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Cell, Tumor, and Stem Cell Biology |
1 Department of Pathology, Albert Einstein College of Medicine, Bronx, New York; 2 Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada; 3 Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania; and 4 Lankenau Institute for Medical Research, Wynnewood, Pennsylvania
Requests for reprints: Rachel B. Hazan, Department of Pathology, Albert Einstein College of Medicine, F529S, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: 718-430-3349; E-mail: rhazan{at}aecom.yu.edu.
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Abstract |
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Introduction |
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N-cadherin proinvasive activity results partly from a synergistic interaction with the fibroblast growth factor (FGF) receptor (FGFR) via their extracellular domains, which stabilizes FGFR-1 and sustains extracellular signal-regulated kinase (ERK) 1/2 activation and matrix metalloproteinase (MMP)-9 production (10, 11). The potentiating effects of N-cadherin on FGFR-1 seem to be unrelated to its adhesive capacity, given that an adhesive-deficient N-cadherin can also stabilize the FGFR-1 (11). It has been suggested that N-cadherin also promotes tumor-host interactions with N-cadherin–expressing tissues, such as the stroma (12, 13) and the vascular endothelium (14), which may support metastatic spread and growth.
Despite the well-documented effects of N-cadherin on tumor cell invasion and its elevated expression in aggressive carcinomas, it remained unknown whether N-cadherin is a cause or consequence of malignant progression in vivo. To determine the effects of N-cadherin on mammary tumorigenesis, we produced bitransgenic mice by mating mouse lines that express N-cadherin and the polyomavirus middle T antigen (PyVmT) in the mammary epithelium, both under the control of the mouse mammary tumor virus (MMTV) long terminal repeat promoter. PyVmT is a tumor promoter that stimulates signaling pathways commonly amplified in human breast cancer, such as the phosphatidylinositol 3-kinase (PI3K)/Akt and RAS-RAF-mitogen-activated protein kinase kinase (MEK)-ERK pathways (15), and induces biomarkers of poor prognosis in breast cancer, such as HER2/neu and cyclin D1 overexpression, as well as loss of estrogen and progesterone receptors (16, 17). Moreover, PyVmT induces a stepwise progression into malignancy similar to that of human breast cancer, featuring hyperplasia (normal/benign), in situ carcinoma (ductal carcinoma in situ), early to late carcinoma with stromal invasion (locally invasive carcinomas), and late carcinoma with distant metastasis (advanced carcinomas with metastasis; refs. 16, 18, 19). Thus, the MMTV-PyVmT mouse is an excellent model to test the effect of tumor-enhancing or tumor-suppressive molecules in breast cancer.
Here, we find that coexpression of PyVmT and N-cadherin in the mammary epithelium produces tumors with greater metastatic potential compared with those induced by PyVmT alone. N-cadherin alone did not cause or enhance tumor formation but did potentiate signaling, resulting in an increase in ERK1/2 phosphorylation in tumors, which was prominent in distant metastases. Consistent with a more metastatic behavior of N-cadherin–positive tumors in vivo, mammary epithelial cell lines derived from PyVmT-N-cadherin tumors displayed increased ERK activation, migration, invasion, and MMP-9 expression compared with cells from PyVmT tumors. MMP-9 production and invasiveness of PyVmT-N-cadherin cells in vitro were suppressed by MEK1 inhibition. Furthermore, inactivation of the FGFR led to reduced motility, invasion, and ERK phosphorylation. These results show an unequivocal effect of N-cadherin on mammary tumor progression leading to metastatic function due to ERK oncogenic signaling involving MMP-9 up-modulation.
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Materials and Methods |
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MMTV-PyVmT mice (strain 634) have been described previously (18). Mice expressing mouse N-cadherin in the mammary epithelium were also described previously (20). Mice used for these studies were in the FVB/N background. Matings were done with MMTV-PyVmT male hemizygous mice. MMTV-PyVmT hemizygote/MMTV-Ncad (+/–) or MMTV-PyVmT hemizygote male mice were interbred with female MMTV-Ncad (+/+), (+/–), or (–/–) mice. Mice used for the tumor study were both female and nonparous, as MMTV-PyVmT mice develop palpable tumors by week 6 to 7 of age. PyVmT-N-cadherin mice were identified by tail PCR using the following primer set: 5'-TGGAGAGACTTCTGAAACAGC-3' and 5'-CCATTCATCAGTTCCATAGGTTG-3'. Transgenic animals were identified by the presence of a distinct 446-bp product.
