Nuclear β-catenin and CD44 upregulation characterize invasive cell populations in non-aggressive MCF-7 breast cancer cells
© Uchino et al; licensee BioMed Central Ltd. 2010
Received: 27 October 2009
Accepted: 10 August 2010
Published: 10 August 2010
In breast cancer cells, the metastatic cell state is strongly correlated to epithelial-to-mesenchymal transition (EMT) and the CD44+/CD24- stem cell phenotype. However, the MCF-7 cell line, which has a luminal epithelial-like phenotype and lacks a CD44+/CD24- subpopulation, has rare cell populations with higher Matrigel invasive ability. Thus, what are the potentially important differences between invasive and non-invasive breast cancer cells, and are the differences related to EMT or CD44/CD24 expression?
Throughout the sequential selection process using Matrigel, we obtained MCF-7-14 cells of opposite migratory and invasive capabilities from MCF-7 cells. Comparative analysis of epithelial and mesenchymal marker expression was performed between parental MCF-7, selected MCF-7-14, and aggressive mesenchymal MDA-MB-231 cells. Furthermore, using microarray expression profiles of these cells, we selected differentially expressed genes for their invasive potential, and performed pathway and network analysis to identify a set of interesting genes, which were evaluated by RT-PCR, flow cytometry or function-blocking antibody treatment.
MCF-7-14 cells had enhanced migratory and invasive ability compared with MCF-7 cells. Although MCF-7-14 cells, similar to MCF-7 cells, expressed E-cadherin but neither vimentin nor fibronectin, β-catenin was expressed not only on the cell membrane but also in the nucleus. Furthermore, using gene expression profiles of MCF-7, MCF-7-14 and MDA-MB-231 cells, we demonstrated that MCF-7-14 cells have alterations in signaling pathways regulating cell migration and identified a set of genes (PIK3R1, SOCS2, BMP7, CD44 and CD24). Interestingly, MCF-7-14 and its invasive clone CL6 cells displayed increased CD44 expression and downregulated CD24 expression compared with MCF-7 cells. Anti-CD44 antibody treatment significantly decreased cell migration and invasion in both MCF-7-14 and MCF-7-14 CL6 cells as well as MDA-MB-231 cells.
MCF-7-14 cells are a novel model for breast cancer metastasis without requiring constitutive EMT and are categorized as a "metastable phenotype", which can be distinguished from both epithelial and mesenchymal cells. The alterations and characteristics of MCF-7-14 cells, especially nuclear β-catenin and CD44 upregulation, may characterize invasive cell populations in breast cancer.
Patients with breast cancer are at risk of metastasis throughout their lifetime because of the heterogeneous nature of breast cancer metastasis. Recent studies focusing on the early steps in the metastatic cascade, such as epithelial-to-mesenchymal transition (EMT) and altered cell adhesion and motility, have demonstrated that aggressive cancer progression is correlated with the loss of epithelial characteristics and the gain of a migratory and mesenchymal phenotype . In fact, the highly aggressive breast cancer cell line MDA-MB-231 exhibits mesenchymal-type behavior, whereas non-aggressive breast cancer cell line MCF-7 has a luminal epithelial-like phenotype [2, 3].
In addition to the heterogeneous nature of metastasis, a solid tumor including breast cancer is comprised of heterogeneous cells in terms of their invasive and metastatic potential, as suggested by in vivo metastasis models  and an in vitro selection process using Matrigel [5, 6]. Tumor heterogeneity has led to the "cancer stem cell hypothesis". Cancer stem cells share common characteristics with normal stem cells: ability to self-renew, differentiate, acquire drug resistance, survive anchorage-independently, and migrate. Furthermore, overlapping sets of molecules and pathways regulate both stem cell migration and cancer metastasis; therefore, cancer stem cells are assumed to contribute to metastasis as well as tumorigenesis . In human breast tumors, the CD44+/CD24-/low phenotype has been reported to have stem cell properties . Cell lines with high CD44+/CD24- cell numbers were basal/mesenchymal or myoepithelial types and more invasive than other cell lines. In contrast, non-aggressive epithelial MCF-7 cells lack a CD44+/CD24- subpopulation. Among CD44+/CD24--positive cell lines, MDA-MB-231 has the unique property of expressing a broad range of genes that favor bone and lung metastasis . Although there remains a need to determine whether CD44+/CD24-/low cells are true breast cancer stem cells across all the various breast cancer subtypes, there seems to be a connection between EMT and CD44/CD24 expression in the mechanisms of breast cancer invasion and metastasis. Indeed, induction of EMT results in the acquisition of the CD44high/CD24low phenotype in immortalized human mammary epithelial cells .
