Affiliations of authors: V. R. J. Schelfhout, E. D. Coene, S. Thys, C. R. De Potter, N. Goormaghtigh Institute for Pathology, University Hospital, Gent, Belgium; B. Delaey, N.V. Innogenetics, Zwijnaarde, Belgium; D. L. Page, Department of Pathology, Vanderbilt University Medical Center, Nashville, TN.
Correspondence to: Christian R. De Potter, M.D., N. Goormaghtigh Institute for Pathology, University Hospital, De Pintelaan 185, B-9000 Gent, Belgium (e-mail: Christian.DePotter{at}rug.ac.be).
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ABSTRACT |
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INTRODUCTION |
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Our aim was to further our understanding of the pathogenesis of Paget's disease by identifying and characterizing the factor(s) released by normal epidermal cells and their target receptors that are involved in the penetration and migration of breast cancer cells through the nipple epidermis.
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SUBJECTS AND METHODS |
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SK-BR-3 cells were grown in minimal essential medium Rega 3 that contains nonessential amino acids (BioWhittaker Inc., Walkersville, MD) supplemented with 10% fetal calf serum, 20 mM HEPES, 14.3 mM sodium bicarbonate, 50 IU/mL penicillin, 50 IU/mL streptomycin, and 2 mM L-glutamine (BioWhittaker Inc.).
Keratinocyte cultures were established from skin specimens of healthy adult donors and cultured on a feeder layer of mitomycin-C-treated Swiss 3T3 fibroblasts (8,9). After two serial passages at 1:3 split ratios, confluent cell layers were allowed to stratify for 12 weeks and form a multilayered epithelium. At the end of the stratification process, keratinocytes were placed in a basal medium containing Dulbecco's modified Eagle medium/Ham's F-12, 3:1 (vol/vol), for 1824 hours; the conditioned medium, containing secreted growth factors, was then collected and stored at -20°C.
Motility and Chemotaxis
SK-BR-3 cells were detached with a solution of 0.05% trypsin and 0.5 mM EDTA in a Ca2+/Mg2+-free balanced salt solution or with 0.5 mM EDTA alone for antibody experiments. Approximately 1.5 x 104 SK-BR-3 cells were seeded per well in a 24-microwell plate in 1 mL of complete culture medium and incubated for 24 hours at 37°C in an atmosphere of 5% CO2/95% air and 100% humidity. Normal keratinocyte-conditioned medium or purified motility factor was added at various dilutions from 1:100 for conditioned medium to 1:10000 for purified factor for 1 hour at 37°C, and then cells were fixed in alcohol and stained with crystal violet. One unit of motility factor activity was defined as the amount of motility factor that causes spreading (i.e., a flat morphology and scattering of 50% of the cells within 1 hour).
To determine whether the HER2/NEU receptor was the target for the motility factor, we used the monoclonal antibody AB2 (Oncogene Research Products; Merck KGaA, Darmstadt, Germany) (10), which is directed against the extracellular domain of the HER2/NEU protein and blocks ligand binding to the receptor. We used AB2 at concentrations from 0.06 to 1 µg/mL and purified motility factor at a dilution that induced maximal spreading, scattering, and motility of SK-BR-3 cells.
Chemotaxis was analyzed in a modified Boyden chamber with the use of 24-microwell plates containing 6.5-mm polycarbonate microporous (pore size, 8 µm) cell culture inserts (TranswellTM; Corning Costar Corp., Cambridge, MA). In this assay, the lower chamber contained 600 µL of culture medium and the upper chamber contained 100 µL of culture medium and 2 x 105 SK-BR-3 cells. Normal human keratinocyte-conditioned medium or purified motility factor was added at various concentrations to the lower chambers for 24 hours. Cells on the filter were then fixed in methanol and stained with 0.1% nuclear fast red in water for 5 minutes. Cells that migrated through the filter were counted over the filter membrane and expressed as the number of cells per filter.
