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. 2015 Jan;19(1):210-26.
doi: 10.1111/jcmm.12418. Epub 2014 Sep 30.

Prostaglandin E2 promotes MYCN non-amplified neuroblastoma cell survival via β-catenin stabilization

Sepp R Jansen  1 , Rian Holman, Ilja Hedemann, Ewoud Frankes, Carolina R S Elzinga, Wim Timens, Reinoud Gosens, Eveline S de Bont, Martina Schmidt
Affiliations

Prostaglandin E2 promotes MYCN non-amplified neuroblastoma cell survival via β-catenin stabilization

Sepp R Jansen et al. J Cell Mol Med. 2015 Jan.

Abstract

Amplification of MYCN is the most well-known prognostic marker of neuroblastoma risk classification, but still is only observed in 25% of cases. Recent evidence points to the cyclic adenosine monophosphate (cAMP) elevating ligand prostaglandin E2 (PGE2 ) and β-catenin as two novel players in neuroblastoma. Here, we aimed to define the potential role of PGE2 and cAMP and its potential interplay with β-catenin, both of which may converge on neuroblastoma cell behaviour. Gain and loss of β-catenin function, PGE2 , the adenylyl cyclase activator forskolin and pharmacological inhibition of cyclooxygenase-2 (COX-2) were studied in two human neuroblastoma cell lines without MYCN amplification. Our findings show that PGE2 enhanced cell viability through the EP4 receptor and cAMP elevation, whereas COX-2 inhibitors attenuated cell viability. Interestingly, PGE2 and forskolin promoted glycogen synthase kinase 3β inhibition, β-catenin phosphorylation at the protein kinase A target residue ser675, β-catenin nuclear translocation and TCF-dependent gene transcription. Ectopic expression of a degradation-resistant β-catenin mutant enhances neuroblastoma cell viability and inhibition of β-catenin with XAV939 prevented PGE2 -induced cell viability. Finally, we show increased β-catenin expression in human high-risk neuroblastoma tissue without MYCN amplification. Our data indicate that PGE2 enhances neuroblastoma cell viability, a process which may involve cAMP-mediated β-catenin stabilization, and suggest that this pathway is of relevance to high-risk neuroblastoma without MYCN amplification.

Keywords: cyclic AMP; neuroblastoma; prostaglandin E2; β-catenin.

