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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Gastric cancer (GC) is one of the most common malignancies worldwide. The epidermal growth factor receptor (EGFR) molecule is very important in GC progression. To examine the correlation between EGFR and GC-related genes, we analyzed gene expression profiles of HT-29 cells treated with EGFR ligands and identified six genes upregulated by epidermal growth factor (EGF) and transforming growth factor (TGF)-α treatment. Among these, we focused on cadherin 17 (CDH17) encoding liver–intestine cadherin (LI-cadherin). Expression of LI-cadherin was induced by both EGF and TGF-α, as detected by quantitative RT-PCR and Western blot analysis. A luciferase assay showed that LI-cadherin promoter activity was enhanced by EGF or TGF-α in both HT-29 cells and MKN-74 GC cells. Immunohistochemical analysis of 152 GC cases showed that out of 58 LI-cadherin-positive cases, 24 (41%) cases were also positive for EGFR, whereas out of 94 LI-cadherin-negative cases, only 9 (10%) cases were positive for EGFR (P < 0.0001). Double-immunofluorescence staining revealed that EGFR and LI-cadherin were coexpressed. Significant correlation was found between LI-cadherin expression and advanced T grade and N grade. Both EGFR and LI-cadherin expression were more frequently found in GC cases with an intestinal mucin phenotype than in cases with a gastric mucin phenotype. These results indicate that, in addition to the known intestinal transcription factor caudal type homeobox 2, EGFR activation induces LI-cadherin expression and participates in intestinal differentiation of GC.

Gastric cancer remains a major public health issue as the fourth most common cancer and the second leading cause of cancer mortalities worldwide.[1] Gastric cancer is assumed to originate from a sequential accumulation of molecular and genetic alterations to stomach epithelial cells.[2] A molecular understanding of the genetics and epigenetics involved in GC pathogenesis may contribute to identifying novel GC biomarkers and highlight potential avenues for targeted therapies.

Epidermal growth factor and TGF-α both phosphorylate the EGFR and stimulate multiple signaling pathways involved in cell proliferation, anti-apoptosis, and other processes.[3-6] The overexpression of EGF and EGFR by various types of malignancies has been shown to correlate with metastasis, apoptosis, resistance to chemotherapy, and poor prognosis.[5, 7, 8] We previously reported that both EGF and EGFR are overexpressed in GC, and play a central role in tumor invasion and metastasis through an autocrine mechanism.[9-12] It is therefore important to gain a functional overview of EGFR signaling in GC.

In the present study, we used an oligonucleotide array analysis to generate a list of genes whose expression was induced by TGF-α or EGF treatment, and found that expression of CDH17 was induced by EGFR ligands. CDH17 was originally cloned from rat liver in 1994.[13] CDH17 encodes LI-cadherin protein, which has similarity to the classic cadherins but is structurally distinct. Although the LI-cadherin name derives from the apparent expression pattern of the gene in the rat, in humans LI-cadherin is expressed almost exclusively in the small intestine and colon, but not in the liver.[13] Liver–intestine cadherin is one of the targets of CDX2, the caudal-related homeobox transcription factor. CDX2 has a key role in intestinal development and differentiation, therefore LI-cadherin may play a role in mediating CDX2 function in intestinal cell fate determination. Expression of TGF-α protein is detected in the top one-third of the intestinal crypt, which is composed only of terminally differentiated cells.[14] There are two major phenotypes of GC that are defined according to the mucin expression profile. We previously reported that LI-cadherin expression is associated with an intestinal mucin phenotype.[15] However, induction of gene expression related to intestinal differentiation of GC by EGFR activation has not been investigated. Here, we used luciferase assays to study whether EGFR activation affects CDH17 transcription. Furthermore, EGFR and LI-cadherin expression were examined in surgically resected GC tissue by immunohistochemistry. The correlation between LI-cadherin expression and clinicopathological characteristics was analyzed.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Plasmids

Plasmids used in the present study were generated and described previously.[16] In brief, genomic DNA sequences corresponding to the promoter and 5′-flanking region of the human CDH17 gene were cloned by PCR using genomic DNA purified from Caco2 cells. The CDH17 PCR products were subcloned into the pGL4 basic vector (Promega, Madison, MD, USA). Polymerase chain reaction-based approaches were used to introduce mutations in the CDX2 binding sites in the pGL4 basic CDH17 reporter gene construct. All inserts were verified by automated sequencing.

