You-Jie Wang

EPHA2/EFNA1 expression in human gastric cancer

The erythropoietin‐producing hepatocellular (EPH)A2 receptor, tyrosine kinase, is overexpressed and phosphorylated in several types of human tumors and has been associated with malignant transformation. A recent report, however, indicated that stimulation of the EPHA2 receptor ligand, ephrinA1 (EFNA1), inhibits the growth of EPHA2‐expressing breast cancer. The authors examined the expression of EPHA2 and EFNA1 using semiquantitative reverse transcription‐polymerase chain reaction (RT‐PCR) in four gastric cancer cell lines and 49 primary gastric cancer samples, as well as in normal gastric tissue. EPHA2 was more highly expressed in tumor tissue than in normal tissue in 27 cases (55%). EFNA1 was overexpressed in tumor tissue in 28 cases (57%). No significant correlation was detected between the expression levels and histologic features such as tumor size, age, vessel invasion, or lymph node involvement. However, EPHA2 overexpression was more prominent in macroscopic type 3 and 4 tumors than in type 1 or 2 advanced gastric cancer. The authors observed EPHA2 expression in three of the four gastric cancer cell lines (AGS, KATO3, and MKN74) that were examined. In one cell line, TMK1, EPHA2 expression was barely detectable using northern blotting, RT‐PCR, and western blotting. In contrast, EFNA1 was detected in all cell lines. In the gastric cancer cell lines that endogenously expressed EPHA2, stimulation with ephrinA1‐Fc led to decreased EPHA2 protein expression and increased EPHA2 phosphorylation. Finally, the growth of EPHA2‐expressing cells was inhibited by repetitive stimulation with soluble ephrinA1‐Fc. Taken together, these findings suggest that EPHA2 and EFNA1 expression may influence the behavior of human gastric cancer. (Cancer Sci 2005; 96: 42–48)

Correlation of EPHA2 overexpression with high microvessel count in human primary colorectal cancer

Evidence suggests that the erythropoietin‐producing hepatocellular (EPH) receptor tyrosine kinases (RTKs) and their ephrin (EFN) ligands are involved in human carcinogenesis. Expression of two of them, EFNA1 ligand and its receptor, EPHA2, has been proposed to contribute to tumor‐induced neovascularization. Colorectal cancers were examined for expressions of EPHA2 and its ligand EFNA1 by semi‐quantitative RT‐PCR, and double‐immunostained for EPHA2 and CD34. Microvessels in the tumors were counted. Double‐staining was also performed in 25 cases of adenoma with focal cancer for comparison. Trends of overexpression of both EPHA2 and EFNA1 was found in tumor tissue compared to the corresponding normal tissue in the same specimen [22/37 (59.5%) and 25/37 (67.5%), respectively; P=0.100 for EPHA2 and P=0.009 for EFNA1]. Overexpression of EPHA2 and EFNA1 was noted more frequently in the early stage than in the late stage [EPHA2, 15/21 (71.4%) vs. 7/16 (43.8%), P=0.007; EFNA1, 15/21 (71.4%) vs. 10/16 (62.5%), P=0.007]. Both EPHA2 and EFNA1 were more frequently overexpressed in smaller tumors (less than 5 cm) than in larger tumors [EPHA2, 15/21 (71.4%) vs. 7/16 (43.8%), P=0.017; EFNA1, 16/21 (76.2%) vs. 8/16 (50%), P=0.001]. Tumors less than 5 cm in diameter and in stages I and II were significantly more likely to overexpress EPHA2 and EFNA1 (P=0.001 for EPHA2, P=0.001 for EFNA1). Microvessel counts (MVCs) after immuno‐staining for CD34 were significantly correlated (r=0.343, P=0.037) with overexpression of EPHA2. EPHA2‐expressing focal cancer also surrounded microvessels in adenomas with focal cancers. These findings suggest an involvement of EPHA2 in colon carcinogenesis, mainly in stages I and II, and probably through their effect on microvessel induction.

Negative regulation of EphA2 receptor by Cbl.

The c-Cbl proto-oncogene product Cbl has emerged as a negative regulator of receptor and non-receptor tyrosine kinases, a function dependent on its recently identified ubiquitin ligase activity. Here, we report that EphA2, a member of Eph receptor tyrosine kinases is negatively regulated by Cbl. The negative regulation of EphA2 mediated by Cbl is dependent on the activity of EphA2, as the kinase inactive mutant of EphA2 cannot be regulated by Cbl. Moreover, a point mutation (G306E-Cbl) in TKB region of Cbl that has been reported to abolish Cbl binding to RTKs and non-receptor tyrosine kinases impaired the binding to active EphA2. The dominant negative mutant 70Z-Cbl, which has a 17-amino acids deletion in the N-boundary of the RING finger domain, defuncted negative regulatory function of Cbl to EphA2. These results demonstrate that the TKB domain and RING finger domain of Cbl are essential for this negative regulation.

