Introduction
The WNT signaling network is complex, with 19 WNT ligands, 10 Frizzled (FZD) receptors, as well as the co-receptors, low-density lipoprotein receptor-related protein (LRP) 5 and LRP6. WNT receptor binding stimulates intracellular signaling, promoting stabilization and nuclear translocation of the key effector of the canonical pathway, β-catenin [
1,
2]. Intracellular mediators of the WNT pathway are mutated in many human cancers. Inactivating mutations in the
APC or
AXIN genes, as well as activating
CTNNB1 mutations, each causes β-catenin stabilization and nuclear accumulation in the absence of WNT ligands. In the nucleus, β-catenin forms functional complexes with transcription factors of the lymphoid enhancer binding factor-1/T-cell factor (TCF) family, activating expression of target genes with cancer-promoting roles [
3]. In addition to activation of the canonical pathway by engagement of FZD and LRP receptors, WNT ligands bind the Ror2 or Ryk receptors to stimulate β-catenin-independent pathways that have been involved with cytoskeletal reorganization and cell migration [
2,
4].
In breast cancer, deregulation of WNT signaling appears to occur by autocrine mechanisms [
5‐
7]. Multiple WNT ligands and FZD receptors are expressed in primary human breast tumors and in breast cancer cell lines [
3,
7‐
9]. Furthermore, most breast tumors show hypermethylation of the promoter region of secreted Frizzled-related protein 1 (sFRP1) and low expression of this negative WNT pathway regulator [
10‐
12]. Interference with autocrine WNT signaling has been shown to block
in vitro proliferation of many human breast cancer cell lines [
6,
7]. We have extended these studies and show in the present article that blocking the WNT pathway in MDA-MB-231 breast cancer cells not only decreases proliferation, but also impairs the motility of the tumor cells. Furthermore, we show that stable expression of sFRP1 in MDA-MD-231 cells has a dramatic effect on the ability of the cells to grow as tumor xenografts in nude mice. The results presented here provide further evidence supporting approaches to target WNT pathway activity in breast cancer.
Materials and methods
Reagents
The following primary antibodies were used: c-Myc (9E10, to detect Myc-tagged protein), DVL2 and DVL3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); DVL1 (R&D Systems, Abingdon, UK); active β-catenin (anti-ABC; Upstate, Billerica, VA, USA); α-tubulin (DM1A) (Neomarkers, Fremont, CA, USA); cyclin D1 (SP4) (Cell MARQUE, Rocklin, CA, USA) for immunohistochemistry (IHC); cyclin D1 (Chemicon, Billerica, MA, USA) for western blotting; bromodeoxyuridine (BrdU) (Roche, Basel Switzerland); p21Cip1 (OP64-100UG) (Oncogene Research Products, Cambridge, MA, USA); ERK and P-ERK (Thr202/Tyr204) (Cell Signaling Technology, Danvers, MA, USA); total β-catenin and CD31 (BD Pharmingen, Franklin Lakes, CA, USA); and active β1-integrin (Clone HUTS-4, MAB2079Z; Chemicon).
As secondary antibodies we used anti-rabbit and anti-mouse antibodies (GE Healthcare, Little Chalfont, UK; LI-COR Bioscience, Lincoln, NE, USA), anti-rat antibody (GE Healthcare) or anti-goat antibody (DAKO A/S, Glostrup, Denmark) coupled to horseradish peroxidase (HRP) or IRDye 800CW.
For IHC we used Biotin-SP-conjugated affinipure donkey anti-rabbit, anti-mouse, anti-rat IgG (Jackson ImmunoResearch, West Grove, PA, USA) and goat anti-rat ALEXA 568 (Molecular Probes, Eugene, OR, USA). Recombinant Wnt3a was purchased from R&D Systems. Y27632 was purchased from Sigma-Aldrich (St. Louis, MO, USA). The cDNA encoding Myc/His-tagged human sFRP1 in pCDNA was provided by Jeffrey Rubin (NCI, Bethesda, MD, USA) and was recloned into the pBabePuro retroviral vector. Conditioned media (CM) from Wnt1-producing cells, from sFRP1-producing cells, and purified sFRP1 were prepared as previously described [
7].