Tumor Onset and Growth Determination
Tumor onset. Mammary gland palpations were begun at 5 weeks of age and done biweekly until the development of mammary gland tumors was detected. In all, 112 animals were analyzed for onset (39 PyVmT and 73 PyVmT-N-cadherin). Data were displayed as mean onset time ±SE. Statistical analyses were done using the Mann-Whitney test, and significant differences were established as P < 0.05.
Tumor growth. Mammary tumors were permitted to grow for varying time points after tumor onset (2, 3.5, 5, 7, or 9 weeks). Mice were then sacrificed and mammary tumors were excised and weighed. Sixty animals were analyzed (26 PyVmT mice: 0 week = 3 mice, 2 weeks = 3 mice, 3.5 weeks = 4 mice, 5 weeks = 2 mice, 6.7 weeks = 10 mice, and 9 weeks = 4 mice; 40 PyVmT-N-cadherin mice: 0 week = 3 mice, 2 weeks = 8 mice, 3.5 weeks = 5 mice, 5 weeks = 6 mice, 6.7 weeks = 10 mice, and 9 weeks = 8 mice). Data are shown as mean tumor mass ± SE. Statistical analyses were done comparing individual time points by the Mann-Whitney test, and significance was established at P < 0.05.
Lung Metastasis Determination
Female PyVmT and PyVmT-N-cadherin mice were sacrificed at various time points after tumor onset. Heart and lungs were exposed by thoracic dissection, and lungs were inflated by tracheal cannulation with slow injection of 1 to 2 mL of 10% neutral-buffered formalin using a 21-gauge syringe. Heart and lungs together were excised and fixed in 10% formalin for 48 h. Formalin-fixed lungs were paraffin embedded and blocks were sectioned at 5 µm. Lungs were serial sectioned through the tissue, and sets of five serial sections, at 50-µm intervals, were captured and mounted on slides. In this manner, 20 to 25 sets of slides were generated per lung. Analysis was done on 3 to 10 random sections after it was determined by inspection that metastases seeded in random locations. After determining metastatic diameters (136 µm, PyVmT; 148 µm, PyVmT-N-cadherin), sections used in the analysis were required to reside more than 150 µm apart in the lung to minimize duplicate analysis. The number of metastases per lung section was normalized to the area (in pixels) of the lung section. Lungs were analyzed in this manner at 0, 2, 3.5, 5, and 7 weeks. Forty-nine lungs were analyzed over these time points (20 PyVmT and 29 PyVmT-N-cad; 20 PyVmT mice: 0 week = 2 mice, 2 weeks = 2 mice, 3.5 weeks = 4 mice, 5 weeks = 4 mice, and 6.7 weeks = 8 mice; 29 PyVmT-N-cadherin mice: 0 week = 2 mice, 2 weeks = 4 mice, 3.5 weeks = 7 mice, 5 weeks = 7 mice, and 6.7 weeks = 9 mice). Data were displayed as mean number of metastases ± SE. Statistical analyses were done comparing individual time points using the Mann-Whitney test, and significant differences were established as P < 0.05.
Antibodies
Mouse monoclonal anti-N-cadherin antibodies (Zymed, San Francisco, CA or BD Biosciences, San Jose, CA) were used for immunofluorescence and Western blots. A rabbit polyclonal anti-E-cadherin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal anti-E-cadherin (BD Biosciences) were used for immunofluorescence and Western blots. Guinea pig N-cadherin antibody (GP1260) was obtained from Drs. David Colman and Weisong Shan (Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada). GP1260 was generated against a glutathione S-transferase-N-cadherin EC1 fusion protein and affinity purified on N-cadherin (EC1) BrCN-activated Sepharose CL-4B, and its specificity and adhesion-blocking properties were fully characterized (21). As control for GP1260, purified guinea pig IgGs (The Jackson Laboratory, Bar Harbor, ME) were used. For Western blotting, we used antibodies to phosphorylated ERK1/2 (Cell Signaling, Danvers, MA), ERK1/2 (Santa Cruz Biotechnology), phosphorylated p38 (Cell Signaling), p38 (Biosource, Camarillo, CA), phosphorylated Akt (Biosource), Akt (Cell Signaling), phosphorylated c-Src (Cell Signaling), c-Src (Santa Cruz Biotechnology), and MMP-9 (Triple Point Biologics, Forest Grove, OR). Anti–phosphorylated ERK1/2 antibody used in immunohistochemistry was purchased from Sigma (St. Louis, MO). A mouse monoclonal anti-PyVmT antibody was a gift from Dr. Steve Dillworth (Imperial College, London, United Kingdom). Secondary FITC- or TRITC-labeled antibodies (Molecular Probes, Invitrogen, Carlsbad, CA) were used in immunofluorescence, and horseradish peroxidase (HRP)-conjugated antibodies (Amersham, Pittsburgh, PA) were used in Western blotting. Enhanced chemiluminescence detection reagents were purchased from Pierce (Rockford, IL).