Cell lines and EGF/TGF-α treatment. The HT-29 colon cancer cell line was obtained from ATCC (Rockville, MD, USA) and maintained in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% FBS in a humidified atmosphere of 5% CO2 and 95% air at 37°C. MKN-74 was kindly provided by Dr Toshimitsu Suzuki (Niigata University School of Medicine, Niigata, Japan). HSC-39 and HSC-57 were established by Dr. Kazuyoshi Yanagihara. The cell line HT-29-CDX2 stably expresses CDX2, and HT-29-neo is the control cell line. These cell lines were maintained as described previously.[16] After 24 h of serum starvation, 1–100 nM concentrations of EGF (Sigma, St Louis, MO, USA) or TGF-α were added. The cells were treated for 48 h, and proteins and RNAs were then extracted.

Oligonucleotide array construction, hybridization, detection, and data analysis

The oligonucleotide array, Genopal (Mitsubishi Rayon, Tokyo, Japan), was prepared as described previously.[17] The array contained 208 genes, including GC-related genes identified by our previous SAGE analysis,[18] known genes related to the development and progression of GC,[19, 20] genes related to DNA damage response and repair, and genes associated with sensitivity to anticancer drugs.[21] A list of the genes on the array is available upon request. Total RNA isolation, quantification and integrity of RNA assessment, hybridization, detection, and data analysis were carried out as described previously.[22]

Quantitative RT-PCR and Western blot analysis

Quantitative RT-PCR was carried out with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) as described previously.[23] Sequence information of primers for CDX2 and CDH17 is available upon request. The ACTB-specific PCR products were amplified from the same RNA samples and served as an internal control. Primer sequences and additional PCR conditions are available upon request.

For Western blot analysis, cells were lysed as described previously.[24] The filter was incubated with primary anti-LI-cadherin antibody (goat polyclonal dilution 1:500 ; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Peroxidase-conjugated anti-goat IgG was used as the secondary probe. Immunocomplexes were visualized with an ECL Western Blot Detection System (Amersham Biosciences, Piscataway, NJ, USA). β-actin antibody (Sigma) was used as a loading control.

Luciferase assay

Forty-eight hours before transfection, cells were plated in 35-mm dishes. Transfection of cells at 30–50% confluency was carried out with 6 μL FuGENE6, 4 μg pGL4 containing LI-cadherin promoter constructs[16] and basic reporter gene construct, and 1 μg phRL-TK Renilla luciferase reporter vector (Promega). At 48 h after transfection, EGF and TGF-α treatment was carried out. At 48 h after treatment, cells were collected and resuspended in reporter lysis buffer (Promega). Luciferase activities were determined with luciferase assay reagent (Promega) and a GloMax luminometer (Promega).

Tissue samples

A total of 152 primary tumor samples were collected from patients diagnosed with GC. Patients were treated at the Hiroshima University Hospital (Hiroshima, Japan) or an affiliated hospital. Tumor staging was according to the TNM classification system. Because written informed consent was not obtained, for privacy protection, identifying information for all samples was removed before analysis. This was in accordance with the Ethical Guidelines for Human Genome/Gene Research of the Japanese Government.

Immunohistochemistry

A Dako LSAB Kit (Dako, Carpinteria, CA, USA) was used for immunohistochemical analysis. In brief, microwave pretreatment in citrate buffer was carried out for 15 min to retrieve antigenicity. After peroxidase activity was blocked with 3% H2O2-methanol for 10 min, sections were incubated with normal goat serum (Dako) for 20 min to block non-specific antibody binding sites. Sections were incubated with mouse monoclonal anti-EGFR (1:20; Novocastra, Newcastle, UK) or goat polyclonal anti-LI-cadherin (1:50; Santa Cruz Biotechnology). After a 10-min incubation with substrate–chromogen solution, sections were counterstained with 0.1% hematoxylin. The percentage of stained cancer cells was evaluated for each antibody. A result was considered positive if at least 10% of the cells were stained. When fewer than 10% of cancer cells were stained, the immunostaining was considered negative.

For double-immunofluorescence staining, Alexa Fluor 546-conjugated anti-goat IgG and Alexa Fluor 488-conjugated anti-mouse IgG (Molecular Probes, Eugene, OR, USA) were used as secondary antibodies.