In the current study, we demonstrated that non-aggressive epithelial MCF-7 cells had rare cell populations with higher Matrigel invasive ability. Throughout the sequential selection process using Matrigel, we obtained MCF-7-14 cells of opposite migratory and invasive capabilities from MCF-7 cells. Thus, what are the potentially important differences between invasive and non-invasive breast cancer cells, and are the differences related to EMT or CD44/CD24 expression? To answer these questions, comparative analysis of epithelial and mesenchymal marker expression was performed between MCF-7, MCF-7-14, and MDA-MB-231 cells. Furthermore, using microarray expression profiles of these cells, we selected differentially regulated genes for their invasive potential and performed pathway and network analysis to better characterize invasive cell populations in breast cancer.
Human breast cancer cell line MCF-7 was obtained from the Institute of Development, Aging and Cancer, Tohoku University. MDA-MB-231 was purchased from the American Type Culture Collection. Both cell lines were maintained in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (FBS) and an antibiotic-antimycotic.
In vitrosequential selection of invasive populations from MCF-7 cells
BD BioCoat Matrigel Invasion Chambers (for a 6-well plate; BD Biosciences, San Jose, CA) were used to select invasive populations from MCF-7 cells. MCF-7 cell suspension (4 × 105 cells/well) in serum-free RPMI1640 was seeded into the upper chamber. After 60 hours' cultivation, cells that migrated through the membrane (named MCF-7-1) were harvested by trypsinization, proliferated on a dish, and then reseeded in a Matrigel Invasion Chamber. The cycle was repeated 14 times, and finally MCF-7-14 cells were obtained. In each cycle, invading cells on the underside of the chamber were fixed with 10% formalin-neutralized buffer, stained with 0.1% crystal violet, and imaged through a microscope (Axiovert 200M; Carl Zeiss, Jena, Germany) with a CCD camera (AxioCam; Carl Zeiss).
Cell proliferation assay
MCF-7 and MCF-7-14 cells (5 × 103 cells/well) were seeded into 96-well plates and cultured. The cells were counted at 24, 48, 72 and 96 h with a Cell Counting Kit-8 (MTT assay; Wako Pure Chemical Industries, Osaka, Japan).
Cell protein lysates were prepared from confluent cell cultures of MCF-7 and MCF-7-14 cells, subjected to SDS-PAGE, and transferred to PVDF membranes. The membranes were blocked (5% skim milk) and incubated with rat anti-human epidermal growth factor receptor 2, HER-2 (GeneTex, Irvine, CA) and mouse anti-estrogen receptor alpha, ER-α (Stressgen/Assay Designs, Ann Arbor, MI) monoclonal antibodies (mAbs), followed by the corresponding HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). Antibody complexes were detected with the ECL detection system (GE Healthcare).
In vitrowound healing assay
Migratory abilities of MCF-7 and MCF-7-14 cells were measured using the in vitro wound healing assay. Cells were plated on 10-cm culture dishes and grown to 100% confluence. Wounds were created by scraping monolayer cells with a sterile pipette tip. At 0, 24, 48, 72 and 96 h after the creation of wounds, three different areas were imaged through an Axiovert 200 M. Wound distances were measured at each time point and expressed as the average percent of wound closure by comparing the zero time.
Animal experiments were performed in accordance with the Guidelines of the Japanese Government for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at Tokyo University of Science and the Ethics Committee of Bio Matrix Research Inc. MCF-7 and MCF-7-14 cells were transfected with pEGFP-C1 (encoding the enhanced green fluorescence protein, EGFP; Clontech Laboratories, Mountain View, CA) using Fugene 6 Transfection Reagent (Roche, Basel, Switzerland) in serum-free medium. MCF-7 and MCF-7-14 cells stably transfected with pEGFP-C1 (named MCF-7-EGFP and MCF-7-14-EGFP, respectively) were selected by G418 treatment (500 μg/ml) for 3 weeks. Suspensions of MCF-7-EGFP and MCF-7-14-EGFP cells (1 × 106 cells/50 μl) were mixed with the same volume of Matrigel, and injected into the fourth mammary fat pad of 10-week-old female BALB/cJ/nu/nu mice.