T-cell factor reporter assay
MDA-MB-231/sFRP1-P1 cells and control-P1 cells were seeded on 12-well plates and were transfected with a mixture of Super TOPFlash plasmid and pRL-CMV (Promega, Madison, WI, USA) to assay TCF promoter activity using Fugene6 (Roche) according to the manufacturer's instructions. Luciferase activities were measured 48 hours later using the Dual-Luciferase Reporter Assay System (Promega) and Mithras LB940 (Berthold Technologies, Bad Wildbad, Germany) according to the manufacturer's instructions. The fold activation was normalized against renilla luciferase. Nine wells were used per condition and the average and standard error are shown in the graph.
Cell culture, transfections, retroviral infections, proliferation and anoikis assays
The human breast cancer cell line MDA-MB-231 (ATCC, Manassas, VA, USA) was cultivated in DMEM, 10% heat-inactivated FCS (Amimed, Allschwil, Switzerland) supplemented with penicillin and streptomycin (Sigma-Aldrich). All transfections were performed using FuGENE 6 Transfection Reagent (Roche) following the manufacturer's guidelines. MDA-MB-231 cells were stably transfected with pCDNA3.1(+) (Invitrogen, Carlsbad, CA, USA) encoding Myc/His-tagged human sFRP1 or empty pCDNA3.1(+) as control. After selection with 1 mg/ml G-418, three clones of MDA-MB-231/sFRP1 and three control clones were isolated. Equal cell numbers of these clones were pooled before some experiments (MDA-MB-231/sFRP1-P1 and MDA-MB-231/control-P1). A second pool of sFRP1-expressing MDA-MB-231 cells (MDA-MB-231/sFRP1-P2) and control cells (MDA-MB-231/control-P2), each representing > 100 clones, was generated by infecting the cells with pBabePuro encoding Myc/His-tagged human sFRP1 or empty pBabePuro followed by selection with 2 μg/ml Puromycin (Sigma-Aldrich).
Cell proliferation was measured either by counting cell numbers with a Vi-Cell XR cell viability analyzer (Beckman Coulter, Fullerton, CA, USA) on selected days after seeding 200,000 cells on six-well plates or using the YOPRO cell viability assay (Invitrogen) 3 days after seeding 1,000 cells on a 96-well plate, according to the manufacturer's instructions. Anoikis was measured by seeding cells in 1% FCS-containing medium on polyHema-coated plates to prevent adhesion. Cells were harvested 24 hours later, stained with propidium iodide. The cell cycle distribution was analyzed with a FACScalibur (Becton Dickinson, San Jose, CA, USA) using the Cellquest software. A representative cell cycle distribution of three MDA-MB-231/sFRP1 clones and three control clones is shown. Unless otherwise noted, P values were calculated using Student's t test.
Protein extraction and western blotting
Cells were lysed in 1% Nonidet P-40, 50 mM Tris pH 7.5, 120 mM NaCl, 5 mM ethylenediamine tetraacetic acid, 1 mM ethylene glycol tetra-acetic acid (EGTA), 2 mM sodium vanadate, 20 mM β-glycerophosphate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 0.5 mM phenylmethanesulphonylfluoride (PMSF), 50 mM NaF, 1 mM dithiothreitol for 5 minutes on ice before collecting lysates. Debris was removed by centrifugation at 4°C and the protein concentration was determined using the Bradford reagent (BioRad, Hercules, CA, USA).
For western blotting, protein loading buffer was added to 30 to 50 μg total protein and the samples were denatured for 10 minutes at 95°C prior to separation on SDS-polyacrylamide gels and blotting by semi-dry transfer for 90 minutes on PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked using 10% horse serum in Tris-buffered saline- Tween buffer for 1 hour (0.2 M NaCl, 25 mM Tris, pH 7.5, 0.5 ml/l Tween-20), except for p21Cip detection where PBS- Tween buffer was used instead of TBS-Tween buffer for blocking. Blots were incubated with primary antibodies at room temperature for 1 hour or at 4°C overnight, followed by 30-minute incubation with secondary antibodies. These antibodies were anti-rabbit-HRP, anti-mouse-HRP (1:5000) or anti-goat-HRP (1:5000) for detection of luminescence, which was carried out using ECL (GE Healthcare) according to the manufacturer's instructions and using X-OMAT LS films (Kodak, New York, NY, USA). Secondary antibodies IRDye 800CW goat anti-rabbit-IgG or anti-mouse-IgG (1:10,000; LI-COR Biosciences) were detected with the LI-COR Odyssey system according to the manufacturer's instructions (LI-COR Biosciences). Quantification of protein expression was carried out using Odyssey 2.1 (LI-COR Biosciences).
