EphrinA5 suppresses colon cancer development by negatively regulating epidermal growth factor receptor stability

Authors

  • Tong-Hong Wang,

    1.  Tissue Bank, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan
    Search for more papers by this author
    • These authors contributed equally to this work

  • Junn-Liang Chang,

    1.  Department of Pathology & Laboratory Medicine, Taoyuan Armed Forces General Hospital, Tao-Yuan, Taiwan
    2.  Biomedical Engineering Department, Ming Chuang University, Tao-Yuan, Taiwan
    Search for more papers by this author
    • These authors contributed equally to this work

  • Jar-Yi Ho,

    1.  Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
    Search for more papers by this author
    • These authors contributed equally to this work

  • Hsiao-Chun Wu,

    1.  Tissue Bank, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan
    Search for more papers by this author
  • Tse-Ching Chen

    1.  Tissue Bank, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan
    2.  Department of Anatomic Pathology, Chang Gung Memorial Hospital, Chang Gung University School of Medicine, Tao-Yuan, Taiwan
    Search for more papers by this author

T.-C. Chen, Department of Anatomic Pathology, Chang Gung Memorial Hospital, 5, Fu-Shin St, Kwei-Shan, Tao-Yuan 333, Taiwan
Fax: +886 3 3280147
Tel: +886 3 3281200 ext. 2730
E-mail: ctc323@adm.cgmh.org.tw

Abstract

Colon cancer is one of the most common human cancers worldwide. Owing to its aggressiveness and lethality, it is necessary to determine the mechanisms regulating the carcinogenesis of colon cancer. EphrinA5 has been reported to act as a putative tumor suppressor in glioma; however, little is known concerning the role of this protein in the context of colon cancer. To elucidate the biological significance of ephrinA5 in colon cancer, we examined ephrinA5 and epidermal growth factor receptor (EGFR) expression profiles in both colon cancer and normal tissues, using immunohistochemistry on a 96-spot tissue microarray. Gain-of-function and loss-of-function experiments were performed on the human colon cancer cell lines SW480 and WiDr to determine the biological effects of ephrinA5 in relation to cell proliferation, survival, and migration. It was found that ephrinA5 mRNA and protein levels were significantly reduced in colon cancer as compared with normal colon tissue specimens. EphrinA5 expression was also negatively associated with tumor differentiation and clinical stage. In colon cancer cell line models, ephrinA5 exerted an inhibitory effect on EGFR by promoting c-Cbl-mediated EGFR ubiquitination and degradation. EphrinA5 did not affect the transcriptional regulation of EGFR mRNA expression in colon cancer cells. Expression of ephrinA5 suppressed colon cancer cell proliferation, migration, and chemotherapeutic resistance. In conclusion, ephrinA5 inhibited colon cancer progression by promoting c-Cbl-mediated EGFR degradation. Our findings identify a novel mechanism that could be utilized to improve the therapeutic efficiency of EGFR-targeting strategies.

Abbreviations
ACP

acid phosphatase

EGFR

epidermal growth factor receptor

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

siRNA

small interfering RNA

Introduction

Colon cancer is the third most common human cancer diagnosed in developed countries [1]. Colon cancer also ranks third among life-threatening and fatal malignancies in Taiwan, with a 5-year survival rate of 53–57% over the past decade. This survival rate, however, declines to < 10% in patients with distant metastases at the time of diagnosis [2]. The underlying pathogenesis of this disease remains poorly understood [1,2]. Previous clinical studies have shown that epidermal growth factor receptor (EGFR) overexpression occurs in the invasive front of high-stage colorectal cancers, indicating that EGFR is a critical risk factor for colon cancer progression [3,4].

The Eph receptors constitute the largest family of receptor protein tyrosine kinases, and interact with their ephrin ligands to form a bidirectional, cell-to-cell signaling communication system designated as ‘forward’ receptor signaling and ‘reverse’ ephrin signaling. The Eph receptors and ephrins are divided into two subclasses (A and B), which are encoded by nine EphA, five EphB, five ephrinA and three ephrinB genes in the human genome. EphrinA ligands are tethered to the outer leaflet of the plasma membrane by a glycosyl-phosphatidylinositol anchor, and ephrinB ligands possess a transmembrane domain and a short cytoplasmic tail [5,6]. Intercellular Eph–ephrin signaling is fundamentally involved in developmental processes that require organized patterning and movement of cells, especially in the development of the central nervous system [7–9] and in the remodeling of blood vessels [10–12]. Eph–ephrin signaling is also essential for the correct formation of crypts and villi in the intestinal epithelium [13–15]. Several lines of evidence also suggest that Eph–ephrin signaling is involved in tumorigenesis, invasiveness, metastasis, and angiogenesis [10,16–18].

