Hijacking of Embryonic Programs by Neural Crest-Derived Neuroblastoma: From Physiological Migration to Metastatic Dissemination

In the developing organism, complex molecular programs orchestrate the generation of cells in adequate numbers, drive them to migrate along the correct pathways towards appropriate territories, eliminate superfluous cells, and induce terminal differentiation of survivors into the appropriate cell-types. Despite strict controls constraining developmental processes, malignancies can emerge in still immature organisms. This is the case of neuroblastoma (NB), a highly heterogeneous disease, predominantly affecting children before the age of 5 years. Highly metastatic forms represent half of the cases and are diagnosed when disseminated foci are detectable. NB arise from a transient population of embryonic cells, the neural crest (NC), and especially NC committed to the establishment of the sympatho-adrenal tissues. The NC is generated at the dorsal edge of the neural tube (NT) of the vertebrate embryo, under the action of NC specifier gene programs. NC cells (NCCs) undergo an epithelial to mesenchymal transition, and engage on a remarkable journey in the developing embryo, contributing to a plethora of cell-types and tissues. Various NCC sub-populations and derived lineages adopt specific migratory behaviors, moving individually as well as collectively, exploiting the different embryonic substrates they encounter along their path. Here we discuss how the specific features of NCC in development are re-iterated during NB metastatic behaviors

Netrin-1 acts as a survival factor for aggressive neuroblastoma

Neuroblastoma (NB), the most frequent solid tumor of early childhood, is diagnosed as a disseminated disease in >60% of cases, and several lines of evidence support the resistance to apoptosis as a prerequisite for NB progression. We show that autocrine production of netrin-1, a multifunctional laminin-related molecule, conveys a selective advantage in tumor growth and dissemination in aggressive NB, as it blocks the proapoptotic activity of the UNC5H netrin-1 dependence receptors. We show that such netrin-1 up-regulation is a potential marker for poor prognosis in stage 4S and, more generally, in NB stage 4 diagnosed infants. Moreover, we propose that interference with the netrin-1 autocrine loop in malignant neuroblasts could represent an alternative therapeutic strategy, as disruption of this loop triggers in vitro NB cell death and inhibits NB metastasis in avian and mouse models

Upstream ORF affects MYCN translation depending on exon 1b alternative splicing

<p>Abstract</p> <p>Background</p> <p>The <it>MYCN </it>gene is transcribed into two major mRNAs: one full-length (<it>MYCN) </it>and one exon 1b-spliced (<it>MYCN</it>^{Δ1<it>b</it>}) mRNA. But nothing is known about their respective ability to translate the MYCN protein.</p> <p>Methods</p> <p>Plasmids were prepared to enable translation from the upstream (uORF) and major ORF of the two <it>MYCN </it>transcripts. Translation was studied after transfection in neuroblastoma SH-EP cell line. Impact of the upstream AUG on translation was evaluated after directed mutagenesis. Functional study with the two <it>MYCN </it>mRNAs was conducted by a cell viability assay. Existence of a new protein encoded by the <it>MYCN</it>^{Δ1<it>b </it>}uORF was explored by designing a rabbit polyclonal antibody against a specific epitope of this protein.</p> <p>Results</p> <p>Both are translated, but higher levels of protein were seen with <it>MYCN</it>^{Δ1<it>b </it>}mRNA. An upstream ORF was shown to have positive cis-regulatory activity on translation from <it>MYCN </it>but not from <it>MYCN</it>^{Δ1<it>b </it>}mRNA. In transfected SH-EP neuroblastoma cells, high MYCN dosage obtained with <it>MYCN</it>^{Δ1<it>b </it>}mRNA translation induces an antiapoptotic effect after serum deprivation that was not observed with low MYCN expression obtained with <it>MYCN </it>mRNA. Here, we showed that MYCNOT: <it>MYCN </it>Overlap Transcript, a new protein of unknown function is translated from the upstream AUG of <it>MYCN</it>^{Δ1<it>b </it>}mRNA.</p> <p>Conclusions</p> <p>Existence of upstream ORF in <it>MYCN </it>transcripts leads to a new level of MYCN regulation. The resulting MYCN dosage has a weak but significant anti-apoptotic activity after intrinsic apoptosis induction.</p

GPC3-Unc5 receptor complex structure and role in cell migration

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

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Interference With Netrin-1 and Tumor Cell Death in Non–Small Cell Lung Cancer

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Inhibition of endothelial cell apoptosis by Netrin-1 during angiogenesis

International audienceNetrin-1 was recently proposed to play an important role in embryonic and pathological angiogenesis. However, data reported led to the apparently contradictory conclusions that netrin-1 is either a pro- or an antiangiogenic factor. Here, we reconcile these opposing observations by demonstrating that netrin-1 acts as a survival factor for endothelial cells, blocking the proapoptotic effect of the dependence receptor UNC5B and its downstream death signaling effector, the serine/threonine kinase DAPK. The netrin-1 effect on blood vessel development is mimicked by caspase inhibitors in ex vivo assays, and the inhibition of caspase activity, the silencing of the UNC5B receptor, and the silencing of DAPK are each sufficient to rescue the vascular sprouting defects induced by netrin-1 silencing in zebrafish. Thus, the proapoptotic effect of unbound UNC5B, and the survival effect of netrin-1 on endothelial cells finely tune the angiogenic process

SlitC-PlexinA1 mediates iterative inhibition for orderly passage of spinal commissural axons through the floor plate

International audienceSpinal commissural axon navigation across the midline in the floor plate requires repulsive forces from local Slit repellents. The long-held view is that Slits push growth cones forward and prevent them from turning back once they became sensitized to these cues after midline crossing. We analyzed with fluorescent reporters Slits distribution and FP glia morphology. We observed clusters of Slit-N and Slit-C fragments decorating a complex architecture of glial basal process ramifications. We found that PC2 proprotein convertase activity contributes to this pattern of ligands. Next, we studied Slit-C acting via PlexinA1 receptor shared with another FP repellent, the Semaphorin3B, through generation of a mouse model baring PlexinA1 Y1815F mutation abrogating SlitC but not Sema3B responsiveness, manipulations in the chicken embryo, and ex vivo live imaging. This revealed a guidance mechanism by which SlitC constantly limits growth cone exploration, imposing ordered and forward-directed progression through aligned corridors formed by FP basal ramifications