In vivo and ex vivoimaging of EGFP-labeled breast cancer cells
At 4, 8 and 12 weeks after implantation of MCF-7-EGFP and MCF-7-14-EGFP cells, mice were sacrificed and scanned by a luminescence/fluorescence imaging analyzer (LAS3000; Fujifilm, Tokyo, Japan) to detect metastatic lesions. Ex vivo fluorescence imaging was also performed on necropsy samples.
Histology and immunohistochemistry
Xenografts and metastatic lesions were rapidly frozen, and 6 μm thick sections were cut on a cryostat. The sections were analyzed by hematoxylin/eosin (HE) and methyl green pyronin (MGP) staining, or unstained sections were analyzed for EGFP fluorescence. For immunostaining epithelial and mesenchymal markers, mouse anti-cytokeratin (CK) 19 (A53-B/A2; 1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-E-cadherin (67A4; 1:25; Santa Cruz Biotechnology) and anti-vimentin (V9; 1:150; Dako, Glostrup, Denmark) mAbs were used. Briefly, frozen sections were treated with 3% H2O2 for 10 min and 5% skim milk in Tris-buffered saline with 0.2% Triton-X for 1 h. Primary antibodies were applied for 1 h at room temperature. Subsequently, the sections were incubated with Simple Stain Mouse MAX PO (Nichirei Biosciences, Tokyo, Japan) or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:200; Molecular Probes, Invitrogen, Carlsbad, CA) at room temperature. Diaminobenzidine (DAB) with 0.1% H2O2 (Dako) was used as the final chromogen and hematoxylin for nuclear counterstaining in immunohistochemistry. In immunofluorescence, cell nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI). Immunostaining of the endothelial marker, CD31, was performed with rat anti-mouse CD31 mAb (MEC 13.3; 1:50; BD Biosciences) and HRP-linked goat anti-rat IgG F(ab')2 fragment (1:200; Amersham ECL, GE Healthcare). Microscopic images were acquired using an Axiovert 200 M equipped with an AxioCam and analyzed by AxioVision software (Carl Zeiss).
MCF-7, MCF-7-14 and MDA-MB-231 cells were plated on coverslips and fixed with 4% paraformaldehyde/phosphate-buffered saline (PBS) for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 2 min, and then incubated in PBS containing 5% skim milk for 1 h at room temperature. Cells were incubated with anti-E-cadherin (1:100), anti-β-catenin (8E7; 1:100; Upstate Biotechnology/Millipore, Billerica, MA), anti-vimentin (1:300) and anti-fibronectin (clone 10; 1:200; BD Biosciences) mAbs for 1 h at room temperature, followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:200-1:1000) and nuclear counterstaining with DAPI.
MCF-7, MCF-7-14 and MDA-MB-231 cells were synchronized in S-phase with the thymidine-hydroxyurea block method. Total RNA was isolated from cells arrested in S-phase using an RNeasy Mini kit (Qiagen, Hilden, Germany).
cDNA was synthesized from total RNA isolated from MCF-7, MCF-7-14 and MDA-MB-231 cells using a One-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA). In vitro transcription reactions were performed using a GeneChip IVT Labeling Kit. Fifteen micrograms of the labeled cRNA was hybridized to a Human Genome U133 Plus 2.0 Array (Affymetrix). The array images were scanned and analyzed using Genechip operating software (GCOS; Affymetrix). The full microarray data set is available in the NCBI Gene Expression Omnibus public database under data series accession number GSE18903.
