Introduction
Breast cancer represents a heterogeneous group of tumors with different morphology, biology and treatment approach [
1]. Triple-negative breast cancers (TNBC), as defined on the basis of immunohistochemistry and for typically being negative for estrogen receptor (ER), progesterone receptor (PR) and HER2, represent approximately 20% of all breast tumors and have a considerable clinical relevance as they primarily affect young women, appear resistant to conventional chemotherapy regimens, have a particularly poor prognosis and a significantly worse clinical outcome than other tumor types [
2]. In the management of patients with TNBC, a promising role seems to be played by the observed relationship between the positivity to the glycosylated trans-membrane protein CD133 and shorter disease free and overall survival, suggesting that CD133 expression may be of help in more accurately predicting the aggressive properties of this neoplasia [
3]. Although a wide range of studies suggest that CD133-positivity identifies cancer stem cells [
4] yet the ability of CD133 to reliably identify breast tumor progenitors is controversial, also due to the use of different antibodies recognizing CD133 splice variants with epitopes of different glycosylation status [
5]. A strong correlation between CD133 expression and aggressive cellular behavior, including resistance to chemotherapy and radiotherapy, was also observed in hepatocellular carcinoma [
6], colon cancers [
7] and malignant gliomas [
8,
9], indicating that, regardless its role as a marker of stemness of tumor cells, CD133 may constitute a prognosticator for a number of different neoplasia.
A functional role of CD133 in tumors is suggested by the evidence that
in vitro targeting of CD133 with a specific binding peptide reduced colon and breast tumor cell motility [
10] and
in vivo down-regulation of CD133 severely impaired the capacity of melanoma cells to metastasize [
11]. Successful immunotoxin targeting of CD133 in hepatocellular and gastric cancer xenografts has also been reported [
6], suggesting that CD133 may be an important cancer therapeutic target. On the contrary, even though recent in vitro data on TNBC correlate CD133 with the inhibitor of cell cycle progression Geminin [
12], at present there is no evidence that associates CD133 to intracellular proteins involved in signalling events promoting breast tumor malignancy and very little is known about the regulation of its expression in breast tumor cells [
13]. A number of signalling molecules are deregulated in breast neoplasias, including specific isoforms of phosphoinositide-dependent phospholipase C (PLC) that resulted variously involved in proliferation, migration and invasiveness of tumor cells [
14‐
17]. We have demonstrated that PLC-β2 expression strongly correlates with a poor prognosis of patients with breast tumors [
18] and that, in breast tumor-derived cells with a triple negative phenotype, this PLC isozyme promotes migration and is necessary to sustain invasion capability [
16].
Aim of this work was to elucidate whether CD133 has a role in determining the malignancy-related properties of TNBC-derived cells. The relationship of CD133 expression with proteins known to be de-regulated in breast neoplasias, particularly with PLC-β2, was also investigated.
Discussion
Initially considered a marker of hematopoietic stem cells, CD133/prominin is a glycosylated trans-membrane protein expressed in various solid tumors, including breast cancer, in which CD133-positivity seems to identify a restricted subgroup of tumor progenitors [
23,
24]. In normal mammary tissue, CD133/prominin is not a marker for stem cells and seems to regulate ductal branching [
25]. Beyond its possible relationship with stemness of tumor cells, CD133 expression in breast cancer significantly correlates with tumor stage, tumor size and occurrence of lymph node metastases [
26]. CD133 is also useful in predicting chemosensitivity to neoadjuvant chemotherapy in breast cancer [
27], suggesting that CD133 expression may be of help in more accurately predicting the aggressive properties and in determining the optimal therapeutic strategy for this neoplasia. A strong correlation of CD133 expression with clinical stage of breast tumor patients was observed in TNBC (ER-, PR- HER2-), a high risk breast neoplasia that lacks the benefit of specific therapy that targets these receptors [
3]. It has been recently demonstrated that the expression of CD133 is associated with markers of hypoxia and/or tumor microvasculature in human breast tumors [
12,
28] and, in TNBC, CD133(+) cells with cancer stem cell characteristics associate with vasculogenic mimicry [
29]. These data suggest that the tumor microenvironment, and in particular hypoxia, induces in breast cancer cells a basal-like phenotype that includes increased expression of CD133 and decreased expression of hormone receptors.
