Neuroblastoma cancer stem cells: The role of NXPH1 and its receptor α-NRXN1

Neuroblastoma (NB) is a devastating paediatric cancer that originates in the developing sympathetic nervous system. These tumours account for 40% of the cases of cancer diagnosed during the first year of life and 8% of the cases detected overall during childhood. They show a striking clinical diversity, ranging from spontaneous remission to fatal metastatic dissemination. To optimize therapeutic approaches, NB patients have been stratified into risk groups. For the high risk (HR} patients, the 5 years overall survival probability remains below 30-40%. The failure to successfully treat these HR patients mainly results from the therapy-resistance and metastatic potential harboured by these tumours. The large intra-tumour heterogeneity of the HR tumours seems to be a key factor contributing to tumour progression. Thereby, there is an urgent need to understand the biological diversity of the HR-NBs in order to develop efficient therapies for these patients. lnterestingly, NBs show a marked scarcity of recurrent genetic changes even during relapse, pointing towards a relevant role of non-genetic sources of tumour heterogeneity. Compelling evidences suggest the existence of cells possessing a tumour-propagating capacity, called cancer stem cells or tumour-propagating cells (eses/TPes}, in these HR-NB tumours. Defining the heterogeneity of NBs and their CSCs: their self-renewal ability. The general aim of this doctoral thesis was to identify a genetic signature for neuroblastoma cancer stem cells (NBcsc), with the ultimate goal of providing new specific molecular markers of these NBcsc and candidate genes that might be useful for the development of future therapeutic strategies targeting the NBcsc in HR-NBs. lt is assumed that NBcsc share many similarities with their normal stem cells counterparts, the neural crest cells (NCCs), which represents transient embryonic population of pluripotent stem cells that give rise to the peripheral nervous system. Therefore, we compared the transcriptomic signature of NCCs with the transcriptomic profiles established for clinically relevant groups of NB patients. Among the candidates identified by our double screening, we retrieved Neurexophilin 1 (NXPHl }: an extracellular glycoprotein known to bind to the transmembrane receptors of the alpha-Neurexin (a-NRXN) family. To determine whether NXPHl/a-NRXNs activity is effectively related to NBcsc, we analysed their expression in a panel of human NB cell lines in conditions of stem cell enrichment. The marked induction of a-NRXNl/2 mRNA levels in the stem cell-enriched fraction suggested that they might be NBcsc markers. We identified the existence of a low subpopulation of a-NRXNl+ cells in samples from human NB cell lines and patient-derived xenografts. We further established that these a-NRXNl+ cells represents functionally discrete subpopulation of NB cells characterized by: 1} an active cycling behaviour, 2} an increased self-renewal capacity, and 3) a higher probability to survive upon chemotherapeutic insult. Our data suggest that these a-NRXNl+ NB cells are endowed with a tumour-propagating capacity and that they are required to sustain tumour growth in vivo. In parallel, we investigated whether NXPH1 is implicated in NB malignancy. Our data revealed that NXPH1 promotes NB growth, possibly by stimulating the proliferation of actively dividing NB cells and by increasing the proportion of NB cells expressing the NCC stem cell marker p75NTR. Finally, we showed that inhibiting NXPH1 or a-NRXN1 activity abrogates NB tumour growth in xenograft models. Our work provides the first experimental evidence that NXPH1, probably acting through its receptor a-NRXN1, exerts a functional role in NB progression. We suggest that NXPH1, present in the tumour microenvironment, controls the tumour-propagating capacity of NBcsc and that its reduced activity might facilitate the malignant progression of these tumours by enabling NBcsc to acquire a metastatic capacityEl neuroblastoma (NB) es un tumor pediátrico del sistema nervioso simpático en desarrollo. La probabilidad de supervivencia de los pacientes de alto riesgo no supera el 30-40%. El estudio de la heterogeneidad celular del NB revela la probable existencia de células con capacidad de propagación tumoral (en inglés cancer stem cells, CSCs). Su identificación y caracterización es fundamental en la búsqueda de nuevos fármacos para dichos pacientes. El objeto de esta tesis doctoral ha sido la identificación de la huella genética de las CSCs en el NB con la finalidad de identificar marcadores moleculares específicos y nuevas dianas terapéuticas. Para ello, partimos de la premisa de que las CSCs de los NBs deben de compartir ciertas características con sus equivalentes en el tejido sano, las células de la cresta neural (en inglés neural crest cells, NCCs), una población embrionaria de células madre que origina el sistema nervioso periférico. Al comparar el transcriptoma de las NCCs con el extraído de una comparación entre grupos de pacientes con relevancia clínica, seleccionamos la Neurexofilina 1 (NXPHl), un ligando extracelular de las Neurexinas alfa (a-NRXN). Para determinar si ambos están relacionados con el fenotipo de eses, analizamos su expresión en una colección de líneas celulares en condiciones de enriquecimiento para dicho fenotipo. El marcado aumento en los niveles de RNA mensajero de a-NRXNl/2 sugirió que podrían fun­ cionar como marcadores de las CSCs . Hemos confirmado la existencia de una población a-NRXNl+ caracterizada por una mayor capacidad de proliferación, de replicación y de pervivencia a quimioterapia. Las células a-NRXNl+ presentan características de eses y son requeridas para el crecimiento tumoral. En paralelo vemos que NXPHl promueve el crecimiento tumoral estimulando la proliferación y la presencia de células p75NTR+, marcador asociado a las NCCs . Además, la inhibición de NXPHl o a-NRXNl impide el crecimiento tumoral en modelos experimentale . Este trabajo constituye la primera evidencia funcional del papel de NXPHl en la progresión del NB. Sugerimos que NXPHl , probablemente a través de a-NRXNl, participa en la regulación de la capacidad de propagación tumoral y que su pérdida favorece la adquisición de rasgos asociados a pacientes de alto riesgo