MOXD1 is a lineage-specific gene and a tumor suppressor in neuroblastoma.

Neuroblastoma is a childhood developmental cancer; however, its embryonic origins remain poorly understood. Moreover, in-depth studies of early tumor-driving events are limited because of the lack of appropriate models. Herein, we analyzed RNA sequencing data obtained from human neuroblastoma samples and found that loss of expression of trunk neural crest-enriched gene MOXD1 associates with advanced disease and worse outcome. Further, by using single-cell RNA sequencing data of human neuroblastoma cells and fetal adrenal glands and creating in vivo models of zebrafish, chick, and mouse, we show that MOXD1 is a determinate of tumor development. In addition, we found that MOXD1 expression is highly conserved and restricted to mesenchymal neuroblastoma cells and Schwann cell precursors during healthy development. Our findings identify MOXD1 as a lineage-restricted tumor-suppressor gene in neuroblastoma, potentiating further stratification of these tumors and development of novel therapeutic interventions.

Hypoxia inducible factor-2α importance for migration, proliferation, and self-renewal of trunk neural crest cells.

BACKGROUND: The neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)-2α is associated with neural crest stem cell appearance and aggressiveness in tumors. However, little is known about its role in normal neural crest development. RESULTS: Here, we show that HIF-2α is expressed in trunk neural crest cells of human, murine, and avian embryos. Knockdown as well as overexpression of HIF-2α in vivo causes developmental delays, induces proliferation, and self-renewal capacity of neural crest cells while decreasing the proportion of neural crest cells that migrate ventrally to sympathoadrenal sites. Reflecting the in vivo phenotype, transcriptome changes after loss of HIF-2α reveal enrichment of genes associated with cancer, invasion, epithelial-to-mesenchymal transition, and growth arrest. CONCLUSIONS: Taken together, these results suggest that expression levels of HIF-2α must be strictly controlled during normal trunk neural crest development and that dysregulated levels affects several important features connected to stemness, migration, and development.

ARNT-dependent HIF-2 transcriptional activity is not sufficient to regulate downstream target genes in neuroblastoma.

BACKGROUND: Hypoxia-inducible factor (HIF)-2α associates with poor outcome in neuroblastoma and glioblastoma, and gain-of-function mutations in the EPAS1 gene (encoding HIF-2α) have been reported in paragangliomas and pheochromocytomas. Specific targeting of a druggable hydrophobic pocket in the HIF-2α PAS-B domain with PT2385 have demonstrated promising clinical results for clear cell renal cell carcinoma (ccRCC). Here, we investigated the effect of PT2385-mediated inhibition of ARNT dependent HIF-2 activity. METHODS: Neuroblastoma patient-derived xenograft (PDX) cells were treated with PT2385 and analyzed for HIF-2-dependent gene expression, HIF activity, HIF-2α protein localization, response to chemotherapy and orthotopic tumor growth in vivo. Two-sided student t-test was used. RESULTS: We detected high levels of HIF-2α protein in perivascular niches in neuroblastoma PDXs in vivo and at oxygenated conditions in PDX-derived cell cultures in vitro, particularly in the cytoplasmic fraction. Nuclear HIF-2α expression was reduced following PT2385 treatment, but surprisingly, virtually no effects on tumor growth in vivo or expression of canonical HIF downstream target genes in vitro were observed. In coherence, RNA sequencing of PT2385-treated PDX cells revealed a virtually unaffected transcriptome. Treatment with PT2385 did not affect cellular response to chemotherapy. In contrast, HIF-2α protein knockdown resulted in profound downregulation of target genes. CONCLUSIONS: The lack of effect from PT2385 treatment in combination with high cytoplasmic HIF-2α expression at normoxia suggest that HIF-2α have additional roles than acting as an ARNT dependent transcription factor. It is important to further unravel the conditions at which HIF-2α has transcriptional and non-transcriptional roles in neuroblastoma.

Unique animal friendly 3D culturing of human cancer and normal cells.

Two-dimensional cell culturing has proven inadequate as a reliable preclinical tumour model due to many inherent limitations. Hence, novel three-dimensional (3D) cell culture models are needed, which in many aspects can mimic a native tumour with 3D extracellular matrix. Here, we present a 3D electrospun polycaprolactone (PCL) mesh mimicking the collagen network of tissue. The naturally hydrophobic PCL mesh was subjected to O2 plasma treatment to obtain hydrophilic fibres. Biocompatibility tests performed using L929 fibroblasts show that the 3D PCL fibre unit compartments were non-toxic. The human breast cancer cell lines MCF-7 and JIMT-1, the normal-like human breast cell line MCF-10A, and human adult fibroblast were cultured in PCL meshes and cell proliferation was monitored using the AlamarBlue® assay. Confocal microscopy and cryosectioning show that the cells penetrated deep into the fibre mesh within 1 week of cell culturing. The cancer cells form spheroids with the cells attaching mainly to each other and partly to the fibres. The human adult fibroblasts stretch out along the fibres while the MCF-10A cells stretch between fibres. Overall, we show that normal and cancer cells thrive in the 3D meshes cultured in fetal bovine free medium which eliminates the use of collagen as an extracellular matrix support.