Zhong-You Li

HOGG1 Ser326Cys polymorphism and modification by environmental factors of stomach cancer risk in Chinese

Oxidative stress is involved in many types of DNA damage, e.g., resulting in 8‐hydroxyguanine adducts. Since a human counterpart exists for the yeast gene OGG1 (hOGG1) encoding an enzyme that repairs 8‐hydroxyguanine, its polymorphism, Ser326Cys, might have potential as a genetic marker for cancer susceptibility. To investigate its association with stomach cancer risk and possible interactions with environmental factors, we conducted a case‐control study of 101 stomach cancer cases and 198 controls using PCR‐single‐strand conformation polymorphism and a questionnaire approach. The proportional distribution of the Cys/Cys alleles did not differ between stomach cancer cases and controls, but subgroup analyses revealed that a frequent drinking habit elevated the odds ratio (OR) for stomach cancer in Cys/Cys compared to Ser/Ser and Ser/Cys carriers. The ORs with frequent consumption of pickled vegetables and meat tended to be higher in Cys/Cys than in Ser/Ser and Ser/Cys carriers, these interactions being on the borderline of statistical significance. Our findings suggest that the hOGG1 Ser326Cys polymorphism may alter the impact of some environmental factors on stomach cancer development. For confirmation, an additional study with a larger number of subjects is now required.

Glutathione-S-transferases M1 (GSTM1) and GSTT1 genotype, smoking, consumption of alcohol and tea and risk of esophageal and stomach cancers: a case-control study of a high-incidence area in Jiangsu Province, China

To evaluate interactions between lifestyle factors and glutathione-S-transferases M1 (GSTM1) and GSTT1 genotypes with reference to development of esophageal and stomach cancers, we conducted a case-control study of 141 cases of esophageal cancer, 153 cases of stomach cancer and 223 population-based controls in Huaian City of Jiangsu Province, China. GSTM1 and GSTT1 genotypes were identified by multiplex polymerase chain reaction. The GSTM1 null genotype was associated with an increased odds ratio for esophageal cancer (2.17, 95% confidence interval=1.35-3.50), but not for stomach cancer. A combined effect was also observed between smoking and the GSTM1 null genotype with regard to esophageal risk. Tea drinking was a protective factor for both cancers, its effect being independent of the GSTT1 and GSTM1 genotypes. These findings suggest the GSTM1 polymorphism is involved in the susceptibility to esophageal cancer development, and tea consumption reduces the risk of esophageal and stomach cancers.

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.

EphA2 Up-regulation induced by deoxycholic acid in human colon carcinoma cells, an involvement of extracellular signal-regulated kinase and p53-independence

PurposeThe EphA2 receptor protein tyrosine kinase gene has been shown to be over-expressed or functionally altered in a number of human tumors, including colon cancer, but little is known about the regulation of this new oncoprotein. In order to explore the mechanism of EphA2 up-regulation in cancer cells, we examined the change of expression of EphA2 gene induced by deoxycholic acid (DCA) and elucidated its possible pathways in human colon cancer cells.MethodsWestern blot and RT-PCR were used to assess the protein expression and messenger RNA in several colon cancer cell lines, which harbor various p53 status. The inhibition study to interfere the MAPK pathway was performed by using various chemicals and by transfecting dominant negative mutant plasmids.ResultsUp-regulation of EphA2 induced by DCA was observed in a dose- and time-dependent fashion both in mRNA and protein levels. This regulation is constant regardless of p53 status including wild, mutant or knocked out in the colon cell lines used. This induction was in part blocked by either erk1/2 inhibitors or dominant negative mutants erk1/2 plasmids.ConclusionsThese results suggest that DCA induced up-regulation of EphA2 in colon cancer cells is due to activation of erk1/2 cascade, and is p53-independent. Taken together with the roles of EphA2 and DCA in tumorigenesis, which have been independently reported, our observation will provide a new mechanistic basis of DCA commitment in carcinogenesis.

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.