Wound healing assay
Cells were seeded on six-well plates and grown to confluency. Monolayers were scratched, and in the indicated experiments the media were changed to Wnt1 CM or control CM. When using recombinant Wnt3a and Y27632, 90 minutes before the scratch was made the media were changed to DMEM 10% FBS containing 100 μg/ml Wnt3a (R&D Systems) and/or 5 μM Y27632 (Sigma-Aldrich). Pictures of randomly-chosen nine wound edges per condition were taken at time 0 and at the indicated time points using Nikon DIAPHOT (Nikon, Tokyo, Japan). The recovered area was calculated using ImageQuant TL (GE Healthcare). In some experiments, purified sFRP1 was added to the CM [
7].
Fluorescence-activated cell sorting analysis for active β1-integrin
MDA-MB-231/sFRP1-P1 and control-P1 cells (0.3 × 106) were incubated for 30 minutes on ice with an antibody recognizing active β1-integrin (final concentration 20 ng/μl) in HEPES/NaCl buffer. This was followed by incubation for 30 minutes on ice with fluorescein isothiocyanate-conjugated (FITC) donkey anti-mouse IgG secondary antibody (Jackson Laboratories) (diluted 1:250) in Flow PBS (1 × PBS, 2% horse serum, 0.1% sodium azide). Fluorescence was measured using a FACSCalibur machine (Becton Dickinson) and the percentage of gated cells stained with active β1-integrin was calculated.
In vivoexperiments with MDA-MB-231 cells
Female Balb/c nude mice 7 to 10 weeks old were obtained from Charles River Laboratories (L'Arbresle, France) and were maintained in accordance with the Swiss guidelines for animal safety. Mammary tumors were established in mice (5 to 8 mice per group) by injecting 0.5 to 1.0 × 10
6 control or sFRP1-expressing MDA-MB-231 cell lines in 100 to 150 μl PBS into the fourth right-side mammary fat pad. The tumor size was measured two or three times per week using a gage, and the volume was calculated considering the tumor as an oval according to the formula:
Statistical analyses were performed with two-way repeated-measures analysis of variance (repeated measures ANOVA (RM ANOVA)).
To assay tumor cell proliferation, 100 μg/g body weight of BrdU (Cell Proliferation Kit II; Roche) was intraperitoneally injected into tumor-bearing mice that were sacrificed 2 hours later. Tumors were excised and washed with PBS before fixation in 4% paraformaldehyde (PFA) at 4°C for 24 hours, and BrdU detection was performed as previously described [
13]. To examine experimental metastasis, 1.0 × 10
6 MDA-MB-231/sFRP1-P2 cells or control-P2 cells were injected into the tail vein of female Balb/c nude mice (5 to 6 mice per group). Fifty-three days after the injection, the mice were sacrificed, lungs were dissected and the total number of surface lung metastases was determined. For western analyses, excised tumors were snap frozen and pulverized in liquid nitrogen and lysed in SDS buffer (100 mM Tris- HCl pH 7.6, 2% SDS, 10 mM dithiothreitol, 2 mM sodium vanadate, 0.5 mM ethylenediamine tetraacetic acid) by incubation at 95°C for 10 minutes.
