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Neuroblastoma is a pediatric malignancy of the sympathetic ganglia and adrenal glands, hypothesized to originate from progenitors of the developing sympathetic nervous system. Amplification of the MYCN oncogene is a genetic marker of risk in this disease. Understanding the impact of oncogene expression on sympathoadrenal progenitor development may improve our knowledge of neuroblastoma initiation and progression. We isolated sympathoadrenal progenitor cells from the postnatal murine adrenal gland by sphere culture and found them to be multipotent, generating differentiated colonies of neurons, Schwann cells, and myofibroblasts. MYCN overexpression in spheres promoted commitment to the neural lineage, evidenced by an increased frequency of neuron-containing colonies. MYCN promoted proliferation of both sympathoadrenal progenitor spheres and differentiated neurons derived from these spheres, but there was also an increase in apoptosis. The proliferation, apoptosis, and neural lineage commitment induced by MYCN are tumor-like characteristics and thereby support the hypothesis that multipotent adrenal medullary progenitor cells are cells of origin for neuroblastoma. We find, however, that MYCN overexpression is not sufficient for these cells to form tumors in nude mice, suggesting that additional transforming mutations are necessary for tumorigenesis.
CacyBP/SIP [calcyclin-binding protein/Siah-1 [seven in absentia homolog 1 (Siah E3 ubiquitin protein ligase 1)] interacting protein] is a multifunctional protein whose activity includes acting as an ERK1/2 phosphatase. We analyzed dimerization of mouse CacyBP/SIP in vitro and in mouse neuroblastoma cell line (NB2a) cells, as well as the structure of a full-length protein. Moreover, we searched for the CacyBP/SIP domain important for dimerization and dephosphorylation of ERK2, and we analyzed the role of dimerization in ERK1/2 signaling in NB2a cells. Cell-based assays showed that CacyBP/SIP forms a homodimer in NB2a cell lysate, and biophysical methods demonstrated that CacyBP/SIP forms a stable dimer in vitro. Data obtained using small-angle X-ray scattering supported a model in which CacyBP/SIP occupies an anti-parallel orientation mediated by the N-terminal dimerization domain. Site-directed mutagenesis established that the N-terminal domain is indispensable for full phosphatase activity of CacyBP/SIP. We also demonstrated that the oligomerization state of CacyBP/SIP as well as the level of post-translational modifications and subcellular distribution of CacyBP/SIP change after activation of the ERK1/2 pathway in NB2a cells due to oxidative stress. Together, our results suggest that dimerization is important for controlling phosphatase activity of CacyBP/SIP and for regulating the ERK1/2 signaling pathway.
Microarray-based molecular signatures have not been widely integrated into neuroblastoma diagnostic classification systems due to the complexities of the assay and requirement for high-quality RNA. New digital technologies that accurately quantify gene expression using RNA isolated from formalin-fixed paraffin embedded (FFPE) tissues are now available. In this study, we describe the first use of a high-throughput digital system to assay the expression of genes in an "ultra-high risk" microarray classifier in FFPE high-risk neuroblastoma tumors. Customized probes corresponding to the 42 genes in a published multi-gene neuroblastoma signature were hybridized to RNA isolated from 107 FFPE high-risk neuroblastoma samples using the NanoString nCounter™ Analysis System. For classification of each patient, the Pearson's correlation coefficient was calculated between the standardized nCounter™ data and the molecular signature from the microarray data. We demonstrate that the nCounter™ 42-gene panel sub-stratified the high-risk cohort into two subsets with statistically significantly different overall survival (p = 0.0027) and event-free survival (p = 0.028). In contrast, none of the established prognostic risk markers (age, stage, tumor histology, MYCN status, and ploidy) were significantly associated with survival. We conclude that the nCounter™ System can reproducibly quantify expression levels of signature genes in FFPE tumor samples. Validation of this microarray signature in our high-risk patient cohort using a completely different technology emphasizes the prognostic relevance of this classifier. Prospective studies testing the prognostic value of molecular signatures in high-risk neuroblastoma patients using FFPE tumor samples and the nCounter™ System are warranted.
Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
High-risk neuroblastoma is an aggressive malignancy, with high rates of treatment failure. We evaluated genetic variants associated with in vitro sensitivity to two derivatives of cyclophosphamide for association with clinical response in a separate replication cohort of neuroblastoma patients (n = 2,709). To determine sensitivity, lymphoblastoid cell lines (LCLs) were exposed to increasing concentrations of 4-hydroperoxycyclophosphamide (4HC; n = 422) and phosphoramide mustard (PM; n = 428). Genome-wide association studies were performed to identify single-nucleotide polymorphisms (SNPs) associated with sensitivity to 4HC and PM. SNPs consistently associated with LCL sensitivity were analyzed for associations with event-free survival (EFS) in patients. Two linked SNPs, rs9908694 and rs1453560, were found to be associated with (i) sensitivity to PM in LCLs across populations and (ii) EFS in all patients (P = 0.01) and within the high-risk subset (P = 0.05). Our study highlights the value of cell-based models to identify candidate variants that may predict response to treatment in patients with cancer.
Neuroblastoma is characterized by florid vascularization leading to rapid tumor dissemination to distant organs; angiogenesis contributes to tumor progression and poor clinical outcomes. We have previously demonstrated an increased expression of gastrin-releasing peptide (GRP) and its receptor, GRPR, in neuroblastoma and that GRP activates the PI3K-AKT pathway as a proangiogenic factor during tumor progression. Interestingly, AKT activation phosphorylates MTOR, a critical negative regulator of autophagy, a cellular process involved in the degradation of key proteins. We hypothesize that inhibition of GRPR enhances autophagy-mediated degradation of GRP and subsequent inhibition of angiogenesis in neuroblastoma. Here, we demonstrated a novel phenomenon where targeting GRPR using shRNA or a specific antagonist, RC-3095, decreased GRP secretion by neuroblastoma cells and tubule formation by endothelial cells in vitro. Furthermore, shGRPR or RC-3095 treatment enhanced expression of proautophagic proteins in human neuroblastoma cell lines, BE(2)-C, and BE(2)-M17. Interestingly, rapamycin, an inhibitor of MTOR, enhanced the expression of the autophagosomal marker LC3-II and GRP was localized within LC3-II-marked autophagosomes in vitro as well as in vivo, indicating autophagy-mediated degradation of GRP. Moreover, overexpression of ATG5 or BECN1 attenuated GRP secretion and tubule formation, whereas opposite effects were observed with siRNA silencing of ATG5 and BECN1. Our data supported the role of autophagy in the degradation of GRP and subsequent inhibition of angiogenesis. Therefore, activation of autophagy may lead to novel antivascular therapeutic strategies in the treatment of highly vascular neuroblastomas.
We have previously demonstrated the role of gastrin-releasing peptide (GRP) as an autocrine growth factor for neuroblastoma. Here, we report that GRP silencing regulates cell signaling involved in the invasion-metastasis cascade. Using a doxycycline inducible system, we demonstrate that GRP silencing decreased anchorage-independent growth, inhibited migration and neuroblastoma cell-mediated angiogenesis in vitro, and suppressed metastasis in vivo. Targeted inhibition of GRP decreased the mRNA levels of oncogenes responsible for neuroblastoma progression. We also identified PTEN/AKT signaling as a key mediator of the tumorigenic properties of GRP in neuroblastoma cells. Interestingly, PTEN overexpression decreased GRP-mediated migration and angiogenesis; a novel role for this, otherwise, understated tumor suppressor in neuroblastoma. Furthermore, activation of AKT (pAKT) positively correlated with neuroblastoma progression in an in vivo tumor-metastasis model. PTEN expression was slightly decreased in metastatic lesions. A similar phenomenon was observed in human neuroblastoma sections, where, early-stage localized tumors had a higher PTEN expression relative to pAKT; however, an inverse expression pattern was observed in liver lesions. Taken together, our results argue for a dual purpose of targeting GRP in neuroblastoma--1) decreasing expression of critical oncogenes involved in tumor progression, and 2) enhancing activation of tumor suppressor genes to treat aggressive, advanced-stage disease.