Chromatography
We used a total of 178 L of conditioned medium from cultured human keratinocytes. First, 10-L batches of medium were thawed, cleared by filtration over a 0.22-µm (pore size) filter, and adjusted to 10 mM sodium phosphate and 0.05% CHAPS (i.e., 3-[(3-cholamidopropyl)demethylammonio]-1-propanesulfonate) at pH 7.3. The medium was then fractionated over an 80-mL Biogel HTP hydroxyapatite column (Bio-Rad Laboratories, Hercules, CA), and the material was eluted with a phosphate buffer step gradient ranging from 50 to 400 mM. Active fractions eluted at the 400-mM step were further fractionated on a 4-mL Poros heparin column (50-µm beads; Perseptive Biosystems, Framingham, MA), and activity was eluted with a linear gradient of 150750 mM NaCl, followed by a 1 M NaCl step. SK-BR-3-positive fractions that eluted between 700 and 750 mM NaCl were pooled, diluted 1:2 in high-pressure liquid chromatography-quality water, and adjusted to 0.2% trifluoroacetic acid. Batches of this material equivalent to 2.5 L of conditioned medium were loaded onto a 75-µL Zorbax microbore reversed-phase C8 column (5-µm bead size; LC Packings, San Francisco, CA), and material was eluted with a linear acetonitrile gradient of 6.4%80%. SK-BR-3-positive fractions were eluted in a sharp peak at 27%28% acetonitrile, with a minor part of the activity trailing at 28%30% acetonitrile.
Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis
Purified material from the reverse-phase C8 column step was vacuum dried, resuspended in Laemmli sample buffer, and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) on the PHASTTM system (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) in 20% homogeneous polyacrylamide gels. The purified protein had an apparent molecular mass of 50 kd, as detected by silver staining. Preparative SDSPAGE was performed on the PHAST system with an amount of purified material corresponding to 2.5 L of conditioned medium (divided over five lanes). This gel was stained with Coomassie brilliant blue R250.
Isolation, Purification, and In Situ Digestion of Motility Factor With Trypsin for Mass Spectrometry
The 50-kd protein band containing motility factor was excised from the preparative PHAST gel and destained in 50% acetonitrile in water. Gel pieces were completely dried in a vacuum centrifuge (Speed Vac; Savant Instruments, Inc., Farmingdale, NY) and immersed in 30 µL of digest buffer containing 0.08 µg of trypsin, 25 mM NH4HCO3, and 10% acetonitrile in water at pH 8.0 for 18 hours at 37°C. The resulting peptides were subsequently extracted with 50% acetonitrile and 0.1% trifluoroacetic acid in water, pooled, vacuum dried, dissolved in 20 µL of 0.1% formic acid, and analyzed by nano-liquid chromatography (NanoLC)tandem mass spectrometry.
Identification of Motility Factor Use of NanoLCTandem Mass Spectrometry
NanoLCtandem mass spectrometry was performed by on-line coupling of a FAMOSTM (LC Packings) controlled liquid chromatography system (Kontron, Milan, Italy) and Q-TOFTM (Micromass, Wytenshaw, U.K.) quadrupole-time-of-flight hybrid tandem mass spectrometer adapted with a Z-spay interface.
NanoLC separation was done on a PepMapTM (LC Packings) C18 column 150 mm long with an internal diameter of 75 µm by use of a column-switching technique. Peptides are first captured by a PepMap C18 µ-precolumn 2 mm long with an internal diameter of 0.8 mm at a flow rate of 20 µL/minute, after which the bound peptides were back flushed and gradually eluted and separated on the PepMap column with an internal diameter of 75 µm at a flow rate of 230 nL/minute. On-line mass spectrometry and tandem mass spectrometry spectra were acquired on Q-TOF mass spectrometer. Automated switching between mass spectrometry and tandem mass spectrometry was monitored by the MassLynxTM version 3.2 (Micromass) software. All obtained mass spectrometry and tandem mass spectrometry spectra were manually processed and screened for identity against the National Center for Biotechnology Information release nr 19990501 by use of the MASCOT software (http://www.matrixscience.com/).
Immunocytochemistry and Immunohistochemistry
We investigated the expression of HER1 (BioGenex Laboratories, San Ramon, CA), HER2/NEU (Calbiochem Corp., La Jolla, CA), HER3 (Novocastra Laboratories Ltd., New Castle, U.K.), and HER4 (Probio, Kent, U.K.) in SK-BR-3 cells and in biopsy specimens from 30 patients with Paget's disease by the use of specific antibodies from the suppliers indicated. Cells were grown on chamber slides and fixed in methanol at -20°C for 5 minutes, followed by acetone at 4°C for 2 minutes; immunocytochemistry was then performed. Biopsy specimens from 30 patients with Paget's disease of the nipple were formalin fixed, paraffin embedded, sectioned, and deparaffinized; immunohistochemistry was then performed. HER1, HER2/NEU, HER3, and HER4 were detected by the biotinstreptavidinperoxidase method. For the immunochemistry of HER1, HER3, and HER4, deparaffinized sections were digested with 0.4% pepsin in 0.02 N HCl for 2 hours at 37°C.