Immunohistochemistry and functional vessel analysis on tumor sections
To detect functional vessels in tumors, 100 μl of 2 μg/μl solution of fluorescein-labeled
Lycopersicon esculentum lectin (Vector Labs, Burlingame, CA, USA) was injected into tail veins of tumor-bearing mice [
14], and the mice were sacrificed 5 minutes later. Tumors were excised, fixed in 4% paraformaldehyde in PBS for 48 hours at 4°C, followed by an overnight incubation in 30% sucrose in PBS at 4°C, and then embedded in tissue-Tec O.C.T. Compound 4583 (Sakura, Tokyo, Japan). Frozen sections (9 μm) were subjected to IHC analysis to detect tumor-associated vessels using rat anti-mouse CD31 (diluted 1:100; BD Pharmingen) and goat anti-rat ALEXA 568 (diluted 1:200; Molecular Probes). Staining was performed using Discovery XT (Ventana Medical Systems, Inc., Tucson, AZ, USA). Pictures were taken with a Z1 microscope (Carl Zeiss, Jena, Germany) and were analyzed with IMARIS (Bitplane, Zurich, Switzerland) to calculate the co-localized area. For detection of cyclin D1, frozen tumor sections (9 μm) were subjected to IHC using SP4 (diluted 1:100) and Biotin-SP-conjugated affinipure donkey anti-rabbit IgG (diluted 1:100). Staining was carried out using Discovery XT with sCC1 pretreatment. Pictures were taken with an Eclipse E600 (Nikon) and were analyzed with IMARIS (Bitplane) to calculate the signal intensity.
RNA isolation, real-time PCR and microarray analyses
Cultured cells were collected when plates were 70 to 80% confluent and total RNA was extracted using the RNeasy Mini kit (Qiagen, Venlo, The Netherlands). To extract total RNA from tumors, dissected tumors were put into RNAlater (Qiagen) overnight at 4°C, followed by RNA extraction using TRIzol reagent (Invitrogen) and washing using the RNAeasy Mini kit according to the manufacturer's instructions. RNA from mammary tumors (six MDA-MB-231/sFRP1-P1 tumors and five control tumors) and cultured cells (three MDA-MB-231/sFRP1 clones and three control clones) were individually amplified and labeled using the Ambion MesageAMP III RNA Amplification Kit (Applied Biosystems, Austin, TX, USA). Biotinylated, fragmented cRNA was hybridized to Affymetrix U133 plus 2.0 human GeneChips™ (Affymetrix, Santa Clara, CA, USA).
Expression values were estimated using the GC-RMA implementation found in Genedata's Refiner 4.5 software (Genedata AG, Basel, Switzerland). Quantile normalization and median scaling were performed in order to standardize array signal distributions to facilitate the comparison between in vitro cultured cells and in vivo tumor samples. Probesets showing statistically different expression profiles (one-way analysis of variance with P < 0.01; Benjamini and Hochberg Q values determined to minimize the false discovery rate) and specific pairwise fold changes were clustered by rank correlation with R > 0.8 for the first criterion and R > 0.885 for the second criterion using the Profile Distance Search function of Genedata's Analyst 4.5 tool (Genedata AG, Basel, Switzerland). All of the microarray data are stored in Gene Expression Omnibus [GEO:GSE13806].
For the quantitative real-time PCR, each sample cDNA was made from 2.5 μg RNA using Ready-To-Go™ You-Prime First-Strand Beads (GE Healthcare). Quantitative real-time PCR was performed with ABI Prism 7000 (Applied Biosystems, Austin, TX, USA) using ABsolute SYBR Green ROX Mix (THERMO Scientific, Waltham, MA, USA) following the manufacturer's guidelines. The primer sequences used for quantitative real-time PCR are as follows: human c-Myc forward, 5'-CCTACCCTCTCAACGACAG-3'; human c-Myc reverse, 5'-CTTGTTCCTCCTCAGAGTCG-3'; human β-actin forward, 5'-TGTCCACCTTCCAGCAGATGT-3'; and human β-actin reverse, 5'-CGCAACTAAGTCATAGTCCGCC-3'.
Discussion
Aberrant activation of WNT signaling plays an important role in many types of human cancer, warranting therapeutic approaches to target the pathway [
26]. Wnt1 was the first identified oncogene activated by mouse mammary tumor virus insertional mutagenesis [
27], establishing the potential of aberrant WNT expression to promote mammary cancer. Currently, it is well documented that multiple WNT ligands and FZD receptors are expressed in primary human breast tumors and breast cancer cell lines [
3,
7‐
9], making it difficult to identify an individual ligand/receptor complex that could serve as a cancer target. Using broad antagonists – including the cysteine-rich domain of the FZD8 receptor [
28] or sFRP1 [
6,
7,
29] – to interfere with WNT/FZD binding, however, the potential of targeting WNT binding to FZD as a therapeutic approach in breast cancer [
6,
7] and in other cancers [
28,
30] has been demonstrated.
