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

Perioperative detection of circulating tumour cells in patients with lung cancer

© 2017 Chudasama et al. Lung cancer is a leading cause of mortality and despite surgical resection a proportion of patients may develop metastatic spread. The detection of circulating tumour cells (CTCs) may allow for improved prediction of metastatic spread and survival. The current study evaluates the efficacy of the ScreenCell® filtration device, to capture, isolate and propagate CTCs in patients with primary lung cancer. Prior to assessment of CTCs, the present study detected cancer cells in a proof-of-principle- experiment using A549 human lung carcinoma cells as a model. Ten patients (five males and five females) with pathologically diagnosed primary non-small cell lung cancer undergoing surgical resection, had their blood tested for CTCs. Samples were taken from a peripheral vessel at the baseline, from the pulmonary vein draining the lobe containing the tumour immediately prior to division, a further central sample was taken following completion of the resection, and a final peripheral sample was taken three days post‑resection. A significant increase in CTCs was observed from baseline levels following lung manipulation. No association was able to be made between increased levels of circulating tumour cells and survival or the development of metastatic deposits. Manipulation of the lung during surgical resection for non-small cell lung carcinoma results in a temporarily increased level of CTCs; however, no clinical impact for this increase was observed. Overall, the study suggests the ScreenCell® device has the potential to be used as a CTC isolation tool, following further work, adaptations and improvements to the technology and validation of results

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

LRP2, an auxiliary receptor that controls sonic hedgehog signaling in development and disease

To fulfill their multiple roles in organ development and adult tissue homeostasis Hedgehog morphogens (HH) act through their receptor Patched (PTCH) on target cells. However, HH actions also require HH binding proteins, auxiliary cell surface receptors that agonize or antagonize morphogen signaling in a context-dependent manner. Here, we discuss recent findings on the LDL receptor-related protein 2 (LRP2), an exemplary HH binding protein that modulates sonic hedgehog activities in stem and progenitor cell niches in embryonic and adult tissues. LRP2 functions are crucial for developmental processes in a number of tissues, including the brain, the eye, and the heart, and defects in this receptor pathway are the cause of devastating congenital diseases in humans

SAMMSON fosters cancer cell fitness by concertedly enhancing mitochondrial and cytosolic translation

Synchronization of mitochondrial and cytoplasmic translation rates is critical for the maintenance of cellular fitness, with cancer cells being especially vulnerable to translational uncoupling. Although alterations of cytosolic protein synthesis are common in human cancer, compensating mechanisms in mitochondrial translation remain elusive. Here we show that the malignant long non-coding RNA (lncRNA) SAMMSON promotes a balanced increase in ribosomal RNA (rRNA) maturation and protein synthesis in the cytosol and mitochondria by modulating the localization of CARF, an RNA-binding protein that sequesters the exo-ribonuclease XRN2 in the nucleoplasm, which under normal circumstances limits nucleolar rRNA maturation. SAMMSON interferes with XRN2 binding to CARF in the nucleus by favoring the formation of an aberrant cytoplasmic RNA-protein complex containing CARF and p32, a mitochondrial protein required for the processing of the mitochondrial rRNAs. These data highlight how a single oncogenic lncRNA can simultaneously modulate RNA-protein complex formation in two distinct cellular compartments to promote cell growth

Down-Regulation of Neogenin Accelerated Glioma Progression through Promoter Methylation and Its Overexpression in SHG-44 Induced Apoptosis

Dependence receptors have been proved to act as tumor suppressors in tumorigenesis. Neogenin, a DCC homologue, well known for its fundamental role in axon guidance and cellular differentiation, is also a dependence receptor functioning to control apoptosis. However, loss of neogenin has been reported in several kinds of cancers, but its role in glioma remains to be further investigated.Western blot analysis showed that neogenin level was lower in glioma tissues than in their matching surrounding non-neoplastic tissues (n = 13, p<0.01). By immunohistochemical analysis of 69 primary and 16 paired initial and recurrent glioma sections, we found that the loss of neogenin did not only correlate negatively with glioma malignancy (n = 69, p<0.01), but also glioma recurrence (n = 16, p<0.05). Kaplan-Meier plot and Cox proportional hazards modelling showed that over-expressive neogenin could prolong the tumor latency (n = 69, p<0.001, 1187.6 ± 162.6 days versus 687.4 ± 254.2 days) and restrain high-grade glioma development (n = 69, p<0.01, HR: 0.264, 95% CI: 0.102 to 0.687). By Methylation specific polymerase chain reaction (MSP), we reported that neogenin promoter was methylated in 31.0% (9/29) gliomas, but absent in 3 kinds of glioma cell lines. Interestingly, the prevalence of methylation in high-grade gliomas was higher than low-grade gliomas and non-neoplastic brain tissues (n = 33, p<0.05) and overall methylation rate increased as glioma malignancy advanced. Furthermore, when cells were over-expressed by neogenin, the apoptotic rate in SHG-44 was increased to 39.7% compared with 8.1% in the blank control (p<0.01) and 9.3% in the negative control (p<0.01).These observations recapitulated the proposed role of neogenin as a tumor suppressor in gliomas and we suggest its down-regulation owing to promoter methylation is a selective advantage for glioma genesis, progression and recurrence. Furthermore, the induction of apoptosis in SHG-44 cells after overexpression of neogenin, indicated that neogenin could be a novel target for glioma therapy