Immunoblotting
Cells or tissues were extracted in solubilization buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.5 mmol/L MgCl2, 0.2 mmol/L EGTA, 1% Triton X-100], including protease inhibitors. Protein (30 µg) was loaded on 7.5% SDS-polyacrylamide gels and transferred to Immobilon membranes. Blots were probed with indicated antibodies and developed by chemiluminescence. Immunoblot signals were quantitated using densitometry and ImageJ software (version 1.34s; by Wayne Rasband, NIH, Bethesda, MD), and data were analyzed by Excel (Microsoft, Redmond, WA).
Immunohistochemistry and Immunofluorescence
Tissue staining. Formalin-fixed/paraffin-embedded tissues were sectioned at 5-µm thickness, deparaffinized in xylene, and rehydrated in a series of decreasing ethanol/H20 solutions. Antigens were retrieved by steam heat for 20 min in a 0.01 mol/L trisodium citrate buffer (pH 6.0) followed by 1 h blocking in 5% serum/2% albumin in PBS. Tissues were incubated with primary antibodies diluted in blocking buffer for 1 h at room temperature or overnight at 4°C. Secondary antibodies were incubated for 1 h at room temperature. Antibody binding was revealed by secondary antibodies coupled to either FITC, TRITC, or HRP. Sections using fluorescent secondary detection were counterstained with 4',6-diamidino-2-phenylindole (DAPI) to visualize nuclei. Sections using HRP secondary detection were developed in 3,3'-diaminobenzidine (DAB) chromogen according to DAKO (Carpinteria, CA), and tissues were counterstained with Harris hematoxylin.
Cell staining. Cells were plated on collagen-coated coverslips, fixed in 3.7% paraformaldehyde for 15 min, permeabilized for 2 min in 0.1% Triton X-100, blocked in 2% BSA/PBS, incubated with 2 µg/mL of primary antibody for 1 h at room temperature, washed and incubated for 1 h with 1:5,000 FITC- or TRITC-conjugated secondary antibodies, and counterstained with DAPI. Images were captured using a Zeiss Axioskop 2 microscope (Oberkochen, Germany).
Phosphorylated ERK1/2 Analysis in Tissue Sections
Formalin-fixed, paraffin-embedded mammary tumors (seven tumor sections each, PyVmT and PyVmT-N-cadherin, 7 weeks after tumor onset) and lungs (four PyVmT and four PyVmT-N-cadherin, 7 weeks after tumor onset) were stained with a phosphorylated-specific ERK1/2 antibody and visualized by HRP/DAB reaction. Images were captured under similar lighting and exposure times. From stained mammary tumor sections, images of randomly selected, equally spaced fields were captured (162 fields in total, >90 x 103 cells). For each tumor section, 7,000 cells were scored. Mammary tumor cells were scored as either positive or negative and graphically displayed as percentage positive. From lung, 80 metastases (40 each PyVmT and PyVmT-N-cadherin) were picked at random and analyzed. DAB signal was scored by pixel intensity and divided into five levels of increasing staining intensity (darker DAB signal). Data bars are the mean of 40 metastatic lesions and displayed as the percentage of each DAB level present in the lesions. Analysis and scoring was done using ImageJ software and a color deconvolution plug-in. Data were graphed and statistically analyzed in Excel.
