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Published online before print May 24, 2007
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From the Growth Factor Division,* Central Animal Laboratory, National Cancer Center Research Institute, Tokyo, Japan
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Abstract |
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The biological functions of Ephs and ephrins in epithelial cells and tumors have recently been highlighted.4-9 For example, EphB receptors and ephrin-B ligands are expressed in normal intestinal epithelium, which contributes for the restriction of cell migration and positioning along the crypt-villus axis.4 Overexpression of B-type ephrin in cancer cells correlates with poor prognosis characterized by high invasion and high vascularity of the tumors.10-15 Expression of ephrin-B2 has been reported in invasive tumor cells and is often highly expressed in the peripheral region of the tumors especially at the front of the invasion.11 Ephrin-B1 is frequently overexpressed in gastrointestinal tumors, especially in poorly differentiated invasive tumor cells.15 Although an accumulating number of reports have suggested that expression of B-type ephrin is closely associated with tumor cell invasion, whether ephrin-B modifies tumor invasion in vivo has not been well established.
Ephrin-Bs are tyrosine phosphorylated via Src family kinases in response to the interaction with EphB receptors, which serves as a docking site for Src homology 2 domain of adaptor protein Grb4, and transduce intracellular signaling.16,17 We have also demonstrated that ephrin-B1 is phosphorylated independently of Eph receptors through association with an intercellular adhesion molecule, which leads to attenuation of cell-cell adhesion.7 In our recent observations, signaling mediated by ephrin-B1 promoted the process of intracellular transport and secretion of matrix metalloproteinase (M. Tanaka, K. Sasaki, R. Kamata, and R. Sakai, unpublished data), which led us to examine in this study whether disruption of ephrin-B1-mediated signaling, especially through the tyrosine phosphorylation of ephrin-B1, could suppress tumor cell invasion.
Scirrhous gastric carcinoma diffusely infiltrates a broad region of the stomach and frequently associates with metastasis to lymph nodes and peritoneal dissemination and, therefore, has the worst prognosis among various types of gastric cancers. We previously established two cell lines of human gastric scirrhous carcinoma possessing high infiltrative potential by repeating cycles of orthotopic transplantation in nude mice and collecting cancer cells from the ascitic fluid formed as a result of cancerous peritonitis.18,19 In this study, we show that reduction of ephrin-B1 expression or blocking of tyrosine phosphorylation of ephrin-B1 inhibits tumor invasion of these highly invasive gastric cancer cells. Our results suggest that ephrin-B1 represents a rational therapeutic target and that suppression of its phosphorylation is a strategy for modulating the invasion of some types of cancers.
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Materials and Methods |
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Plasmids encoding full-length cDNAs of human ephrin-B1 and ephrin-B1 with mutations of four tyrosine residues in the cytoplasmic domain (Y313, 317, 324, and 329) ephrin-B1 4YF have already been described.7
To generate the recombinant retrovirus, cDNAs were subcloned into the vector pDON-AI (Takara, Kyoto, Japan). The rabbit polyclonal antibodies for ephrin-B1 (C18) and 
-tubulin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal antibody against tyrosine-phosphorylated ephrin-B1 (ephrin-B1 pY317, amino acids 314 to 321, which is identical to corresponding region of ephrin-B2, 301 to 308, and ephrin-B3, 308 to 315) was raised in rabbits and affinity-purified as described previously.7
The monoclonal antibodies for phosphotyrosine (4G10) and Rac1 were from Upstate Biotechnology (Lake Placid, NY). The polyclonal antibodies for EphB2 and EphB4 were from R&D Systems (Minneapolis, MN). Fibronectin (bovine), collagen type I, and Matrigel basement membrane matrices were purchased from Sigma (St. Louis, MO), Nitta Gelatin, Inc. (Osaka, Japan), and BD Biosciences (San Jose, CA), respectively.
Cell Culture and Transfection
Gastric carcinoma cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Mouse fibroblast L cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. L cells stably expressing EphB2 (L EphB2) were established through transfection of a plasmid encoding human ephrin-B1 in parent L cells, which do not express cognate receptors for ephrin-B1 as previously described,7 and selection in medium containing hygromycin B at a concentration of 400 µg/ml. Recombinant retroviral plasmid pDON-AI was cotransfected with pCL-10A1 retrovirus packaging vector (Imgenex, San Diego, CA) into 293gp cells to allow the production of retroviral particles. Gastric cancer cells stably expressing ephrin-B1 4YF were established by infecting cancer cells with retroviruses and selected in the medium containing G418 at a concentration of 500 µg/ml for 3 weeks. The mixture of selected cells was used for the experiments.
