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Nuclear GRP75 binds retinoic acid receptors to promote neuronal differentiation of neuroblastoma.

ABSTRACT: Retinoic acid (RA) has been approved for the differentiation therapy of neuroblastoma (NB). Previous work revealed a correlation between glucose-regulated protein 75 (GRP75) and the RA-elicited neuronal differentiation of NB cells. The present study further demonstrated that GRP75 translocates into the nucleus and physically interacts with retinoid receptors (RAR? and RXR?) to augment RA-elicited neuronal differentiation. GRP75 was required for RAR?/RXR?-mediated transcriptional regulation and was shown to reduce the proteasome-mediated degradation of RAR?/RXR?in a RA-dependent manner. More intriguingly, the level of GRP75/RAR?/RXR? tripartite complexes was tightly associated with the RA-induced suppression of tumor growth in animals and the histological grade of differentiation in human NB tumors. The formation of GRP75/RAR?/RXR? complexes was intimately correlated with a normal MYCN copy number of NB tumors, possibly implicating a favorable prognosis of NB tumors. The present findings reveal a novel function of nucleus-localized GRP75 in actively promoting neuronal differentiation, delineating the mode of action for the differentiation therapy of NB by RA.

SUBMITTER: Shih YY

PROVIDER: S-EPMC3194821 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Nuclear GRP75 binds retinoic acid receptors to promote neuronal differentiation of neuroblastoma.

Shih Yu-Yin YY Lee Hsinyu H Nakagawara Akira A Juan Hseuh-Fen HF Jeng Yung-Ming YM Tsay Yeou-Guang YG Lin Dong-Tsamn DT Hsieh Fon-Jou FJ Pan Chien-Yuan CY Hsu Wen-Ming WM Liao Yung-Feng YF

PloS one 20111014 10

Retinoic acid (RA) has been approved for the differentiation therapy of neuroblastoma (NB). Previous work revealed a correlation between glucose-regulated protein 75 (GRP75) and the RA-elicited neuronal differentiation of NB cells. The present study further demonstrated that GRP75 translocates into the nucleus and physically interacts with retinoid receptors (RARα and RXRα) to augment RA-elicited neuronal differentiation. GRP75 was required for RARα/RXRα-mediated transcriptional regulation and w ...[more]

PMID: 22022577

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Project description:BackgroundChromodomain-helicase DNA binding protein 5 (CHD5) is an important tumor suppressor gene deleted from 1p36.31 in neuroblastomas (NBs). High CHD5 expression is associated with a favorable prognosis, but deletion or low expression is frequent in high-risk tumors. We explored the role of CHD5 expression in the neuronal differentiation of NB cell lines.MethodsNB cell lines SH-SY5Y (SY5Y), NGP, SK-N-DZ, IMR5, LAN5, SK-N-FI, NB69 and SH-EP were treated with 1-10 μM 13-cis-retinoic acid (13cRA) for 3-12 days. qRT-PCR and Western blot analyses were performed to measure mRNA and protein expression levels, respectively. Morphological differences were examined by both phase contrast and immunofluorescence studies.ResultsTreatment of SY5Y cells with 13cRA caused upregulation of CHD5 expression in a time- and dose-dependent manner (1, 5, or 10 μM for 7 or 12 days) and also induced neuronal differentiation. Furthermore, both NGP and SK-N-DZ cells showed CHD5 upregulation and neuronal differentiation after 13cRA treatment. In contrast, 13cRA treatment of IMR5, LAN5, or SK-N-FI induced neither CHD5 expression nor neuronal differentiation. NB69 cells showed two different morphologies (neuronal and substrate adherent) after 12 days treatment with 10 μM of 13cRA. CHD5 expression was high in the neuronal cells, but low/absent in the flat, substrate adherent cells. Finally, NGF treatment caused upregulation of CHD5 expression and neuronal differentiation in SY5Y cells transfected to express TrkA (SY5Y-TrkA) but not in TrkA-null parental SY5Y cells, and both changes were blocked by a pan-TRK inhibitor.ConclusionsTreatment with 13cRA induces neuronal differentiation only in NB cells that upregulate CHD5. In addition, NGF induced CHD5 upregulation and neuronal differentiation only in TrkA expressing cells. Together, these results suggest that CHD5 is downstream of TrkA, and CHD5 expression may be crucial for neuronal differentiation induced by either 13cRA or TrkA/NGF signaling.

Project description:BackgroundNeuroblastoma (NB), a childhood tumor derived from the sympathetic nervous system, presents with heterogeneous clinical behavior. While some tumors regress spontaneously without medical intervention, others are resistant to therapy, associated with an aggressive phenotype. MYCN-amplification, frequently occurring in high-risk NB, is correlated with an undifferentiated phenotype and poor prognosis. Differentiation induction has been proposed as a therapeutic approach for high-risk NB. We have previously shown that MYCN maintains an undifferentiated state via regulation of the miR-17 ~ 92 microRNA cluster, repressing the nuclear hormone receptors (NHRs) estrogen receptor alpha (ERα) and the glucocorticoid receptor (GR).MethodsCell viability was determined by WST-1. Expression of differentiation markers was analyzed by Western blot, RT-qPCR, and immunofluorescence analysis. Metabolic phenotypes were studied using Agilent Extracellular Flux Analyzer, and accumulation of lipid droplets by Nile Red staining. Expression of angiogenesis, proliferation, and neuronal differentiation markers, and tumor sections were assessed by immunohistochemistry. Gene expression from NB patient as well as adrenal gland cohorts were analyzed using GraphPad Prism software (v.8) and GSEA (v4.0.3), while pseudo-time progression on post-natal adrenal gland cells from single-nuclei transcriptome data was computed using scVelo.ResultsHere, we show that simultaneous activation of GR and ERα potentiated induction of neuronal differentiation, reduced NB cell viability in vitro, and decreased tumor burden in vivo. This was accompanied by a metabolic reprogramming manifested by changes in the glycolytic and mitochondrial functions and in lipid droplet accumulation. Activation of the retinoic acid receptor alpha (RARα) with all-trans retinoic acid (ATRA) further enhanced the differentiated phenotype as well as the metabolic switch. Single-cell nuclei transcriptome analysis of human adrenal glands indicated a sequential expression of ERα, GR, and RARα during development from progenitor to differentiated chromaffin cells. Further, in silico analysis revealed that patients with higher combined expression of GR, ERα, and RARα mRNA levels had elevated expression of neuronal differentiation markers and a favorable outcome.ConclusionTogether, our findings suggest that combination therapy involving activation of several NHRs could be a promising pharmacological approach for differentiation treatment of NB patients.

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Nuclear GRP75 binds retinoic acid receptors to promote neuronal differentiation of neuroblastoma.

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Nuclear GRP75 binds retinoic acid receptors to promote neuronal differentiation of neuroblastoma.

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