EphrinA5 was recently identified as a tumor suppressor that is significantly downregulated in several types of human carcinoma [19–23]; however, ephrinA5 was also reported to be overexpressed in breast cancer carcinoma cell lines [24], and was associated with poor prognosis in both ovarian cancer [25] and pancreatic ductal adenocarcinoma [26]. Recently, ephrinA5 has been shown to regulate EGFR in gliomas by promoting c-Cbl-mediated EGFR degradation [21]. Dysregulated EGFR signaling in various types of malignancy, either through overexpression or constitutive activation, promotes cancer cell proliferation, survival, invasiveness, metastasis, and angiogenesis. Misregulation of EGFR signaling is associated with poor prognosis in many human malignancies, and several therapeutic remedies have been developed to suppress EGFR signaling [27–33].

In this study, we investigated the association between ephrinA5 and colon cancer progression. Our data identified mutually exclusive expression profiles of ephrinA5 and EGFR in colon cancer tissues. We also found that ephrinA5 can suppress EGFR function through c-Cbl-mediated EGFR ubiquitination and degradation, and reduce colon cancer cell proliferation, migration, and chemotherapeutic resistance.

Results

EphrinA5 is downregulated in colon cancer

In order to elucidate the biological significance of ephrinA5 in the progression of colon cancer, we analyzed ephrinA5 expression levels with immunohistochemistry on a 96-spot tissue microarray slide composed of 10 normal colon tissues, 14 benign colon lesions (adenomas and polyps), and 72 colon malignancies (68 colon adenocarcinomas and four non-Hodgkin lymphomas). EphrinA5 expression levels were significantly reduced in cancer tissue as compared with normal tissue or benign lesions (P < 0.001). Although ephrinA5 was slightly downregulated in benign lesions, there was no significant difference between normal colon tissues and benign lesions (Table 1). Figure 1A is representative of immunohistochemical staining for ephrinA5 and EGFR in a colon adenocarcinoma. In contrast, EGFR was significantly upregulated in colon cancer as compared with normal tissue or benign lesions (P = 0.001). We also examined the mRNA expression levels of ephrinA5 in 14 paired frozen colon adenocarcinoma specimens. As shown in Fig. 1B, ephrinA5 mRNA levels were significantly reduced by up to 50–90% in tumor tissues as compared with normal colon tissue (P = 0.04).

Table 1.    Association between ephrinA5, EGFR expression and clinicopathological parameters. CI, confidence interval; HR, hazard ratio; IHC, immunohistochemistry. P-values: benign lesion or colon cancer versus normal. The statistical significance (P < 0.05) is shown in bold.
Subject characteristicsNormal (n = 10)Benign lesion (n = 14)UnivariateaMultivariateaCancer (n = 72)UnivariatebMultivariateb
HR (95% CI)PHR (95% CI)PHR (95% CI)PHR (95% CI)P
  1. a Cancer versus normal. b IHC data were analyzed as categorized variables on the basis of Cox’s hazard regression model. Comparisons were performed among three groups: (a) colon cancer (n = 72); (b) benign lesions (adenomas/polyps) (n = 14); (c) normal (n = 10).

Age (years) (mean)49.6 ± 18.5 41.1 ± 17.1 0.97 (0.92–1.02)0.2420.98 (0.92–1.03)0.38160.3 ± 13.2 1.05(1.01–1.10)0.0281.070(1.01–1.13)0.019
SexMale (8)Female (2)Male (10)Female (4)1.60 (0.23–11.08)0.6341.43 (0.19–10.99)0.734Male (36)Female (36)4.00 (0.79–20.15)0.0934.25 (0.72–25.27)0.111
EphrinA5 IHC intensityPositive (2+, 3+)Negative (0, 1+)Positive (2+, 3+)Negative (0, 1+)    Positive (2+, 3+)Negative (0, 1+)    
82685.33 (0.82–34.83)0.0804.00 (0.56–28.46)0.16624488.00(1.575–40.63)0.0126.09(1.02–36.26)0.047
EGFR IHC intensityNegative (0, 1+)Positive (2+, 3+)Negative (0, 1+)Positive (2+, 3+)          
82863.00 (0.46–19.59)0.2512.28 (0.31–16.72)0.41828446.29(1.24–31.78)0.0263.902 (0.66–22.95)0.132
Figure 1.