In Vitro Short Interfering RNA (siRNA) Treatment
Two sets of Stealth siRNAs of ephrin-B1 were synthesized as follows (Invitrogen, Carlsbad, CA): ephrin-B1 sense 1, 5'-UAAGGGAAUGAUGAUGUCGCUGGGC-3'; ephrin-B1 antisense 1, 5'-GCCCAGCGACAUCAUCAUUCCCUUA-3'; ephrin-B1 sense 2, UAGUCCGUAAGGGAAUGAUGAUGUC-3'; and ephrin-B1 antisense 2, GACAUCAUCAUUCCCUUACGGACUA-3'. The control siRNA (scramble II duplex, 5'-GCGCGCUUUGUAGGAUUCGdTdT-3') was purchased from Dharmacon (Lafayette, CO). siRNAs were incorporated into cells using Lipofectamine2000 according to the manufacturer’s instructions (Invitrogen). Assays were performed 72 hours after treatment.
Immunoprecipitation
Cell lysates were prepared with protease inhibitors in PLC buffer [50 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5, 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L ethylene glycol bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 10% glycerol, 100 mmol/L NaF, 1 mmol/L Na3VO4, and 1% Triton X-100]. To precipitate the proteins, 1 µg of affinity-purified polyclonal antibody was incubated with 500 µg of cell lysate for 2 hours at 4°C and then precipitated with protein G agarose for 1 hour at 4°C. Immunoprecipitates were extensively washed with PLC buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotted.
Affinity Precipitation
Affinity precipitation with GST-PBD (p21-binding domain of p21-activated kinase 1) was performed as described previously.20 In brief, cells were lysed in the lysis buffer [50 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5, 150 mmol/L NaCl, 10 mmol/L MgCl2, 1 mmol/L ethylene glycol bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 10% glycerol, 100 mmol/L NaF, 1 mmol/L Na3VO4, and 1% Triton X-100] and incubated with GST-PBD on Sepharose for 1 hour at 4°C. Precipitants were washed three times in the same buffer, and endogenous Rac1 was detected by immunoblotting with anti-Rac1 antibody.
5-Bromo-2'-Deoxyuridine Incorporation
Cell proliferation was assessed by measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation into the DNA with Cell Proliferation enzyme-linked immunosorbent assay, BrdU (colorimetric) kit (Roche, Basel, Switzerland). In brief, gastric cancer cells were plated onto 96-well plates (1 x 104 cells/well) 48 hours after treatment of siRNAs and further incubated for 24 hours before the addition of BrdU. Cells were reincubated for 6 hours, and incorporated BrdU was detected with peroxidase-labeled anti-BrdU antibody and developed with tetramethyl-benzidine as a chromogenic substrate according to the manufacturer’s instructions (Roche). The absorbance of the samples was measured at the wavelength of 450 nm using a microplate reader (model 550; Bio-Rad, Hercules, CA).
Apoptosis Assays
Gastric cancer cells were plated in triplicate onto 96-well plates (1 x 104 cells) 48 hours after treatment of siRNAs and incubated for 24 hours. Cells were lysed to detect apoptosis by measurement of nucleosomes in the cytoplasm of apoptotic cells using a Cell Death enzyme-linked immunosorbent assay kit according to the manufacturer’s instructions (Roche Molecular Biochemicals). In brief, nucleosomes in cell lysates were detected with peroxidase-labeled anti-DNA antibody and developed with 2,2'-azino-di[3-ethylbenzthiazolin-sulfonate] as a chromogenic substrate. The absorbance of the samples was measured at the wavelength of 405 nm using a microplate reader (model 550; Bio-Rad).
Cell Attachment Assay
Cancer cells were detached by phosphate-buffered saline(–) [PBS(–)] containing ethylenediamine tetraacetic acid (2 mmol/L) and replated on the chamber slides coated with either collagen type I (100 µg/ml; Nitta Gelatin, Inc.), fibronectin (50 µg/ml; Sigma), or Matrigel (85 µg/ml; Asahi Techno Glass Co., Tokyo, Japan). After incubation for 30 minutes, unattached cells were removed by washing the slides in PBS(–) several times, and the remaining cells were stained with Giemsa’s solution. The number of attached cells on each substrate was counted.
