Abstract
Objective To investigate the relationship of the EphA2 gene with the occurrence, invasion and metastasis of esophageal carcinoma.
Methods The expression of EphA2 mRNA was detected by RT-PCR and the EphA2 protein was estimated by immunohistochemistry (SP method) in both esophageal cancerous tissues and normal epithelial tissues.
Results The expression of EphA2 mRNA showed no difference between esophageal cancerous tissues and normal epithelium, and there appeared to be no correlation with differentiation of the cancerous tissues, the depth of infiltration or lymph node metastasis (P>0.05). However, the expression of the EphA2 protein was significantly higher in cancerous tissues compared to normal epithelial tissues (P<0.05). The expression of the EphA2 protein in a deeper invasive group and in a group with lymph node metastasis was significantly higher compared to a superficially invasive group and a group without lymph node metastasis (P<0.05). Its expression did not appear to be correlated with differentiation of cancerous tissues (P>0.05).
Conclusion The occurrence of esophagus carcinoma and the formation of invasion and metastasis may be related to overexpression of the EphA2 protein but not to the level of mRNA, a finding which may due to up-regulation at the translation level or by increased protein stability.
keywords
The Eph receptors are the largest known family of RTKs (receptor tyrosine kinases). The signal transduction caused by interaction of Eph receptors and their membrane-attached ligands, ephrins, play important roles in many developmental processes such as cell proliferation, differentiation and migration.[1,2] EphA2 is widely expressed in epithelial tissues including skin epithelia, intestine and lung mucosas and the ovary. Recent studies have demonstrated that EphA2 maybe involved in carcinogenesis, aggression and metastasis.[3] Eph overexpression has been observed in many human cancerous tissues such as lung, mammary, colon cancers and melanoma.[4-7] EphA2 overexpression was shown to be related to the reduction of cell-cell adhesion in invasive mammary cancer[4] and its expressional level is higher in distant metastatic focus compared to that of the primary focus in melanoma tissues.[5] Our study was performed to investigate the relationship between EphA2 overexpression and the clinicopathological factors in esophageal squamous cell carcinoma (ESCC) since it has rarely been reported.
Materials and Methods
Patients and tissue samples
The tissue specimens were removed from 60 patients with esophageal cancer who had undergone curative surgery at the First Affiliated Hospital of Zhengzhou University and PLA. No. 460 hospital between 2002 and 2003. All 60 patients had squamous cell carcinoma according to pathological diagnosis. Degree of tumor differentiation: high (Grade I, n=18), low (Grade II and Grade III, n=42); invasiveness: superficially invasive (invase to the submucosa or low musularis, n=25), deeper invasive (invasive to the deep musularis, n=35); lymph node metastasis: non-metastatic (n=15) and metastatic (n=45).
A total of 100 cases of conserved paraffin-embedded specimens of ESCC were collected from the First Affiliated Hospital of Zhengzhou University in the years of 1997, 2002 and 2003. There were 90 cases who had corresponding normal epithelium as internal control among the 100 cases. All the specimens were squamous cell carcinomas: high differentiation (n=39), low differentiation (n=61); superficial invasive (n=30), deeper invasive (n=70); non-metastatic (n=59) and metastatic (n=41).
Treatment of specimens
Fresh specimens were resected from cancerous and normal epithelial tissues and immediately separated and stored at -70°C until used for RNA extraction. Remaining tissues were fixed in a 10% formaldehyde solution and embedded in paraffin and sectioned for pathological diagnosis and immunohistochemical (I-HC) staining. From the 100 cases of conserved paraffinized specimens, sections were made directly for H&E (hematoxylin/eosin) and IHC staining.
Main reagents
Primers for the EphA2 gene: 5’-GCC AGG CAG GCT ACG AGA AGG TG-3’ and 5’-CTC CAG GAG ACG CTA AGC GAG GTG-3’, primers for the p-actin gene: 5’-CAA GGC CAA CCG CGA GAA GAT G-3’ and 5-GTC CAG GGC GAC GTA GCA CAG C-3’. Polyclonal antibody specific for EphA2 was purchased from Santa Cruz Biotechnology, Inc, CA, USA.
Process of RT-PCR (reverse transcription-polymerase chain reaction) and IHC
Total RNA was extracted from frozen tissues and cD-NA synthesis was performed by RT for PCR amplification: 25°C 10 min, 42°C 1 h, 70°C 10 min. β-actin was used as the internal control in every sample to guarantee the same condition. Then the process of PCR was performed: 94°C 5 min, 94°C 30 s, 60°C 30 s, 72°C40 s recycling 30 times, 72°C 5 min to 4°C. The PCR products were subjected to 2% agarose gel electrophoresis and were visualized and photographed by BIO IMAGING SYSTEM. EphA2 cDNA fragment was 586 bp and P-actin fragment was 330 bp (Fig.1).
Expression of EphA2 mRNA in esophageal carcinoma tissues and normal epithelial tissues.