 (A) Representative immunohistochemical results for ephrinA5 and EGFR from tissue microarray analysis of colon cancer. EphrinA5 and EGFR were expressed with mutually exclusive distributions within colon cancer tissue and normal colon tissue, respectively. White arrows indicate normal colonic mucosa. (B) Relative ephrinA5 mRNA levels of 14 pairs of colon adenocarcinoma tissues normalized with GAPDH. EphrinA5 was also transcriptionally downregulated in most colon cancer specimens to a greater extent than in normal colon tissues; only sample CO001 had modestly higher ephrinA5 expression in tumor specimens.

EphrinA5 downregulation and EGFR upregulation are correlated with tumor differentiation and clinical stage

To investigate the clinical significance of ephrinA5 downregulation, we analyzed the association between ephrinA5 and the clinicopathological parameters of the 10 normal colon tissues, 14 benign colon lesions (adenomas and polyps), and 72 colon cancer samples. Univariate and multivariate analyses were performed with Cox’s hazard regression model (Table 1). EphrinA5 downregulation, EGFR upregulation and older age were positively associated with colon cancers; however, only ephrinA5 and age were identified as independent risk factors by the multivariate regression model. This indicated that ephrinA5 was significantly downregulated during the genesis of colon cancer. EGFR levels, however, were not statistically significant according to multivariate analysis, indicating that EGFR may interact with other risk factors during the process of carcinogenesis.

To determine the role of ephrinA5 downregulation in the process of cancer progression, we further analyzed the correlation between ephrinA5 and cancer prognostic factors in 68 colon adenocarcinomas, including 24 grade 1, 26 grade 2 and six grade 3 adenocarcinomas. Of these tumors, 38 were at stage I, four were at stage II, and 26 were at stage III. EphrinA5 downregulation was significantly correlated with higher stage (P = 0.047) and poorer differentiation (P = 0.012) by Cox regression model analysis (Table 2). Additionally, most (24/26) of the stage III samples exhibited reduced ephrinA5 expression as compared with stage I + II samples (23/42). As expected, ephrinA5 levels were not associated with patient age or gender (Table 2). EGFR overexpression was not observed to be associated with colon cancer differentiation (P = 0.206) or clinical stage (P = 0.756) in these samples (Cox’s regression model).

Table 2.   Association between ephrinA5 expression and cancer prognostic factors (for carcinoma patients only). On the basis of Cox regression model analysis, the statistical significance (P < 0.05) is shown in bold. CI, confidence interval; HR, hazard ratio; IHC, immunohistochemistry.
EphrinA5 IHC intensityNegative (0, 1+) (n = 47)Positive (2+, 3+) (n = 21)UnivariateMultivariate
HR (95% CI)PHR (95% CI)P
  1. a There were 12 mucinous adenocarcinomas without grade records.

Age (years) (mean)60.66 ± 14.0360.71 ± 12.311.000 (0.962–1.039)0.9881.011 (0.959–1.066)0.680
Sex
 Male22101 (ref.)   
 Female25111.033 (0.369–2.895)0.9513.858 (0.655–22.720)0.136
Gradea
 I12121 (ref.)   
 II + III2571.890 (1.059–3.373)0.0313.006 (1.274–7.091)0.012
Stage
 I + II23191 (ref.)  
 III2423.148 (1.440–6.886)0.0042.438 (1.012–5.871)0.047