Cell Staining
Cells were fixed for 5 minutes at room temperature with 4% paraformaldehyde in PBS and permeabilized for 10 minutes with 0.2% Triton X-100. The cells were preincubated in 2% bovine serum albumin for 0.5 hour and incubated with Alexa546-conjugated phalloidin (Molecular Probes, Eugene, OR) for 1 hour at room temperature. Photos were taken with a Radiance 2100 confocal microscope (Bio-Rad).
Overlay Tumor Invasion Assay
Invasion of tumor cells into the monolayer of stromal cells was monitored basically as described previously.20 Gastric cancer cells treated with ephrin-B1 siRNA or control siRNA were labeled with 2 mmol/L lipophilic tracer DiO (Molecular Probes) and then detached with Hanks’ balanced salt solution– containing 2 mmol/L ethylenediamine tetraacetic acid and seeded on the confluent monolayer of parent L cells or L EphB2 cells. After being cultured in the medium with 10% fetal bovine serum for 15 hours, the cells were fixed with 4% paraformaldehyde in PBS, and the number of invasion foci of cancer cells was counted using fluorescence microscopy.
In Vivo Tumor Cell Invasion Assay
The animal experimental protocols were approved by the Committee for Ethics of Animal Experimentation, and the experiments were conducted in accordance with the guidelines for Animal Experiments in the National Cancer Center. Peritoneal dissemination of tumors was examined by intraperitoneal injection of 5 x 106 gastric cancer cells suspended in 0.3 ml of RPMI 1640 medium into 6-week-old BALB/c nude mice (CLEA Japan, Inc., Tokyo, Japan). The mice were sacrificed 2 weeks after injection, and peritoneal dissemination was evaluated. Orthotopic implantation of gastric cancer cells into BALB/c nude mice has been described previously.18,19 In brief, 1 x 106 cells were inoculated into the middle wall of the greater curvature of the glandular stomach by using a 30-gauge needle. The mice were sacrificed at different time points after the orthotopic transplantation of the cancer cells and subjected to macroscopic and histopathological examination of the tumors.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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To examine the involvement of ephrin-B1 for invasion of tumors, we analyzed cell lines of scirrhous gastric carcinoma, which is characterized as reduced cell-cell adhesion with high invasion potential. HSC-44PE and HSC-58 were originally established from the patients of scirrhous gastric carcinoma, and highly invasive sublines were further selected from these parent cells (44As3 from HSC-44PE; 58As1 and 58As9 from HSC-5818,19
). Both expression and phosphorylation levels of ephrin-B1 were higher in cells of invasive sublines than in corresponding parent cell lines, whereas the expression level of control -tubulin was not altered in these cell lines (Figure 1a)
. In addition, EphB2 was expressed in all of these cell lines, and HSC-44PE and 44As3 cells also expressed EphB4, showing the existence of cognate receptors (Figure 1a)
.
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. In addition, phosphorylation of B class ephrin was greatly reduced by knocking down ephrin-B1, as judged by the antibody recognizing phosphorylation of all three members of ephrin-Bs at the tyrosine in the cytoplasmic region (Figure 1b)
. From the analysis of in vitro Transwell assay, reducing the amount of ephrin-B1 in 44As3 cells inhibited migration and invasion through the extracellular matrix (Figure 2a)
. Similar results were also observed in 58As9 cells (Figure 2a)
. On the other hand, proliferation and apoptosis of these cells were not significantly affected (Figure 2b)
. In addition, reduction of ephrin-B1 expression did not cause remarkable change in the adhesion of 44As3 and 58As9 cells on different extracellular matrices, including type I collagen, fibronectin, and Matrigel (Figure 2c)
. We further examined whether overexpression of ephrin-B1 is sufficient to promote the migration and invasion of cancer cells by stably expressing ephrin-B1 in one parental cell line HSC-58. The migration and invasion through extracellular matrix was apparently increased by the overexpression of ephrin-B1 (Figure 2d)
.
