, P-IkBα, P-p38, P-Erk, and P-Akt were observed in FL-RANK when compared with parental cells (Supplementary Fig. S1C), indicating that the pathway is constitutively active. Parental cells showed highly organized cell–cell adhesion with cobble stone–like appearance at confluence, whereas FL-RANK MCF10A cells had an elongated appearance and loss of cell–cell contacts with a spindle-like fibroblast morphology, suggesting that the cells may be undergoing EMT (Fig. 1B). In monolayer cultures, parental MCF10A cells show high levels of the epithelial protein E-cadherin, low levels of vimentin, and no expression of fibronectin (mesenchymal proteins). In contrast, undetectable E-cadherin and strong staining of vimentin and fibronectin was observed in FL-RANK MCF10A cells, supporting an EMT phenotype (Fig. 1C). Quantitative real-time reverse transcriptase (RT-PCR) analyses confirmed changes observed for fibronectin, vimentin, and E-cadherin (73- and 25-fold increase, 13-fold decrease, respectively) and increased N-cadherin expression in FL-RANK cells (Fig. 1D). We observed a clear increase in mRNA levels of transcription factors known as repressors of E-cadherin promoter activity (13, 14), including, Snail (23-fold), Twist (3-fold), Zeb1 (6-fold), Zeb2 (10-fold), and Slug (2-fold) in FL-RANK MCF10A (Fig. 1D). The increased expression in EMT-related genes was confirmed using independently transduced pools of RANK expressing MCF10A and also in a distinct, nontransformed breast cell line, HMECS immortalized with telomerase (Supplementary Fig. S2A–C). Treatment of FL-RANK MCF10A cells with hRANKL for 48 hours led to further increases in EMT genes such as Vimentin (1.4 fold) or Snail (7-fold), suggesting that RANKL stimulation further induces the EMT characteristics of the FL-RANK cells (Supplementary Fig. S2D). In summary, analyses of morphologic and molecular changes indicated that RANK overexpression causes EMT in human mammary epithelial cells.

RANK overexpression induces stemness in nontranformed human mammary epithelial cells

It has been shown that mammary cells undergoing EMT exhibit stem cell markers and properties of stem cells (10). We therefore looked at markers that have been related to stem cells and cancer stem cells in the human breast such as CD44, CD24, EpCAM, CD10, CD49f, and CD133 (15–20). We found that most FL-RANK MCF10A cells (82.6 ± 2.9%) were CD44+CD24 (Fig. 2A, Supplementary Fig. S3A), compared with only 3.1 ± 0.15% parental cells. In correlation with their mesenchymal phenotype, most FL-RANK cells (99.6%) were negative for the luminal marker EpCAM and 95.6% positive for the basal marker CD10, whereas parental cells were EpCAM+ (87.7%), CD10+ (64.4%); lower levels of CD49f, and a slight increase in CD133 expression was observed in FL-RANK cells (Fig. 2B and Supplementary Fig. S3A). Both phenotypes related with basal/stem-like cells (16, 17, 20, 21). RANKL stimulation for 48 hours further enhanced the changes induced by RANK, including the increased frequency of CD44+CD24 (10% more) and CD10 cells, and the decreased EpCAM and CD49fhi (Supplementary Fig. S3B). FL-RANK cells also show significantly higher levels of mRNA expression of SOX2, NANOG, and OCT4 (fold change 3×, 3×, and 2×, respectively), transcription factors expressed in mammary stem cells and breast tumor cells (ref. 22; Fig. 2C). To evaluate the in vivo stem cell activity, we used the cleared fat pad transplant assay (23, 24). FL-RANK MCF10A cells, in contrast to parental, were detected in the fat pad 4 months after injection, as revealed by bioluminiscence analyses (Fig. 2D); moreover, they formed small outgrowths showing that, in correlation with their stem characteristics, FL-RANK MCF10A cells were able to repopulate the mammary epithelia in vivo (Fig. 2E and F; Supplementary Fig. S3C). In summary, these results showed that in human mammary cells RANK overexpression induces the expression of mammary stem cell markers and transcription factors and the ability to generate mammary outgrowths in vivo, a function of stem cells.

