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Chromogranin A regulates neuroblastoma proliferation and phenotype.

ABSTRACT: Neuroblastoma is a commonly encountered solid tumor in early childhood with high neuroplasticity, and differentiation therapy is hypothesized to lead to tumor mass shrinkage and/or symptom relief. CgA is a tissue specific protein restricted to the diffuse neuroendocrine system, and widely expressed in neuroblastomas. Using knockdown and knockout approaches to deplete CgA levels, we demonstrated that CgA loss inhibits SH-SY5Y cell proliferation and leads to a morphological shift with increased expression of Schwann and extracellular matrix specific molecules, and suppression of chromaffin features. We further confirmed the effects of CgA in a series of neuroblastoma cells with [BE(2)-M17 and IMR-32] and without (SK-N-SH) N-Myc amplification. We demonstrated that CgA depletion reduced IGF-II and IGFBP-2 expression, increased IGFBP-3 levels, and suppresses IGF downstream signaling as evidenced by reduced AKT/ERK pathway activation. This was further supported by an increased anti-proliferative effect of the ERK inhibitor in the CgA depleted cells. In an in vivo xenograft neuroblastoma model, CgA knockdown led to increased S-phenotypic marker expression at both protein and mRNA levels. Together these results suggest that CgA maintains IGF secretion and intracellular signaling to regulate proliferation and differentiation in neuroblastomas.

SUBMITTER: Zhang D

PROVIDER: S-EPMC6451332 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Chromogranin A regulates neuroblastoma proliferation and phenotype.

Zhang Dongyun D Babayan Lilit L Ho Hillary H Heaney Anthony P AP

Biology open 20190304 3

Neuroblastoma is a commonly encountered solid tumor in early childhood with high neuroplasticity, and differentiation therapy is hypothesized to lead to tumor mass shrinkage and/or symptom relief. CgA is a tissue specific protein restricted to the diffuse neuroendocrine system, and widely expressed in neuroblastomas. Using knockdown and knockout approaches to deplete CgA levels, we demonstrated that CgA loss inhibits SH-SY5Y cell proliferation and leads to a morphological shift with increased ex ...[more]

PMID: 30833285

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Project description:Neuroblastoma is believed to arise from sympathetic neuroblast precursors that fail to engage the neuronal differentiation programme, but instead become locked in a pro-proliferative developmental state. Achaete-scute homolog 1 (ASCL1) is a proneural master regulator of transcription which modulates both proliferation and differentiation of sympathetic neuroblast precursor cells during development, while its expression has been implicated in the maintenance of an oncogenic programme in MYCN-amplified neuroblastoma. However, the role of ASCL1 expression in neuroblastoma is not clear, especially as its levels vary considerably in different neuroblastoma cell lines. Here, we have investigated the role of ASCL1 in maintaining proliferation and controlling differentiation in both MYCN amplified and Anaplastic Lymphoma Kinase (ALK)-driven neuroblastoma cells. Using CRISPR deletion, we generated neuroblastoma cell lines lacking ASCL1 expression, and these grew more slowly than parental cells, indicating that ASCL1 contributes to rapid proliferation of MYCN amplified and non-amplified neuroblastoma cells. Genome-wide analysis after ASCL1 deletion revealed reduced expression of genes associated with neuronal differentiation, while chromatin accessibility at regulatory regions associated with differentiation genes was also attenuated by ASCL1 knock-out. In neuroblastoma, ASCL1 has been described as part of a core regulatory circuit of developmental regulators whose high expression is maintained by mutual cross-activation of a network of super enhancers and is further augmented by the activity of MYC/MYCN. Surprisingly, ASCL1 deletion had little effect on the transcription of CRC gene transcripts in these neuroblastoma cell lines, but the ability of MYC/MYCN and CRC component proteins, PHOX2B and GATA3, to bind to chromatin was compromised. Taken together, our results demonstrate several roles for endogenous ASCL1 in neuroblastoma cells: maintaining a highly proliferative phenotype, regulating DNA binding of the core regulatory circuit genes to chromatin, while also controlling accessibility and transcription of differentiation targets. Thus, we propose a model where ASCL1, a key developmental regulator of sympathetic neurogenesis, plays a pivotal role in maintaining proliferation while simultaneously priming cells for differentiation in neuroblastoma.

Project description:Targeting glutamine metabolism has emerged as a potential therapeutic strategy for Myc overexpressing cancer cells. Myc proteins contribute to an aggressive neuroblastoma phenotype. Radiotherapy is one of the treatment modalities for high-risk neuroblastoma patients. Herein, we investigated the effect of glutamine deprivation in combination with irradiation in neuroblastoma cells representative of high-risk disease and studied the role of Myc member interplay in regulating neuroblastoma cell radioresistance. Methods: Cell proliferation and viability assays were used to establish the effect of glutamine deprivation in neuroblastoma cells expressing c-Myc or MycN. Gene silencing and overexpression were used to modulate the expression of Myc genes to determine their role in neuroblastoma radioresistance. qPCR and western blot investigated interplay between expression of Myc members. The impact of glutamine deprivation on cell response following irradiation was explored using a radiobiological 3D colony assay. DNA repair gene pathways as well as CSC-related genes were studied by qPCR array. Reactive Oxygen Species (ROS) and glutathione (GSH) levels were detected by fluorescence and luminescence probes respectively. Cancer-stem cell (CSC) properties were investigated by sphere-forming assay and flow cytometry to quantify CSC markers. Expression of DNA repair genes and CSC-related genes was analysed by mining publicly available patient datasets. Results: Our results showed that glutamine deprivation decreased neuroblastoma cell proliferation and viability and modulated Myc member expression. We then demonstrated for the first time that combined glutamine deprivation with irradiation led to a selective radioresistance of MYCN-amplified neuroblastoma cells. By exploring the underlying mechanism of neuroblastoma radioresistance properties, our results highlight interplay between c-Myc and MycN expression suggesting compensatory mechanisms in Myc proteins leading to radioresistance in MYCN-amplified cells. This result was associated with the ability of MYCN-amplified cells to dysregulate the DNA repair gene pathway, maintain GSH and ROS levels and to increase the CSC-like population and properties. Conversely, glutamine deprivation led to radiosensitization in non-MYCN amplified cell lines through a disruption of the cell redox balance and a trend to decrease in the CSC-like populations. Mining publicly available gene expression dataset obtained from pediatric neuroblastoma patients, we identified a correlation pattern between Myc members and CSC-related genes as well as a specific group of DNA repair gene pathways. Conclusions: This study demonstrated that MycN and c-Myc tightly cooperate in regulation of the neuroblastoma CSC phenotypes and radioresistance upon glutamine deprivation. Pharmacologically, strategies targeting glutamine metabolism may prove beneficial in Myc-driven tumors. Consideration of MycN/c-Myc status in selecting neuroblastoma patients for glutamine metabolism treatment will be important to avoid potential radioresistance.

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Chromogranin A regulates neuroblastoma proliferation and phenotype.

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Chromogranin A regulates neuroblastoma proliferation and phenotype.

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