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Because the level of tyrosine phosphorylation of ephrin-B1 was higher in invasive sublines of gastric cancer cells, we next examined whether blocking of ephrin-B1 phosphorylation in these cells attenuates their migration and invasion. The stable expression of ephrin-B1 with mutations of four tyrosine residues in the cytoplasmic domain (Y313, 317, 324, and 329) ephrin-B1 4YF reduced the tyrosine phosphorylation level of ephrin-B1 in 44As3 and 58As9 cells, because overexpression of ephrin-B1 4YF prevents endogenous ephrin-B1 from association with EphB receptors expressed in these cells (Figure 3a)
. From the analysis of in vitro Transwell assay, migration and invasion of cancer cells stably expressing ephrin-B1 4YF mutant (44As3 4YF and 58As9 4YF cells) were decreased compared with the control cells expressing mock vector (44As3 mock and 58As9 mock cells) (Figure 3b)
. On the other hand, expression of ephrin-B1 4YF did not affect cell proliferation under usual two-dimensional cell culture condition (Figure 3c)
. When these cancer cells expressing mock vector or ephrin-B1 4YF were implanted subcutaneously in nude mice, the mean size and weight of the tumors were not significantly different (Figure 3d)
. To understand the mechanism by which ephrin-B1 4YF attenuates the cell migration, we examined activity of Rac1 GTPase, which is a critical molecule controlling the organization of actin cytoskeleton. The activation of Rac1 was examined by affinity precipitation of GTP-bound Rac1 with the GST-fusion protein of the p21-binding domain of p21-activated kinase 1.21
The activated Rac1 was apparently reduced in 44As3 or 58s9 cells expressing ephrin-B1 4YF compared with the cells expressing mock vector (Figure 3e)
. When the appearance of cytoskeleton of these cancer cells was examined, formation of large lamellipodia was observed in most of the mock-containing 44As3 and 58As9 cells, whereas it was less frequently observed in 44As3 4YF and 58As9 4YF cells (Figure 3f)
, which may be consistent with the reduced Rac1 activity in cancer cells expressing ephrin-B1 4YF.
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. The formation of such tumor islands is the sign of penetration of tumor cells into the sheet of stromal cells, as used for the evaluation of cancer cell invasion through endothelial cells or mesothelial cells.20,22,23
The invasion of 44As3 cells into the fibroblasts monolayer was more evident when the cancer cells were plated onto L cells stably expressing EphB2 (L EphB2) than on the parent L cells, which do not express receptors for ephrin-B17
(Figure 4b)
. The invasion foci of 44As3 in monolayer of L EphB2 cells were decreased in number and size when expression of ephrin-B1 in cancer cells was reduced (Figure 4a)
. In addition, the invasion of 44As3 4YF cells into the monolayer of L EphB2 cells was also decreased compared with 44As3 mock cells (Figure 4, a and b)
. These results indicate that activation of the signaling mediated by ephrin-B1 phosphorylation in cancer cells induced by the interaction with EphB2 receptor expressed in stromal cells enhanced the tumor invasion.
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, top).19
Innumerable whitish nodules were observed in the mesentery of almost all mice injected with 58As9 mock cells (Figure 5a
, top left) and 44As3 mock cells (data not shown). In addition, many tumor nodules of 44As3 mock cells were observed in the peritoneal cavity, including the rectouterine region with invasion into the retroperitoneum (Figure 5a
, top right). On the other hand, dissemination of cancer cells expressing ephrin-B1 4YF was apparently modest. Tumor nodules of 58As9 4YF cells in the mesentery were small in size and number compared with those of control 58As9 mock cells (Figure 5b
; Table 1
). Such reduction of tumor nodules in the mesentery was also observed in 44As3 cells expressing ephrin-B1 4YF (Table 1)
. In addition, the tumor volume involving the rectouterine region was reduced in the mice injected with 44As3 4YF cells (Figure 5c)
. These results suggest that ephrin-B1 promotes peritoneal dissemination of 58As9 and 44As3 gastric cancer cells through signaling mediated by its tyrosine phosphorylation.
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To further evaluate the effect of ephrin-B1 on the process of tumor invasion, we implanted gastric cancer cells orthotopically in the gastric submucosa of nude mice. At 15 days after implantation, 70% of 44As3 tumors expressing the mock vector formed a large tumor mass involving the greater omentum because of the disruption of gastric serosa and invasion into the surrounding fat tissue (Figure 6a)
. On the other hand, such local invasion into the omentum was less frequently observed (18%) in mice implanted with 44As3 4YF cells, and most tumors remained within the gastric wall at day 15 (Figure 6a
; Table 2
). The dissemination of 44As3 mock cells was also observed in several tissues, including the liver surface and mesenteric sheets (Figure 6, b and c)
. However, we rarely observed cancer dissemination in the peritoneal cavity in mice implanted with 44As3 4YF cells (Figure 6, b and c
; Table 2
).