Phenotypic analysis of GC

Gastric cancers were classified into four phenotypes: G type; I type; GI type; and N type. For phenotypic expression analysis of GC, we analyzed immunohistochemistry (as described above) with four antibodies, anti-MUC5AC, anti-MUC6, anti-MUC2, and anti-CD10 (all Novocastra). Gastric cancers in which more than 10% of cells in the section expressed at least one gastric epithelial cell marker (MUC5AC or MUC6) or intestinal epithelial cell marker (MUC2 or CD10) were classified as G type or I type cancers, respectively; sections that showed both gastric and intestinal phenotypes were classified as GI type; and those that lacked both gastric and intestinal phenotypes were classified as N type.

RNA interference and cell growth and in vitro invasion assays

To knock down endogenous CDH17, RNAi was carried out using CDH17 and negative control siRNA oligonucleotides (Invitrogen). For MTT assays to monitor cell growth, cells were seeded at 2000 cells per well in 96-well plates.[25] Modified Boyden chamber assays were carried out to examine invasiveness. Transiently transfected cells were plated at 1 × 106 cells per well in RPMI-1640 medium with no serum in the upper chamber of a Transwell insert (8-μm pore diameter; Chemicon, Temecula, CA, USA) coated with Matrigel. Medium containing 10% serum was added in the bottom chamber. After incubation at 37°C for 24 and 48 h, cells in the upper chamber were removed by scraping, and the cells remaining on the lower surface of the insert were stained with CyQuant GR dye (Chemicon, Temecula, CA, USA) to assess the number of cells.

Statistical methods

Correlations between clinicopathologic parameters and LI-cadherin protein expression were analyzed by Fisher's exact test. P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Oligonucleotide microarray analysis

To identify GC-related genes whose expression was regulated by EGFR activation, we analyzed the gene expression profiles from TGF-α and EGF-treated HT-29 and non-treated HT-29. Previously, we showed activation of LI-cadherin expression by forced expression of CDX2 in the HT-29 colon cancer cell line;[16] however, in GC cell lines, forced expression of CDX2 could not activate LI-cadherin expression (Naohide Oue, unpublished data, 2010). Therefore, the correlation between EGFR and GC-related genes was investigated in HT-29 cells. Expression levels of 208 individual genes were compared between these two profiles. Six genes were identified that were expressed significantly higher (more than twice) in HT-29 cells treated with TGF-α or EGF, than in non-treated cells (Table 1). In EGFR ligand-treated HT-29 cells, no gene showed lower expression than in non-treated HT-29.

Table 1. Six genes upregulated by both epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) treatment in HT-29 cells
SymbolDescriptionGenbank accession no.IntensityFold changeIntensityFold change
No treatTGF-αNo treatEGF
  1. No treat, control cells that did not receive treatment.

VEGFVascular endothelial growth factorNM_003376.33.99.12.43.917.34.5
CDH17Cadherin 17, LI cadherin (liver–intestine)NM_0040632.76.22.32.77.72.9
CDKN1ACyclin-dependent kinase inhibitor 1A (p21, Cip1)NM_00038915.239.82.615.241.82.7
IL8Interleukin 8NM_000584.27.726.33.47.716.22.1
CTSLCathepsin LNM_0019125.514.52.65.511.12.0
ABTB2Ankyrin repeat and BTB (POT) domain containing 2NM_145804.16.321.23.46.312.52.0