M:Standard 100 bp DNA ladder; A: EphA2 gene, 586 bp; B: β-actin gene, 330 bp; 1,3,5: Expression in normal epithelial tissues; 2,4: Expression in nonmetastatic carcinoma tissues. 6: Expression in metastatic carcinoma tissues.
IHC staining was performed by the standard SP (streptavidin/peroxidase) method. Briefly, after antigen restoration and covering with serum, the tissue sections were incubated with anti-EphA2 antibody at a dilution of 1:400 at 4°C overnight. Then the sections were incubated with secondary antibody for 30 min, stained with DAB (diaminobenzidine) and lightly counterstained with hematoxylin. Negative controls were prepared by substituting normal serum for primary antibody, and positive controls were prepared using known positive tissue sections.
Evaluation of staining results
Immunostaining of EphA2 was detected in the cytoplasm and cell membrane of the basal cells in normal esophageal epithelium and mainly in the cytoplasm in cancer cells (Figs.2~4). Evaluation of staining results was formulated based on previous reports.[5,7] A staining index value (0 to 9), was calculated as a product of staining intensity (0 to 3) and the proportion of immunopositive cells of interest (<10%=1, 10 to 50%=2, >75%=3). The staining result was divided into 3 levels: negative expression (index<3), positive expression (index 4~6) and intensely positive expression (index 7~9).
Positive expression of the EphA2 protein in normal esophageal epithelium, SP method (× 200).
Positive expression of the EphA2 protein in esophageal carcinoma tissue, SP method (x 400).
Positive expression of the EphA2 protein in esophageal carcinoma tissue, SP method (x 400).
Statistical analysis
Statistical analysis was performed by using the χ2 test by SPSS 11.0 software. A P value <0.05 was adopted as a significant difference.
Results
Expression of EphA2 mRNA
The positive rates of EphA2 mRNA in normal epithelium and carcinoma tissues were 28% (17/60) and 32% (19/60) showing that there was no significant difference between them. In addition, the expression of EphA2 mRNA did not appear to be correlated with tumor differentiation, the depth of infiltration or lymph node metastasis (P>0.05, Table.1).
Expression of the EphA2 protein
The relationship between the EphA2 protein expression and clinicopathological factors of patients is summarized in Table 2. The expression of EphA2 protein was greater in cancerous tissues than that in normal epithelial tissues (P<0.05). The EphA2 protein was expressed at a higher level in deeper invasive tissues and metastatic cases compared to superficially invasive tissues and non-metastic cases (P<0.05). It did not appear to be correlated with tumor differentiation (P>0.05).
The relationship between EphA2 protein expression and clinicopathological features in ESCC
Discussion
RTKs are the key proteins for signal transduction and play important roles in many developmental processes, including embryo development, cell proliferation, malignant transformation and tumor aggression. Eph is the largest family of RTKs and Eph overexpression has been observed in many human cancerous tissues or cell lines including melanoma, mammary, lung and esophageal cancers.[4-7] In order to investigate the relationship between EphA2 expression and clinicopathological features of ESCC, the expression of EphA2 mRNA and EphA2 protein was examined in both carcinoma and normal epithelial tissues by the RT-PCR and IHC. The results showed that EphA2 protein expression was correlated with carcinogenesis, invasion and metatasis of ESCC. EphA2 overexpression could make malignant cells to be more aggressive and to become prone to metastasis. However there was no relationship between EphA2 overexpression and tumor differentiation. EphA2 overexpression was not demonstrated at the mRNA level which may due to up-regulation at the point of translation or by increased protein stability.
With normal expression of Eph receptors, proper interaction with ephrins results in a balance between cell-cell and cell-ECM (extracelluar matrix) adhesions via cellular signal-transducting regulation. But in carcinoma tissues, aberrant signal transduction caused by an absence of normal interaction of Eph receptors with their ephrins could reduce adhesion of malignant cells making them more isolated and aggressive.[8-10] Zelinski et al. observed that EphA2 overexpression induced mammary epithelial cell transformation and reduction of tumor cell-cell attachment while strengthening tumor cell-ECM adhesion and increasing invasion of tumor cells into the matrix.[4] An important characteristic of tumor cells such as enhancement of aggressive ability indicates that EphA2 overexpression increases tumor invasiveness as has been demonstrated by many reports relating to tumor prognosis. It was reported by Kinch et al. that EphA2 overexpression in early lung cancerous tissues predicted a high probability of brain metastasis and poor survival rate.[6] Similarly, Miyazaki et al. showed that EphA2 overexpression indicated a poor prognosis but the expression of EphA2 mRNA in different 7 ESCC cell lines did not show a significant difference. Therefore it was concluded that the regulation of EphA2 overexpression was at the point of translation.[7] Now more and more evidence has indicated that EphA2 overexpression is caused by up-regulation at the translational level or by increased protein stability, instead of an up-regulation of transcription.[11] This view is consistent with our investigation.
- Received February 22, 2006.
- Accepted March 24, 2006.
- Copyright © 2006 by Tianjin Medical University Cancer Institute & Hospital and Springer
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