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. However, invasion of control 44As3 mock cells into lymphatic vessels in subserosa and metastasis to the regional lymph nodes was more frequently observed than 44As3 4YF cells at day 10 after tumor implantation (70 and 18%, respectively; Figure 7, e–g
; Table 3
). Histological analysis also revealed that the tumor had already reached the outside of the serosal surface and invaded into the surrounding fat tissue in 80% of mice implanted with 44As3 mock cells, whereas it was of low frequency in mice implanted with 44As3 4YF cells (27%) (Figure 7, h and i
; Table 3
). These phenotypes, including lymphatic vessel invasion, lymph node metastasis, and perforation of gastric serosa, were developed in the same mice, and all three phenotypes are overlapped in 7 of 10 mice in the mock group and in 2 of 10 mice in the 4YF group, suggesting that these are sequential or correlated events. These results indicate that the disruption of signaling mediated by phosphorylation of ephrin-B1 suppressed the invasion and peritoneal dissemination of scirrhous gastric carcinomas.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Association with EphB receptors triggers tyrosine phosphorylation of ephrin-B1, including Tyr317 (corresponding to Tyr298 of Xenopus ephrin-B1), by Src family kinases, which is a critical requirement for interaction with an Src homology 2/Src homology 3 adaptor, Grb4, to transduce signaling.16
In the physiological conditions, phosphorylation of ephrin-B1 is induced by the contact of ephrin-B1-expressing cells with heterologous cells expressing EphB receptors. On the other hand, ephrin-B1 also promotes migration of cancer cells in a cell-autonomous mechanism as observed in Transwell assay (Figures 2 and 4b)
. Because EphB2 receptor was expressed in 44As3 and 58As9 cells and expression of EphB4 was also detected in 44As3 cells (Figure 1a)
, there is a possibility that ephrin-B1 in these cancer cells may be constitutively stimulated by EphB receptors coexpressed in the same cell surface or contacting neighboring cancer cells. This conclusion may be consistent with the observation that the basal phosphorylation level of ephrin-B1 is elevated in 44As3 and 58As9 cells under the usual two-dimensional culture condition in vitro (Figure 1a)
. Overexpression of ephrin-B1 4YF may suppress tumor invasion in vivo by blocking cell autonomous phosphorylation of ephrin-B1 in cancer cells and the induction of ephrin-B1 phosphorylation through the interaction with stromal cells. There is still the possibility that expression of ephrin-B1 4YF may also block the signaling mediated by other members of ephrin-Bs that also bind the similar group of EphB receptors. However, knocking down of ephrin-B1 by siRNA greatly reduced the phosphorylation of total ephrin-Bs (Figure 1b)
, suggesting that ephrin-B1, but not ephrin-B2 or ephrin-B3, is the major member of B-class ephrin, which is phosphorylated in these cells. Consistent with this conclusion, at least treatment of 44As3 and 58As9 cells with ephrin-B2 siRNA did not affect the phosphorylation level of the corresponding tyrosine residue of ephrin-Bs examined by anti-ephrin-B1 pY317 antibody (data not shown).
The ephrin/Eph interaction provides both ephrin-B1-mediated reverse signaling and EphB-mediated forward signaling. The expression of ephrin-B1 4YF may suppress the invasion of gastric cancer cells by stimulation of EphB receptor-mediated forward signaling in cancer cells through acting as a stimulator of EphB receptors, because EphB receptor-mediated forward signaling inhibited migration of colorectal tumors in a recent report.24 However, cancer cell invasion was also inhibited by treatment with ephrin-B1 siRNA, which should cause reduction of forward signaling. Therefore, such inhibitory effect of forward signaling caused by the overexpression of the ligand, even if it exists, does not seem to be strong. In addition, we previously observed that overexpression of ephrin-B1 in Panc1 and Capan1 pancreas cancer cells, in which EphB2 is endogenously expressed, promoted the peritoneal dissemination of these cells by the similar experiment in this study (data not shown), which may also support this conclusion.
