Notch activation stimulates migration of breast cancer cells and promotes tumor growth
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Dysregulated NOTCH receptor activity has been implicated in breast cancer but the mechanisms by which NOTCH contributes to transformation are not yet clear, as it has context-dependent effects on the properties of transformed cells.
We have used various in vitro and in vivo carcinogenic models to analyze the impact of Notch signaling in the onset and progression of breast tumors.
We found that ectopic expression of the Notch1 intracellular domain (N1ICD) in MCF-7 breast adenocarcinoma cell line caused reduction and delocalization of E-CADHERIN levels and increased migratory and invasive abilities. Notch inhibition in the invasive breast cancer cell line MDA-MB-231 resulted in increased E-CADHERIN expression and a parallel reduction in their invasive capacity. The growth of subcutaneous xenografts produced with MCF-7 cells was boosted after N1ICD induction, in a cell autonomous manner. In vivo Notch1 activation in the mammary gland using the MMTV-Cre driver caused the formation of papillary tumors that showed increased Hes1 and Hey1 expression and delocalized E-cadherin staining.
These results confirm NOTCH1 as a signal triggering epithelial-mesenchymal transition in epithelial cancer cells, which may have implications in tumor dissemination, metastasis and proliferation in vivo. The identification of specific factors interacting with NOTCH signaling could thus be relevant to fully understanding the role of NOTCH in breast neoplasia.
KeywordsMammary tumor MCF-7 HT-29 MDA-MB-231 NOTCH E-CADHERIN EMT migration growth
CSL: Suppressor of Hairless
CBF1: Suppressor of Hairless: Lag-1
N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester
estrogen receptor alpha
Hematoxylin and eosin
hairy and enhancer of split 1
hairy/enhancer-of-split related with YRPW motif one
Human colon adenocarcinoma grade II cell line
Insulin growth factor one
Michigan Cancer Foundation-7 breast cancer cell line
Breast cancer cell line derived from metastatic site (pleural effusion)
Mouse mammary Tumor Virus-driven Cre recombinase
Notch one intracellular domain
standard error of the mean
Structural Maintenance of Chromomosomes-3 protein
Stromal cell Derived factor 1α
Notch is a fundamental signaling pathway that regulates embryonic cell fate specification, proliferation and patterning [1, 2]. In addition to its central role in development, Notch signaling is deregulated in a number of cancers . Notch1 mutations lead to oncogene expression in certain T cell acute lymphoblastic leukemias  and a subset of breast carcinomas ; deregulated Notch activity might also affect cell transformation , regulation of the cell cycle , progenitor/stem cell maintenance  and the outcome of breast cancer .
The mammalian Notch proteins (Notch1 to 4) are membrane-bound type I receptors with a large extracellular domain involved in ligand binding, and a cytoplasmic domain responsible for signal transduction. The Notch ligands Delta-like 1, 3 and 4 and Jagged 1 and 2 are also membrane-bound. Ligand-receptor interactions between neighboring cells trigger Notch signaling, which leads to a sequence of proteolytic cleavage events in the receptor. The last of these is mediated by γ-secretase activity, generating the Notch intracellular domain (NICD), which translocates to the nucleus and binds the CSL transcription factor. The NICD/CSL complex induces expression of target genes, including those of the hairy/enhancer of split (Hes) family [1, 2], the cell cycle regulator p21  and cyclin D1 .
Many studies focus on the role of Notch1 in mammary tumorigenesis. Hyperactivated Notch1 signaling was first implicated in mammary tumorigenesis in studies of the MMTV model, which showed that N1ICD expression in MMTV-Neu mammary tumors is due to an MMTV insertion in the Notch1 locus . Other reports indicated that transgenic activation of N1ICD in mammary glands leads to development of lactation-dependent tumors that regress at weaning [11, 12]. These findings link aberrant Notch activation in the murine mammary gland to adenocarcinoma. Experimental evidence shows that altered Notch1 signaling leads to direct transcriptional regulation of c-myc, which is crucial in MMTV-N1ICD-induced murine mammary tumorigenesis . NOTCH1 is also involved in human mammary tumorigenesis as a downstream effector of oncogenic Ras .