RANK overexpression increases migration and induces hallmarks of transformation in MCF10A cells

The main functional consequence of EMT is enhanced cell scattering and migration. Thus, in wound healing assays, FL-RANK MCF10A cells migrated faster than the parental cells, both in the presence or absence of EGF. RANKL significantly increased the motility of FL-RANK as compared with EGF, and the most motile cells were the FL-RANK cells treated with EGF plus RANKL (Fig. 3A).

We next asked whether RANK overexpression could induce transformation. MCF10A cells grown in Matrigel with EGF, recapitulate several features of breast epithelium in vivo, including the formation of acinus-like spheroids with a hollow lumen, apicobasal polarization, and growth arrest (12, 25), whereas transformed mammary epithelial cells show a multiacinar phenotype, filled lumen, and absence of proliferative arrest (25). In contrast to parental, FL-RANK MCF10A cells formed large and disrupted structures at 20 days of culture (Fig. 3B). RANKL treatment resulted in aberrantly elongated tubular like structures (day 2) that by day 20 looked like aggregates (Fig. 3B). Confocal cross-sections revealed in FL-RANK cultures abnormal structures often composed of cells filling the luminal space and occasionaly showing protrusions into the Matrigel, characteristic of invasive cells (Fig. 3C). In FL-RANK structures E-cadherin expression was hardly detected in the cell–cell contacts and decreased expression or mislocalization of the basal marker CD49f was often observed in FL-RANK cells indicating loss of polarization (Fig. 3C). Unlike parental cells, FL-RANK acini/aggregates were not growth arrested after 20 days of culture as shown by the presence of ki67 and activated caspase 3 (Supplementary Fig. S4A). Moreover, FL-RANK overexpressing cells cultured with RANKL formed acini with filled lumens in the absence of EGF, whereas parental cells were unable to grow in these conditions (Supplementary Fig. S4B). Consistent with their nontransformed phenotype, parental MCF10A cells failed to grow in soft agar whereas FL-RANK MCF10A cells formed numerous foci (Fig. 3D), indicating that RANK expression conferes the cells the ability to grow in the absence of anchorage, a characteristic of tumor or stem cells. We concluded that RANK overexpression in human mammary epithelial cells resulted in hallmarks of transformation, including, increased motility, inability to respond to growth arrest signals, impaired polarization, luminal filling, and growth in soft agar. Despite in vitro observations consistent with the RANK-dependent transformation of MCF10A cells, these cells although able to grow in vivo (Fig. 2D and E) were nontumorigenic when injected in the cleared fat pad of immunodeficient mice and did not form lung metastasis when injected in the tail vein (Supplementary Fig. S4C).

RANK overexpression increases the frequency of CD44+CD24 cells in BRCA1-deficient cell lines

We aim to evaluate whether RANK can cooperate with other mutations relevant in breast cancer such as BRCA1 deficiency (26) during tumorigenesis and metastasis. We therefore overexpressed RANK in breast cancer cells defective for BRCA1, MDA-MB-436, HCC1937, and UACC3199 cells (27) and confirmed its functionality (Fig. 4A and Supplementary Fig. S5A and B). RANK expression in the parental lines was low or undetectable by our methods; RANKL expression was low and comparable between both phenotypes (Fig. 4A, Supplementary Fig. S5A). As in MCF10A cells, RANK overexpression increased the frequency of CD44+CD24 cells in all 3 cell lines due to reduced CD24 levels as compared with parental cell lines (Fig. 4B, Fig. S5C and D); a further reduction in CD24 expression was observed after RANKL stimulation in most cells (Supplementary Fig. S5C). Lower frequency of EpCAM+ (13% reduction) and CD49fhi (17% reduction) cells in FL-RANK MDA-MB-436 and higher of CD10+ in UACC3199-FL-RANK, as compared with the correspondent parental lines was observed (Supplementary Fig. S5C and D). Consistent with their mesenchymal morphology, MDA-MB-436 cells express fibronectin, vimentin, N-cadherin, and the transcription factors, Snail, Twist, Zeb1 and Zeb2, and even higher levels upon RANK overexpression (Fig. 4C, Supplementary Fig. S5E). In FL-RANK HCC1937 and UACC3199, the expression levels of EMT mesenchymal markers or transcription factors, and the frequency of EpCAM+ was similar to the corresponding parental lines (Fig. 4C, Supplementary Fig. S5D and E). These results showed that RANK induces CD44+CD24 phenotype in breast cancer cell lines concomitantly with or separately from EMT.

