ABSTRACT
Childhood neuroblastoma has a remarkable variability in outcome. Age at diagnosis is one of the most important prognostic factors, with children less than 1 year old having favorable outcomes. Here we study single-cell and single-nuclei transcriptomes of neuroblastoma with different clinical risk groups and stages, including healthy adrenal gland. We compare tumor cell populations with embryonic mouse sympatho-adrenal derivatives, and post-natal human adrenal gland. We provide evidence that low and high-risk neuroblastoma have different cell identities, representing two disease entities. Low-risk neuroblastoma presents a transcriptome that resembles sympatho- and chromaffin cells, whereas malignant cells enriched in high-risk neuroblastoma resembles a subtype of TRKB+ cholinergic progenitor population identified in human post-natal gland. Analyses of these populations reveal different gene expression programs for worst and better survival in correlation with age at diagnosis. Our findings reveal two cellular identities and a composition of human neuroblastoma tumors reflecting clinical heterogeneity and outcome.
Subject(s)
Adrenal Gland Neoplasms/genetics , Adrenal Glands/metabolism , Membrane Glycoproteins/genetics , Neoplasm Proteins/genetics , Neuroblastoma/genetics , Receptor, trkB/genetics , Transcriptome , Adrenal Gland Neoplasms/metabolism , Adrenal Gland Neoplasms/mortality , Adrenal Gland Neoplasms/pathology , Adrenal Glands/pathology , Animals , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Differentiation , Cell Nucleus/genetics , Cell Nucleus/metabolism , Child, Preschool , Chromaffin Cells/metabolism , Chromaffin Cells/pathology , Early Diagnosis , Female , Gene Expression Regulation, Neoplastic , Humans , Infant , Male , Membrane Glycoproteins/metabolism , Mice , Neoplasm Proteins/classification , Neoplasm Proteins/metabolism , Neuroblastoma/metabolism , Neuroblastoma/mortality , Neuroblastoma/pathology , Receptor, trkB/metabolism , Risk Assessment , Single-Cell Analysis , Species Specificity , Survival AnalysisABSTRACT
The WNT/ß-catenin signaling pathway plays a central role in the biology of the periodontium, yet the function of specific extracellular WNT ligands remains poorly understood. By using a Wnt1-inducible transgenic mouse model targeting Col1a1-expressing alveolar osteoblasts, odontoblasts, and cementoblasts, we demonstrate that the WNT ligand WNT1 is a strong promoter of cementum and alveolar bone formation in vivo. We induced Wnt1 expression for 1, 3, or 9 wk in Wnt1Tg mice and analyzed them at the age of 6 wk and 12 wk. Micro-computed tomography (CT) analyses of the mandibles revealed a 1.8-fold increased bone volume after 1 and 3 wk of Wnt1 expression and a 3-fold increased bone volume after 9 wk of Wnt1 expression compared to controls. In addition, the alveolar ridges were higher in Wnt1Tg mice as compared to controls. Nondecalcified histology demonstrated increased acellular cementum thickness and cellular cementum volume after 3 and 9 wk of Wnt1 expression. However, 9 wk of Wnt1 expression was also associated with periodontal breakdown and ectopic mineralization of the pulp. The composition of this ectopic matrix was comparable to those of cellular cementum as demonstrated by quantitative backscattered electron imaging and immunohistochemistry for noncollagenous proteins. Our analyses of 52-wk-old mice after 9 wk of Wnt1 expression revealed that Wnt1 expression affects mandibular bone and growing incisors but not molar teeth, indicating that Wnt1 influences only growing tissues. To further investigate the effect of Wnt1 on cementoblasts, we stably transfected the cementoblast cell line (OCCM-30) with a vector expressing Wnt1-HA and performed proliferation as well as differentiation experiments. These experiments demonstrated that Wnt1 promotes proliferation but not differentiation of cementoblasts. Taken together, our findings identify, for the first time, Wnt1 as a critical regulator of alveolar bone and cementum formation, as well as provide important insights for harnessing the WNT signal pathway in regenerative dentistry.
Subject(s)
Cementogenesis , Dental Cementum , Animals , Mice , Osteogenesis , Periodontal Ligament , X-Ray MicrotomographyABSTRACT
AIM: In this study, we analysed the post-translational modification of receptor tyrosine kinase-like orphan receptor (Ror1). Ror1 is highly upregulated in B cells of patients with chronic lymphocytic leukaemia (CLL). Molecularly, Ror1 acts as the Wnt receptor in the non-canonical Wnt pathway. METHODS: The level of Ror1 glycosylation in HEK293 cells and in primary human CLL cells was analysed by treatment of inhibitors interfering with different steps of glycosylation process and by direct treatment of cell lysates with N-glycosidase. Ror1 ubiquitination was determined by ubiquitination assay. Functional consequences of post-translational modifications were analysed by immunohistochemistry and by analysis of cell surface proteins. Differences in Ror1 glycosylation were confirmed by analysis of 14 samples of B cells from CLL patients. RESULTS: We demonstrate that Ror1 is extensively modified by N-linked glycosylation. Glycosylation produces several variants of Ror1 with electrophoretic migration of approx. 100, 115 and 130 kDa. Inhibition of glycosylation interferes with cell surface localization of the 130-kDa variant of Ror1 and prevents Ror1-induced formation of filopodia. Moreover, we show that 130-kDa Ror1 is mono-ubiquitinated. Furthermore, individual CLL patients show striking differences in the electrophoretic migration of Ror1, which correspond to the level of glycosylation. CONCLUSION: Our data show that Ror1 undergoes complex post-translational modifications by glycosylation and mono-ubiquitination. These modifications regulate Ror1 localization and signalling, and are highly variable among individual CLL patients. These may suggest that Ror1 signals only in a subset of CLL patients despite Ror1 levels are ubiquitously high in all CLL patients.