Epidermal growth factor receptor ligands induce LI-cadherin expression

Among six genes whose expression was upregulated in both TGF-α- and EGF-treated cells, we focused on CDH17. As shown in Figure 1(a), LI-cadherin expression detected by Western blotting was induced by EGF or TGF-α treatment in both HT-29 and MKN-74 cell lines. It has been reported that CDX2 regulates LI-cadherin expression, however, the potential interplay between CDX2 and EGFR signaling pathways has not been investigated. Expression of LI-cadherin in both HT-29-neo and HT-29-CDX2 cell lines was induced after TGF-α or EGF treatment; basal levels of LI-cadherin expression were higher in HT-29-CDX2 than in HT-29-neo cells (Fig. 1b). Quantitative RT-PCR analyses also revealed CDX2 expression was reduced and not induced after treatment with TGF-α or EGF. However, LI-cadherin expression was induced by treatment with TGF-α or EGF (Fig. 2a). A similar tendency was observed in HT-29-neo (data not shown) and HT-29-CDX2 (Fig. 2a) cells. We also examined MUC2, villin, and CD10 expression in HT-29 with TGF-α or EGF treatment. However, induction of these intestinal differentiation markers was not detected. These findings suggest that induction of LI-cadherin expression induced by TGF-α or EGF treatment may occur in a CDX2-independent manner. The induction of LI-cadherin in response to TGF-α or EGF treatment was then studied at a transcriptional level with an LI-cadherin promoter luciferase assay. HT-29 cells were cotransfected with a 0.5-kb human LI-cadherin promoter-driven luciferase construct and an SV40-directed Renilla construct as a control. At 48 h after transfection, cells were stimulated with TGF-α or EGF, resulting in a three and fourfold increase, respectively, in LI-cadherin promoter activity. In our previous observations, four CDX2 binding sites were found in the 0.5-kb human LI-cadherin promoter.[16] We also reported that when human LI-cadherin promoter-driven luciferase constructs with mutations in all four CDX2 binding sites were transfected, luciferase activity was eliminated. To investigate CDX2-independent induction of LI-cadherin by TGF-α or EGF treatment, HT-29 cells were transfected with a LI-cadherin promoter-driven luciferase construct with mutations in all four CDX2 binding sites. Following this transfection, cells stimulated with TGF-α or EGF also showed a three and fourfold increase, respectively, in LI-cadherin promoter activity (Fig. 2b). The similar upregulation of LI-cadherin promoter activity was observed in case of using HT-29-CDX2 (data not shown). We also examined LI-cadherin promoter activity using MKN-74 under the same conditions, and found that cells stimulated with TGF-α or EGF also showed a three and fourfold increase, respectively, in LI-cadherin promoter activity. This implies that EGFR signaling induced LI-cadherin transcription independently of CDX2.

image

Figure 1. Induction of liver–intestine cadherin (LI-cadherin) expression in cancer cells treated with epidermal growth factor receptor (EGFR) ligands. (a) LI-cadherin expression in HT-29 and MKN-74 cell lines after epidermal growth factor (EGF)/transforming growth factor-α (TGF-α treatment. (b) LI-cadherin expression in HT29-neo and HT29-caudal type homeobox 2 (CDX2) cell lines after EGF/TGF-α treatment. Western blot analysis confirmed temporal induction of LI-cadherin after EGF/TGF-α treatment, regardless of CDX2 expression.

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image

Figure 2. Effect of epidermal growth factor (EGF)/transforming growth factor-α (TGF-α) treatment on cadherin 17 (CDH17) and caudal type homeobox 2 (CDX2) expression. (a) Quantitative RT-PCR analysis. Strong induction of CDH17 was observed in both HT-29 and HT-29-CDX2 cells after EGF or TGF-α treatment without the upregulation of CDX2. (b) Liver–intestine cadherin (LI-cadherin) promoter reporter assays. Bars and error bars, mean and SE, respectively, of three different experiments. No treat, negative control. In the right-hand graph, luciferase activity of EGF and TGF-α were standardized by normalizing the strain that did not receive treatment (no treat) at 1.0. Empty, empty vector transfected HT-29 cells; Mutant, HT-29 or MKN-74 transfected with a LI-cadherin promoter-driven luciferase construct with mutations in all four CDX2 binding sites; Wild type, HT-29 or MKN-74 transfected with a LI-cadherin promoter-driven luciferase construct with no mutations in all CDX2 binding sites.

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Expression and distribution of EGFR and LI-cadherin in GC tissue

To examine the correlation between EGFR and LI-cadherin expression in GC tissue, we carried out immunohistochemical staining of LI-cadherin and EGFR in 152 surgically resected GC tissue samples. Staining of both LI-cadherin and EGFR occurred at the cell membrane. Of 152 GC cases, LI-cadherin and EGFR were expressed in 58 (38%) and 33 (22%) cases, respectively (Fig. 3a,b). In relation to CDX2, CDX2 expression was observed in almost all LI-cadherin-positive cases, and almost all gastric cancer cells simultaneously expressed CDX2 and LI-cadherin. However, LI-cadherin positive cell contained about a few CDX2 negative cells, CDX2-negative cells frequently expressed EGFR (Fig. 3a–c). In light of these findings, we suspect that EGFR contribute to induce LI-cadherin expression in GC at least partially. Coexpression of LI-cadherin and EGFR was observed in some GC cells by double-immunofluorescence staining (Fig. 3d). In EGFR-positive GC cells, expression of LI-cadherin was frequently found. However, in LI-cadherin-positive GC cells, EGFR was not always detected. In total, of 33 EGFR-positive cases, 24 (73%) cases were LI-cadherin-positive, whereas of 119 EGFR-negative cases, only 34 (29%) cases were LI-cadherin-positive (P < 0.0001, Table 2).