RANK overexpression increases invasiveness and promotes tumorigenesis and metastasis in BRCA1-deficient cell lines

We next investigated the functional consequences of RANK overexpression in BRCA1-defective breast cancer cell lines. Monolayer cultures revealed a modest increase in growth of the 3 FL-RANK BRCA1-defective cell lines (Supplementary Fig. S6A). Protusions into the Matrigel, indicative of an invasive phenotype, were often observed in MDA-MB-436 FL-RANK acini but, not in parental, and even more prominent in the presence of RANKL (Supplementary Fig. S6B). Quantification of proliferation in acinar cultures revealed a small increase upon RANK overexpression in some cell lines. FL-RANK cells showed lower levels of apoptosis when cultured in the presence of RANKL (Supplementary Fig. S6C).

To investigate whether RANK expression in BRCA1-defective cells promotes tumorigenesis and metastasis in vivo, we used the MDA-MB-436 cells in which EMT was enhanced upon RANK overexpression and UACC3199 cells in which RANK increased the frequency of CD44+CD24 cells but no EMT was observed. After injection in the mammary fat pad, FL-RANK MDA-MB-436 and UACC3199 cells gave rise to tumors with faster growth and/or a shorter latency than parental cells (Fig. 5A and B). We next determined whether RANK overexpression would enhance the metastatic properties of these cells. A total of 6.5 weeks after tail vein injection, frequency and size of lung metastasis were higher in mice inoculated with FL-RANK cells as compared with parental MDA-MB-436 cells (Fig. 5C). By 12 weeks metastatic lesions in mice injected with FL-RANK MDA-MB-436 cells colonized most of the lung area as compared with smaller metastatic foci formed by parental cells (Fig. 5D; Supplementary Fig. S6D). UACC3199 cells were poorly metastatic as compared with MDA-MB-436, in correlation with the absence of EMT in these cells. Micrometastases were observed in 50% mice injected with FL-RANK UACC3199 cells (up to 56 metastatic foci) and in 33% (n = 6) mice injected with the parental UACC3199 (up to 3 lesions per mouse; Supplementary Fig. S6D). In summary, RANK overexpression enhanced tumorigenesis and metastasis in BRCA1-deficient cells.

High RANK/RANKL expression levels are found in aggressive and metastatic adenocarcinomas

To investigate the potential clinical relevance of RANK pathway in human breast cancer, we analyzed RANK and RANKL mRNA expression levels in adenocarcinomas considering their pathologic characteristics. We found significantly higher levels of mRNA RANK in human tumors that lack expression of the hormonal receptors for estrogen and progesterone (ER and PR) as compared with (ER+PR+) tumors that usually bear a better prognosis, and in tumors with high proliferation index (more than 40% of ki67+ cells) and high pathologic grade (Fig. 6A). Similar epithelial and leucocyte content was found within all groups (Supplementary Fig. S7A). To confirm the mRNA results, we analyzed RANK protein expression by IHC in a second collection of breast carcinomas. Consistent with our previous study (7), RANK protein expression was found within the carcinoma element of some tumors and a subset of infiltrating macrophages but was not observed on stromal cells, fiboblasts, or lymphocytes (Fig. 6B). A higher incidence of RANK protein expression was observed in ER/PR (50%) versus ER+/PR+ (18%) and in grade III (32%) versus grade I (13%) or grade II (18%; Fig. 6C).