CD133 is expressed at the surface of several cancer cells, not only with characteristics of stemness [
30], but a direct function of CD133 in determining specific features of tumor cells was not described. In particular, nothing is known about the role of CD133 in determining the biological properties of TNBC cells. This issue was tentatively addressed with the highly tumorigenic and moderately metastatic MDA-MB-231 cells [
31], which show an ER-, PR-, Her2- immunoprofile, mimicking the condition that is characterized by a low response to chemotherapy and worst prognosis in breast tumor patients [
32]. Here we show that the cytofluorimetrical analysis with anti CD133 antibodies identifies, in the bulk of the cell population, a low basal CD133 expression, and in a small percentage of cells (2-3%), a much higher expression level, making this cell line useful to compare TNBC cells with different levels of CD133 expression. By using antibodies directed against different CD133 epitopes [
33,
34] and Tunicamycin we ruled out the potential bias arising from variable glycosylation levels and from glycosylation-dependent epitopes in the extracellular portion of CD133 that it was reported to be potentially lost upon differentiation of tumor cells [
5]. We also extended the analysis to intracellular CD133 that allowed to definitely confirm the existence, in MDA-MB-231 cells, of a small but stable subpopulation expressing high levels of CD133 in both membrane and cytoplasm compartments. A comparison between cells expressing either low or high levels of CD133 indicates that CD133
high cells show lower proliferation and migration rate together with a larger adhesion area, consistent with a more undifferentiated tumoral phenotype. Interestingly, CD133
high cells exhibit a higher invasion capability through Matrigel, suggestive of higher metastatic potential. This is consistent with the data obtained in triple negative tumors, in which CD133 expression levels positively correlate with metastatization to lymph nodes [
3].
Protein profiles of CD133
low and CD133
high cells were compared by means of 2D analysis followed by mass spectrometry, showing that a number of proteins already known to be de-regulated in breast cancer [
22,
35‐
42] are differentially expressed between the two sub-populations. In particular, CD133
low cells that proliferate and migrate faster than CD133
high cells, show higher expression of proteins regulating cell motility. Interestingly, CD133
high cells, which exhibit a more invasive phenotype, show higher expression of the actin-binding protein Tm4, that was reported to be up-regulated in highly metastatic breast cancer cell lines and to be associated with the presence of lymph node metastasis of breast tumors [
22]. Tms are a family of cytoskeletal proteins present in virtually all eukaryotic cells, where they bind actin filaments and stabilize their structure [
43]. Changes in the expression of specific Tms are commonly found in malignantly transformed cells and overexpression of Tm4 in breast cancer cells is related to metastatic behaviour and may be a useful marker for predicting distant metastasis [
32]. In comparison to CD133
low cells, CD133
high cells also express higher levels of AdoHcyase, known to play a key role in the control of methylation [
44] and that, in breast cancer, seems to be involved in regulation of histone methylation via the 2 member enhancer of zeste homolog 2 (EZH2) [
39]. Since inhibition of AdoHcyase results in G2/M cell cycle arrest, apoptosis and cellular differentiation of breast tumor cells, including MDA-MB-231 [
39], targeting of this enzyme might be of therapeutic value in breast cancer.