Mammary Tumor Cultures
Primary mammary tumor cultures were prepared as follows. Each gram of tumor material was incubated with 10 mL of digestion medium (2 mg/mL collagenase, 2 mg/mL of BSA in Medium 199). The suspension was incubated at 37°C for 3 h with shaking, digested material was spun at 1,000 rpm for 5 min, and pelleted cells were plated at high density (1 x 107 per 10-cm dish) in DMEM/10% fetal bovine serum (FBS) supplemented with 10 µg/mL insulin and epidermal growth factor (EGF). Fibroblasts were depleted from epithelial tumor cells by magnetic separation using CD90 (Thy 1.2) MicroBeads (magnetic-activated cell sorting) according to the supplier's protocol. CD90-negative cells (epithelial cells) were collected and plated in DMEM/10% FBS supplemented with 10 µg/mL insulin and EGF. Epithelial cells were cultured for several weeks until they reached crisis and adapted to in vitro culture conditions (22, 23). The resulting cell lines were used in the analysis.
Signaling Pathway Inhibitors
Cells were plated at 3 x 105/mL in growth medium/10% FBS in 35-mm Petri dishes, allowed to adhere for 18 h, and then treated for 16 h in the same medium with either DMSO or various concentrations of U0126 (Promega, Madison, WI), SB 203508 (Calbiochem, San Diego, CA), or PD173074 (Pfizer, New York, NY) diluted in DMSO. Single-cell suspensions were tested in migration or invasion assays (11). Cell lysates were controlled for pathway inhibition using anti–phosphorylated ERK or phosphorylated p38 immunoblotting.
In vitro Cell Migration and Invasion Assays
Tumor cell migration and invasion assays were done in a Boyden chamber using 24-well culture plates fitted with 8-µm Transwell filters as described (11, 24). Filters were coated either with (invasion) or without (migration) 5 µg Matrigel. Fibroblast conditioned medium was used as a chemoattractant in the lower compartment of the Boyden chamber. Trypsin-made cell suspensions (1 x 105 cells/0.5 mL DMEM, 0.1% BSA) were plated onto filters for 18 h. Cells on top of the filters were removed, and cells penetrating the filters were stained with 0.5% crystal violet and imaged by bright-field microscopy. Invaded or migrated cells were expressed as the average number of migrated cells bound per microscopic field over four fields per assay in triplicate experiments. Statistical significance was determined by one-tailed t test.
Measurement of MMP-9 Secretion
Cells were plated at 3 x 105/mL in growth medium/10% FBS in 35-mm Petri dishes for 18 h and then treated for 16 h in the same medium containing DMSO, U0126, or SB 203508 diluted in DMSO. Conditioned medium was obtained by removal of growth medium and addition of 0.75 mL serum-free DMEM per dish. After 24 h, conditioned medium was removed and spun, and 30 µL conditioned medium from each treatment was mixed with Laemmli sample buffer, boiled, applied on SDS-PAGE, and immunoblotted with an anti-MMP-9 antibody. Cell monolayers were lysed and protein was determined to control for similar 
-tubulin loading in each sample.
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Results |
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We mated MMTV-PyVmT mice with MMTV-N-cadherin mice and compared bitransgenic PyVmT-N-cadherin mice with PyVmT littermates. Primary tumors excised at 7 weeks after tumor onset were analyzed by Western blotting. Bitransgenic PyVmT-N-cadherin mouse tumors expressed high levels of N-cadherin (Fig. 1A, lanes 8–14 ), whereas in tumors from PyVmT mice, N-cadherin was undetectable (Fig. 1A, lanes 1–7). Tumors from both mice continued to express E-cadherin and contained similar levels of PyVmT (Fig. 1A). By immunostaining, N-cadherin was absent in primary tumors (Fig. 1B, a), whereas in PyVmT-N-cadherin tumors, N-cadherin expression was abundant in most of the cells (Fig. 1B, d) and was localized at cell-cell interfaces. E-cadherin was detected at cell-cell junctions in tumors from both PyVmT and PyVmT-N-cadherin mice (Fig. 1B, b and e, respectively).