Our results with cell lines indicated that activation of RANK pathway enhances migration and metastasis. In human breast adenocarcinomas using a multivariant ANOVA analyses, we found that RANK/RANKL mRNA expression levels were able to discriminate between nonmetastatic (N0) and metastatic tumors (N1; either to the lymph nodel or other sites; Fig. 6D; Supplementary Fig. S7B–E). These results indicated that high RANK expression levels correlate with tumor aggressiveness and that RANK/RANKL expression in the primary tumor may indicate its metastatic behavior.

The RANK/RANKL signaling pathway, although dispensable for the initial development of the mammary gland in mice (2), controls the expansion of the stem cell compartment in adults (5, 6). Similarly, it has been recently shown that RANK is expressed in several stem cells of the skin, and that RANK signaling activate the hair cycle and epidermal growth (28). The mechanism responsible for this RANK-induced expansion of stem cells remains unknown. We show that RANK overexpression in human MCF10A cells results in constitutive activation of the pathway shown by the increased levels of RANK downstream targets (P-Iκβα, P-p38, P-Akt, and P-Erk). It is well established that overexpression of any TNF receptor family member leads to ligand-independent receptor oligomerization and ligand-independent activation of signal transduction pathways (29–32). RANK overexpression generates a EpCAM-/CD10+/CD49f lo phenotype, markers used to identify populations enriched in mammary stem cells in the healthy breast (15–17) and provides the MCF10A with the ability to reconstitute the cleared fat pad of immunodeficient mice in contrast to parental MCF10A that are unable to grow in vivo (33). Moreover, RANK overexpression increases the frequency of CD44+CD24lo/, a phenotype ascribed to human breast cancer stem cells, in MCF10A and in breast cancer cell lines with nonfunctional BRCA1. Plasticity of breast cancer cells with respect to CD24, CD44, EpCAM, and CD49f has been reported (18, 34, 35). Although we cannot rule out that RANK is promoting proliferation or survival of stem cells, the rapid acquisition of stem markers by the entire MCF10A population upon RANK overexpression suggests that activation of RANK signaling may induce a dedifferentiation process.

An association between RANK expression and EMT has been reported in prostate cancer (36). Here we show that RANK overexpression directly induces a strong EMT phenotype in MCF10A cells (and HMECs). EMT induced by twist/snail generates cells with cancer stem cell properties (10), and usually CD44+CD24 cells have a mesenchymal phenotype as compared with the luminal phenotype of CD24+ cells, leading to the belief that CSC and EMT are interchangeable events. However, despite the gain in CD44+CD24 phenotype induced by RANK in all BRCA1-deficient cell lines, an increase in the mesenchymal markers is observed only in MDA-MB-436 cells, similar to results reported with urokinase receptor signaling (37). Thus, RANK overexpression induces CD44+CD24 phenotype in nontransformed mammary epithelial cells and in breast cancer cell lines that can be accompanied by EMT depending on the cell of origin.