Also the expression levels of a member of the eukaryotic initiation factor eIF3 family is higher in CD133
high than in CD133
low cells. eIF3 complex is essential for initiation of protein synthesis and the β subunit was already reported to be over-expressed in human breast carcinoma [
42]. Data on glioblastoma cells suggested for eIF3β an oncogenic role since its down-modulation inhibited cell proliferation and increased the apoptosis rate [
45]. This evidence indicates that, at least in TNBC cells, high expression of CD133 identifies cells with a peculiar protein expression pattern which accounts for their relatively differentiated tumoral phenotype together with high metastatic potential. Concerning the signalling molecules known to modulate proliferation/motility of breast tumor cells, no differences have been observed between CD133
high and CD133
low cells in the expression and activation levels of Akt, whose activity seems to have dichotomous effects on neoplastic progression of breast cancer [
19]. Also expression and activation levels of PLC-γ1, correlated with distant metastases of early breast tumors [
21] and involved in metastatic properties of TNBC cells [
46] were investigated. However, no difference between the two sub-populations expressing different levels of CD133 was found. On the contrary, CD133
high cells express PLC-β2 at levels significantly lower than CD133
low cells, in accordance with our previous data indicating that, in breast tumor-derived cells, PLC-β2 amount positively correlates with proliferation rate and motility [
16]. In particular, our previous studies on MDA-MB-231 cells, 98% of which express basal levels of CD133, have demonstrated that the down-modulation or the over-expression of PLC-β2 respectively reduces or increases their proliferation and migration rate [
16]. On the other hand, we have demonstrated that the silencing of PLC-β2 decreases invasion capability of MDA-MB-231 but its overexpression fails to affect their invasion capability through Matrigel [
16], indicating that the sole PLC-β2 is necessary but not sufficient to sustain the metastatic potential of TNBC cells. Here we show a peculiar role of PLC-β2 in cells expressing high levels of CD133. In fact, the over-expression of PLC-β2 in CD133
high cells, which contain relatively low levels of the protein, is able to induce a significant decrease of their invasion capability, in parallel with a reduced expression of CD133, at both membrane and cytoplasm levels. When the expression of PLC-β2 was down-modulated in CD133
low cells, containing relatively high levels of the protein if compared with CD133
high cells, a significant decrease of invasion capability was observed, according with our data previously obtained on the entire MDA-MB-231 cell population (accounting for about 98% of CD133
low cells) [
16]. The lack of effects of PLC-β2 down-modulation on CD133 expression in CD133
low cells confirms that the two sub-populations expressing different CD133 levels correspond to different stages of tumor differentiation, in which different signalling mechanisms take place. In this context, while PLC-β2 promotes the conversion of CD133
high to CD133
low cells, its silencing in cells showing a more differentiated tumoral phenotype (CD133
low) is not sufficient to revert the phenomenon.
A reduction of invasiveness trough Matrigel of CD133
high cells was found also when CD133 expression was forcedly down-modulated by specific siRNAs, indicating that CD133 is primarily involved in invasion capability of TNBC-derived cells. The mechanism may be correlated with the preferential localization of CD133 in plasma membrane protrusions, ended to regulate lipid composition and membrane topology [
4]. By establishing and maintaining membrane protrusions, CD133 may be involved in cell polarity and migration and may regulate the invasive properties of TNBC cells. On the other hand, the decreased expression of Tm4 observed after down-modulation of CD133 in highly expressing cells allows to speculate on a more specific mechanism by which CD133 can promote invasiveness of tumor cells, taking into account that the expression of specific isoforms of the Tms family correlates with the metastatic potential of TNBC-derived cells [
22].
The results indicating that up-regulation of PLC-β2 in cells expressing high levels of CD133 reduces the expression of this glysosylated protein in parallel with the invasion capability of CD133
high cells was confirmed in MDA-MB-468, a triple negative cell line expressing CD133 at high levels [
47] and almost negative for PLC-β2. The overall results indicate that, in TNBC cells, the increased expression of PLC-β2 down-regulates invasiveness only in cells with high levels of CD133 since this PLC isozyme negatively modulates the expression of CD133, in turn involved in determining the invasive properties of CD133
high cells.
Materials and methods
Cell culture and reagents
All reagents were from Sigma (St Louis, Mo., USA) unless otherwise indicated.
The breast cancer-derived cell line MDA-MB-231 and MDA-MB-468 and the human colon cancer cell line Caco-2 were purchased from the American Type Culture Collection (Rockville, MD). MDA-MB-231 and MDA-MB-468 cells were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM, Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco Laboratories). Caco-2 cells were cultured in DMEM with 1% Non-essential Amino Acid (NEAA, Lonza Sales Ltd, Basel, CH), 1% Sodium Pyruvate (Gibco Laboratories), 1% Penicillin-streptomycin solution (Lonza) and 10% FBS. All cell lines were grown at 37°C in a humidified atmosphere of 5% CO2 in air.