We observed that ephrin-B1-expressing cancer cells invaded more frequently into the monolayer of fibroblasts expressing EphB2 receptor than into the parent fibroblasts in overlay invasion assay. This result suggests that cancer cells expressing ephrin-B1 may actively invade into the stromal tissues that express EphB receptors, and such mechanism may also be involved in the process of peritoneal dissemination of ephrin-B1-expressing cancer cells in vivo. For example, stromal cells composed of mouse mesentery sheets actually expressed cognate receptors for ephrin-B1 (Supplemental Figure 1 at http://ajp.amjpathol.org). Again, there is a possibility that EphB2-mediated forward signaling in L EphB2 fibroblasts also contributes to the formation of invasion foci of cancer cells in this overlay assay. However, expression of ephrin-B1 4YF prevented the invasion of 44As3 cells into the monolayer of L EphB2 cells, suggesting that this invasion is considered to depend predominantly on the ephrin-B1-mediated reverse signaling in cancer cells rather than the forward signaling in fibroblasts.
Tumor cells expressing ephrin-B1 may gain invasiveness in multiple steps of the cell invasion process. Phosphorylation of ephrin-B1 leads to the recruitment of scaffolding protein disheveled via Grb4, which results in aberrant activation of RhoA.25,26 Ephrin-B1 makes a complex with Tiam1 to induce Rac1 activation,20 and we showed blocking of tyrosine phosphorylation of ephrin-B1 decreased the Rac1 activity in this study. Ephrin-B1 is also phosphorylated on tyrosine residues through physical association with cell adhesion proteins such as claudin, which attenuates the cell-cell adhesion.7 In addition, EphB2-induced phosphorylation of ephrin-B1 has been reported to modify the cell-to-substrate adhesion,17 although reduction of ephrin-B1 with siRNA did not significantly affect cell adhesion to several substrates in vitro under the condition that ephrin-B1 was not stimulated with exogenous EphB2 receptor. Therefore, phosphorylation of ephrin-B1 may regulate cell motility also by affecting the cell adhesion to substrates in the process of stromal invasion of tumors in vivo. Blocking of ephrin-B1 phosphorylation by the ephrin-B1 4YF mutant may inhibit tumor invasion through blocking such multiple events.
We cannot exclude a possibility that phosphorylation-independent signaling of ephrin-B1 is also involved in the regulation of cancer cell invasion. For example, proteins containing PDZ domains make a stable complex with ephrin-B1 via PDZ domain-binding motif -YXV located at the carboxyl terminus of ephrin-B1.27 It may be important to examine the phosphorylation state of ephrin-B1 in vivo in a wide range of cancers to estimate what types of cancers will be sensitive for blocking ephrin-B1 phosphorylation on tumor suppression. For example, phosphorylation of B-type ephrins in invading glioblastoma has been reported recently.14
We performed intraperitoneal inoculation of cancer cells into nude mice as a model of peritoneal dissemination of the tumor, as also reported by others.28,29 Although many gastric cancer cell lines do not show typical histological appearance of human scirrhous carcinoma when inoculated into nude mice, our system of orthotopic transplantation of 44As3 cells is a highly reproducible animal model of peritoneal dissemination of human scirrhous gastric carcinoma.18,19,30 Because early clinical diagnosis of scirrhous gastric carcinoma is difficult, peritoneal dissemination or metastasis to lymph nodes has frequently occurred by the time the diagnosis is made. We observed that expression of ephrin-B1 4YF suppressed not only local invasion of 44As3 cells in the gastric wall but also the infiltration of cancer cells into lymphatic vessels and lymph node metastasis. These results suggest that ephrin-B1 phosphorylation-mediated signaling may modulate the process of the invasion of cancer cells to lymphatic vessels. It may be useful to examine the expression and phosphorylation level of ephrin-B1 in a surgical specimen of scirrhous gastric carcinomas to predict lymphatic metastasis of tumors. Dissemination is a frequent form of the recurrence of scirrhous gastric carcinoma, which serves as a major factor determining the prognosis. Ephrin-B1 is considered to be a prognostic factor of gastric scirrhous carcinoma, and the inhibition of a specific cellular signal originating in ephrin-B1 phosphorylation may be a good candidate for regulating its invasion and dissemination.
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Footnotes |
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Supported by the Program for the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan.
Supplemental material for this article can be found on http://ajp.amjpathol.org.
Accepted for publication April 10, 2007.
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