EphrinA5 reduces EGFR protein expression and phosphorylation

EphrinA5 has been reported to be downregulated in glioma cells and to function as a tumor suppressor by negatively regulating EGFR expression [21]. To determine whether a similar regulatory mechanism between ephrinA5 and EGFR exists in colon cancer, we examined the protein expression levels of ephrinA5 and EGFR with immunohistochemistry. As shown in Fig. 1A, ephrinA5 was highly expressed in normal colon tissues and downregulated in cancerous lesions. In the same regions, EGFR was observed to be overexpressed. We found there were 45.5% (31/68) colon cancer specimens exhibiting a mutually exclusive expression pattern of ephrinA5 and EGFR. Additionally, ephrinA5 expression was also significantly negatively correlated with EGFR levels in those samples (Spearman’s rho correlation = −0.367, P < 0.0001). We therefore utilized a gain-of-function and a loss-of-function experimental strategy to verify the clinical observation of ephrinA5–EGFR interaction within colon cancer cell line models. Human ephrinA5 was cloned into the pIRESneo mammalian expression vector for transfection of ectopic human ephrinA5 into the colon cancer cell lines. Forced ectopic ephrinA5 expression significantly reduced endogenous EGFR protein and phosphorylation levels in cancer cell lines as compared with a vector control, as detected by both western blot and immunofluorescence staining (Fig. 2A,B). We further examined the influence of ephrinA5 on EGFR expression at the transcriptional level. Forced ectopic ephrinA5 expression did not alter endogenous EGFR mRNA expression (Fig. 2C). We also observed that ephrinA5 knockdown by specific small interfering RNA (siRNA) did not influence EGFR mRNA expression in colon cancer cell lines (Fig. 2C). These results indicate that ephrinA5 downregulates EGFR expression at the protein level and not at the mRNA level.

Figure 2.

 Human ephrinA5 was constructed in the pIRESneo vector and transfected into SW480 cells at 3 × 105 cells per well. The cells were seeded in a six-well plate and transfected with 1 μg pIRESneo-ephrinA5 plasmid. After 48 h of incubation, cells were harvested for western blotting or immunofluorescence staining. Ectopic ephrinA5 reduced EGFR expression as compared with the vector control according to both (A) western blot and (B) immunofluorescence staining. The phosphorylation level of EGFR was also examined by western blot, and β-actin was used as the loading control. CD71 was a marker for membranous localization in immunofluorescence staining. (C) Gain-of-function and loss-of-function experiments with ephrinA5 revealed no transcriptional modification of EGFR. Gain-of-function testing (left) was performed in pIRESneo-ephrinA5-transfected SW480 cells in comparison with vector control. Loss-of-function testing (right) was performed with ephrinA5 siRNA-treated SW480 cells in comparison with scrambled siRNA-treated controls. GAPDH was used as a loading control for quantitative real-time PCR.

EphrinA5 reduces EGFR expression by promoting c-Cbl-mediated EGFR degradation

The E3 ubiquitin ligase c-Cbl is required for the ubiquitin-dependent degradation of EGFR, and ephrinA5 may enhance c-Cbl binding to EGFR to promote ubiquitination and degradation of the receptor. We therefore examined the regulatory effects of ephrinA5 on c-Cbl in colon cancer cell lines. Forced ectopic ephrinA5 expression resulted in a greater than two-thirds reduction in endogenous EGFR protein levels. In the reverse experiment, c-Cbl knockdown by siRNA significantly rescued EGFR protein levels (Fig. 3A,B). EphrinA5 overexpression did not alter c-Cbl mRNA levels (Fig. 3C,D), indicating that ephrinA5 promotes c-Cbl binding to EGFR to facilitate EGFR ubiquitination and degradation in colon adenocarcinomas.

Figure 3.

 (A) Ectopic ephrinA5 reduced endogenous EGFR protein expression in SW480 cells, and EGFR protein expression was rescued after c-Cbl knockdown by siRNA. (B) Quantitative western blot data. Student’s t-test was employed to compare the differences between treated and untreated groups. Experiments for each group were performed in triplicate. (C) Ectopic ephrinA5 did not affect c-Cbl mRNA levels in RT-PCR. (D) Quantitative RT-PCR data. Student’s t-test was employed to compare the differences between treated and untreated groups. Experiments for each group were performed in triplicate. β-Actin and GAPDH were used as loading controls for western blotting and RT-PCR, respectively.

EphrinA5 inhibits cancer cell proliferation and migration

EGFR overexpression was observed in several types of cancer and was associated with cancer cell growth. We also found that EGFR expression was significantly increased in colon cancers. EphrinA5 reduced EGFR protein levels by facilitating its degradation, indicating that ephrinA5 may function to repress cancer cell growth. Here, we examined the inhibitory effects of ephrinA5 on colon cancer cells. First, we analyzed the effect of ephrinA5 expression on colon cancer cell growth with the acid phosphatase (ACP) assay. Forced expression of ephrinA5 significantly reduced cellular proliferation in the colon cancer cell lines SW480 and WiDr (Fig. 4A). We observed similar suppressive effects in HEK cells (data not shown). Ectopic expression of ephrinA5 also increased doxorubicin sensitivity in SW480 cells, and flow cytometric analysis revealed a sub-G1 increment (Fig. 4B). A reduction in the expression of ephrinA5 could therefore increase cancer cell survival under conditions of cytotoxic stress.