To inhibit N-glycosylation, Caco-2 and MDA-MB-231 cells were cultured in the presence of 2.5 μg/ml Tunicamycin or vehicle (medium containing 0.1% DMSO) for 24 hours.
Evaluation of CD133 expression
CD133 surface expression was evaluated by means of flow cytometry by direct staining of the cells with phycoerythrin (PE)-conjugated anti-CD133/1 (AC133) and anti-CD133/2 (293C3) mouse monoclonal antibodies (Miltenyi Biotec, Bologna, I), as suggested by manufacter's protocol, and by indirect labelling with a hybridoma supernatant (clone 7) containing a monoclonal antibody directed against unmodified CD133 epitopes, kindly provided by Dr. Panyam and Ohlfest (University of Minnesota) and used as described by Swaminathan et al. [
33]. In particular, 5×10
5 cells were stained with 100 μl of clone 7 hybridoma supernatant and reacted with a secondary anti-mouse-PE antibody (Becton-Dickinson, San José, CA).
For analysis of intracellular amounts of CD133, Perm and Stab Solutions Kit (Instrumentation Laboratory S.p.A, Milan, I) was used, performing the staining with anti-CD133/2-PE antibody, as suggested by manufacturers.
All the samples were analyzed by a FACSCalibur flow cytometer (Becton-Dickinson) with CellQuest Pro 6.0 software (Becton-Dickinson). Data collected from 10 000 cells are shown as percentage of positive cells or as mean fluorescence intensity (MFI) values.
Immunomagnetic separation
MDA-MB-231 cells were resuspended in PBS containing 0.5% bovine serum albumin and 2 mmol/L EDTA. For magnetic labeling, CD133/1 Micro Beads were used (Miltenyi Biotech) and positive magnetic cell separation was done using MACS SD columns (Miltenyi Biotech), according to manufacturer's instructions. CD133low and CD133high subpopulations were cultured in the same above reported medium and subjected to morphological analysis, to xCELLigence RTCA assays and to modulation of PLC-β2 and CD133 expression.
Two-dimensional gel electrophoresis and mass spectrometry
2-DE was performed essentially as described by Bertagnolo et al. [
48], with some modifications. Briefly, CD133
low and CD133
high cells were lysed with 2 M thiourea, 7 M urea, 4% CHAPS, 1% DTT, 2% IPG buffer pH 3–10 (Bio-Rad, Hercules, CA), benzonase and protease inhibitors, followed by heating for 30 min at 30°C, sonication and centrifugation at 21 000 ×
g for 60 min at 4°C. Supernatant containing 400
μ g of proteins was used to rehydrate 17 cm pH 4–7 IPG gel strips (Bio-Rad) for 16 h at 20°C. Focusing was carried out on PROTEAN IEF cell (Bio-Rad) using the following conditions: 250 V (60 min), 500 V (60 min), 1000 V (60 min), 5000 V (60 min), 10 000 V (60 min) and 10 000 V for the additional time required to reach a total of 80 kVh. The separation in the second dimension was performed using 1 mm thick, 12% constant vertical SDS-PAGE in PROTEAN II xi apparatus (Bio-Rad) at constant 35 mA/gel. The gels were stained with Coomassie Brilliant Blue G-250 (Bio-Rad) and scanned using a Pharos-FX Molecular Imager (Bio-Rad). The acquired maps were analyzed using the PDQuest Basic Version 8.0 software (Bio-Rad), as previously reported [
48]. A difference in intensity of 200% between spots of two compared gels was considered significant. Spots of interest were excised using a sterile blade and subjected to mass spectrometry analysis essentially as described by Bavelloni et al. [
49]. For peptide sequence searching, monoisotopic peptide mass lists were submitted to Mascot v.2.1 (Matrix Science, London, UK) against the UniProtKB-SwissProt database (April 23, 2012, total of 535 698 entries). The search parameters were as follows: two missed cleavage allowed, carbamidomethylation of cysteine as fixed modification, oxidation of methionines as variable modification, precursor ion mass tolerance 50 ppm and fragment ion tolerance 1 Da.