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N-cadherin potentiates oncogenic signaling in vivo. PyVmT is a membrane-anchored intracellular oncoprotein that transduces signals leading to tumor formation (15). PyVmT activates c-Src, which in turn phosphorylates PyVmT on two tyrosine residues, causing subsequent activation of PI3K and ERK signaling (15, 25). We sought to determine whether PyVmT signaling is affected by N-cadherin expression in mammary tumors. Tissue extracts from PyVmT and PyVmT-N-cadherin mammary tumors were subjected to Western blot analysis using antibodies to phosphorylated or total ERK1/2, p38, c-Jun NH2-terminal kinase, Akt, and c-Src (Fig. 3A ). Signals were quantified by densitometry and expressed as fold change in PyVmT-N-cadherin relative to PyVmT tumors (Fig. 3B and C). PyVmT-N-cadherin tumors exhibited increased ERK1, ERK2 and p38 phosphorylation (Fig. 3B, right) when compared with PyVmT tumors, with no significant change in total levels of these molecules (Fig. 3B, left). We tested for potential N-cadherin–stimulated changes in phosphorylation of Akt or c-Src, as the latter is known for activating PyVmT downstream signaling (15, 25). In PyVmT-N-cadherin tumors, levels of phosphorylated (Fig. 3C, right) or total (Fig. 3C, left) Akt or Src were not significantly different from those in PyVmT controls.
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3-fold increase (P = 0.03) in the number of cells (Fig. 4D, right). Interestingly, in both primary tumors and metastases, highest phosphorylated ERK staining was observed in tumor cells at the interface with the surrounding host tissue (Fig. 4A and C), suggesting the possibility that phosphorylated ERK increases in tumor cells in response to the microenvironment.
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Cell lines derived from PyVmT-N-cadherin tumors retain their metastatic properties. To further characterize the signaling effects of N-cadherin overexpression on tumor cell behavior, we derived primary mammary epithelial cell lines from PyVmT and PyVmT-N-cadherin tumors (22, 23). Cell lines were double immunostained for E-cadherin and N-cadherin. PyVmT-N-cadherin cells expressed both E-cadherin and N-cadherin at cell-cell contacts (Fig. 5A, a and b ), whereas many PyVmT cells expressed E-cadherin but not N-cadherin (Fig. 5A, c and d).
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Treatment of PyVmT-N-cadherin cells with the MEK1 inhibitor U0126 at concentrations that suppressed phosphorylated ERK (see Fig. 6D ) reduced invasivity, but not motility, of the cells (Fig. 5B and C). By comparison, U0126 had no effect on the relatively low invasion of PyVmT cells (Fig. 5B) but reduced basal motility of these cells (P = 0.013). In contrast, inhibition of p38 signaling with SB 203580 did not affect invasion (Fig. 5B) or motility (Fig. 5C) of both cell types. These results show that potentiation of the ERK pathway by N-cadherin contributes to the invasive phenotype, likely to reflect its metastasis-promoting effect in vivo.
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We further determined whether the enhanced Matrigel invasion of PyVmT-N-cadherin tumor cells is due to increased production of MMPs, which might also be dependent on ERK activity (11, 26). We evaluated MMP-9 secretion in PyVmT and PyVmT-N-cadherin cells, as this MMP has been reported to play an important role in cancer promotion and in pulmonary metastasis (27, 28). Whereas conditioned medium from PyVmT-N-cadherin cells contained high levels of MMP-9, which is detected as a murine dimeric form of 210 kDa (Fig. 6C, middle, lanes 2 and 3; ref. 29), PyVmT cells produced low levels of MMP-9 (Fig. 6C, middle, lane 1). N-cadherin levels in PyVmT-N-cadherin cells (Fig. 6C, top, lanes 2 and 3) and -tubulin loading controls for the amount of cells that produced the conditioned medium represented in these lanes are shown (Fig. 6C, bottom, lanes 1–3).
Inhibition of ERK phosphorylation with U0126 in PyVmT-N-cadherin cells (Fig. 6D, middle, lanes 2–4) caused a sharp reduction in MMP-9 production (Fig. 6D, top, lanes 2–4). Total ERK1/2 expression in PyVmT-N-cadherin cells was unaffected by U0126 treatment (Fig. 6D, bottom). Thus, ERK activation and MMP expression are up-modulated in PyVmT-N-cadherin cells, and taken together with the enhanced ERK phosphorylation in vivo, we suggest that activation of the ERK-MMP pathway underlies the mechanism behind the enhancement of metastasis in the PyVmT-N-cadherin mouse.