Expression profile of EFNB1, EFNB2, two ligands of EPHB2 in human gastric cancer

Abstract Purpose. We have previously reported that EPHB2 is overexpressed in gastric cancer; however, the expression profiles of its ligands, EFNB1 and EFNB2, are unknown. This study was designed to investigate the expression of EPHB2 and its ligands, EFNB1 and EFNB2, in human gastric cancer. Methods. Semi-quantitative RT-PCR using 32P was performed on human gastric cancer tissues and corresponding normal tissues (29 gastric cancer patients). Results. EPHB2 was more highly expressed in tumor tissues than in normal tissues in 21 out of 29 (72.4%), in gastric cancer patients (P = 0.01); EFNB1 and EFNB2 were highly expressed in 21 out of 29 (72.4%) (P = 0.037) and 14 out of 29 (48.3%) patients, respectively. The overexpression of EPHB2 was frequently detected in both well-differentiated adenocarcinoma and poorly differentiated adenocarcinoma [10/13 (76.9%) and 9/14 (64.3%), respectively]. On the other hand, the overexpression of EFNB1 was more frequently detected in poorly differentiated adenocarcinoma than in well-differentiated adenocarcinoma [12/14 (85.7%) and 7/13 (53.8%), respectively. P =0.027]. Elevated levels of EPHB2 and EFNB1 were detected in substantial subsets of early gastric cancers. Genomic alterations to these three genes in gastric cancer specimens were either not found or rarely found except for several rare variations of EPHB2. Conclusions. These findings suggest that not only the expression of EPHB2, but the expression of its ligand EFNB1 may have some relation with the oncogenesis of gastric cancer.

Genomic structure and loss of heterozygosity of EPHB2 in colorectal cancer

EphB2, a member of the Eph receptor protein-tyrosine kinase family, is overexpressed in several human gastrointestinal tumors. Furthermore, the EphB2 gene is localized at 1p35-p36.1, a frequently deleted region in colon and other cancers. So, despite its overexpression in some kind of tumors, we decided to study the possibility of involvement in the EphB2 gene (EPHB2) mutation in colon cancers, because some of the well known tumor suppressor genes (e.g. p53) is overexpressed (really accumulated) in tumors. Fifty colon tumor samples of matched with their respective normal tissues, were studied for mutation of the EPHB2. Analysis of the genomic structure of EphB2 and survey of all 16 exons revealed an infrequent polymorphism (intron 2) and mutation (intron 8). Another polymorphism in exon 6, localized at nucleotide 1359 (A-->G) was found to be rather frequent in Japanese and Chinese subjects, but very rare in Caucasians. Taking advantage of this polymorphism within EPHB2, we surveyed the loss of heterozygosity (LOH) status of this gene in Japanese colorectal tumors. Among the 50 samples analyzed, 24 were informative, and LOH was found in five of the15 (33.3%) informative rectal cancer cases. Mutation analysis covering all 16 exons in the remaining allele did not reveal any mutations. Thus, EPHB2 is not a classical tumor suppressor gene.

Genomic structure of human alpha-pix, and variable deletions in a poly (T) tract in gastric cancer tissue.

PAK-interacting exchange factor (PIX) has been reported to mediate the recruitment of PAK into focal adhesions and activate Rac, thus creating a feedback loop that stimulate PAK and other targets. This pathway is thought to be related to cellular changes, such as transformation and migration, that are often encountered in cancer cells. Here, we report the genomic structure of alpha-PIX, one of the PAK- interacting exchange factors, including the identification of the promoter region, which consisted 772 amino acids in 22 exons, spanning about 100 kb on genome of X chromosome. All splice sites conformed to the GT-AT rule. To investigate the role of alpha-PIX in carcinogenesis, we screened 60 cases of gastric cancer for mutations and polymorphisms using an intron-primer that covered all the exons, but no mutations or polymorphisms were found in the coding region. However an 18 bp repeat of thymidine tract was present in 50 bp downstream from exon 12 and the deletion of variable numbers of mononucleotide repeats was observed in seven out of the 60 gastric cancer tissue specimens that were examined. These seven cases all exhibited a mutator phenotype, suggesting that the deletions are passenger mutations. Thus our results revealed that alpha-PIX probably does not play any primary role in human gastric carcinogenesis.

Segregation Analysis of Gastric Cancer in a Japanese Population

Abstract Though its incidence and mortality have declined, gastric cancer is still the most common type of cancer in Japan. The cause of gastric cancer appears to be both genetic and environmental, with the possibility that the differentiated (internal) type is more environmentally determined than the non-differentiated (diffuse) type. Prior to this study, no formal segregation analysis of gastric cancer in Japan has been performed. Segregation analysis of gastric cancer that allowed for variable age of onset was performed on 851 two-generational pedigrees ascertained via gastric cancer probands collected from the Hospital based Epidemiological Research Program in the Aichi region of Japan. Families were classified based on the proband’s histopathological classification of having either differentiated type or nondifferentiated type of cancer. A random no major effect hypothesis was rejected, as was a purely recessive or dominant hypothesis. The most parsimonious model was one of purely multifactorial inheritance with males having a higher susceptibility than females. Under a model where genotype influences age of onset, a dominant or recessive mode of inheritance with multifactorial effects also fitted the data. In addition, the analyses were performed separately for the differentiated type and the non-differentiated type and homogeneity was not rejected.