Figure 4.

 (A) Determination of cell proliferation of colon cancer cell lines SW480 and WiDr by ACP assay. One microgram of pIRESneo-ephrinA5 or pIRESneo vector was transfected into 3 × 105 cells per well, and analysis was performed at the indicated time points. The expression of ephrinA5 was also analyzed by western blot at 48 h after tansfection. Ectopic forced ephrinA5 expression significantly reduced cell growth in both SW480 and WiDr cells as compared with the vector control or mock controls (all P < 0.05, Student’s t-test). (B) Cell apoptosis assays were evaluated with propidium iodide staining and flow cytometry to compare the sub-G1 groups. Ectopic forced ephrinA5 expression increased the doxorubicin sensitivity of SW480 cells, which showed a significant difference in sub-G1 groups between pIRESneo control (sub-G1: 14.82%) and pIRESneo-ephrinA5 (sub-G1: 36.28%). (C) Expression patterns of Eph receptors in colon cancer tissues. Colon cancer cell lines, primary colon cancers and paratumoral tissues were analyzed by RT-PCR. EphA2 expression was found in all tumor and paratumoral colon tissues. EphB2 and A3 were also present in the majority of colon cancers. (D) Wound-healing was compared between pIRESneo-ephrinA5-transfected SW480 cells and vector controls. More than 90% confluent cells were scraped with a p200 tip, and photographed at 0, 24, and 48 h. Ectopic ephrinA5 inhibited the wound-healing ability of SW480 cells. (E) Transwell assay was compared between pIRESneo-ephrinA5-transfected SW480 cells and their vector controls. A total of 5 × 104 cells were plated in transwell inserts and cultured for 24 h. Data were analyzed with Student’s t-tests; ectopic ephrinA5 expression significantly reduced the cell migratory ability of SW480 cells. DOX, doxorubicin.

Next, we analyzed the expression of EphB2, EphA2, EphA3, and EphA5, the preferred receptors for ephrinA5, in the SW480 and WiDr cell lines. Expression of EphB2, EphA2, and EphA3, but not of EphA5, was detected in both cell lines. EphA2 expression was also found in all tumor and paratumoral colon tissues (Fig. 4C). EphB2 and EphA3 were present in the majority of colon cancers. This suggests that ephrinA5 is able to exhibit its suppressive effect through its Eph receptors in colon cancer.

In addition to promoting cell growth, EGFR has also been reported to be involved in cancer cell migration and invasiveness [31,32]. Here, we examined the regulatory role of ephrinA5 in cancer cell migration. We found that ephrinA5 inhibited cancer cell migration as detected by both wound healing (Fig. 4D) and transwell migration assays with SW480 cells (Fig. 4E). Taken together, these results suggest that ephrinA5 represses both cancer cell proliferation and migration. Thus, ephrinA5 may function as a tumor suppressor by decreasing EGFR stability and negatively regulating the progression of colon carcinomas.

Discussion

Eph/ephrin family members are well documented as being important for the development of the neural system. In this study, we found that ephrinA5 was significantly downregulated at both the transcriptional and translational levels in colon cancers. EphrinA5 downregulation was significantly associated with poor tumor differentiation and higher clinical stage. EphrinA5 has been reported to function as a putative tumor suppressor in glioma [21], chondrosarcoma [22], and leukemia [23]. In contrast, ephrinA5 was reported to be upregulated in breast cancer cell lines [24] and osteosarcoma [34], and ephrinA5 overexpression was associated with poor prognosis in both ovarian cancer [25] and pancreatic adenocarcinoma [26]. Therefore, the clinical significance of ephrinA5 appears to be dependent upon cancer type. In this study, we are the first to report ephrinA5 downregulation as an independent risk factor in colon cancer, where its clinical significance seems to be more important than that of EGFR. Additionally, we found that ephrinA5 was gradually downregulated during tumor progression. Therefore, ephrinA5 may act as one of the important checkpoints for cell proliferation and differentiation in colon cancers.