Microarray data analysis
Using GeneSpring GX 7.3.1 software (Agilent Technologies, Santa Clara, CA), microarray data of each chip were normalized to the 50th percentile of the measurements on that chip. For per-gene normalization, the measurements of each probe were normalized to the median of the measurements of that probe in MCF-7, MCF-7-14 and MDA-MB-231 cell lines. Average linkage hierarchical clustering was carried out on genes filtered on detection flags ("present" in two or more cell lines) and signal intensity (>50 in all cell lines). To identify potentially important differences in biological mechanisms regarding their invasive potential, genes up- or down-regulated >2-fold in both MCF-7-14 and MDA-MB-231 cells compared with MCF-7 cells were selected from the filtered genes. Ingenuity Pathway Analysis software (IPA 5.0; Ingenuity Systems, Redwood City, CA) was also utilized to identify the top significant canonical pathways for these selected genes and to functionally link the most differentially expressed genes.
cDNA was synthesized from 2 μg total RNA using SuperScript III reverse transcriptase (Invitrogen). Reverse transcription (RT) was run for 1 h at 50°C and stopped by heating for 5 min at 85°C. PCR was conducted in a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). A 10 μl reaction containing 0.2 μl cDNA, 0.5 μM of each primer and 2.5 μl Power SYBR Green PCR Master Mix (Applied Biosystems) was used to monitor double-strand DNA synthesis. Primer sequences are provided in Additional file 1. Quantitative RT-PCR (qRT-PCR) was carried out following the recommended thermal profile: 95°C for 10 min (pre-incubation) followed by 40 cycles of 95°C for 15 sec (denaturation) and 60°C for 1 min (annealing and elongation). Fluorescence intensity of the amplified products was measured at the end of each PCR cycle. Two runs were performed with each data point run in triplicate. Results were normalized to internal control GAPDH mRNA and if necessary, represented relative to mRNA levels of MCF-7 cells.
MCF-7 and MCF-7-14 cells were plated on 96-well plates at a concentration of a single cell per well, which was confirmed visually. Wells containing either none or more than one cell were excluded from further analysis. Single-cell clones were cultured to allow the growth of individual colonies, which were picked and expanded in culture, and analyzed by Matrigel invasion assay and flow cytometry.
Three human breast cancer cell lines, MCF-7, MCF-7-14 and MDA-MB-231, and single-cell clones derived from the MCF-7 and MCF-7-14 cell lines were used in flow cytometry analysis. Cells were harvested with TrypLE Express (Invitrogen), and then suspended (2 × 106 cells/100 μl) with Stain Buffer containing 1% FBS (BD Biosciences). Phycoerythrin (PE)-conjugated mAbs against human CD44 (G44-26; BD Biosciences) or CD24 (ML5; BD Biosciences) were added to the cell suspension at the concentrations recommended by the manufacturer and incubated at 4°C in the dark for 60 min. The labeled cells were fixed in 100% methanol on ice for 5 min. Flow cytometry analysis were performed in triplicate using a FACSCalibur system (BD Biosciences).
Function-blocking antibody treatment
Cells (1 × 106 cells) were incubated with rat anti-human CD44 mAb (IM7; BD Biosciences) or normal rat IgG at 400 μg/ml for 20 min. After preincubation, a cell proliferation assay, cell migration assay and Matrigel invasion assay were performed. Cell proliferation was assessed by MTT assay. The cell migration assay was performed using an Oris Cell Migration Assay (Platypus Technologies, Madison, WI), which comprises a 96-well plate with silicone stoppers (2 mm diameter) in each well. Following cell seeding and cell attachment (2 × 104 cells), the stoppers were removed and migration proceeded for 72 h. Cells were then stained with crystal violet and imaged through a microscope. Matrigel Invasion Chambers (for a 24-well plate; BD Biosciences) were used to examine cell invasion. Pretreated cells (5 × 104 cells) in serum-free RPMI1640 were seeded into the upper chamber and invading cells were fixed and stained after 60 h cultivation.
Data are the means ± standard deviation (SD). Values were compared between MCF-7 and MCF-7-14 cells and between normal IgG and anti-CD44 mAb using Student's t test. Comparisons among cell lines were made using analysis of variance (ANOVA) and Tukey-Kramer multiple comparison, where appropriate. P < 0.05 was considered significant.
Selection and morphological characterization of MCF-7-14 cells
Migratory and metastatic ability of MCF-7-14 cells
Epithelial and mesenchymal marker expression in MCF-7-14 cells
144 unique genes differentially expressed in both MCF-7-14 and MDA-MB-231 cells
Top canonical pathways and functional networks for differentially expressed genes
Top signaling pathways for the 163 differentially expressed probe sets in MCF-7-14 and MDA-MB-231 cells.