N-cadherin–induced motility and invasiveness depends on the FGFR. Because N-cadherin invasive function was shown to involve a synergistic interaction with the FGFR (11), we determined whether N-cadherin is dependent on the FGFR to promote invasiveness of tumor cells. We tested whether inactivation of FGFR by a FGFR kinase inhibitor PD173074 (30, 31) suppresses tumor cell motility or invasion. Treatment of PyVmT-N-cadherin cells with 0.5 µmol/L PD173074 caused a dramatic reduction in both migration (64%) and invasion (56%) but had no effect on these processes in PyVmT control cells (Fig. 7A ). Consistent with these observations, 0.5 µmol/L PD173074 suppressed ERK phosphorylation in PyVmT-N-cadherin cells compared with DMSO-treated cells (Fig. 7B, top, lanes 1 and 2) and had no effect on total ERK levels (Fig. 7B, bottom, lanes 1 and 2). As control, inhibition of the EGF receptor (EGFR) in PyVmT-N-cadherin cells with 0.5 µmol/L ZD1839 (Iressa) did not reduce constitutive ERK phosphorylation in these cells (Fig. 7B, top, lane 3). These findings suggest that N-cadherin sensitizes tumor cells to FGFR inhibition, thus implicating the N-cadherin FGFR axis in enhancement of ERK signaling, leading to the invasive phenotype.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N-cadherin is normally expressed in neuronal and mesenchymal tissues (32, 33) but is absent from the breast ductal epithelium (5), where E-cadherin is abundantly expressed. N-cadherin expression is associated with a more aggressive behavior of cell lines and tumors. We and others have found that N-cadherin is up-regulated in invasive breast cancer cell lines (12) and in aggressive tumors from breast (5) and prostate (13, 34). In human breast cancer, high levels of N-cadherin expression were observed in most high-grade invasive ductal carcinomas and levels were further increased in metastasizing subtypes, such as the micropapillary breast cancers (5). Consistently, forced expression of N-cadherin in breast tumor cell lines was shown to have dramatic effects on cell migration (7–11), invasion (9, 11), and metastasis of tumor xenografts (9), but it remained to be determined whether N-cadherin affects tumor progression in vivo.
Here, we show that N-cadherin coexpression with the PyVmT antigen in the mammary epithelium results in the production of tumors with high metastatic potential. Our findings suggest that N-cadherin contributes primarily to metastasis by potentiating ERK signaling. PyVmT-N-cadherin mice produced mammary tumors with high levels of phosphorylated ERK relative to PyVmT mice, and metastases from both mouse lines exhibited an increase in phosphorylated ERK staining relative to primary tumors. This relationship implies that phosphorylated ERK signaling may select tumor cells with metastatic potential. In both PyVmT and PyVmT-N-cadherin mice, phosphorylated ERK was primarily increased at the peripheral zone of individual metastases, suggesting a response to the surrounding tissue, and this response was significantly greater in the PyVmT-N-cadherin mice. This suggests that signaling from the microenvironment may stimulate ERK, thus dynamically regulating tumor invasion.
In further support of ERK function in tissue invasiveness, phosphorylated ERK, MMP-9, and invasion were increased in PyVmT-N-cadherin cells relative to PyVmT controls and suppressed by ERK inactivation with U0126. The partial effect of U0126 on invasiveness of PyVmT-N-cadherin cells may stem from its limiting effects on N-cadherin enhancement of motility, which was not blocked by U0126. Because PyVmT cell migration was inhibited by U0126, we speculate that N-cadherin activates an ERK-independent pathway to support cell migration. Consistent with these findings, similar differential effects of ERK inhibition on invasion and motility of N-cadherin–transfected MCF-7 cells were noted (9) and may be discriminate signaling pathways governing these events.
The ERK signaling pathway is known to regulate diverse cellular processes, such as proliferation, survival, differentiation, motility, and invasion (11, 35–37). It has been shown that ERK regulates these processes through modulation of strength or duration of ERK activation (35, 38, 39). Moreover, subtle differences in ERK phosphorylation kinetics can result in different biological outcomes (39). In agreement with these findings, we found that FGF-2 caused sustained ERK activation in N-cadherin–expressing MCF-7 cells, resulting in MMP-9 expression and invasion but not in control MCF-7 in which FGF-2 induced transient ERK phosphorylation (11). These observations show that transient and sustained ERK kinetics have different outcomes, and the latter is selectively associated with an invasive phenotype.