Here we used various in vitro and in vivo models to analyze the impact of Notch signaling in breast tumor onset and progression. We find that stable or inducible N1ICD expression in the poorly invasive MCF-7 breast adenocarcinoma cell line causes a reduction and delocalization of E-CADHERIN levels, suggesting a disassembly of adherens junctions that correlates with enhanced cell migratory and invasive abilities. These properties may be extended to other epithelial tumor cell lines as we have made similar observations in the colon cancer cell line HT-29 stably expressing N1ICD. To the contrary, Notch inhibition in the highly invasive cell line MDA-MB-231 resulted in increased E-CADHERIN expression and a parallel reduction in their invasive capacity. Notch1 activation in the mouse mammary gland using the MMTV-Cre driver caused the formation of papillary tumors that showed increased Hes1 and Hey1 and delocalized E-cadherin expression. We also found that the growth of subcutaneous xenografts produced with MCF-7 cells was boosted after N1ICD induction, in a cell autonomous manner. These results confirm Notch1 as an epithelial-to-mesenchymal transition (EMT) inducer in breast cancer cells, which may have implications in tumor dissemination and metastasis.
The human breast cancer cell lines MCF-7 (ATCC® HTB-22™) and MDA-MB-231 (ATCC® HTB-26™), and the human colorectal adenocarcinoma cell line HT-29 (ATCC® HTB-38™) were used. For culture conditions see Additional file 1, Supplementary Materials and methods.
Transfection of MCF-7 and HT-29 cells
A cDNA fragment encoding the active version of mouse Notch1 (N1ICDΔOP) was used . The Tet-Off system was employed to obtain transfectants of MCF-7 with inducible N1ICD expression. In this system, gene expression is turned on when doxycycline (DOXY; a tetracycline derivative) is removed from the culture medium. For details see Additional file 1, Supplementary Materials and methods.
Western blot analysis
For details see Additional file 1, Supplementary Materials and methods.
Semi-quantitative RT-PCR and real-time quantitative PCR
Total RNA was extracted with Trizol reagent (Life Technologies, NY, USA) and cDNA was synthesized with SuperScript III First Strand kit (Life Technologies, NY, USA). N-Cadherin primers were 5´-CACCCAACATGTTTACAATCAACAATGAGAC-3 (forward) and 5´-CTGCAGCAACAGTAAGGACAAACATCCTATT-3 (reverse) . Commercial β-actin primers were used (Stratagene, La Jolla, CA, USA). Quantitative PCR was performed with Power SYBR Green Master Mix (Applied Biosystems, NY, USA, 4367659) and commercial primers for HEY1, HES1, cMYC, NOTCH1, NOTCH4, SNAI1, ECAD, VIMENTIN and HPRT1 (Sigma, St. Louis, MO, USA) were used.
Promoter activity assays
Hes1-Luc promoter activity  was measured in MCF-7 cells expressing N1ICD in a constitutive or inducible manner. The activity of the artificial promoter 10XCBF1  was measured after transient transfection of MDA-MB-231 cells. Briefly, cells were co-transfected with the plasmid containing the promoter 10XCBF1-Luc and pcDNA3-CBF1-VP16 or pcDNA3-DN-CBF1/RBPJK. The plasmid pTK-RL (Promega, Madison, WI, USA)
was also included as a control of transfection efficiency. When indicated, cells were treated for the indicated period of time with DOXY 2 μg/ml or with the γ-Secretase Inhibitors DAPT (N-(N-(3,5-Difluorophenacetyl)-L-alanyl)-S-phenylglycine t-butyl ester, 10 to 50 μM; 565770, Calbiochem, Millipore, MA, USA) and RO4929097 ((2,2-dimethyl-N-(S)-6-oxo-6,7-dihydro-5H-dibenzo(b,d)azepin-7-yl)-N'-(2,2,3,3,3-pentafluoro-propyl)-malonamide)), 10 to 20 μM; S1575, Selleckchem, Houston, TX, USA) for 48 h. After transfection cells were lysed with passive lysis buffer (Promega, Madison, WI, USA) and firefly and renilla luciferase were measured with the "Dual-luciferase reporter assay" (Promega, Madison, WI, USA). The activity in MCF-7 clones or in MDA-MB-231 treated cells was referred to the activity in control cells or cells transfected with the empty vector (pcDNA3).