Aberrant methylation of the sFRP1 promoter is one of the most consistent alterations in human cancer. In addition to breast tumors that have low sFRP1 levels [
10‐
12,
29,
31], sFRP1 suppression has been described in colon tumors [
32], ovarian tumors [
33], bladder tumors [
34] and prostate tumors [
35]. Based on its widespread loss, interest in testing the effects of sFRP1 treatment in tumor models has been high. Indeed, sFRP1 has also been shown to impact on transforming properties of breast cancer cells [
6,
29] and cervix cancer cells [
36]; while sFRP2 has been shown to block proliferation of gastric cancer cells [
37]. We have previously shown that proliferation of the estrogen-receptor-positive MCF7 and T47D, and the ErbB2-overexpressing JIMT-1, SKBR3 and BT474 breast tumor cell lines is decreased following treatment with sFRP1 [
7]. In the current study we tested the impact of ectopic sFRP1 expression in the aggressive, basal-like [
15] MDA-MB-231 breast tumor cells. The results presented show that ectopic sFRP1 expression in MDA-MB-231 tumor cells blocks the migratory ability and the proliferative potential of the tumor cells, both
in vitro and
in vivo, supporting the proposal that blockade of WNT signaling with sFRP1 might be a general approach to target not only breast, but also other types of cancer.
In addition to testing sFRP1, we also examined the effects of specific Wnt ligands on motility and found that the canonical ligands Wnt1 and Wnt3a stimulate MDA-MB-231 cells in a wound closure assay. These results correlate well with the stimulatory effects of both Wnt ligands in other cellular models [
38,
39]. The Wnt/PCP pathway is considered the major mediator of cell motility. Indeed, this pathway stimulates many cytoskeleton regulators, including Rho family GTPases and Rho kinase [
40]. Both Wnt1 and Wnt3a have been shown to activate RhoA [
38,
39], whereas the non-canonical Wnt5a promotes melanoma migration via RhoB [
41]. Furthermore, Rho kinase inhibition has been shown to block the effects of Wnt3a [
39]. We have also observed that the Rho kinase inhibitor Y27632 blocks Wnt3a-induced MDA-MB-231 wound closure (data not shown). In contrast to the positive effects of Wnt ligands on motility, we show here that sFRP1-mediated blockade of endogenous WNT signaling not only reduced the basal motility of the MDA-MB-231 cells, but also impaired the ability of the cells to respond to Wnt1 in a wound closure assay. sFRP1 has also been shown to block motility [
39] and invasion [
36] of other types of tumor cells. Importantly, the negative impact of sFRP1 on MDA-MB231 motility translated,
in vivo, to a block in the metastatic potential of these aggressive breast tumor cells. In comparison with control MDA-MB-231 cells, we observed a 20-fold decrease in the number of lung metastasis arising from sFRP1-expressing MDA-MB-231 cells.
MDA-MB-231/sFRP1 cells also proliferated more slowly than control cells; however, the effect of sFRP1 was more striking
in vivo than
in vitro. Following injection of MDA-MB-231/sFRP1 cells into mammary glands of nude mice, the time to appearance of the tumors was consistently longer than that observed with control MDA-MB-231 cells. Furthermore, tumors generated by the sFRP1-expressing cells not only grew more slowly than control tumors, but there were threefold more tumor-free mice at the end of each experiment in this group. Since sFRP1 is a secreted protein, it could act extrinsically on cells in the tumor environment. We concentrated in particular on tumor-associated vessels based on the reported ability of sFPR1 to block
in vivo neovascularization [
20]. Neither the vessel number nor their functionality differed, however, in tumors generated by sFRP1-expressing cells in comparison with those of control MDA-MB-231 cells. The impact of sFRP1 on WNT signaling and downstream transcription in the MDA-MB-231 cells might therefore more probably explain the protein's strong
in vivo effects. Indeed, there were 3.7-fold more genes whose transcription was altered by sFRP1 expression
in vivo compared with
in vitro (1,246 vs. 332). Furthermore, only 54 genes overlapped in the two lists. Taken together, these results demonstrate the strong impact of tumor environment on gene expression.