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Figures

Fig. 1
Fig. 1
Effect of PGE2 and forskolin on SK-N-AS cell viability. (A) Cell viability after 24- and 48-hr incubation with 10 μM forskolin or 3 μM PGE2. (B) cAMP formation under basal (1.8 ± 0.38 pmol/min/mg/protein), forskolin (23.04 ± 0.31/pmol/min/mg protein) and PGE2 (4.79 ± 0.64 pmol/min/mg protein)-treated conditions. (C) Representative Western blot images showing expression of cyclin D1 after 8-hr incubation with forskolin or PGE2. (D) qRT-PCR detection of EP1-4 mRNA expression (Cq values) and representative bands on agarose gel. (E) Cell viability after 48-hr incubation with 10 μM AH6809 and 3 μM L-161,982 in combination with PGE2. Data represent mean ± SE of the mean of four separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to DMSO-treated cells.
Fig. 2
Fig. 2
Effect of specific COX-2 inhibition on SK-N-AS cell viability. (A) Cell viability after 48-hr incubation with indicated concentrations of niflumic acid (IC50 SK-N-AS 355 μM). (B) Cell viability after 48-hr incubation with 50 μM celecoxib. (C and D) Representative pictures of a colony formation assay. Data represent mean ± SE of the mean of 4–10 separate experiments. **P < 0.01, ***P < 0.001 compared to DMSO-treated cells.
Fig. 3
Fig. 3
COX-2 inhibition decreases cell cycle progression and induces apoptotic events in SK-N-AS cells. (A) Cell cycle analysis after 200 μM niflumic acid. (B) Niflumic acid-induced apoptosis was analysed by annexin V labelling. Lower left: viable cells; lower right: early apoptotic cells; upper right: late apoptotic/necrotic cells. (C) Mitochondrial membrane polarization was measured with JC-1 in cells incubated with niflumic acid. (D) qPCR data showing N-Myc mRNA expression after 16-hr incubation with niflumic acid. (E) Representative Western blot images showing niflumic acid-induced PARP cleavage and expression of Cyclin D1 after 24 hrs. Data represent mean ± SE of the mean of 4–10 separate experiments. ***P < 0.001 compared to DMSO-treated cells.
Fig. 4
Fig. 4
Attenuated cell viability of SK-N-AS cells in response to COX-2 inhibition can be restored by PGE2. (A) Viability after 48-hr incubation with niflumic acid in the absence or presence of the indicated concentrations of PGE2. (B) cAMP formation in response to niflumic acid with and without PGE2. (C) Representative pictures of a colony formation assay. (D) Cell viability after 48-hr incubation with 25 μM celecoxib and PGE2. Data represent mean ± SE of the mean of four separate experiments. *P < 0.05, **P < 0.01 compared to DMSO-treated cells, #P < 0.05, ##P < 0.01 compared to COX-2 inhibitor-treated cells.
Fig. 5
Fig. 5
β-Catenin activity is enhanced by PGE2 and forskolin in SK-N-AS cells. (A) Representative Western blot images of active β-catenin, p-β-catenin (ser675) and p-GSK3β (ser9) in cells incubated with forskolin or PGE2 for 30 min. (B) Representative Western blot images of cytosolic (C) and nuclear fractions (N) of active β-catenin and p-β-catenin (ser675) in cells incubated with forskolin or PGE2 for 30 min. (C) Representative immunofluorescence images of p-β-catenin (ser675) in response to forskolin or PGE2 for the indicated periods of time. White arrows indicate presence at (peri)nuclear regions. (D) TOPFlash assay of cells incubated with forskolin or PGE2. Data represent mean ± SE of the mean of four separate experiments. *P < 0.05, ***P < 0.001 compared to DMSO-treated cells.
Fig. 6
Fig. 6
Overexpression of a degradation-resistant mutant of β-catenin (β-cateninS33Y) in SK-N-AS cells and inhibition of β-catenin by XAV939. (A) Representative Western blot images of β-cateninS33Y expression. (B) TOPFlash assay of cells expressing of β-cateninS33Y. (C) Representative Western blot images of β-catenin expression in cells after 1 μM XAV939 treatment for 24 hrs. (D) Cell viability of cells expressing of β-cateninS33Y. (E) Cell viability of cells incubated with PGE2 absence or presence of XAV939. Data represent mean ± SE of the mean of four separate experiments. *P < 0.05, ***P < 0.001 compared to DMSO-treated cells, #P < 0.05 compared to PGE2-treated cells.
Fig. 7
Fig. 7
Viability of SK-N-SH human neuroblastoma cells after modulation by PGE2 and β-catenin. (A) Cell viability after 48 hrs of cells incubated with forskolin or PGE2 and L-161,982. (B) Cell viability of cells expressing of β-cateninS33Y. (C) Cell viability after 48-hr incubation with indicated concentrations of niflumic acid. (D) Cell viability of cells incubated with PGE2 in the absence or presence of XAV939. Data represent mean ± SE of the mean of 4–10 separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to DMSO-treated cells, #P < 0.05 compared to PGE2-treated cells.
Fig. 8
Fig. 8
β-Catenin expression is increased in high-risk neuroblastoma tumours without amplification of MYCN. (A) Characteristics of study population. For this study, we focused on tumours that have no MYCN amplification. (B) Representative IHC pictures of low and high β-catenin expression. (C) Quantification of average β-catenin intensity in tumours without amplification of MYCN. Boxes represent Q1, median (Q2) and Q3. Whiskers represent minimum and maximum. *P < 0.05 (Kruskal–Wallis).
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