An increased frequency of metastasis development high mRNA expression levels of the two RTKs EPHB6 and DKFZ1 indicated a reduced risk for metastasis. Recently, we identified EPHB6 as an epigenetically silenced metastasis suppressor in NSCLC, and expression of EPHB6 prevented metastasis formation in a xenograft metastasis model. Here, we scrutinized the EPHB6 variation by DNA sequencing, and characterized the functional RO5185426 Raf inhibitor consequences of EPHB6 mutations in vivo and in vitro with regard to their potential role in NSCLC metastasis. Ephrin – Eph receptor interactions are frequently deregulated in cancer. In current study we identified mutations of EPHB6 as a pro-metastatic feature in non-small cell lung cancer. One mutation, del915-917, was also present in matched normal tissue, strongly suggesting a germline alteration. Germline alterations have previously been described for EPHB6 in familial colorectal cancer To date, the functional consequences of these genetic alterations on a cellular level are unknown. Alterations of Eph receptors frequently occur in lung cancer. One large scale sequencing study found mutations in 10 out of 13 Eph receptor genes in lung adenocarcinoma. Due to the multiplicity of Eph receptor associated signaling events and the complex networking of receptors, the functional outcome of Eph receptor aberrations remain unclear. For most of the Eph receptor alterations identified to date, functional consequences have not been studied. Several somatic mutations of the EPHB6 gene have been previously identified in lung cancer, colorectal cancer, ovarian cancer and glioma. In this study, screening of 80 NSCLC patient samples and 3 NSCLC cell lines identified 3 previously unknown mutations for the EPHB6 gene. One of this mutations, del915-917, resides in the domain between the tyrosine kinase and the sterile alpha motif domain, where 2 somatic mutations were recently identified in colorectal cancer. The function of this domain is suggested to be related to cancer, and our findings in this work do support this suggestion. The in vivo experiments show clearly that expression of the mutated EPHB6 enhanced metastasis. In addition EPHB6-mutant expressing cells showed a threefold enhanced transwell migration towards a serum gradient. These results are consistent with our in vivo results. Mice transplanted with EPHB6-mut cells developed significantly more lung metastases as mice transplanted with EPHB6-wt cells. In addition to the altered functions of the EPHB6 del mutant, a few aspects might also suggest a gain of function. For example, the patterns of wound healing differed between EPHB6 wildytpe and mutant. It is possible that signaling differences exist between the wildtype and the mutant receptor. On the other hand, it might also be interesting to speculate that the mutant receptor might act dominant negative towards other inhibitory EPH receptors. This dominant negative activity might lead to the observation of potential gain of function potency. Clearly, future studies might reveal the underlying differences in signaling and the influence of other member of the EPH and EPH-receptor networks. Future studies might also reveal the functional effects of the non-del915-917 mutations. It is likely that these also inactivate EPHB6 but this needs to be confirmed in the future. Recently, we could demonstrate that EPHB6 is frequently silenced by epigenetic mechanisms in lung cancer, and others could show the same inactivation mechanism in breast cancer. Our studies also indicated that loss of EPHB6 induces a highly metastatic phenotype.