Western Blotting
Nuclear and cytoplasmic fractions of SK-BR-3 cells were separated by use of the Pierce nuclear and cytoplasmic extraction reagent kit (Pierce Chemical Co., Rockford, IL). Cytoplasmic fractions were subjected to electrophoresis in 3%8% Trisacetate gels and Trisacetate SDS running buffer (Novex, San Diego, CA) at 150 V for 2 hours. The proteins were transferred to a nitrocellulose membrane (Novex) in Trisacetate transfer buffer (Novex) for 1 hour at 30 V. The membranes were blocked for 23 hours with a solution of 10% milk, phosphate-buffered saline (PBS), and 0.1% Triton X-100 at room temperature and then incubated with the primary antibodies to HER1 (1:100 dilution), HER2/NEU (1:5000 dilution), HER3 (1:20 dilution), or HER4 (1:400 dilution) overnight at 4°C. Membranes were washed in PBS and 0.1% Triton X-100 and blocked with 10% milk, PBS, and 0.1% Triton X-100. Sheep anti-mouse immunoglobulin G (IgG) peroxidase at 120 U (1:1000 dilution; Roche Molecular Biochemicals, Mannheim, Germany) was used for HER1, HER2/NEU, and HER3. Sheep anti-rabbit IgG peroxidase, 200 U (1:4000 dilution; Roche Molecular Biochemicals), was used for HER4. Proteins were detected by the biochemiluminescence technique (Roche Molecular Biochemicals) (11) and hyperfilm enhanced chemiluminescence development (Amersham Pharmacia Biotech, Uppsala, Sweden).
In Situ Hybridization
To evaluate messenger RNA (mRNA) expression, 10 randomly selected specimens were subjected to mRNA in situ hybridization. The following two DNA probes were used to visualize heregulin- mRNA: 5`-FLUO-GCTCTCGGCGCAGGCGAGTTTGGTCCAAGGGCTCGGATCG-3` and 5`-FLUO-GCTCGGACATCTCGCCGGAGACGGAGCGCTCTACGCGGACG-3` (where FLUO is fluorescein isothiocyanate), corresponding to the predicted sequence of base pairs 62101 and 102142, respectively. These sequences were specific for heregulin-
, as shown by a thorough search of public sequence databases.
Five-micrometer sections were deparaffinized and incubated with 4% paraformaldehyde in PBS containing 1% diethylpyrocarbonate (DEPC) followed by 0.1 M glycine in PBS/DEPC for 5 minutes. The slides were incubated with 0.3% Triton X-100 in PBS/DEPC for 10 minutes, and proteins were digested with 10% proteinase K (Dako, Glostrup, Denmark) for 15 minutes. Slides were then postfixed in 4% paraformaldehyde. The slides were prehybridized with hybridization solution (BioGenex Laboratories) for 90 minutes at 42°C and hybridized with probe at 2.84 pg/mL preheated to 95°C for 10 minutes. The slides were then heated to 80°C for 2 minutes and incubated in a moist chamber at 37°C overnight. Between each step, the slides were rinsed with PBS and DEPC and, after hybridization, the slides were washed at 40°C in standard saline citrate. The fluorescein isothiocyanate (FITC)-coupled probe was detected with mouse anti-FITC (BioGenex Laboratories) for 20 minutes and amplified with anti-mouse biotin for 20 minutes, streptavidine peroxidase (Dako) for 20 minutes, and 3-amino-9-ethylcarbazole chromogen solution (Dako) for 10 minutes. After each step, slides were washed in PBS. The slides were counterstained with hematoxylin and mounted in Aquatex (Merck KGaA).
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RESULTS |
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In the absence of motility factor, cultured SK-BR-3 breast cancer cells attached to a culture plate and remained spherical for 3 days. When human keratinocyte-conditioned medium or purified motility factor is added, cells rapidly became motile by flattening, forming long thin plasma membrane protrusions and pseudopodia, and moving apart (Fig. 1). Cells began to spread within 15 minutes of the addition of conditioned medium or purified factor and completed spreading 12 hours later.