Immunofluorescence and immunohistochemistry
For details see Additional file 1, Supplementary Materials and methods.
In situ hybridization was performed as described in . Details of probes will be provided on request.
In vitrocell chemotaxis
Cell migration was performed in Transwell (Corning, Tewksbury, MA, USA) with 8 μm pore filters coated with 20 μg/ml collagen type IV (Sigma, St. Louis, MO, USA). Cells were pretreated for the time indicated with DOXY (MCF-7) or DAPT/RO4929097/DMSO (MDA-MB-231), trypsinized and added to the upper chamber in basal medium with 0.5% BSA and the additives. The lower chamber was replenished with basal medium with BSA and the chemoattractant (IGF-1, 50 ng/ml, R&D Systems, Minneapolis, MN, USA or SDF1α, PeProTech (New Jersey, USA). After 18 h incubation, the upper chamber was emptied and cells remaining are removed. Cells in the filter are fixed with PFA and then stained with violet crystal (Sigma-Aldrich). Cell counts were obtained by counting two (MDA-MB-231) or four (MCF-7) grids using a microscope fitted with a grid eyepiece at a total magnification of 100X.
For details see Additional file 1, Supplementary Materials and methods.
In vivoexperiments with mice
All animal procedures were approved by the Institutional Committee for the Care and Use of Laboratory Animals of the Centro Nacional de Investigaciones Cardiovasculares (CNIC, Madrid, Spain) and Centro Nacional de Biotecnología (CNB-CSIC, Madrid, Spain). Animal procedures conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC, regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 1201/2005.
MCF-7/TetOff and B12, M5 and M20 derivatives' clones, growing in culture without DOXY for 25 days, were inoculated s.c in both flanks (1.5 or 2.4 × 106 cells) in BALBc/SCID mice treated with 17α-ethylenestradiol 1 μg/ml (Sigma) provided in the drinking water from one week before cells were injected. Tumor size was monitored weekly and tumor volume estimated with a caliper by measuring the width (a) and the length (b) and applying the formula (a2 × b)/2. Once finished with the period of treatment, mice were sacrificed and tumors were extracted for further analysis. MCF-7/TetOff and B12 were transduced with recombinant retrovirus to express luciferase activity. Plasmid pRV-luc-IRES-CopGreen was used to obtain the retroviral supernatants (Genetrix S.L., Madrid, Spain) and transduced cells were sorted according to the associated green fluorescence. BALBc/SCID mice (Harlan Laboratories, Indianapolis, IN, USA) were injected in the two inguinal mammary glands with 2.5 × 106 cells and mice were treated as above. After injection, half of the mice were treated also with DOXY 2 mg/ml provided in the drinking water. Tumoral growth rate was analyzed by bioluminescence at different weeks after cell inoculation. Briefly, mice were injected with luciferin with the general anesthetic and luciferase activity expressed by cells was detected with a CCD camera placed in a dark box (Hamamatsu Photonics, Shizuoka, Japan). Images were processed with the software provided and luminescence units were represented. Tumor size was estimated as above and once finished with the period of treatment, tumors were excised, weighted and preserved adequately to make further analysis.
