Autocrine Prostaglandin E2 Signaling Promotes Tumor Cell Survival and Proliferation in Childhood Neuroblastoma

Background: Prostaglandin E2 (PGE2) is an important mediator in tumor-promoting inflammation. High expression of cyclooxygenase-2 (COX-2) has been detected in the embryonic childhood tumor neuroblastoma, and treatment with COX inhibitors significantly reduces tumor growth. Here, we have investigated the significance of a high COX-2 expression in neuroblastoma by analysis of PGE2 production, the expression pattern and localization of PGE2 receptors and intracellular signal transduction pathways activated by PGE2. Principal Findings: A high expression of the PGE2 receptors, EP1, EP2, EP3 and EP4 in primary neuroblastomas, independent of biological and clinical characteristics, was detected using immunohistochemistry. In addition, mRNA and protein corresponding to each of the receptors were detected in neuroblastoma cell lines. Immunofluorescent staining revealed localization of the receptors to the cellular membrane, in the cytoplasm, and in the nuclear compartment. Neuroblastoma cells produced PGE2 and stimulation of serum-starved neuroblastoma cells with PGE2 increased the intracellular concentration of calcium and cyclic AMP with subsequent phosphorylation of Akt. Addition of 16,16-dimethyl PGE 2 (dmPGE2) increased cell viability in a time, dose- and cell line-dependent manner. Treatment of neuroblastoma cells with a COX-2 inhibitor resulted in a diminished cell growth and viability that was reversed by the addition of dmPGE2. Similarly, PGE 2 receptor antagonists caused a decrease in neuroblastoma cell viability in a dose-dependent manner

Tumor Development, Growth Characteristics and Spectrum of Genetic Aberrations in the TH-<em>MYCN</em> Mouse Model of Neuroblastoma

<div><h3>Background</h3><p>The TH-<em>MYCN</em> transgenic neuroblastoma model, with targeted MYCN expression to the developing neural crest, has been used to study neuroblastoma development and evaluate novel targeted tumor therapies.</p> <h3>Methods</h3><p>We followed tumor development in 395 TH-<em>MYCN</em> (129X1/SvJ) mice (125 negative, 206 hemizygous and 64 homozygous mice) by abdominal palpations up to 40 weeks of age. DNA sequencing of <em>MYCN</em> in the original plasmid construct and mouse genomic DNA was done to verify the accuracy. Copy number analysis with Affymetrix® Mouse Diversity Genotyping Arrays was used to characterize acquired genetic aberrations.</p> <h3>Results</h3><p>DNA sequencing confirmed presence of human <em>MYCN</em> cDNA in genomic TH-<em>MYCN</em> DNA corresponding to the original plasmid construct. Tumor incidence and growth correlated significantly to transgene status with event-free survival for hemizygous mice at 50%, and 0% for homozygous mice. Hemizygous mice developed tumors at 5.6–19 weeks (median 9.1) and homozygous mice at 4.0–6.9 weeks (5.4). The mean treatment window, time from palpable tumor to sacrifice, for hemizygous and homozygous mice was 15 and 5.2 days, respectively. Hemizygous mice developing tumors as early as homozygous mice had a longer treatment window. Age at tumor development did not influence treatment window for hemizygous mice, whereas treatment window in homozygous mice decreased significantly with increasing age. Seven out of 10 analysed tumors had a flat DNA profile with neither segmental nor numerical chromosomal aberrations. Only three tumors from hemizygous mice showed acquired genetic features with one or more numerical aberrations. Of these, one event corresponded to gain on the mouse equivalent of human chromosome 17.</p> <h3>Conclusion</h3><p>Hemizygous and homozygous TH-<em>MYCN</em> mice have significantly different neuroblastoma incidence, tumor growth characteristics and treatment windows but overlap in age at tumor development making correct early genotyping essential to evaluate therapeutic interventions. Contrasting previous studies, our data show that TH-<em>MYCN</em> tumors have few genetic aberrations.</p> </div

EC₅₀ of EP1-4 receptor antagonists on neuroblastoma cell viability <i>in vitro</i>.

<p>Abbreviations: EC₅₀; effective concentration decreasing neuroblastoma cell viability with 50%,</p>a<p>MYCN amplification;</p>b<p>Multidrug-resistant phenotype.</p

dmPGE₂ increases intracellular Ca²⁺ and cAMP concentrations followed by phosphorylation of Akt.

