Receptor protein tyrosine kinase EphB4 is up-regulated in colon cancer

Abstract

Background: We have used commercially available cDNA arrays to identify EphB4 as a gene that is up-regulated in colon cancer tissue when compared with matched normal tissue from the same patient.

Results: Quantitative RT-PCR analysis of the expression of the EphB4 gene has shown that its expression is increased in 82% of tumour samples when compared with the matched normal tissue from the same patient. Using immunohistochemistry and Western analysis techniques with an EphB4-specific antibody, we also show that this receptor is expressed in the epithelial cells of the tumour tissue and either not at all, or in only low levels, in the normal tissue.

Conclusion: The results presented here supports the emerging idea that Eph receptors play a role in tumour formation and suggests that further elucidation of this signalling pathway may identify useful targets for cancer treatment therapies.

Figure 1

Expression of EphB4 and CK19 in ten colon cancer cell lines -LIM2099, LIM2412, LIM1863, SW48, LIM1899, LIM1215, SW480, HT29, LIM2405 and SW620. Sizes of the amplified products are indicated on the right. RT-ve – RT negative control, PCR -ve – PCR negative control, M – pUC19 HpaII marker.

Figure 2

Semiquantitative analysis of EphB4 gene expression in five tumour (T) and normal (N) pairs, one of which includes a liver metastasis consistent with the colorectal primary from this patient (LM) and a normal liver sample (NL), by comparison with CK19 expression. Sizes of the amplified products are indicated on the right. C1 – colon cancer cell line LIM2405, C2 – colon cancer cell line SW480, RT- RT negative control, P – PCR negative control, M -pUC19 HpaII marker.

Figure 3

Relative RT-PCR comparing expression of EphB4 (1187 bp) and internal 18S rRNA (489 bp) in the same five tumour (T)/ normal (N) pairs shown in Fig 4. C1 – colon cancer cell line LIM2405, C2 – colon cancer cell line SW480, RT- RT negative control, P – PCR negative control, M – pUC19 HpaII marker.

Figure 4

(A) Coomassie stained duplicate gel for loading comparison. (B) Western analysis of EphB4 protein expression in colon tumour (T) and matched normal mucosa (N) from five different patients. The size markers in kDa are shown on the left. The arrow indicates the normal EphB4 protein.

Figure 5

Immunohistochemical localisation of EphB4 expression in three different colon cancers and matched normal mucosa. The brown stain from the biotinylated secondary antibody indicates the EphB4 protein. Nuclei are stained with Harris haematoxylin and appear blue. High power (100X) magnification images of three different adenocarcinomas (well differentiated, moderately well differentiated and poorly differentiated) and their matched normal mucosa are shown. Strong staining of the tumour tissue and very weak, diffuse staining of normal tissue was evident for each sample set. There was no cross-reactivity with the secondary antibody alone (result not shown).