In this study, we also found that ephrinA5 suppressed EGFR function through c-Cbl-mediated EGFR degradation, resulting in reduced cellular proliferation, migration, and increased doxorubicin-induced apoptosis in colon cancer cells. These results indicate that ephrinA5 functions as a tumor suppressor in the context of colon cancer. EGFR dysregulation, either by overexpression or constitutive activation, has been widely observed in several types of cancer. Small-molecule tyrosine kinase inhibitors and mAbs targeting EGFR have therefore been adopted in clinic settings to block EGFR signaling [29–33]. EGFR degradation provides intrinsic negative feedback regulation following receptor activation. The c-Cbl E3 ubiquitin ligase is involved in EGFR ligand–receptor interactions [35,36], and is responsible for EGFR ubiquitination and subsequent internalization into early endosomes for degradation [37,38]. c-Cbl phosphorylation and activation have been shown to be negatively associated with cancer cell aggressiveness [39–41]. In this study, we found that ephrinA5 was involved in c-Cbl activation and required for EGFR degradation. EphrinA5 was reported to partially colocalize with EGFR and to stereochemically induce Fyn activation and c-Cbl phosphorylation by ephrinA5-mediated reverse signaling [21,42]. EphA receptor forward signaling has also been implicated in facilitating EGFR ubiquitinylation and degradation [43]. Cellular adhesion to the extracellular matrix and EGFR activation both induced EphA2 upregulation in several cancer cell lines, resulting in activation of Ras–mitogen-activated protein kinase signaling and cellular proliferation [44]. Thus, ephrinA5 reverse signaling may function synergistically with Eph receptor forward signaling to negatively regulate EGFR degradation. Our results also indicated that ephrinA5–EphA synergistic signaling may function as a negative feedback mechanism for EGFR-induced EphA forward signaling. We also observed that ephrinA5 expression did not influence EGFR at the transcriptional level (Fig. 3); however, modest transcriptional suppression has been observed in gliomas [21]. This suggests that ephrinA5 might regulate EGFR transcription to different degrees, depending upon the cancer type.

We found that ephrinA5 was downregulated at the transcriptional level (Fig. 1B); however, the upstream regulatory mechanism responsible for this remains undefined. Epigenetic regulation of ephrinA5 has been studied in several types of cancer. EphrinA5 is epigenetically silenced in lymphocytic leukemia [20,45], and these malignancies exhibit EGFR accumulation during cancer progression. In contrast, no significant epigenetic modification was observed in chondrosarcoma [22]. More clinical evidence is necessary to evaluate the epigenetic effects of ephrinA5 in cancer biology.

In conclusion, we found that mutually exclusive expression between ephrinA5 and EGFR occurs in the majority of colon adenocarcinomas, and we observed that ephrinA5 expression was negatively correlated with tumor differentiation and clinical stage. EphrinA5 exerted a suppressive effect on EGFR stability in colon cancer cell lines, and significantly inhibited cancer cell proliferation and migration. Ours is the first study to address the role of ephrinA5 in the context of colon cancer. The data presented in this study provide valuable information concerning the regulation of EGFR stability, and provide a basis for the development of novel strategies that could be utilized to enhance EGFR-targeted treatments.

Experimental procedures

Tissue samples and immunohistochemistry

The tissue microarray slides used for immunohistochemistry were purchased from Pantomics (San Francisco, CA, USA), and were composed of 10 normal colon tissues, 14 benign colon lesions (adenomas and polyps), and 72 colon malignancies (56 adenocarcinomas, 12 mucinous adenocarcinomas, and four non-Hodgkin lymphomas). Slides were first incubated at 65 °C for 30 min, and then subjected to deparaffinization in xylene, rehydration in graded ethanol solutions, and boiling in Trilogy reagent (Cell Marque, Rocklin, CA, USA) for 10 min in a microwave oven for antigen retrieval. After being washed with 1× NaCl/Pi, the slides were immersed in 3% hydrogen peroxide for 10 min to suppress endogenous peroxidase activity. After triple rinses with 1× NaCl/Pi, sections were exposed to mouse anti-ephrinA5 IgG or anti-EGFR IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. After triple rinses with 1× NaCl/Pi, the slides were incubated with biotinylated secondary antibody (Dako, Glostrup, Denmark) for 25 min. Following triple rinses with 1× NaCl/Pi, horseradish peroxidase-conjugated streptavidin was added for 25 min at room temperature. The peroxidase activity was detected with AEC+ substrate chromogen (Dako) at room temperature. The slides were then counterstained with hematoxylin.

