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 T he erythropoietin-producing hepatocellular (EPH ) receptors represent the largest known family of receptor tyrosine kinases and are activated by interaction with the cell-surface ligands, ephrins (EFN). There is evidence to suggest that some members of the EPH family and their EFN ligands are involved in angiogenesis and oncogenesis through cell adhesion, morphogenesis, capillary sprouting, and chemoattraction.(1−5) EPH receptors have been classified into two subfamilies, EPHA and EPHB. EPHA receptors bind mainly to glycosylphosphatidylinositolanchored EFNA ligands, and EPHB receptors bind to transmembrane EFNB ligands. The expression of EPH family transcripts has been documented in some melanomas and carcinomas.(6,7) Overexpression of EPHA2 is believed to be sufficient to confer malignant/tumorigenic potential on nontransformed mammary epithelial cells.(8) Esophageal squamous cell carcinomas that overexpress EFNA2 have a poorer prognosis than those that do not. (9)

A significant reduction of EphA7 expression in human colorectal cancers was shown using semiquantitative reverse transcription-polymerase chain reaction analysis in 59 colorectal cancer tissues, compared to corresponding normal mucosas (P ¼ 0.008), and five colon cancer cell lines. To investigate the mechanism of EphA7 downregulation in colorectal cancer, we examined the methylation status of the 5 0 CpG island around the translation start site in five colon cancer cell lines using restriction enzymes, methylation-specific PCR, and bisulfite sequencing and found evidence of aberrant methylation. The expression of EphA7 in colon cancer cell lines was restored after treatment with 5-aza-2 0 -deoxycytidine. Analysis of methylation status in totally 75 tumors compared to clinicopathological parameters revealed that hypermethylation of colorectal cancers was more frequent in male than in female (P ¼ 0.0078), and in moderately differentiated than in well-differentiated adenocarcinomas (P ¼ 0.0361). There was a tendency that hypermethylation in rectal cancers was more frequent than in colon cancers (P ¼ 0.0816). Hypermethylation was also observed in colorectal adenomas. This is the first report describing the downregulation of an Eph family gene in a solid tumor via aberrant 5 0 CpG island methylation. It provides the evidence that EphA7 gene is involved in human colorectal carcinogenesis.

Bidirectional signals mediated by Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, play pivotal roles in the formation of neural networks by induction of both collapse and elongation of neurites. However, the downstream molecular modules to deliver these cues are largely unknown. We report here that the interaction of a Rac1-specific guanine nucleotide-exchanging factor, Tiam1, with ephrin-B1 and EphA2 mediates neurite outgrowth. In cells coexpressing Tiam1 and ephrin-B1, Rac1 is activated by the extracellular stimulation of clustered soluble EphB2 receptors. Similarly, soluble ephrin-A1 activates Rac1 in cells coexpressing Tiam1 and EphA2. Cortical neurons from the E14 mouse embryos and neuroblastoma cells significantly extend neurites when placed on surfaces coated with the extracellular domain of EphB2 or ephrin-A1, which were abolished by the forced expression of the dominant-negative mutant of ephrin-B1 or EphA2. Furthermore, the introduction of a dominant-negative form of Tiam1 also inhibits neurite outgrowth induced by the ephrin-B1 and EphA2 signals. These results indicate that Tiam1 is required for neurite outgrowth induced by both ephrin-B1-mediated reverse signaling and EphA2-mediated forward signaling.

Neural epidermal growth factor-like like (NELL) 1 and 2 constitute a family of multimeric and multimodular extracellular glycoproteins. Although the osteogenic effects of NELL1 and functions of NELL2 in neural development have been reported, their expression and functions in cancer are largely unknown. In this study, we examined expression of NELL1 and NELL2 in renal cell carcinoma (RCC) using clinical specimens and cell lines. We show that, whereas NELL1 and NELL2 proteins are strongly expressed in renal tubules in non-cancerous areas of RCC specimens, their expression is significantly downregulated in cancerous areas. Silencing of NELL1 and NELL2 mRNA expression was also detected in RCC cell lines. Analysis of NELL1/2 promoter methylation status indicated that the CpG islands in the NELL1 and NELL2 genes are hypermethylated in RCC cell lines. NELL1 and NELL2 bind to RCC cells, suggesting that these cells express a receptor for NELL1 and NELL2 that can transduce signals. Furthermore, we found that both NELL1 and NELL2 inhibit RCC cell migration, and NELL1 further inhibits RCC cell adhesion. These results suggest that silencing of NELL gene expression by promoter hypermethylation plays roles in RCC progression by affecting cancer cell behavior.

Background: Nel is a multimodular glycoprotein and plays important roles in neural development and functions. Results: The N-terminal thrombospondin-1 domain is involved in multimer formation and heparin-and retinal axon-binding. Cysteine-rich domains bind to and inhibit retinal axons. Conclusion: Different molecular interactions and functions are mediated by distinct domains of Nel. Significance: The findings provide insights into how Nel exerts diverse functions.