Transgenic N1ICD expression in the mammary gland
The transgenic lines MMTV-Cre  and Rosa26N1ICD  were bred to generate MMTV-Cre/+; Rosa26N1ICD/+ double transgenic mice. For primers and conditions of mouse genotyping see [20, 21]. Mice were subjected to several rounds (a median of four) of pregnancy and lactation, and when a breast tumor arose, mice were euthanized and the breast tumor excised and processed for further analysis. Tumor samples were fixed with 10% buffered formalin (Sigma-Aldrich) for 48 h and afterward were paraffin-embedded. Staining of Hes1, ERα, p63, E-cadherin and Ki67 was performed in 5 μm sections of paraffin samples following standard techniques. For details see Additional file 1, Supplementary Materials and methods.
N1ICD expression enhances the invasive capacity of the breast cancer cell line MCF-7
We generated MCF-7 clones stably expressing a myc-tagged N1ICD version (Figure 1B). MCF-7 cells have a typical cobblestone phenotype (not shown) and express the epithelial cell marker E-CADHERIN (Figure 1B, C). N1ICD expression caused a reduction in total E-CADHERIN levels in MCF-7 clones E8 and F7 but not in clone F5 (Figure 1B, C). The levels of Notch activity in MCF-7/N1ICD cells measured by a luciferase reporter assay using a fragment of the mouse Hes1 promoter , revealed an evident activation of the Notch pathway in comparison with control, mock-transfected MCF-7 cells (Figure 1D). qPCR analysis revealed a marked up-regulation of the NOTCH target genes HEY1, HES1 and C-MYC while the epithelial marker E-CADHERIN was down-regulated and SNAIL1 and VIMENTIN were not significantly changed (Figure 1E). We also examined NOTCH1 and NOTCH4 expression because of their role in mouse breast cancer malignancy  and their overexpression in triple-negative breast cancer subtypes . NOTCH4 expression was almost undetectable in MCF-7 cells (Additional file 2, Figure S1A) while NOTCH1 expression was unaffected (Additional file 2, Figure S1B and not shown), as previously reported . Semi-quantitative RT-PCR analysis of various MCF-7-N1ICD expressing clones revealed no variation in JAG1 and TWIST1 expression (Additional file 2, Figure S1B). The lack of response of TWIST1 to N1ICD expression is in agreement with previous findings showing that during developmental EMT, Twist1 is induced by Bmp2  but does not respond to Notch . Immunofluorescence analysis confirmed that forced N1ICD expression caused a reduction in membranous E-CADHERIN staining (Figure 1Fa-i). Cells with strong nuclear N1ICD staining showed mostly nuclear E-CADHERIN expression, suggesting a disassembly of adherens junctions , which contrasted with its accumulation in the membrane at the cell-cell contacts of MCF-7 cells that did not express N1ICD (Figure 1Fe). Concomitant to the reduction in E-CADHERIN levels, N1ICD expression endowed MCF-7 cells with increased chemotactic ability towards IGF-1 (Figure 1G, H), a chemo-attractant for this cell line ; some N1ICD-expressing MCF-7 clones showed an increased migratory capacity, even in basal medium (Figure 1Gb, c). To test if the reduction of E-CADHERIN upon N1ICD expression could be extended to other epithelial tumor cell lines, we transfected N1ICD into the HT-29 colon adenocarcinoma cell line and generated stable clones. We chose HT-29 cells because, similarly to the mammary gland, they derive from a tissue in which NOTCH has an oncogenic role [31, 32]. Additional file 3, Figure S2A, B shows that HT-29 cells stably expressing N1ICD down-regulate E-CADHERIN expression.
Inducible N1ICD expression in MCF-7 cells leads to E-cadherindown-regulation and increased migratory capacity
The effect of DOXY retrieval in N1ICD induction was measured by luciferase assay upon transfection of a Hes1 reporter. There was clear reporter activation after N1ICD induction in the different clones studied, especially in clone B12 at 48 h (Figure 2B). This enhanced N1ICD-induced transcriptional activity correlated with the increase of N1ICD expression in the different clones upon doxycycline withdrawal (Figure 2C). Concomitantly to N1ICD induction, there was an increase in the migratory capacity of these cells (Figure 2D, E).