The intensity of the membranous and cytoplasmic immunostaining was scored as follows: 0, negative; 1+, weak; 2+, moderate; and 3+, strong. The immunoreactivity and histological appearance of all tissue microarray slides was examined in triplicate and scored by two pathologists who were blinded to the experimental or associated clinicopathological data.

Cell lines, siRNAs, and plasmids

The human colorectal carcinoma cell lines SW480 and WiDr were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 100 IU·mL−1 penicillin, and 100 μg·mL−1 streptomycin (Invitrogen), and cultured at 37 °C in a humidified atmosphere containing 5% CO2. HEK-293T cells were maintained in DMEM with the same supplements and culture conditions. All siRNAs targeting ephrinA5 and c-Cbl were purchased from Santa Cruz Biotechnology. pIRESneo-ephrinA5, cytomegalovirus-based expression and neomycin-selective plasmids containing ephrinA5 cDNA were constructed in the laboratory by processing with an ephrinA5 cloning primer set (forward, 5′-CATAAGCTTCCACCATGTTGCACGTGGAGATGTT-3′; reverse, 5′-ATCGGATCCTGACTCATGTACGGTGTC-3′).

Transient transfection of plasmids and siRNAs

SW480 cells were seeded into six-well plates at a concentration of 3 × 105 cells per well, and 1 μg of pIRESneo-ephrinA5 plasmid or 3 μg of siRNA was added to RPMI medium with Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. After 48 h of incubation, cells were harvested and subjected to RT-PCR, real-time PCR, or western blot analyses.

RNA preparation, RT-PCR, and quantitative real-time PCR

Total RNA was extracted with Trizol reagent (Invitrogen), and DNA contamination was prevented by treatment with RQ1 RNase-free DNase (Promega, Madison, WI, USA). Both assays were performed according to the manufacturer’s instructions. After calculation of the concentration of each RNA sample with a Nanodrop detector (Thermo Scientific, Wilmington, DE, USA), first-strand cDNA was synthesized from 2 μg of total RNA with poly(deoxythymidine nucleotide) (dT14) and the SuperScript III system (Invitrogen). Specimens for RNA analyses were obtained from the tissue bank at Chang Gung Memorial Hospital. This study was approved by the hospital institutional review board.

RT-PCR was performed with 2 μL of cDNA in a 25-μL aliquot containing 5 pmol of primer, 2 units of Taq (Invitrogen), 1× reaction buffer and 200 nm dNTP for 30 cycles, which consisted of denaturation at 94 °C, annealing at 58 °C, and elongation at 72 °C. PCR products were analyzed by agarose gel electrophoresis, and then visualized with ethidium bromide staining. The primer sets were as follows: EphA2-forward, 5′-TCAGCAGCAGCGACTTCGAGGCA-3′; EphA2-reverse, 5′-CAGTGGCCAGGGAAGGTGCA-3′; EphA3-forward, 5′-ATGTTTCCAGACACGGTACC-3′; EphA3-reverse, 5′-CCATCTTCCTGAGTAGAACTGTGAGG-3′; EphA5-forward, 5′-CCTTCTGTGGTACGACACTTG-3′; EphA5-reverse, 5′-GGTCTGCACACTTGACAGGTG-3′; EphB2-forward, 5′-ATGGCGCCCCTCTCCTCTGGCATCA-3′; EphB2-reverse, 5′-ACCGCTTGGTTCTTCCCGTG-3′; c-Cbl-forward, 5′-CGCTAAAGAATAGCCCACCTTAT-3′; c-Cbl-reverse, 5′-ATGGCCTCCAGCCCAGAACTGAT-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-forward, 5′-TGCACCACCAACTGCTTAGC-3′; and GAPDH-reverse, 5′-GGCATGGACTGTGGTCATGAG-3′.

Quantitative real-time PCR was processed with an ABI-3700 machine, using a Quantifast syber green PCR kit (Qiagen, Valencia, CA, USA), and GAPDH mRNA was used as an internal control. Real-time PCR products were also analyzed by gel electrophoresis to confirm a single PCR product. The primer sets were as follows: ephrinA5-forward, 5′-ACCAACAAATAGCTGTATGA-3′; ephrinA5-reverse, 5′-TCGGCTGACTCATGTACGGT-3′; EGFR-forward, 5′-CGGGACATAGTCAGCAGTG-3′; EGFR-reverse, 5′-GCTGGGCACAGATGATTTTG-3′; GAPDH-forward, 5′-AGCCTCAAGATCATCAGCAA-3′; and GAPDH-reverse, 5′-GGCATGGACTGTGGTCATGAG-3′.