image

Figure 3. Immunohistochemical analysis of epidermal growth factor receptor (EGFR) and liver–intestine cadherin (LI-cadherin) in gastric cancer (GC) and phenotypic analysis of EGFR- and LI-cadherin-positive GC cases. Expression pattern of EGFR (a), caudal type homeobox 2 (CDX2) (b), and LI-cadherin (c) (original magnification, ×400). Arrows indicate EGFR-positive (a), LI-cadherin-positive (b), and CDX2-negative (c) GC cells. (d) Double immunofluorescence staining shows that EGFR and LI-cadherin were coexpressed in some GC cells. Blue, DAPI; green, EGFR; red, LI-cadherin. (e) Distribution of gastric (G), intestinal (I), gastric and intestinal mixed (GI), and unclassified (N) phenotypes of GC in EGFR- and LI-cadherin-positive cases. Both EGFR and LI-cadherin expression were more frequently found in GC with intestinal features (I or GI type) than the others. P-values were analyzed by Fisher's exact test.

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Table 2. Expression of liver–intestine cadherin (LI-cadherin) and epidermal growth factor receptor (EGFR) in gastric cancer tissue
 EGFRP-valuea
PositiveNegative
  1. a

    Fisher's exact test.

LI-cadherin
Positive2434<0.0001
Negative985

Clinicopathological characteristics of LI-cadherin-positive and EGFR-positive GC. The relationship of LI-cadherin staining to clinicopathological characteristics were investigated (Table 3). The LI-cadherin staining was observed more frequently in stage I/II cases (16/41, 39%) than in stage III/IV cases (6/39, 15%; P = 0.0243, Fisher's exact test). Moreover, LI-cadherin staining was detected more frequently in intestinal-type GC (20/46, 46%) than in diffuse-type GC (2/34, 6%; P < 0.0001, Fisher's exact test). In addition to the Lauren histology-based classification, GC can be subdivided into four phenotypes according to mucin expression. Gastric and intestinal markers were detected in 67 of 152 (44%) cases for MUC5AC, 16 (11%) cases for MUC6, 46 (31%) cases for MUC2, and 16 (11%) cases for CD10. We further investigated the association between LI-cadherin expression and mucin phenotype, because LI-cadherin was detected in intestinal metaplasia of the stomach and colon. Expression of both EGFR and LI-cadherin was found more frequently in GC of I and GI types than GC of G and GI types (Fig. 3d). In the group of 59 advanced GC patients, EGFR expression had significant prognostic impact. However, no significant prognostic impact was found for LI-cadherin expression in GC cases (data not shown).

Table 3. Relationship between liver–intestine cadherin (LI-cadherin) or epidermal growth factor receptor (EGFR) and clinicopathologic characteristics of gastric cancer
 LI-cadherinEGFR
Positive (%)NegativeP-valueaPositive (%)NegativeP-valuea
  1. a

    Fisher's exact test.

T grade
T110 (23)330.02540 (0)43<0.0001
T2/3/448 (44)5133 (30)76
N grade
N017 (27)460.01866 (10)570.0025
N1/2/341 (46)4827 (30)62
Stage
I/II25 (31)550.068910 (13)700.0054
III/IV33 (46)3923 (32)49
Histology
Intestinal33 (43)430.242411 (14)650.0481
Diffuse25 (33)5122 (29)54

Effect of LI-cadherin inhibition on cell growth and invasive activity

The LI-cadherin-positive GC cases were observed more frequently in stage III/IV than in stage I/II, suggesting that LI-cadherin could be associated with tumor progression. However, the biological significance of LI-cadherin in GC has not been studied. We carried out an MTT assay 8 days after LI-cadherin siRNA transfection in the HSC-57 cell line. HSC-57 cells were selected for high LI-cadherin expression. We confirmed that LI-cadherin siRNA-transfected HSC-57 cells showed significantly reduced LI-cadherin expression (data not shown). Cell viability was not significantly different between LI-cadherin siRNA-transfected and negative control GC cells (data not shown). To determine the possible role of LI-cadherin in GC cell invasiveness, a Transwell invasion assay was carried out in the HSC-57 cell line. There was no significant difference in invasion between LI-cadherin knockdown and negative control GC cells (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

In the present study, by using a customized oligonucleotide microarray, we identified six GC-related genes (VEGF, CDH17, CDKN1, IL8, CTSL, and ABTB2) whose expression was upregulated by EGFR activation. Among these genes, we successfully showed that EGFR activation induced LI-cadherin expression. In addition, LI-cadherin induction occurred in a CDX2-independent manner, and additional induction of LI-cadherin expression was observed in CDX2-transfected cells. These results suggest that high expression of LI-cadherin is required for EGFR activation as well as CDX2 expression. Because the EGFR signaling pathway forms a wide-ranging network, it remains unclear which component of the signaling pathway directly regulates LI-cadherin. This requires further study.

