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Although deregulation of EPHB signaling has been shown to be an important step in colorectal tumorigenesis, the role of EPHB6 in this process has not been investigated. We found here that manipulation of EPHB6 levels in colon cancer cell lines has no effect on their motility and growth on a solid substrate, soft agar or in a xenograft mouse model. We then used an EphB6 knockout mouse model to show that EphB6 inactivation does not efficiently initiate tumorigenesis in the intestinal tract. In addition, when intestinal tumors are initiated genetically or pharmacologically in EphB6 +/+ and EphB6 −/− mice, no differences were observed in animal survival, tumor multiplicity, size or histology, and proliferation of intestinal epithelial cells or tumor cells. However, reintroduction of EPHB6 into colon cancer cells significantly reduced the number of lung metastasis after tail-vein injection in immunodeficient mice, while EPHB6 knockdown in EPHB6-expressing cells increased their metastatic spread. Consistently, although EPHB6 protein expression in a series of 130 primary colorectal tumors was not associated with patient survival, EPHB6 expression was significantly lower in lymph node metastases compared to primary tumors. Our results indicate that the loss of EPHB6 contributes to the metastatic process of colorectal cancer.

EPH signaling deregulation has been shown to be important for colorectal carcinogenesis and genome-wide sequencing efforts have identified EPHA3 as one of the most frequently mutated genes in these tumors. However, the role of EPHA3 in colorectal cancer has not been thoroughly investigated. We show here that ectopic expression of wild type EPHA3 in colon cancer cells did not affect their growth, motility/invasion or metastatic potential in vivo. Moreover, overexpression of mutant EPHA3 or deletion of the endogenous mutant EPHA3 in colon cancer cells did not affect their growth or motility. EPHA3 inactivation in mice did not initiate the tumorigenic process in their intestine, and had no effects on tumor size/multiplicity after tumor initiation either genetically or pharmacologically. In addition, immunohistochemical analysis of EPHA3 tumor levels did not reveal associations with survival or clinicopathological features of colorectal cancer patients. In conclusion, we show that EPHA3 does not play a major role in colorectal tumorigenesis. These results significantly contribute to our understanding of the role of EPH signaling during colorectal carcinogenesis, and highlighting the need for detailed functional studies to confirm the relevance of putative cancer driver genes identified in sequencing efforts of the cancer genome

Los receptores EPH (receptores de efrina) constituyen la subfamilia más grande de los receptores tirosina quinasas en mamífero. Se unen a las efrinas, ancladas a las membranas celulares mediante un enlace glicosilfosfatidilinositol (GPI) (efrinas de tipo A) o mediante una dominio transmembrana seguido por un dominio citoplasmático (efrinas de tipo B). El complejo receptor-ligando activa una cascada de señalización tanto en las células que expresan los receptores EPH como en las células adyacentes, que expresan las efrinas. Este mecanismo se denomina "forward" y "reverse signalling", respectivamente. La interacción entre receptor y su ligando puede desencadenar una amplia gama de respuestas celulares incluyendo la adhesión, repulsión y la segregación/posicionamiento celular durante la separación de los tejidos en las primeras fases de desarrollo embrionario. Los mecanismos que regulan estas distintas respuestas dependen en gran medida del tipo celular y del tejido. Aunque, los receptores EPH han sido ampliamente caracterizados para sus funciones durante el desarrollo embrionario, se encuentran re-expresados en una variedad de tumores malignos humanos. No obstante, dependiendo del tipo de tumor, de su estadio y de la actividad quinasa, los receptores EPH pueden actuar induciendo o inhibiendo la progresión del cáncer. Los receptores EPH de tipo B están involucrados en la homeostasis del epitelio intestinal y la pérdida de función de estos receptores es un evento clave en la progresión del cáncer de colon, sobretodo de adenoma a carcinoma. El receptor EPHA3, identificado y aislado por primera vez ellas membranas de células pre-B de leucemia linfoblástica aguda, se encuentra ampliamente expresado durante el desarrollo embrionario, particularmente en el tejido nervioso y corazón. Tal como se espera de su primera identificación, se ha demostrado que también está involucrado en muchos tipos de cáncer. Los efectos inactivantes de distintas mutaciones somáticas encontradas en, EPHs, which make up the largest family of mammalian receptor tyrosine kinases, bind cell surface–associated Ephrins that have either a glycosylphosphatidylinositol (GPI) membrane anchor (A-type Ephrins) or a transmembrane region followed by a conserved cytoplasmatic tail (B-type Ephrins). Clustering of EPH receptors and their ligand at the surface of adjacent cells activates downstream signaling in both the EPH- and Ephrin-expressing cells, termed forward and reverse signaling, respectively. Eph–ephrin interactions can trigger a wide array of cellular responses including cell adhesion, repulsion and cell sorting/positioning during tissue boundary and the mechanisms leading to these different responses are highly dependent on the cell type and tissue context. Although most extensively characterized for their roles in development, Eph receptors are re-expressed in a variety of human malignancies. However, depending on the tumor type, disease stage and kinase function, EPH overexpression can promote or inhibit tumor progression. The role of EPHB receptors was discovered to be involved in the homeostasis of the normal intestinal epithelium and loss of function of B-type EPHs is a key event of colorectal cancer progression, mainly from adenoma to carcinoma. The EPHA3 receptor, originally recognized and isolated from membranes of pre-B acute lymphoblastic leukemia cells, is widely expressed during embryonic development, with the highest levels occurring in the nervous system and heart. As expected from its first identification, it was then implicated as having a key role in many cancers. The inactivating effects of many somatic mutations found in different cancers, strongly suggest that wild-type EPHA3 receptor suppresses the malignant properties of cancer cells in an ephrin-and kinase-dependent manner. Two independent studies of next-generation DNA sequencing using large cohorts of human colorectal cancer samples showed unexpected high mutation frequency in EPHA3 and, im