We also performed an in-depth analysis to identify genes that were only affected
in vivo, in the sFRP1-expressing tumors, with the intention of finding potential targets that could account for the strong effect of sFRP1 on MDA-MB-231 tumor-forming potential. There were a total of 168 genes (106 downregulated genes and 62 upregulated genes) that were only affected
in vivo in sFRP1-expressing tumors (see Additional data files
5 and
6). Changes in expression of two
in vivo identified genes,
CCND1 and
CDKN1A, encoding important cell cycle regulators were confirmed by IHC and immunoblotting on tumor cells. Cyclin D1 and p21
Cip1 were found to be downregulated and upregulated, respectively, only in sFRP1-expressing tumors, which might be one reason why the impact of sFRP1 expression is stronger
in vivo. These results raise the question as to why
CCND1 and
CDKN1A were only affected
in vivo. While we can only speculate at this time, two explanations are worth discussion. First, c-Myc, which controls expression of both genes, was only downregulated in the sFRP1-expressing tumors; second, in the tumors there were major changes in expression of probesets for extracellular matrix proteins.
MYC is WNT pathway target [
23,
24]. We did not detect changes in
MYC expression, however, either in the microarrays (see Additional data files
5 and
6) or by quantitative real-time PCR (see Additional data file
7). Interestingly, c-Myc protein was low in all of the sFRP1-expressing tumors (see Additional data file
7). C-Myc is subject to control at many levels [
42,
43] and the fact that c-Myc protein only changed in the sFRP-1-expressing tumors could be a reflection of the
in vivo environment. Irrespective of the mechanism underlying these results, the fact that c-Myc stimulates cyclin D1 expression [
22] and is a repressor for p21
Cip1 [
25] suggests that lower c-Myc levels could contribute to the altered expression of both cell cycle regulators in sFRP1-expressing tumors.
Turning to the extracellular matrix components, probesets for fibronectin, laminins and collagens were found to be strongly altered (see Additional data file
8, 29 probesets for the extracellular matrix). As expected,
FN1 – a WNT pathway target (see Additional data files
3 and
4) – was decreased in sFRP1-expressing cells and tumors. The most striking difference, however, was seen when comparing the signal of the 29 probesets in cultured cells versus tumors. On the one hand, 25 out of the 29 probesets (see Additional data file
8) were strongly increased
in vivo in control tumors, showing the impact of the tumor environment on expression of the encoded genes. Moreover, 21 out of these 25 were downregulated in sFRP1-expressing tumors.
Fibronectin, laminin and collagen are ligands for β
1-integrin, which was also suppressed in sFRP1-expressing cells (see Additional data file
8). The decrease in β
1-integrin RNA was confirmed at the protein level by performing a fluorescence-activated cell sorting analysis on intact cells with an antibody that recognizes active β
1-integrin (see Additional data file
9). Integrin engagement is therefore likely to be decreased in sFRP1-expressing MDA-MB-231 cells, which in turn is likely to influence their proliferation. On the one hand fibronectin has been shown to stimulate proliferation of cancer cells, and this was associated with increased cyclin D1 and decreased p21
Cip1 levels [
44]. Moreover, we have previously shown that siRNA-mediated knockdown of β
1-integrin in MDA-MB-231 cells increases p21
Cip1 expression and leads to a proliferative decrease [
13]. We propose that integrin engagement would be more strongly affected
in vivo since not only the receptor but also many of its extracellular matrix binding partners are decreased
in vivo.
In summary, the results presented here show that sFRP1-mediated blockade of WNT signaling in MDA-MB-231 breast cancer cells has an impact on the in vitro proliferation and motility of the cancer cells. The in vivo effects of WNT pathway blockade were even more dramatic since we observed a strong decrease in the mammary tumor-forming potential and an impairment of lung metastases. In summary, blockade of the WNT-FZ interaction using sFRP1 has a strong effect on breast tumor growth.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YM planned and performed the experiments and participated in writing the paper. TS planned and discussed the experiments and performed the experiment shown in the supplemental figure in Additional data file
1. EJO performed the array and data analyses. AB performed the experiment shown in Figure
5c. NEH planned and discussed the experiments and wrote the paper.