We have previously shown that RANK overexpression promotes mammary tumorigenesis in mice (7), but the relevance of this pathway in human breast disease remained largerly unknown, except for a single-nucleotide polymorphism in the locus of the TNFRSF11A gene (RANK) that associates with breast cancer risk (38). Now we show that in nontransformed mammary epithelial cells, RANK expression enhances migration, disrupts acinar formation, and allows growth in soft agar, a classical transformation assay. Changes observed in FL-RANK MCF10A acini are consistent with those described in mouse MMTV-RANK MECs (4, 7) and may reflect an imbalance response between proliferation and apoptosis that together with the impaired polarization results in increased organoid size and luminal filling. Despite these changes associated with transformation, FL-RANK MCF10A cells do not form tumors or metastasis when injected in immunodeficient mice. Similar results have been observed with several oncogenes, such as ErbB2/neu, that are able to disrupt acinus formation and transform MCF10A or HMLE in vitro (39) but fail to convey tumor growth as xenografts (10, 40). It has been shown that RANK signaling induces migration and bone metastasis in human breast cancer lines (41). Here, we provide a putative mechanism by which RANK promotes tumorigenesis, migration, and metastasis. The enhanced motility and ability to seed tumors in other locations provided by the CD44+CD24 phenotype in cells with nonfunctional BRCA1, together with the EMT phentoype when exisiting, indicates that metastasis may not be restricted to the bone. In fact, pharmacologic inhibition of RANKL or genetic ablation of RANK significantly reduced the incidence and multiplicity of lung metastasis in MMTV-neu mice (7, 42). These findings show that RANK overexpression in human breast cancer cells increases their aggressiveness and may result in poorer clinical outcome, as recently shown (43). In fact, we observe that RANK expression progressively increases with pathologic grade and significantly associates with a high proliferative index in clinical samples of breast cancer patients. Higher levels of RANK mRNA expression were found in (ER PR) tumors, which were consistent with the higher incidence of RANK protein expression in these tumors. ERPR tumors are more aggressive, show a higher incidence of metastasis and worse prognosis than luminal tumors, and contain a higher frequency of CD44+/CD24 cells (21, 44). Moreover, RANK/RANKL mRNA expression levels allow discrimination between metastatic and nonmetastatic adenocarcinomas.

In summary, we show that RANK overexpression in human mammary epithelial cells generates a “stem” phenotype in nontransformed basal human mammary epithelial cells and human breast cancer cells that can be accompanied by EMT. Our results highlight the relevance of RANK signaling pathway in human mammary tumorigenesis, and human mammary stem cells in correlation with the results previously obtained in mice, and suggest that RANK promotes tumor initiation, progression, and metastasis in breast cancer.

M. Tometsko, D. Branstetter, and WC. Dougall are Amgen employees and stockholders. No potential conflicts of interest were disclosed.

Conception and design: W.C. Dougall, E. González-Suárez

Development of methodology: M. Palafox, I. Ferrer, P. Pellegrini, S. Hernandez-Ortega, D. Branstetter, W.C. Dougall, E. González-Suárez

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Palafox, A. Urruticoechea, F. Climent, M.T. Soler, M. Tometsko, D. Branstetter, W.C. Dougall, E. González-Suárez

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Palafox, I. Ferrer, P. Pellegrini, M. Tometsko, D. Branstetter, W.C. Dougall, E. González-Suárez

Writing, review, and/or revision of the manuscript: M. Palafox, I. Ferrer, P. Pellegrini, A. Urruticoechea, P. Muñoz, F. Viñals, W.C. Dougall, E. González-Suárez

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P. Pellegrini, S. Vila

Study supervision: E. González-Suárez

The authors thank M.A. Pujana, C. Maxwell, and M. Esteller for reactives and helpful advice, K. Rohrbach, R. Soriano, and L.Y. Huang for expert technical support of the IHC procedures, the IDIBELL animal facility service, histology service, and UB-SCT for technical support; X. Sole and V. Navarro for statistic analyses; O. Casanovas, C. Muñoz Pinedo, and members of the laboratory for useful discussions.

