2051

EPHB6

EPH receptor B6;EPHB6;EPH receptor B6

ephrin type-B receptor 6|human kinase-defective Eph-family receptor protein|tyrosine-protein kinase-defective receptor EPH-6

7q33-q35

protein-coding

PURPOSE: Loss of EPHB6 receptor tyrosine kinase expression in early-stage non-small cell lung carcinoma (NSCLC) is associated with the subsequent development of distant metastasis. Here, we analyzed the regulation and function of EPHB6 in lung cancer metastasis. EXPERIMENTAL DESIGN: The expression levels of EPHB6 were compared among normal lung tissue (n = 9), NSCLC without metastasis (n = 39), and NSCLC with metastasis (n = 39) according to the history of the patients. In addition, EPHB6 expression levels of matched tumor-normal pairs from 24 NSCLC patients were analyzed. The promoter DNA methylation status and its association with the expression levels of EPHB6 were determined among 14 pairs of tumor-normal samples. metastatic potential of EPHB6 was assessed in vitro and in vivo in a metastasis mouse model. Overexpression and RNA interference (RNAi) approaches were used for analysis of the biological functions of EPHB6. RESULTS: EPHB6 mRNA and protein levels were significantly reduced in NSCLC tumors compared with matched normal lung tissue. Decreased EPHB6 expression levels were associated with an increased risk for metastasis development in NSCLC patients. Loss of expression correlated with EPHB6 hypermethylation. EPHB6 expression was induced by 5-aza-2'-deoxycytidine treatment in an NSCLC cell line. Restoration of EPHB6 expression in lung adenocarcinoma cells increased adhesion and decreased migration. Reexpression of EPHB6 in lung cancer cells almost entirely abolished metastasis formation in non obese diabetic (NOD)/severe combined immunodeficient mice. CONCLUSIONS: Taken together, these analyses show that EPHB6 is a metastasis inhibitory gene that is frequently silenced by hypermethylation of its promoter in NSCLC.

Overexpression of various members of the Eph receptor tyrosine kinases and their ephrin ligands has been frequently reported in cancer. In contrast, a loss of EphB6 gene expression has been correlated with a poor prognosis in human neuroblastoma, suggesting a distinct role for this receptor compared to other family members. More recently, an important role of EphB6 signalling in T-cells has been described, suggesting possibly deleterious immunologic effects of a loss of EphB6 in cancer progression. We investigated the expression of EphB6 in melanocytic tumors. EphB6 mRNA of 22 microdissected tissues (7 benign nevi, 7 melanomas, 8 metastases) and 10 different cell lines (normal melanocytes, non-metastatic/metastatic melanoma cell lines) were measured by quantitative real-time RT-PCR. For visualization of EphB6 protein expression, immunohistochemistry of 32 melanocytic lesions were performed. On the mRNA level, the benign nevi revealed the highest EphB6 expression (mean = 1.43), while melanomas (mean = 0.63) and metastases (mean = 0.08; p=0.001) displayed a progressive and significant reduction of EphB6 expression. Accordingly, established melanoma cell lines with metastatic potential showed low EphB6 expression in comparison to normal melanocytes and to most of the melanoma cell lines. Immunohistochemistry revealed homogeneous staining in common nevi, whereas in malignant melanomas and metastases a heterogeneously positive to completely negative EphB6 staining was observed. Remarkably, Spitz nevi stained similarly to ordinary melanocytic nevi. Taken together, we show that melanoma progression to metastatic disease is associated with a significant reduction of EphB6 gene expression which may have considerable consequences for the prognosis of malignant melanoma patients and possible gene-therapeutic approaches.

metastatic dissemination drives the high mortality associated with melanoma. However, difficulties in visualizing in vivo cell dynamics during metastatic invasion have limited our understanding of these cell behaviors. Recent evidence has revealed that melanoma cells exploit portions of their ancestral embryonic neural crest emigration program to facilitate invasion. What remains to be determined is how embryonic microenvironmental signals influence invasive melanoma cell behavior, and whether these signals are relevant to human disease. To address these questions, we interrogated the role of the neural crest microenvironment in dictating the spatiotemporal pattern of melanoma cell invasion in the chick embryo using 2-photon time-lapse microscopy. Results reveal that both permissive and inhibitory neural crest microenvironmental signals regulate the timing and direction of melanoma invasion to coincide with the neural crest migration pattern. These cues include bidirectional signaling mediated through the ephrin family of receptor tyrosine kinases. We demonstrate that EphB6 reexpression forces metastatic melanoma cells to deviate from the canonical migration pattern observed in the chick embryo transplant model. Furthermore, EphB6-expressing melanoma cells display significantly reduced metastatic potential in a chorioallantoic membrane (CAM) metastasis assay. These data on melanoma invasion in the embryonic neural crest and CAM microenvironments identify EphB6 as a metastasis suppressor in melanoma, likely acting at the stage of intravasation. IMPLICATIONS: This article links cellular metastasis to behaviors observed in the ancestrally related embryonic neural crest and demonstrates the powerful influence of the embryonic microenvironment in regulating cell migratory behavior.

PURPOSE: Loss of EPHB6 receptor tyrosine kinase expression in early-stage non-small cell lung carcinoma (NSCLC) is associated with the subsequent development of distant metastasis. Here, we analyzed the regulation and function of EPHB6 in lung cancer metastasis. EXPERIMENTAL DESIGN: The expression levels of EPHB6 were compared among normal lung tissue (n = 9), NSCLC without metastasis (n = 39), and NSCLC with metastasis (n = 39) according to the history of the patients. In addition, EPHB6 expression levels of matched tumor-normal pairs from 24 NSCLC patients were analyzed. The promoter DNA methylation status and its association with the expression levels of EPHB6 were determined among 14 pairs of tumor-normal samples. metastatic potential of EPHB6 was assessed in vitro and in vivo in a metastasis mouse model. Overexpression and RNA interference (RNAi) approaches were used for analysis of the biological functions of EPHB6. RESULTS: EPHB6 mRNA and protein levels were significantly reduced in NSCLC tumors compared with matched normal lung tissue. Decreased EPHB6 expression levels were associated with an increased risk for metastasis development in NSCLC patients. Loss of expression correlated with EPHB6 hypermethylation. EPHB6 expression was induced by 5-aza-2'-deoxycytidine treatment in an NSCLC cell line. Restoration of EPHB6 expression in lung adenocarcinoma cells increased adhesion and decreased migration. Reexpression of EPHB6 in lung cancer cells almost entirely abolished metastasis formation in non obese diabetic (NOD)/severe combined immunodeficient mice. CONCLUSIONS: Taken together, these analyses show that EPHB6 is a metastasis inhibitory gene that is frequently silenced by hypermethylation of its promoter in NSCLC.