Gene mapped to canonical pathways
Xenobiotic Metabolism Signaling
ALDH3B2, MAOB, PIK3R1, UGT1A6
Axonal Guidance Signaling
BMP7, CFL2, CXCL12, PIK3R1, SEMA6D
Acute Phase Response Signaling
PIK3R1, SOCS2, TCF3
Leukocyte Extravasation Signaling
CD44, CXCL12, PIK3R1
LPS/IL-1 Mediated Inhibition of RXR Function
ALDH3B2, MAOB, SLC27A2
Serotonin Receptor Signaling
Cell Cycle: G2/M DNA Damage Checkpoint Regulation
Evaluation of selected interesting genes: CD44 upregulation and CD24 downregulation in MCF-7-14 cells
Effects of anti-CD44 antibody treatment on cell migration and invasion
MCF-7-14 cells as a novel model for breast cancer metastasis without requiring constitutive EMT
We obtained MCF-7-14 cells that had a significantly higher proportion of invasive cells throughout the in vitro sequential selection process, although parental MCF-7 cells had few Matrigel invasive cells. In addition, we demonstrated that EGFP-labeled MCF-7-14 cells metastasized to the pancreas and peritoneum in a xenograft model. In mouse orthotopic xenograft models of breast cancer, the pancreas seems to be a relatively common metastatic site. When injected into the mammary fat pad of nude mice, N-cadherin-expressing cells, but not control MCF-7 cells, metastasized to the liver, pancreas, salivary gland, omentum, lung, lymph nodes, and lumbar spinal muscle . Murine 4T1 mammary cancer cells (expressing the pcDNA-Neo expression vector as a control; 4T1-Neo cells) implanted into the axillary mammary gland of BALB/c mice metastasized to the mesentery adjacent to the pancreas . Despite differences in invasive ability, MCF-7-14 cells, similar to MCF-7 cells, expressed E-cadherin, but neither vimentin nor fibronectin. In the cell migration assay, MCF-7-14 and its clone CL6 cells moved as a sheet, in contrast to the more individual movements of mesenchymal MDA-MB-231 cells. Cell-sheet movement is a typical feature of epithelial cells during embryonic development and wound healing ; therefore, MCF-7-14 cells may be a novel model for breast cancer metastasis without requiring constitutive EMT. Complete and constitutive EMT seems not to be required for breast cancer metastasis [1, 14, 15]. In particular, Lou et al.  reported that 4T1 cells, which express E-cadherin and ZO-1, are migratory, invasive, and metastasize to multiple sites, and that 4T1-derived (67NR) cells, which form primary tumors but fail to metastasize, express vimentin and N-cadherin, but not E-cadherin. The metastatic ability of breast cancer cells does not seem to strictly correlate with the genotypic and phenotypic properties of EMT per se. In addition, Tarin et al.  suggested that the fundamental premise that EMT occurs in real cancers is very much in doubt because of the lack of convincing evidence for the conversion of epithelial cells into mesenchymal cell lineages in vivo; however, it is possible that MCF-7 and MCF-7-14 cells can make transient EMT in vitro in a reversible fashion. For example, estrogen treatment promoted reversible EMT-like changes and collective motility in ER-positive breast cancer cells . The transitory acquisition of mesenchymal characteristics could explain the difficulty in observing EMT in cancer development.
Nuclear β-catenin expression and "metastable phenoptype" in MCF-7-14 cells
On the other hand, loss of E-cadherin-β-catenin complex is an important step in the progression of many epithelial malignancies . β-catenin has a dual role in the EMT; it enhances cell-cell adhesion when bound to cadherin complexes in adherens junctions and also functions as a transcriptional coactivator upon entry into the nucleus . Nuclear import of β-catenin is another important player in EMT [21, 22]. Indeed, in MDA-MB-231 cells, β-catenin was mainly located in the nucleus, whereas in MCF-7 cells, β-catenin was mostly expressed on the cell membrane. Nuclear β-catenin induces a gene expression pattern favoring tumor invasion and proceeds with transition to the mesenchymal phenotype of epithelial tumor cells . In MCF-7-14 cells, β-catenin was expressed not only on the cell membrane but also in the nucleus; therefore, enhanced invasive potential of MCF-7-14 cells may result from "incomplete" EMT, altering some signaling pathways. Several studies [24, 25] have identified a hybrid cell showing both epithelial and mesenchymal traits. These cells are referred to as a "metastable phenotype" and distinguished from both epithelial and mesenchymal cells by characteristics such as residual E-cadherin, nuclear β-catenin and sheet movement [13, 26]. According to this categorization, MCF-7-14 cells may be just in a "metastable" cell state.
