2050

EPHB4

HTK|MYK1|TYRO11;EPH receptor B4;EPHB4;EPH receptor B4

ephrin receptor EphB4|ephrin type-B receptor 4|hepatoma transmembrane kinase|soluble EPHB4 variant 1|soluble EPHB4 variant 2|soluble EPHB4 variant 3|tyrosine-protein kinase TYRO11|tyrosine-protein kinase receptor HTK

7q22

protein-coding

The family of receptor tyrosine kinases EPH and their Ephrin ligands regulate cell proliferation, migration, and attachment. An important role in colorectal carcinogenesis is emerging for some of its members. In this study, we evaluate the role of EPHB4 in colorectal cancer and its value as a prognostic marker. EPHB4 levels were assessed by immunohistochemical staining of tissue microarrays of 137 colorectal tumors and aberrant hypermethylation of the EPHB4 promoter was investigated using methylation-specific PCR. We found that EPHB4 expression is frequently reduced or lost in colorectal tumors. Patients with low EPHB4 tumor levels had significantly shorter survival than patients in the high EPHB4 group (median survival, 1.8 and >9 years, respectively; P < 0.01, log-rank test), and this finding was validated using an independent set of 125 tumor samples. In addition, we show that EPHB4 promoter hypermethylation is a common mechanism of EPHB4 inactivation. Moreover, reintroduction of EPHB4 resulted in a significant reduction in the clonogenic potential of EPHB4-deficient cells, whereas abrogation of EPHB4 in cells with high levels of this receptor lead to a significant increase in clonogenicity. In summary, we identified EPHB4 as a useful prognostic marker for colorectal cancer. In addition, we provide mechanistic evidence showing that promoter methylation regulates EPHB4 transcription and functional evidence that EPHB4 can regulate the long-term clonogenic potential of colorectal tumor cells, revealing EPHB4 as a potential new tumor suppressor gene in colorectal cancer.

Colorectal cancer is the second cause of cancer-related death in the western world, and although the genetic and molecular mechanisms involved in the initiation and progression of these tumors are among the best characterized, there are significant gaps in our understanding of this disease. The role of EPHB signaling in colorectal cancer has only recently been realized. Here, we use animal models to investigate the role of EphB4 in intestinal tumorigenesis. Modulation of EPHB4 levels in colon cancer cell lines resulted in significant differences in tumor growth in a xenograft model, with low levels of EPHB4 associated with faster growth. In addition, using a genetic model of intestinal tumorigenesis where adenomatous polyposis coli (Apc) mutations lead to initiation of the tumorigenic process (Apc(min) mice), we show that inactivation of a single allele of EphB4 results in higher proliferation in both the normal epithelium and intestinal tumors, significantly larger tumors in the small intestine, and a 10-fold increase in the number of tumors in the large intestine. This was associated with a 25% reduction in the lifespan of Apc(min) mice (P < 0.0001). gene expression analysis showed that EphB4 mutations result in a profound transcriptional reprogramming, affecting genes involved in cell proliferation, remodeling of the extracellular matrix, and cell attachment to the basement membrane among other functional groups of genes. Importantly, in agreement with the expression profiling experiments, using an in vitro assay, we show that loss of EPHB4 in colon cancer cells results in a significantly increased potential to invade through a complex extracellular matrix. Collectively, these results indicate that EphB4 has tumor suppressor activities and that regulation of cell proliferation, extracellular matrix remodeling, and invasive potential are important mechanisms of tumor suppression.

Eph receptors and their ephrin ligands are involved in normal hematopoietic development and tumorigenesis. Using methylated CpG island amplification/DNA promoter microarray, we identified several EPH receptor and EPHRIN genes as potential hypermethylation targets in acute lymphoblastic leukemia (ALL). We subsequently studied the DNA methylation status of the Eph/ephrin family by bisulfite pyrosequencing. Hypermethylation of EPHA2, -A4, -A5, -A6, -A7, -A10, EPHB1, -B2, -B3, -B4, EFNA1, -A3, -A5, and EFNB1 and -B2 genes was detected in leukemia cell lines and primary ALL bone marrow samples. expression analysis of EPHB4, EFNB2, and EFNA5 genes demonstrated that DNA methylation was associated with gene silencing. We cloned the promoter region of EPHB4 and demonstrated that promoter hypermethylation can result in EPHB4 transcriptional silencing. Restoration of EPHB4 expression by lentiviral transduction resulted in reduced proliferation and apoptotic cell death in Raji cells in which EPHB4 is methylated and silenced. Finally, we demonstrated that phosphorylated Akt is down-regulated in Raji cells transduced with EPHB4. These results suggest that epigenetic silencing by hypermethylation of EPH/EPHRIN family genes contributes to ALL pathogenesis and that EPHB4 can function as a tumor suppressor in ALL.