These data indicated that N1ICD expression in MCF-7 cells, either in an inducible or stable manner, leads to a reduction in E-CADHERIN levels, suggesting that these cells began to lose their epithelial phenotype. During EMT there is a progressive cadherin switch, such that E-cadherin expression is reduced and N-cadherin expression is increased . Figure 3G shows a semi-quantitative RT-PCR analysis of N-CADHERIN expression in clone B12. Upon DOXY withdrawal there was a progressive increase in N-CADHERIN expression that was at a maximum after seven days of culture (Figure 3G, H), suggesting that B12 cells acquired a mesenchymal phenotype. Moreover, we found that N1ICD induction enhanced VIMENTIN expression although it did not change Twist1 mRNA levels (Additional file 4, Figure S3).
NOTCH inhibition reduces the migratory ability of MDA-MB-231 cells
Inducible Notch1 activation in MCF-7 cells stimulates tumor growth in vivo
The growth curves shown in Figure 5A, B suggest also that estrogens may be a limiting factor in Notch-mediated tumor formation and growth. Western blot analysis of xenografts generated after 12 weeks of N1ICD induction revealed strong N1ICD expression (Figure 5C).
Next, we analyzed growth of orthotopic tumors formed by the clone B12 transduced with a luciferase-expressing retrovirus to monitor tumor evolution by chemoluminiscence. After cell injection, mice were separated in two groups, receiving (N1ICD off) or not (N1ICD on) DOXY in the drinking water. In agreement with our previous results (Figure 5A), clone B12 with induced N1ICD expression gave rise to tumors significantly larger than those generated when N1ICD was not expressed (Figure 5D, E). DOXY treatment did not affect the growth of tumors formed by control MCF-7 cells (Figure 5D, F). These results suggested that N1ICD induction might be directly responsible for MCF-7 tumor formation.
N1ICD expression in the mammary gland leads to tumor formation and reduction in estrogen receptor and E-cadherin expression
There is considerable recent interest in understanding how NOTCH signaling affects the development or progression of breast cancer. Notch is critical in mammary gland development, probably by regulating mammary stem cell function . In addition, NOTCH activity has been associated with a number of pro-tumorogenic activities in breast cancer cell lines, and could cause mammary hyperplasia and carcinogenesis in mice [11, 40, 41, 42]. This evidence strongly pinpoints NOTCH receptors and/or ligands as targets in breast cancer.
Here we used three different in vitro and in vivo models to analyze the impact of NOTCH signaling in the onset and progression of breast tumors. We found a positive association of NOTCH activity with cancer growth or initiation. In agreement with others [43, 44, 45, 46], one of the most consistent observations along our study was the association between NOTCH1 activity and E-CADHERIN down-regulation. First, stable expression of activated Notch (N1ICD) was associated with a reduction and delocalization of E-CADHERIN in most of the MCF-7 cell clones analyzed; second, the data with the inducible MCF-7-N1ICD clone B12 clearly established a causal relationship between active NOTCH1 and reduced E-CADHERIN levels; third, inhibition of endogenous NOTCH activation with DAPT in MDA-MB-231 cells, a highly invasive cell line that expresses high NOTCH1 levels, resulted in an increase of E-CADHERIN expression; and fourth, papillary tumors raised in MMTV-Cre/+;N1ICD/+ transgenic mice expressing high levels of Hes1 also showed, at least, a delocalization of E-cadherin in the epithelium. Repression and/or delocalization of E-CADHERIN is usually associated with adherens junctions disassembly  and enhanced cell invasiveness . Concurrently, we observed that N1ICD-induced E-CADHERIN repression correlates with enhanced motility in transwell assays, whereas inhibition of Notch signaling, either by DAPT or RO inhibitors treatment or DN-CBF1 overexpression, reduced the motility of the invasive MDA-MB-231 cells. Collectively, these results indicate that NOTCH1 activation could induce EMT in epithelial tumor cells and, consequently, to favor tumor metastasis .