Activation of the Hedgehog (Hh) signaling pathway has been implicated in a variety of malignancies including neuroblastoma. Expression of Gli1, a downstream effector of Hh, correlates with a favorable prognosis in patients with neuroblastoma. Moreover, Gli1 overexpression reduces mitotic index and induces transcription of genes involved in the differentiation of neuroblastoma cells; however, much remains unknown regarding the regulation of Gli1 transcriptional activity. Here, we report a novel negative regulation of Gli1 transcriptional activity by PI3K/AKT2 signal transduction pathway. Constitutively active PI3K subunit, p110α, inhibited Gli1 transcriptional activity in neuroblastoma cells, whereas, overexpression of an inactive form of PI3K subunit, p85, enhanced its activity. Specifically, the AKT2 isoform inhibited Gli1 luciferase activity. Silencing AKT2 using siRNA increased Gli1 transcriptional activity and conversely, overexpression of constitutively active AKT2 (myr-AKT2) decreased Gli1 transcriptional activity. Furthermore, Gli1 overexpression-mediated decrease in anchorage-independent growth was rescued by AKT2 overexpression. We also demonstrated that AKT2 overexpression regulates the nuclear-cytoplasmic distribution of exogenous Gli1 protein in neuroblastoma cells by relieving a GSK3β-mediated destabilization of SUFU, a negative regulator of Gli1 nuclear translocation. Inhibition of nuclear Gli1 accumulation may explain for the suppression of the tumor-suppressive function of Gli1. Collectively, our findings suggest an important role of Gli1 as a tumor suppressor in neuroblastoma, and offer a mechanism by which AKT2 regulates the subcellular localization, and in turn, inhibits the tumor-suppressive function of Gli1 in neuroblastoma.
BACKGROUND - Gastrin-releasing peptide (GRP) and its receptor, GRP-R, are critically involved in neuroblastoma tumorigenesis; however, the molecular mechanisms and signaling pathways that are responsible for GRP/GRP-R-induced cell migration and invasion remain unclear. In this study, we sought to determine the cell signals involved in GRP/GRP-R-mediated neuroblastoma cell migration and invasion.
METHODS - Human neuroblastoma cell lines SK-N-SH, LAN-1, and IMR-32 were used for our study. Transwell migration and invasion assays were performed after GRP (10(-7) M) stimulation. The cDNA GEArray Microarray kit was used to determine GRP-R-induced gene expression changes. Protein and membrane expression of integrin subunits were confirmed by Western blotting and flow cytometry analysis. siRNA transfection was performed using Lipofectamine 2000. For scratch assay, a confluent monolayer of cells in 6-well plates were wounded with micropipette tip and observed microscopically at 24 to 72 h.
RESULTS - GRP increased neuroblastoma cell migration and expressions of MMP-2 whereas the TIMP-1 level decreased. GRP-R overexpression stimulated SK-N-SH cell migration and upregulated integrin α2, α3, and β1 protein as well as mRNA expression. Targeted silencing of integrin β1 inhibited cell migration.
CONCLUSION - GRP/GRP-R signaling contributes to neuroblastoma cell migration and invasion. Moreover, the integrin ß1 subunit critically regulates GRP-R-mediated neuroblastoma cell migration and invasion.
Copyright © 2013 Mosby, Inc. All rights reserved.
BACKGROUND - microRNA (miRNA) functions broadly as post-transcriptional regulators of gene expression, and disproportionate miRNAs can result in dysregulation of oncogenes in cancer cells. We have previously shown that gastrin-releasing peptide receptor (GRP-R) signaling regulates tumorigenicity of neuroblastoma cells. Herein, we sought to characterize miRNA profile in GRP-R silenced neuroblastoma cells, and to determine the role of miRNAs on tumorigenicity and metastatic potential.
METHODS - Human neuroblastoma cell lines, BE(2)-C and SK-N-SH, were used for our study. Stably transfected GRP-R silenced cells were assessed for miRNA profiles. Cells were transfected with miR-335, miR-363, or miR-CON, a nontargeting control, and in vitro assays were performed. In vivo functions of miR-335 and miR-363 were also assessed in a spleen-liver metastasis murine model.
RESULTS - GRP-R silencing significantly increased expression of miR-335 and miR-363 in BE(2)-C cells. Overexpression of miR-335 and miR-363 decreased tumorigenicity as measured by clonogenicity, anchorage-independent growth, and metastasis determined by cell invasion assay and liver metastasis in vivo.
CONCLUSION - We report, for the first time, that GRP-R-mediated tumorigenicity and increased metastatic potential in neuroblastoma are regulated, in part, by miR-335 and miR-363. A better understanding of the anti-tumor functions of miRNAs could provide valuable insights to discerning molecular mechanisms responsible for neuroblastoma metastasis.
Copyright © 2013 Mosby, Inc. All rights reserved.
Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.
Copyright © 2013 Elsevier Inc. All rights reserved.