Analysis of adhesion area
The morphology of CD133
low and CD133
high MDA-MB-231 cells was analyzed with an inverted phase-contrast microscope (Nikon Eclipse TE2000-E; Nikon, Florence, I) and cell images were acquired by the ACT-1 software with a DXM1200F digital camera (Nikon) and analyzed with the ImageJ software, as previously reported [
50].
Real-time cell proliferation, migration and invasion assays
Cell proliferation, migration and invasiveness were evaluated by means of the xCELLigence RTCA System (Real-Time Cell Analyzer System, Roche Applied Science, Mannheim, D), developed to monitor cell events in real time by measuring the electrical impedance produced by cells. The employed procedures were essentially those described by Stander et al. [
51] for proliferation kinetics and by Mandel et al. [
52] for migration and invasiveness assays. In particular, to measure cell proliferation, 5000 cells ⁄well were used with a programmed signal detection every 15 min for a total of 96 h. For migration assays, 4 × 10
4 cells∕well were seeded onto the top chambers of CIM-16 plates (Roche) and the bottom chambers were filled with medium containing 5% serum. The setup for analysis of invasiveness was the same described for migration except that the upper side of the membranes was covered with a layer of Matrigel (BD Biosciences, San Josè, CA) diluted 1:20 and the bottom chambers were filled with 10% serum containing medium. For both migration and invasion assays, the signal detection was programmed every 15 min for a total of 24 h. Impedance values were expressed as a dimensionless parameter (cell index, CI).
Modulation of PLC-β2 and CD133 expression
PLC-β2 over-expression was performed by transient transfection with a plasmid expressing an Enhanced Green Fluorescent Protein (EGFP)-tagged full-length human PLC-β2, as previously reported [
16].
The down-modulation of CD133 and of PLC-β2 was performed by silencing the proteins with specific siRNAs (Santa Cruz Biotechnology, Santa Cruz, CA), following a previously described procedure [
16]. As a control of transfection efficiency a non-silencing fluorescein-labeled duplex RNA, purchased from Qiagen (Milan, I), was used. The transfected cells were incubated at 37°C in a 5% CO
2 atmosphere for 48 h and then subjected to immunochemical and cytofluorimetrical analysis and to xCELLigence RTCA assays.
Immunoprecipitation and immunochemical analysis
PLC-β2 was immunoprecipitated from CD133
low and CD133
high MDA-MB-231 cells and CD133 was immunoprecipitated with an anti-CD133/1 (W6B3C1, Miltenyi) from MDA-MB-231, CD133
high MDA-MB-231 and Caco-2 cells following a previously reported procedure [
16].
Total lysates and immunoprecipitates were separated on 7.5% polyacrylamide denaturing gels and blotted to nitrocellulose membranes (GE Healthcare Life Science, Little Chalfont, UK). The membranes were then incubated with antibodies directed against pY783-PLCγ1, PLC-γ1, PLC-β2, 14-3-3ϵ, eIF3β, AdoHcyase and Akt (Santa Cruz Biotechnology), pS473-Akt and Tm4 (Millipore S.p.A., Milan, I), CD133/1 (W6B3C1, Miltenyi Biotec) and β-tubulin (Sigma). The chemiluminescence derived bands were acquired with ImageQuant™ LAS 4000 biomolecular imager (GE Healthcare) and the densitometric analysis was performed by means of Image Quant TL software (GE Healthcare).
Statistical analysis
The results were expressed as means ± standard deviations of three independent experiments. Statistical analysis was performed by using the two-tailed Student's t-test for unpaired data. P values ≤0.05 were considered statistically significant.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
FB and VB participated in the design of the overall study. FB carried out the cytofluorimetrical and Real-time cell analysis. SG performed mono-dimensional and bi-dimensional protein analysis. MP1 and EN carried out transfection experiments. MP2 and AB performed mass spectra analysis. FB and VB conceived the study and interpreted the results. VB and SC wrote the paper and all authors read and approved the final manuscript.