Western blot analysis

Cultured cells were washed twice with ice-cold NaCl/Pi and lysed in lysis buffer (50 mm Tris/HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.2 mm Na3VO4, 1 mm dithiothreitol, and 1× protease inhibitor cocktail) (Roche, Branchburg, NJ, USA) for 30 min on ice. Cell lysates were clarified by centrifugation at 15 000 g for 15 min, and protein concentrations were determined with the Bradford Reagent (Bio-Rad, Hercules, CA, USA). Next, 100 μg of each lysate was separated by 10% SDS/PAGE and transferred to Immobilon membranes (Millipore, Bedford, MA, USA) and immunoblotted with the indicated antibodies. All immunoblots were visualized with the NEN ECL system (Life Science, Little Chalfont, UK) and developed on X-ray films. The intensity of each band was quantified with imagequant 5.2 (GE Healthcare, Piscataway, NJ, USA).

Cell proliferation assay

Cell growth was determined with the ACP assay, as described previously [46]. In brief, cells were transfected with pIRESnro-ephrinA5 plasmid or siRNA for 48 h, and subsequently washed twice with 1× NaCl/Pi and subjected to the ACP assay to determine the proliferation rate.

Cell apoptosis assay and flow cytometry

For determination of ephrinA5-induced cell apoptosis, > 70% confluent pIRESneo-ephrinA5-transfected cells and empty vector cells were treated with 2 μm doxorubicin in fresh culture medium. After 48 h, subconfluent cells were trypsinized, washed with 1× NaCl/Pi, and resuspended at 2 × 106 cells·mL−1. A total of 1 × 106 cells were fixed with 100% EtOH for 10 min, and then incubated with 1 mg·mL−1 propidium iodide for 10 min at room temperature. Cells were analyzed within 20 min of staining with a BD FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA).

Immunofluorescence staining

A total of 1 × 104 cells were cultured on each slide. Forty-eight hours after transfection, cells were harvested and fixed in 10% paraformaldehyde for 2 h. The slides were washed twice with 1× NaCl/Pi, blocked with 5% goat serum, incubated with antibodies against EGFR and CD71 (Santa Cruz Biotechnology), labeled with Alexa Fluor 488 goat anti-(rabbit IgG) and Alexa Fluor 546 goat anti-(mouse IgG) (Invitrogen), and counterstained with 4′,6-diamidino-2-phenylindole. The cells were then analyzed by confocal fluorescence microscopy.

Migration assays

For wound-healing experiments, cells were plated in six-well plates and cultured to 90% confluence. Cells were scraped with a p200 tip (time 0), transferred to low-serum culture medium, and treated as indicated. The distances of migrating cells were measured from pictures (five fields) taken at the indicated time points. The transwell assay was performed in 24-well PET inserts (Falcon, 8.0-μm pore size) for the indicated treatments. In brief, 5 × 104 cells were plated in transwell inserts (in triplicate for each sample). Cells were plated in the transwells in 10% serum, which was subsequently replaced with 0.2% serum. After 24 h, the cells in the upper part of the transwells were removed with a cotton swab, and migrated cells were fixed in methanol and stained with hematoxylin. Filters were photographed, and the total number of cells was counted. Each experiment was repeated at least three times independently.

Data analysis

The intensity of immunostaining was subclassified into two categories: 0 and 1 were categorized as negative, and 2 and 3 were categorized as positive. These data were examined with Cox’s hazard regression model. Western blot and migration assay data were recorded as continuous variables, and analyzed with Student’s t-test or ANOVA. Real-time PCR results for ephrinA5 were analyzed with the one-sample t-test. All statistical analyses were performed with spss 16.0 and Excel 2007. All statistical tests were two-sided, and the level of significance was set at < 0.05 (*), < 0.01 (**), or < 0.001 (***).

Acknowledgements

This work was supported by grants from the Department of Health (DOH99-TD-C-111-006) and the Research Foundation of Taoyuan Armed Forces General Hospital (TAFGH-9910), and by Chang Gung Medical research grants (CMRPG391051 and CMRPG380372) from Chang Gung Memorial Hospital, Lin-Kou, Taiwan. This work was approved by the Chang Gung Memorial Hospital institutional review board (CGMH-IRB-99-0721B).

Ancillary

Advertisement