The Netrin/ Neogenin-1 complex promotes cell migration by activating Integrin β1 through FAK, in human neuroblastoma cells. (El complejo Netrina/ Neogenina-1 promueve la migración celular al activar a Integrina β1 a través de FAK, en células de neuroblastoma humano)

Abstract

Cell adhesion to the extracellular matrix (ECM) is key to the regulation of cellular processes such as proliferation, survival, and migration. Mesenchymal cell adhesion requires the formation of focal adhesions, defined as multiprotein complexes that allow cell binding to the ECM. Focal adhesions involve the congregation of more than 150 proteins, which include integrins, the main ECM receptors, and the Focal Adhesion Kinase (FAK), which has as a fundamental role in the turnover of focal adhesions. Formation and disassembly of focal adhesions are dynamically regulated during cell migration, and numerous studies show that increased expression or activity of focal adhesion proteins is associated with many human diseases. For example, increased levels and/or activities of FAK have been associated with oncogenic transformation in a variety of tissues and organs. Aside the roles commonly described for FAK in activating integrins, such as integrin β1 (ITGB1), and modulating integrin downstream signaling, this kinase has been shown to behave as an intracellular effector of multiple signaling pathways, including Neogenin-1 (NEO1). NEO1 is a transmembrane receptor involved in tissue growth during embryogenesis, although it is also broadly expressed in adulthood. More recently, overexpression of NEO1 has been observed in a wide variety of human cancers including those of the breast, pancreas, cervix, colon and medulloblastoma. Despite the latter, its role in tumorigenesis is still controversial and remains to be elucidated. In the context of neural development NEO1 promotes neuronal migration and axonal guidance through interaction with the extracellular Netrin (NTN) ligand family. Interestingly, NEO1 is strongly expressed in neuroblastoma (NB), a cancer derived from neural progenitors of the sympathoadrenal lineage. Therefore, it will be relevant xii determining if NEO1, by interacting with NTNs, could promote the migration and metastasis of NB cells, and if this phenomenon is mediated through the activation of FAK, thus promoting the intracellular activation of the ITGB1. It was shown that both the NEO1 receptor and its main NTN ligands family members, Netrin-1 (NTN1) and Netrin-4 (NTN4), are expressed in NB patient samples. We reveal that NEO1, in addition to promoting chemotactic migration in association with NTN4 in vitro, promotes metastasis in vivo, as demonstrated in the chicken embryo model. Recent data from the literature supports our results and allow us to suggest that the interaction between NTN4 and NEO1 might not be direct, but rather mediated by a signaling complex also made up by laminin g1 and ITGB1. NTN1, on the other hand, acts as a chemoattractant molecule and binds directly to NEO1. In addition, we show that FAK is an downstream effector of NEO1, and that the NTN1/ NEO1 signaling complex interacts with ITGB1, in the SK-N-SH NB cells. Thus, our results suggest that NTN1/ NEO1 promotes the activation of ITGB1 through FAK, most likely through the formation of a macromolecular complex, driving cell migration. All these results are consistent with our in vivo observations, which show that NEO1 promotes the metastasis of SK-N-SH cells in an immunodeficient mouse model. In summary, this thesis shows that NEO1 promotes the migration of NB cells and that its mechanism of action is via interaction with ITGB1, with FAK being an intracellular mediator of this signaling pathway. NEO1, by promoting cell migration, could play a key role in the process of NB metastasis. Therefore, the signaling complex xiii conformed by NTNs / NEO1 is proposed as a possible prognostic marker of the progression of NB.