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A modified Boyden chamber assay was used to investigate the role of the motility factor in chemotaxis. The number of SK-BR-3 cells moving through the filter increased in a concentration-dependent fashion when the concentration of purified factor in the lower compartment was increased (Fig. 2). After a 30-minute preincubation with monoclonal antibody AB2 (1 µg/mL), cell migration was almost completely inhibited (Fig. 2
).
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Motility factor was purified from 178 L of human primary keratinocyte-conditioned medium by hydroxyapatite chromatography, heparin-affinity chromatography, and reversed-phase chromatography. Throughout purification, motility factor activity was monitored with the SK-BR-3 spreading assay. Motility factor activity in the reverse-phase C8 purified fractions corresponded to a 50-kd protein band on SDSPAGE gels (Fig. 3). The total amount of purified motility factor protein (estimated at <2 µg) was too low for sequencing by Edman degradation, and thus mass spectrometry was used to identify and sequence material in the 50-kd band (12). A small amount of purified material (corresponding to 2.5 L of conditioned medium) was separated by preparative SDSPAGE on the PHAST system (Pharmacia LKB Biotechnology AB). In total, five lanes were loaded, with a per lane amount corresponding to approximately 0.5 L of unpurified conditioned medium (or 2277 U of activity). After electrophoresis, the 50-kd motility factor bands were excised and pooled. Material in the band was digested with trypsin, and all tryptic peptides were analyzed by NanoLCtandem mass spectrometry.
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To determine whether Paget cells and SK-BR-3 cells express heregulin receptors HER3 and HER4, as well as their co-receptor HER2/NEU, 30 tissue specimens from patients with Paget's disease of the breast were investigated by immunohistochemistry and SK-BR-3 cells were investigated by immunocytochemistry.
HER2/NEU, HER3, and HER4 were detected in SK-BR-3 cells (Fig. 4). HER4 and particularly HER2/NEU were detected mainly on small dot-like extensions of the cell, consistent with a previous report (16). HER3 was detected more diffusely throughout the cell. HER1 was not detected in SK-BR-3 cells (Fig. 4
).
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Western Blotting
Western blot analysis of SK-BR-3 cells showed the absence of HER1, a strong 185-kd band for HER2/NEU, a faint 170-kd band for HER3, and a strong 180-kd band for HER4 (Fig. 4).
In Situ Hybridization
To confirm that the normal epidermal cells surrounding the penetrating Paget cells in the nipple could express the motility factor heregulin-, we used in situ hybridization to detect heregulin-
mRNA in sections containing Paget's disease tissue and normal tissue. In all cases, normal epidermal cells and normal ductal cells expressed heregulin-
mRNA (Fig. 5
), but Paget cells expressed little or no heregulin-
mRNA (Fig. 5
). These findings are consistent with the production and release of heregulin-
by cultured epidermal cells.
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DISCUSSION |
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As in extramammary Paget's disease (3), four of 30 specimens of Paget's disease did not overexpress HER2/NEU. These cases may represent another pathogenesis that does not require overexpression of HER2/NEU and members of the HER family for migration of adenocarcinoma cells in the epidermis. Alternatively, these cases might be artifacts, reflecting false-negative tests in poorly fixed material.
We cannot exclude the possibility that other members of the NDF/heregulin family of ligands for HER, generated by alternative splicing of pro-NDF/heregulin mRNA, may be involved in the pathogenesis of mammary Paget's disease. This could occur if these ligands were much less abundant in keratinocyte-conditioned medium and not detected by SDSPAGE. However, if this were the case, the pathogenesis of Paget's disease would remain the same (28,29). Additional experiments will be required to clarify this issue.
In conclusion, heregulin-, a motility factor released by keratinocytes of the nipple, plays a key role in the pathogenesis of Paget's disease by attracting breast cancer cells to spread throughout the nipple epidermis. The motility factor acts on its receptors HER3 or HER4 or both, as well as on their co-receptor HER2/NEU, which are expressed by Paget cells, to induce chemotaxis and subsequent spread of Paget cells throughout the nipple epidermis.
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Manuscript received September 21, 1999; revised January 12, 2000; accepted January 28, 2000.
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