How N-cadherin regulates ERK phosphorylation in vivo remains to be determined. It is possible that increases in phosphorylated ERK result from N-cadherin activation of the FGFR. N-cadherin is known to bind and stabilize the FGFR, thus promoting enhanced ERK signaling and invasive capacity (11). In support of this mechanism, we found that inhibition of FGFR activity in PyVmT-N-cadherin cells caused a dramatic reduction in motility and invasiveness, although it had no effect on control PyVmT cells (see Fig. 7A). Consistent with a selective effect of FGFR on the N-cadherin invasive phenotype, inhibition of FGFR in PyVmT-N-cadherin cells caused a sharp reduction in phosphorylated ERK levels, whereas inhibition of the EGFR had no effect on constitutive ERK phosphorylation in these cells. These results are consistent with our published observations in which N-cadherin caused FGF-2, but not EGF, to provoke sustained ERK activation and invasion in MCF-7 cells (11). In addition, N-cadherin enhancement of phosphorylated ERK in PyVmT tumors was not accompanied by increases in phosphorylation of c-Src, which is a key transducer of PyVmT downstream signaling (15, 25), thus raising the possibility that N-cadherin might increase ERK phosphorylation through the FGFR.
In contrast to N-cadherin potentiation of ERK signaling, N-cadherin had no significant effect on p38 signaling. Although phosphorylated p38 levels were elevated in PyVmT-N-cadherin tumor extracts, low levels of phosphorylated p38 staining were observed in tumor sections and no differences in phosphorylated p38 levels were observed in cultured cells from PyVmT or PyVmT-N-cadherin tumors.
The mechanism whereby N-cadherin enhances invasion and metastasis remains complex. We hypothesize that N-cadherin has a dual function in metastasis relating to its adhesive and nonadhesive specificity. Our data suggest that N-cadherin adhesion may not be required for invasive activity as an N-cadherin adhesive mutant was able to stabilize the FGFR in L-cells (11). Consistent with this hypothesis, treatment of PyVmT-N-cadherin cells with an N-cadherin adhesion-blocking antibody inhibited partially the motility of the cells, whereas FGFR inactivation led to a dramatic reduction in motility and invasion, implying the FGFR is a pivotal mediator of the N-cadherin invasive phenotype. It remains, however, plausible that N-cadherin contributes to metastasis via its adhesive function, particularly through adhesion of tumor cells to N-cadherin–expressing host cells from the stroma or the vascular endothelium, thus allowing for survival and dissemination.
Finally, our data suggest that invasiveness of N-cadherin–expressing tumors might result from dynamic regulation of E-cadherin expression. Although E-cadherin levels were slightly reduced in PyVmT-N-cadherin tumors, E-cadherin and N-cadherin were coexpressed at sites of adhesion in PyVmT-N-cadherin tumors and cell lines, suggesting that N-cadherin overrides E-cadherin suppressive function. However, we found that tumor areas with high phosphorylated ERK staining were associated with loss of E-cadherin expression, suggesting that localized phosphorylated ERK increases attenuate E-cadherin to enhance the invasive potential, possibly via up-modulation of MMPs, which may cleave E-cadherin from the cell surface (40).
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Drs. Greg Phillips, Joseph Locker, and Jeffrey Segall for excellent discussions and critical readings of the manuscript and Drs. Margaret Wheelock and Keith Johnson for providing the MMTV-N-cadherin mouse.
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Footnotes |
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Received 9/13/06. Revised 1/11/07. Accepted 1/19/07.
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; 3.0 ± 0.2 g at 2 wk, 4.3 ± 1.2 g at 3.5 wk, 7.0 ± 0.9 g at 5 wk, 15.5 ± 1.0 g at 7 wk, and 22.4 ± 1.8 g at 9 wk; n = 37) increased at similar rates as those from PyVmT mice (
; 2.9 ± 0.4 g at 2 wk, 3.7 ± 0.7 g at 3.5 wk, 5.3 ± 1.0 g at 5 wk, 13.7 ± 1.6 g at 7 wk, and 20.7 ± 2.5 g at 9 wk; n = 23). P = 0.001, two-tailed t test.