Alterations in cell migration signaling in MCF-7-14 cells
Here we analyzed which signaling pathways are associated with the enhanced migratory and invasive potential of MCF-7-14 cells. Canonical pathway analysis using IPA 5.0 identified the top 16 signaling pathways for differentially expressed genes in invasive cells. Some of these signaling pathways are involved in regulating cell migration. JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling is necessary for border cell migration in the Drosophila ovary, suggesting its relevance to the progression of cancer . Axon guidance molecules and neuregulin signaling play critical roles in cancer cell invasion as well as in neuronal migration [28, 29]. In addition, leukocytes exhibit the ability to sense and move in the direction of a chemoattractant . Acute-phase response signaling modifies inflammatory responses, thereby contributing to leukocyte extravasation . Directed cell movement or chemotaxis is exhibited during wound healing, angiogenesis, embryonic development, immune function, and during cancer cell metastasis . Pathway and network analysis also identified a set of interesting genes, including PIK3R1, SOCS2 and BMP7. Philp et al.  reported the presence of somatic mutations in PIK3R1, the gene for the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K), in primary human colon and ovarian tumors and cancer cell lines, resulting in the constitutive activation of PI3K. In addition, MCF-7-14 and MDA-MB-231 cells displayed the upregulated expression of SOCS2 and downregulated expression of BMP7 compared with MCF-7 cells. JAK/STAT signaling is negatively regulated by overexpression of SOCS proteins, and SOCS2 interferes with the negative regulatory effects of SOCS1 and SOCS3 [27, 34]. SOCS2 was generally upregulated in primary breast tumors that developed bone metastasis . On the other hand, transforming growth factor beta (TGF-β) is a pro-oncogene in the later stages of tumorigenesis and appears to contribute to metastasis [36, 37]. BMP7 is an antagonist of the TGF-β pathway and can inhibit osteolytic metastasis attributable to prostate cancer . Decreased BMP7 expression during carcinogenesis in the human breast contributes to the acquisition of a bone metastatic phenotype . PI3K, JAK/STAT and TGF-β signaling play key roles in cell migration, and PIK3R1, SOCS2 and BMP7 are regulatory molecules of these signaling pathways, thereby possibly contributing to metastasis development. Consequently, alterations in these signaling pathways may explain the differences in migratory and invasive potential between MCF-7 and MCF-7-14 cells, demonstrating the validity and usefulness of this comparative analysis.
Possible relevance of MCF-7-14 cells to cancer stem cells
Canonical pathway analysis identified xenobiotic metabolism signaling as one of the top signaling pathways in invasive cells. This signaling pathway is possibly responsible for maintenance of the stem cell phenotype and multidrug resistance in breast cancer cells . In addition, some of the top signaling pathways (JAK/STAT, TGF-β, epidermal growth factor and Wnt/β-catenin signaling) overlapped the signaling pathways important for cancer stem-like cells, identified between the MCF-7 side population (SP) and non-SP cells . These signaling pathways are involved in both tumorigenesis in cancer and self-renewal in embryonic stem cells . Although there are no direct evidences yet, MCF-7-14 cells, which have been derived from rare cell populations with higher invasive ability within MCF-7 cells, may be possibly relevant to cancer stem-like cells. MCF-7-14 cells have similar morphological characteristics to those of stem/progenitor or chemotherapy-resistant cells isolated from MCF-7 cells. Cells grown as non-adherent spherical clusters, which were isolated from breast cancer lesions or MCF-7, displayed stem/progenitor cell properties . MCF-7 cells that survived chemotherapeutic treatment formed looser colonies in the monolayer, with cells at the borders tending to have more protruding filopodia . Furthermore, invasive populations seem to generate both invasive and non-invasive populations resembling the cases seen in SP  and tumorigenic CD44+CD24-/low cells  of breast tumor cells, because the numbers of cells invading the Matrigel were very low until the 8th cycle of in vitro sequential selection. Interestingly, MCF-7-14 cells displayed increased expression of CD44 mRNA and downregulated expression of CD24 mRNA and protein. Although MCF-7-14 cells showed cell population heterogeneity in invasiveness and CD44 protein expression, MCF-7-14-derived clone MCF-7-14 CL6, which had a higher number of invading cells, displayed upregulation of CD44 and rapid and extended proliferation. However, there are no overlapping genes between our results and the gene signature identified by gene expression profiling of CD44+CD24- breast cancer stem-like cells ; therefore, our findings should be validated in further studies, for example, analysis of stem cell marker expression.