This work was supported by grants to E. González-Suárez by the Spanish Ministry of Science and Innovation MICINN (SAF2008-01975), by Ramon y Cajal (EGS), by the Scientific Foundation of the AECC (Catalunya), by Concern Foundation, and by institutional funds provided by the Generalitat de Catalunya to the PEBC. M. Palafox is recipient of a FPU grant, P. Pellegrini is recipient of a FPI grant, both from the MICINN.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Boyle
WJ
,
Simonet
WS
,
Lacey
DL
. 
Osteoclast differentiation and activation
.
Nature
2003
;
423
:
337
42
.
2.
Fata
JE
,
Kong
YY
,
Li
J
,
Sasaki
T
,
Irie-Sasaki
J
,
Moorehead
RA
, et al
The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development
.
Cell
2000
;
103
:
41
50
.
3.
Fernandez-Valdivia
R
,
Mukherjee
A
,
Ying
Y
,
Li
J
,
Paquet
M
,
DeMayo
FJ
, et al
The RANKL signaling axis is sufficient to elicit ductal side-branching and alveologenesis in the mammary gland of the virgin mouse
.
Dev Biol
2009
;
328
:
127
39
.
4.
Gonzalez-Suarez
E
,
Branstetter
D
,
Armstrong
A
,
Dinh
H
,
Blumberg
H
,
Dougall
WC
. 
RANK overexpression in transgenic mice with mouse mammary tumor virus promoter-controlled RANK increases proliferation and impairs alveolar differentiation in the mammary epithelia and disrupts lumen formation in cultured epithelial acini
.
Mol Cell Biol
2007
;
27
:
1442
54
.
5.
Asselin-Labat
ML
,
Vaillant
F
,
Sheridan
JM
,
Pal
B
,
Wu
D
,
Simpson
ER
, et al
Control of mammary stem cell function by steroid hormone signalling
.
Nature
2010
;
465
:
798
802
.
6.
Joshi
PA
,
Jackson
HW
,
Beristain
AG
,
Di Grappa
MA
,
Mote
PA
,
Clarke
CL
, et al
Progesterone induces adult mammary stem cell expansion
.
Nature
2010
;
465
:
803
7
.
7.
Gonzalez-Suarez
E
,
Jacob
AP
,
Jones
J
,
Miller
R
,
Roudier-Meyer
MP
,
Erwert
R
, et al
RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis
.
Nature
2010
;
468
:
103
7
.
8.
Schramek
D
,
Leibbrandt
A
,
Sigl
V
,
Kenner
L
,
Pospisilik
JA
,
Lee
HJ
, et al
Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer
.
Nature
2010
;
468
:
98
102
.
9.
Thiery
JP
,
Acloque
H
,
Huang
RY
,
Nieto
MA
. 
Epithelial-mesenchymal transitions in development and disease
.
Cell
2009
;
139
:
871
90
.
10.
Mani
SA
,
Guo
W
,
Liao
MJ
,
Eaton
EN
,
Ayyanan
A
,
Zhou
AY
, et al
The epithelial-mesenchymal transition generates cells with properties of stem cells
.
Cell
2008
;
133
:
704
15
.
11.
Rice
JC
,
Massey-Brown
KS
,
Futscher
BW
. 
Aberrant methylation of the BRCA1 CpG island promoter is associated with decreased BRCA1 mRNA in sporadic breast cancer cells
.
Oncogene
1998
;
17
:
1807
12
.
12.
Debnath
J
,
Muthuswamy
SK
,
Brugge
JS
. 
Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures
.
Methods
2003
;
30
:
256
68
.
13.
Huber
MA
,
Kraut
N
,
Beug
H
. 
Molecular requirements for epithelial-mesenchymal transition during tumor progression
.
Curr Opin Cell Biol
2005
;
17
:
548
58
.
14.
Peinado
H
,
Olmeda
D
,
Cano
A
. 
Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype?
Nat Rev Cancer
