Vasoactive intestinal peptide: autocrine growth factor in neuroblastoma

doi:10.1016/0167-0115(92)90616-3

Regulatory Peptides

Volume 37, Issue 3, 18 February 1992, Pages 213-226

https://doi.org/10.1016/0167-0115(92)90616-3 Get rights and content

Abstract

Neuroblastoma is the most common solid tumor of children less than 5 years of age; yet the biology of this tumor is poorly understood. Neuroblastoma tumors are derived from neural crest precursors; they synthesize both adrenergic and peptidergic neurotransmitters. This study determined VIP receptor expression in primary neuroblastoma tumors prior to chemotherapy. The VIP receptor was expressed in 12 of 15 neuroblastoma tumors as determined by direct binding studies ( $K_{D} = 1.3–12.4 nM$ ) and VIP-mediated stimulation of adenylate cyclase. The VIP stimulation index for adenylate cyclase in the primary tumor was inversely correlated with the VIP content of the tumor, suggesting that VIP regulates its own receptor expression.

Similar observations were made in vitro by comparison of two human neuroblastoma cell lines, IMR32 and SKNSH. Both cell lines were demonstrated to express specific, high affinity VIP receptors ( $K_{D} = 4 nM$ and 2.5 nM for IMR32 and SKNSH, respectively). IMR32 cells contained very low levels of VIP (0.6 pg VIP/10⁶ cells). Exogenous VIP stimulated adenylate cyclase 22-fold over basal activity and VIP inhibited proliferation of IMR32 cells by 49% in 6-day cultures. On the other hand, SKNSH cells synthesized high levels of VIP (6.3 pg/10⁶ cells), metabolized VIP rapidly and demonstrated a low level of VIP-mediated stimulation of adenylate cyclase; their proliferation rate was minimally inhibited by exogenous VIP. These observations help validate the hypothesis that VIP serves as an autocrine growth factor in neuroblastoma.