Nuclear β-catenin and CD44 upregulation characterize invasive cell populations in breast cancer
Mani, Weinberg and colleagues  found that cells that had undergone EMT exhibited CD44high/CD24low expression, suggesting that the EMT might confer malignant properties on breast tumor cells. Although the EMT seems not to be an "all or nothing" event, upregulation of CD44 and downregulation of CD24 seem to participate in the acquisition of invasive and metastatic potential [47–49]. In this study, despite the difference in CD44 expression, treatment with CD44 mAb (clone IM7), which induces CD44 shedding from the cell surface , significantly decreased cell migration and invasion in MCF-7-14 and its clone CL6 cells as well as MDA-MB-231 cells. Although CD44 expression seems to be correlated with mesenchymal marker expression as well as with invasiveness, CD44 may be one of the most important adhesion molecules facilitating cell invasion and metastasis . Furthermore, CD44 expression is, directly or indirectly, regulated by the β-catenin/Tcf-4 signaling pathway, especially in the colorectal cancer precursor lesions, suggesting a role for CD44 in intestinal tumorigenesis [52, 53]. Brabletz et al.  reported that nuclear β-catenin accumulates in dedifferentiated colorectal carcinoma cells at the tumor-host interface, whereas a gradual loss of nuclear β-catenin is seen towards central, well-differentiated areas of the tumor. This heterogeneous pattern of the primary tumor is recapitulated in corresponding liver metastases. Nuclear accumulation of β-catenin in colorectal cancer cells at the invasive front and in the vessels has been suggested to be a powerful predictor of liver metastasis . In breast cancer, Wnt/β-catenin activation is an important feature of basal-like breast cancers and is predictive of worse overall survival . Consequently, nuclear β-catenin and upregulation of CD44 may be potential diagnostic and therapeutic targets for breast cancer metastasis.
Although the MCF-7 cell line has a luminal epithelial-like phenotype and lacks a CD44+/CD24- subpopulation, we obtained MCF-7-14 cells of opposite migratory and invasive capabilities from MCF-7 cells and developed a novel model for breast cancer metastasis without requiring constitutive EMT. This study better characterizes invasive breast cancer cells and provides insight into understanding the biology of breast cancer metastasis. In clinical diagnosis, ER-positive, HER2-negative breast cancer, which is defined as "luminal A" by the specific expression of an intrinsic set of genes , is the most common type of breast cancer and tends to have a better prognosis than the other three types (luminal B, HER-2 overexpressing, basal-like). However, the clinical behavior of luminal-type breast cancer can be markedly heterogeneous despite similar levels of ER expression ; therefore, a set of genes differentially regulated in MCF-7-14 cells (PIK3R1, SOCS2, BMP7, CD44 and CD24) may be useful markers to identify among good prognosis tumors those that will relapse and metastasize. MCF-7-14 cells, in particular, showed nuclear β-catenin expression and a similar phenotype to "metastable" cells, which are distinguished from both epithelial and mesenchymal cells . In addition, MCF-7-14 and its invasive clone CL6 cells displayed increased expression of CD44 and downregulated expression of CD24 compared with MCF-7 cells. The alterations and characteristics of MCF-7-14 cells may characterize invasive cell populations in breast cancer.
analysis of variance
enhanced green fluorescence protein
estrogen receptor alpha
fetal bovine serum
human epidermal growth factor receptor 2
Janus kinase/signal transducer and activator of transcription
methyl green pyronin
quantitative reverse transcription polymerase chain reaction
transforming growth factor beta.
This study is funded by JCR Pharmaceuticals. We are grateful to Mr. Takashi Homma and the members of the Microarray and Bioinformatics Departments for their technical assistance.
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