2007
;
7
:
415
28
.
15.
Al-Hajj
M
,
Wicha
MS
,
Benito-Hernandez
A
,
Morrison
SJ
,
Clarke
MF
. 
Prospective identification of tumorigenic breast cancer cells
.
Proc Natl Acad Sci U S A
2003
;
100
:
3983
8
.
16.
Bachelard-Cascales
E
,
Chapellier
M
,
Delay
E
,
Pochon
G
,
Voeltzel
T
,
Puisieux
A
, et al
The CD10 enzyme is a key player to identify and regulate human mammary stem cells
.
Stem Cells
2010
;
28
:
1081
8
.
17.
Lim
E
,
Vaillant
F
,
Wu
D
,
Forrest
NC
,
Pal
B
,
Hart
AH
, et al
Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers
.
Nat Med
2009
;
15
:
907
13
.
18.
Meyer
MJ
,
Fleming
JM
,
Ali
MA
,
Pesesky
MW
,
Ginsburg
E
,
Vonderhaar
BK
. 
Dynamic regulation of CD24 and the invasive, CD44posCD24neg phenotype in breast cancer cell lines
.
Breast Cancer Res
2009
;
11
:
R82
.
19.
Meyer
MJ
,
Fleming
JM
,
Lin
AF
,
Hussnain
SA
,
Ginsburg
E
,
Vonderhaar
BK
. 
CD44posCD49fhiCD133/2hi defines xenograft-initiating cells in estrogen receptor-negative breast cancer
.
Cancer Res
2010
;
70
:
4624
33
.
20.
Wright
MH
,
Calcagno
AM
,
Salcido
CD
,
Carlson
MD
,
Ambudkar
SV
,
Varticovski
L
. 
Brca1 breast tumors contain distinct CD44+/CD24- and CD133+ cells with cancer stem cell characteristics
.
Breast Cancer Res
2008
;
10
:
R10
.
21.
Park
SY
,
Lee
HE
,
Li
H
,
Shipitsin
M
,
Gelman
R
,
Polyak
K
. 
Heterogeneity for stem cell-related markers according to tumor subtype and histologic stage in breast cancer
.
Clin Cancer Res
2010
;
16
:
876
87
.
22.
Simoes
BM
,
Piva
M
,
Iriondo
O
,
Comaills
V
,
Lopez-Ruiz
JA
,
Zabalza
I
, et al
Effects of estrogen on the proportion of stem cells in the breast
.
Breast Cancer Res Treat
2011
;
129
:
23
35
.
23.
Deome
KB
,
Faulkin
LJ
 Jr
,
Bern
HA
,
Blair
PB
. 
Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice
.
Cancer Res
1959
;
19
:
515
20
.
24.
Smith
GH
. 
Experimental mammary epithelial morphogenesis in an in vivo model: evidence for distinct cellular progenitors of the ductal and lobular phenotype
.
Breast Cancer Res Treat
1996
;
39
:
21
31
.
25.
Schmeichel
KL
,
Bissell
MJ
. 
Modeling tissue-specific signaling and organ function in three dimensions
.
J Cell Sci
2003
;
116
:
2377
88
.
26.
Friedenson
B
. 
The BRCA1/2 pathway prevents hematologic cancers in addition to breast and ovarian cancers
.
BMC Cancer
2007
;
7
:
152
.
27.
Drew
Y
,
Mulligan
EA
,
Vong
WT
,
Thomas
HD
,
Kahn
S
,
Kyle
S
, et al
Therapeutic potential of poly(ADP-ribose) polymerase inhibitor AG014699 in human cancers with mutated or methylated BRCA1 or BRCA2
.
J Natl Cancer Inst
2011
;
103
:
334
46
.
28.
Duheron
V
,
Hess
E
,
Duval
M
,
Decossas
M
,
Castaneda
B
,
Klopper
JE
, et al
Receptor activator of NF-kappaB (RANK) stimulates the proliferation of epithelial cells of the epidermo-pilosebaceous unit
.
Proc Natl Acad Sci U S A
2011
;
108
:
5342
7
.
29.
Anderson
DM
,
Maraskovsky
E
,
Billingsley
WL
,
Dougall
WC
,
Tometsko
ME
,
Roux
ER
, et al
A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function
.
Nature
1997
;
390
:
175
9
.
30.
Chan
FK
. 