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Statistical tests performed are indicated in the figure legends. VIP is able to induce neurite outgrowth in the MYCN-amplified IMR-32 cell line [27,39,40]. Here, this differentiation process was confirmed in other cell lines with MYCN amplification, i.e., SK-N-DZ and Kelly cells.
Neuroblastoma (NB) is a pediatric cancer. New therapies for high-risk NB aim to induce cell differentiation and to inhibit MYCN and ALK signaling in NB. The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP) are 2 related neuropeptides sharing common receptors. The level of VIP increases with NB differentiation. Here, the effects of VIP and PACAP analogs developed for therapeutic use were studied in MYCN-amplified NB SK-N-DZ and IMR-32 cells and in Kelly cells that in addition present the F1174L ALK mutation. As previously reported by our group in IMR-32 cells, VIP induced neuritogenesis in SK-N-DZ and Kelly cells and reduced MYCN expression in Kelly but not in SK-N-DZ cells. VIP decreased AKT activity in the ALK-mutated Kelly cells. These effects were PKA-dependent. IMR-32, SK-NDZ and Kelly cells expressed the genes encoding the 3 subtypes of VIP and PACAP receptors, VPAC1, VPAC2 and PAC1. In parallel to its effect on MYCN expression, VIP inhibited invasion in IMR-32 and Kelly cells. Among the 3 PACAP analogs tested, [Hyp²]PACAP-27 showed higher efficiency than VIP in Kelly cells. These results indicate that VIP and PACAP analogs act on molecular and cellular processes that could reduce aggressiveness of high-risk NB.
Characterization of novel splice variants of the PAC<inf>1</inf> receptor in human neuroblastoma cells: Consequences for signaling by VIP and PACAP
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Expression of VPAC and PAC₁ receptor isoforms was determined in six neuroblastoma cell lines as well as in human embryonic and adult brain using reverse transcriptase PCR and quantitative PCR. PAC₁ receptor splice variants missing a 21 amino acid sequence in the amino terminal domain were found to be the major receptor variants in the neuroblastoma cell lines and also were highly expressed in embryonic brain compared to adult brain. In four of the neuroblastoma cell lines, VIP and PACAP stimulated cyclic AMP production with different potencies and levels of maximal stimulation. High potency and greatest maximal stimulation of cyclic AMP for each peptide were recorded in SH-SY5Y cells, indicating the presence of high affinity VIP and PACAP receptors. Further characterization of specific VPAC and PAC₁ receptor isoforms was carried out in the SH-SY5Y cell line, where along with known PAC₁ receptor splice variants and the VPAC₂ receptor, a number of novel PAC₁ receptor splice variants were identified. The comparatively low level expression of the VPAC₂ receptor along with the poor responsiveness of SH-SY5Y cells to the VPAC₂ receptor-specific agonist Ro 25-1553 indicated that this receptor did not contribute significantly to the observed VIP responses. When the individual PAC₁ receptor isoforms were expressed in COS 7 cells, the ability of VIP to activate cyclic AMP production was increased more than 50-fold at the majority of the PAC₁ receptor variants lacking the 21 amino acid amino terminal domain sequence compared to those with the complete domain. Smaller changes were seen in the potency of PACAP-38. Similar trends were seen with inositol phosphate responses, where in each case agonist potencies were lower than for cyclic AMP production. The results of this study show that the combination of different amino terminal and intracellular loop 3 splicing variants in the PAC₁ receptor dictates the ability of agonists, particularly VIP, to activate signaling pathways. VIP has considerably greater potency at most PAC₁ receptors with the short amino terminal domain, and these therefore may mediate physiological effects of both VIP and PACAP. Furthermore, there may be a phenotypic switch in the expression of different PAC₁ receptor amino terminal splice variants between embryonic and mature nervous system, indicating that regulation of this event may have an important role in VIP/PACAP function, particularly in the developing nervous system.
Vasoactive intestinal peptide-induced neurite remodeling in human neuroblastoma SH-SY5Y cells implicates the Cdc42 GTPase and is independent of Ras-ERK pathway
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Vasoactive intestinal peptide (VIP) is known to regulate proliferation or differentiation in normal and tumoral cells. SH-SY5Y is a differentiated cell subclone derived from the SK-N-SH human neuroblastoma cell line and possess all the components for an autocrine action of VIP. In the present study, we investigated the morphological changes and intracellular signaling pathways occurring upon VIP treatment of SH-SY5Y cells. VIP induced an early remodeling of cell projections: a branched neurite network spread out and prominent varicosities developed along neurites. Although activated by VIP, the Ras/ERK pathway was not required for the remodeling process. In contrast, pull-down experiments revealed a strong Cdc42 activation by VIP while expression of a dominant-negative Cdc42 prevented the VIP-induced neurite changes, suggesting an important role for this small GTPase in the process. These data provide the first evidence for a regulation of the activity of Rho family GTPases by VIP and bring new insights in the signaling pathways implicated in neurite remodeling process induced by VIP in neuroblastoma cells.
Intractable diarrhoea revealing a neuroblastoma hypersecreting the vasoactive intestinal peptide
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Chez l’enfant, le syndrome WDHA (watery diarrhoea–hypokaliema–achlorhydria) est rare et habituellement dû à une tumeur neuroblastique hypersécrétant le vasoactive intestinal peptide (VIP). Nous rapportons le cas d’un syndrome WDHA secondaire à l’hypersécrétion de VIP et révélateur d’un neuroblastome chez une enfant âgée de 13 mois. Une diarrhée sécrétoire, caractérisée par la persistance de la diarrhée en dépit de l’arrêt de l’alimentation orale, a conduit à rechercher une tumeur neuroblastique chez l’enfant. Le taux sérique de VIP s’est rapidement normalisé après l’exérèse chirurgicale de la tumeur.
In children, the watery diarrhoea–hypokalemia–achlorhydria (WDHA) syndrome is uncommon and usually due to a neuroblastic tumour hypersecreting the vasoactive intestinal peptide (VIP). We report a case of WDHA syndrome secondary to hypersecretion of VIP that revealed a neuroblastoma in a 13-month-old girl. A secretory diarrhoea, characterised by the persistence of diarrhoea despite the cessation of oral feeding, led to the search of a neuroblastic tumour in the patient. The serum concentration of VIP decreased to normal values soon after the surgical excision of the tumour.
VIP receptor 1 (VPAC1) promoter targets the expression of a reporter gene to cerebellum and adrenal medulla in transgenic mice
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Vasoactive intestinal peptide (VIP) is a neurotransmitter with neurotropic effects. VIP functions through two distinct G-protein-coupled receptor subtypes (VPAC1 and VPAC2). We have demonstrated expression of VPAC1 in pediatric nervous system tumors, including medulloblastoma arising in the cerebellum and neuroblastoma arising in the adrenal medulla. More recently, we have reported the differentiation of neuroblastoma cells by upregulation of VIP type 1 receptor suggesting a role for VPAC1 in neuronal development.
To understand the molecular mechanisms regulating VPAC1 expression in both cerebellum and adrenal medulla, we have cloned the human VPAC1 gene and sequenced 2.6-kb of the 5′-flanking sequence. Expression of the luciferase reporter gene under the control of this 2.6-kb human VPAC1 promoter was induced 35-fold in a human medulloblastoma cell line (DAOY) and 36-fold in a human neuroblastoma cell line (SKNSH). Analysis of 5′-unidirectional deletion derivatives of the 2.6-kb fragment demonstrated that a 241-bp sequence immediately upstream of the VPAC1 coding region retains high activity, suggesting that it contains the core promoter region. Quantitative RT-PCR analysis demonstrated that VPAC1 is expressed in mouse cerebellar and adrenal tissues. The VPAC1 promoter also directed expression of a reporter gene in cerebellum and adrenal medulla in transgenic mice. Along with our previous findings, these results suggest that VPAC1 may play a functional role in development of both cerebellum and adrenal medulla.
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Vasoactive intestinal peptide: autocrine growth factor in neuroblastoma

Abstract

J. Biol. Chem.

Peptides

Cell

Hum. Pathol.

Neuropeptides

Am. J. Med.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Peptides

Improvements in survival from childhood cancer: results of a population based survey over 30 years

Br. Med. J.

Neuroblastoma: the case for screening infants in North America

Pediatrics

Treatment of neuroblastoma

Cancer

Neuroblastoma

A comparison of four staging systems for localized and regional neuroblastoma: a report from the Children's Cancer Study Group

J. Clin. Oncol.

A pilot study of double A.B.M.T. in advanced neuroblastoma

Intensive chemotherapy for poor prognosis neuroblastoma

Phenotype diversification in human neuroblastoma cells: expression of distinct neural crest lineages

Cancer Res.

Alpha-2-adrenergic and muscarinic cholinergic receptors have opposing actions on cyclic AMP levels in SK-N-SH human neuroblastoma cells

J. Neurochem.

Polypeptide with broad biological activity: isolation from small intestine

Science

Human preprovasoactive intestinal peptide contains a novel PHI-27-like peptide PHM 27

Nature

Rapid changes of serum vasoactive intestinal peptide after removel of ganglioneuroblastoma with watery diarrhea-hypokalemia-achlorhydria syndrome in a child

J. Ped. Gastroent. Nutr.