The role of Notch as a critical inducer of EMT has been demonstrated during the formation of the cardiac valve primordium . In this process, Notch activates Snail expression that in turn down-regulates VE-cadherin . We analyzed Snail1 expression in the MCF-7 clones expressing N1ICD, and found no clear correlation among N1ICD expression, E-CADHERIN down-regulation and SNAIL1 expression. We observed a markedly increased HES1 and HEY1 expression in response to NOTCH1 activation that correlated with a reduction in E-CADHERIN expression in both our cellular and animal models. Interestingly, both HES1 and HEY1 have been implicated as part of the hypoxic response associated to breast cancer progression .
We also analyzed whether NOTCH1 affects the growth and/or the onset of breast tumors. NOTCH signaling regulates the balance between cell proliferation, differentiation and apoptosis  and different reports have demonstrated that NOTCH triggers the proliferation of breast cancer cells [46, 49]. In line with these observations, we found that the growth of subcutaneous and orthotopic xenografts produced with MCF-7-B12 cells was boosted after the induction of N1ICD (Figure 5). This effect was cell autonomous, since silencing of N1ICD-expressing tumors by administration of DOXY stopped the growth of tumors whereas induction of N1ICD by DOXY withdrawal boosted tumor growth, with kinetics compatible with N1ICD induction.
Although induction of N1ICD in MCF-7 fosters tumor growth, this effect was only observed when mice were treated with estrogens; indeed, MCF-7 xenografts did not grow in the absence of estrogens, independently of the induction of N1ICD. These results suggest that N1ICD cooperates with the estrogen receptor (ER) on tumor growth, as recently reported . In agreement with this conclusion, MMTV-Cre/+;N1ICD/+ mice only developed mammary tumors after repeated pregnancies. It is noteworthy to mention that these breast tumors appeared in the lactating gland and regressed after gland involution; the regression was independent of N1ICD activity as determined by the Hes1 expression level. Our results thus resemble those obtained by Kiaris et al. , and contrast with the formation of non-regressing mammary neoplasm in transgenic mice expressing the active forms of Notch1, -3 and -4, reported by others [12, 41, 42]. In summary, our results confirm NOTCH1 as an EMT inducer in breast cancer cells, which may have implications in tumor growth, dissemination and metastasis. The identification of specific factors interacting with NOTCH signaling could thus be relevant to fully understand the role of NOTCH in breast neoplasia.
NOTCH1 activation attenuates E-CADHERIN expression and favors the motility and invasive ability of epithelial human breast cancer MCF-7 cells in vitro. In xenografts and in transgenic mice, NOTCH1 activation caused tumors whose increased growth is NOTCH- and estrogen receptor-dependent. To the contrary, NOTCH inhibition leads to increased E-CADHERIN expression and attenuates the migratory properties of invasive MDA-MB-231 breast cancer cells. Our findings in these mammary tumor models point to NOTCH1 as a potential therapeutic target in breast cancer onset and progression.
We thank Dr. Alberto Muñoz (IIB, CSIC, Madrid, Spain) for the HT-29 cell line and S. Bartlett (CNIC) for English editing. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) or Comunidad de Madrid (CM). The grants were: SAF2010-21205 (MINECO) and MITICP2010/BMD-2502 (CM) to CMA, SAF2011-24453 (MINECO) and INMUNOTHERCAN S2010/BMD-2326 (CM) to SM and SAF2007-62445 and SAF2010-17555 (MINECO) to JLdlP. The CNIC is supported by the MINECO and the Pro-CNIC Foundation.
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