Three is better than one: pre-ligand receptor assembly in the regulation of TNF receptor signaling
.
Cytokine
2007
;
37
:
101
7
.
31.
Galibert
L
,
Tometsko
ME
,
Anderson
DM
,
Cosman
D
,
Dougall
WC
. 
The involvement of multiple tumor necrosis factor receptor (TNFR)-associated factors in the signaling mechanisms of receptor activator of NF-kappaB, a member of the TNFR superfamily
.
J Biol Chem
1998
;
273
:
34120
7
.
32.
Winkles
JA
. 
The TWEAK-Fn14 cytokine-receptor axis: discovery, biology and therapeutic targeting
.
Nat Rev Drug Discov
2008
;
7
:
411
25
.
33.
Dawson
PJ
,
Wolman
SR
,
Tait
L
,
Heppner
GH
,
Miller
FR
. 
MCF10AT: a model for the evolution of cancer from proliferative breast disease
.
Am J Pathol
1996
;
148
:
313
9
.
34.
Chaffer
CL
,
Brueckmann
I
,
Scheel
C
,
Kaestli
AJ
,
Wiggins
PA
,
Rodrigues
LO
, et al
Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state
.
Proc Natl Acad Sci U S A
2011
;
108
:
7950
5
.
35.
Sarrio
D
,
Franklin
CK
,
Mackay
A
,
Reis-Filho
JS
,
Isacke
CM
. 
Epithelial and mesenchymal subpopulations within normal basal breast cell lines exhibit distinct stem cell/progenitor properties
.
Stem Cells
2012
;
30
:
292
303
.
36.
Odero-Marah
VA
,
Wang
R
,
Chu
G
,
Zayzafoon
M
,
Xu
J
,
Shi
C
, et al
Receptor activator of NF-kappaB Ligand (RANKL) expression is associated with epithelial to mesenchymal transition in human prostate cancer cells
.
Cell Res
2008
;
18
:
858
70
.
37.
Jo
M
,
Eastman
BM
,
Webb
DL
,
Stoletov
K
,
Klemke
R
,
Gonias
SL
. 
Cell signaling by urokinase-type plasminogen activator receptor induces stem cell-like properties in breast cancer cells
.
Cancer Res
2010
;
70
:
8948
58
.
38.
Bonifaci
N
,
Palafox
M
,
Pellegrini
P
,
Osorio
A
,
Benitez
J
,
Peterlongo
P
, et al
Evidence for a link between TNFRSF11A and risk of breast cancer
.
Breast Cancer Res Treat
2011
;
129
:
947
54
.
39.
Debnath
J
,
Brugge
JS
. 
Modelling glandular epithelial cancers in three-dimensional cultures
.
Nat Rev Cancer
2005
;
5
:
675
88
.
40.
Giunciuglio
D
,
Culty
M
,
Fassina
G
,
Masiello
L
,
Melchiori
A
,
Paglialunga
G
, et al
Invasive phenotype of MCF10A cells overexpressing c-Ha-ras and c-erbB-2 oncogenes
.
Int J Cancer
1995
;
63
:
815
22
.
41.
Jones
DH
,
Nakashima
T
,
Sanchez
OH
,
Kozieradzki
I
,
Komarova
SV
,
Sarosi
I
, et al
Regulation of cancer cell migration and bone metastasis by RANKL
.
Nature
2006
;
440
:
692
6
.
42.
Tan
W
,
Zhang
W
,
Strasner
A
,
Grivennikov
S
,
Cheng
JQ
,
Hoffman
RM
, et al
Tumour-infiltrating regulatory T cells stimulate mammary cancer metastasis through RANKL-RANK signalling
.
Nature
2011
;
470
:
548
53
.
43.
Santini
D
,
Schiavon
G
,
Vincenzi
B
,
Gaeta
L
,
Pantano
F
,
Russo
A
, et al
Receptor activator of NF-kB (RANK) expression in primary tumors associates with bone metastasis occurrence in breast cancer patients
.
PLoS One
2011
;
6
:
e19234
.
44.
Ricardo
S
,
Vieira
AF
,
Gerhard
R
,
Leitao
D
,
Pinto
R
,
Cameselle-Teijeiro
JF
, et al
Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype
.
J Clin Pathol
2011
;
64
:
937
46
.