In normal colon, expression of EGFR and LI-cadherin was found. Both TGF-α and p21waf1/cip1 protein expression is only detected in the top one-third of the crypt, which is only composed of terminally differentiated cells.[26, 27] In the present study, expression of p21waf1/cip1 was also induced by TGF-α and EGF treatment (data not shown). It has been reported that p21waf1/cip1 is associated with the processes of cell-cycle arrest, apoptosis, and differentiation.[28] Taken together, these results suggest that EGFR activation induces intestinal differentiation. High expression of LI-cadherin may have some effect on terminal differentiation of colonic epithelial cells. However, despite induction of p21waf1/cip1 expression, cell growth activity was upregulated in HT-29 cells (data not shown). To clarify whether p21waf1/cip1 is involved in cell growth inhibition of HT-29, further studies are required.

In GC, LI-cadherin overexpression has been reported to be associated with lymph node metastasis.[29] In contrast, in colorectal cancer, reduced expression of LI-cadherin is frequently found in cases with lymph node metastasis or poor survival. Thus, the functions of LI-cadherin in human cancers are controversial and unclear. In the present study, we carried out MTT and Transwell invasion assays after LI-cadherin knockdown in the HSC-57 cell line. However, cell viability and invasion ability were not significantly altered. To clarify the biological and clinical significance of EGFR and LI-cadherin expression in GC, we examined the expression of these two molecules in GC tissue through immunohistochemistry and compared this with clinicopathologic parameters, prognosis, and mucin phenotype. Our data for the relation between molecular expression and tumor stage in all of our GC cases were consistent with the results of previous studies.[30] With regard to mucin phenotype, both EGFR and LI-cadherin expression were detected more frequently in I-type GC, and this finding is in accordance with previous studies.[15] Many studies have implicated the EGFR signaling pathway in the regulation of intestinal epithelial cell growth and differentiation.[31-33] Animals that are EGFR-null die early in postnatal life and show severe defects in intestinal cell proliferation and organization, along with many other abnormalities.[31] Overexpression or mutation of EGFR has been associated with many different carcinomas, including colonic carcinoma.[34] These observations indicate that the EGFR signaling pathway plays an important role in regulating intestinal epithelial cell production. In the light of these previous studies and ours, it is suspected that EGFR drives GC development through wide-ranging signaling pathways, and also induces intestinal phenotypes in GC by stimulation of LI-cadherin expression.

In summary, this study yielded a list of genes that were upregulated by EGFR activation in GC. We found that LI-cadherin is induced by EGFR, and overexpression of LI-cadherin is associated with the intestinal mucin phenotype. These results suggest that, in addition to CDX2, EGFR activation is involved in LI-cadherin expression in GC, and that EGFR may induce intestinal differentiation through upregulating LI-cadherin expression in a CDX2-independent manner.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

We thank Mr Shinichi Norimura for his excellent technical assistance and advice. This work was carried out with the kind cooperation of the Research Center for Molecular Medicine, Faculty of Medicine, Hiroshima University (Hiroshima, Japan). We thank the Analysis Center of Life Science, Hiroshima University, for the use of their facilities. This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Culture, Science, Sports, and Technology of Japan, in part by a Grant-in-Aid for the Third Comprehensive 10-Year Strategy for Cancer Control and for Cancer Research from the Ministry of Health, Labor and Welfare of Japan, and in part by a grant (07-23911) from the Princess Takamatsu Cancer Research Fund.

Abbreviations
CDH17

cadherin 17

CDX2

caudal type homeobox 2

EGF

epidermal growth factor

EGFR

epidermal growth factor receptor

G type

gastric type

GC

gastric cancer

GI type

gastric and intestinal mixed type

I type

intestinal type

LI-cadherin

liver–intestine cadherin

N type

unclassified type

TGF-α

transforming growth factor-α

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References