<p>(A) Intracellular calcium mobilization in response to dmPGE₂. SK-N-SH cells were loaded with the calcium fluorescent dye Fluo-4/AM before the addition of 1 µM dmPGE₂ or (B) pre-treatment with 2 mM EGTA before exposure to 1 µM dmPGE₂. The fluorescence intensity was followed using a confocal laser scanning microscope and representative single-cell recordings are shown. The arrows indicate when dmPGE₂ is added. (C) Intracellular accumulation of cAMP in response to dmPGE₂. SK-N-SH cells were incubated overnight in a medium without serum before the addition of 1 µM of dmPGE₂. Pretreatment with 10 µM NF 449, which is a Gαs inhibitor, before the incubation in dmPGE₂ for 10 min inhibited the production of cAMP. Forskolin, 10 µM for 10 min, was used as a positive control. The graph shows mean (±SD) in % of untreated control of three independent experiments. A statistical analysis was performed using 2-sided t-test, P<0.05. (D) PGE₂ induces phosphorlyation of Akt. SK-N-BE(2) and SK-N-SH cells were grown in the presence of serum (Ctr) before 24 h of culturing in the absence of serum (0 h) prior to the addition of 1 µM of dmPGE₂. Cells were further incubated in dmPGE₂ for 1, 2, 4, 6, 12 or 24 h and protein extracts were subjected to western blotting to detect phosphorylated Akt(ser473). An antibody detecting unphosphorylated Akt was used to exclude differences in total protein expression. β-actin was used to control for equal protein loading. The western blots are representative of three independent experiments.</p

Neuroblastoma cells produce PGE₂ and dmPGE₂ increases cell viability.

<p>(A) Neuroblastoma cells produce PGE₂. SK-N-BE(2) and SK-N-SH cells were cultured with or without 40 µM of arachidonic acid (AA) for 48 h and 10 ng/mL IL-1β for 12 h. Cell homogenates were incubated with 80 µM of arachidonic acid and the concentration of produced PGE₂ was measured using LC-MS/MS. (B) PGE₂ increases neuroblastoma cell viability. SK-N-BE(2) and SK-N-SH cells were incubated in a serum-free medium for 24 h before adding different concentrations of dmPGE₂. Cell viability was measured using MTT-assay after 24, 48, 72 or 96 h. Values are representative of two independent experiments and data are expressed as mean (±SD) in percentage of control at 24 h. A statistical analysis was performed using 2-way ANOVA p<0.0001 for both concentration and incubation time. (C) PGE₂ rescues neuroblastoma cells from celecoxib induced apoptosis. SK-N-BE(2) cells were incubated in 35 µM celecoxib alone or in combination with 5 µM dmPGE₂. After 48 h cell viability was assessed using MTT-assay. Mean (±SD) of six replicate wells is shown; values are representative of three independent experiments. Statistical analysis was performed using 2-sided t test P<0.0001.</p

Time from a palpable tumor to sacrifice – the treatment window.

<p><b>A.</b> Treatment window for homozygous and hemizygous mice. Mean number of treatment days for homozygous mice was 5.2 days (median 5, n = 30) and for hemizygous mice 15 days (median 11, n = 54). Statistical analysis was performed using two-sided <i>t</i> test on log-transformed data; p<0.01. <b>B.</b> Treatment window for hemizygous mice as a function of age at tumor palpation. Linear regression analysis yielded R² =  0.012 and the correlation was assessed using two-sided Spearman test; p = 0.83. <b>C.</b> Treatment window for homozygous mice as a function of age at tumor palpation. Linear regression analysis yielded R² =  0.30 and the correlation was assessed using two-sided Spearman test; p<0.05. <b>D.</b> Treatment window for homozygous mice versus hemizygous mice with an early tumor development (5.6–7.0 weeks of age). Mean number of treatment days for homozygous mice was 5.2 days (median 5, n = 30) and for hemizygous mice with an early tumor development 15 days (median 18, n = 5). Statistical analysis was performed using two-sided t test on log-transformed data; p<0.01. Box plots show median, inter quartile range (25–75%), min and max, and + represent the mean.</p

Whole genomic profile of two representative cases of the ten analyzed TH-<i>MYCN</i> tumors.

<p>TG1–TG7 showed flat profiles with no segmental or numerical aberrations (TG7 is shown here). TG8, TG9 and TG10 showed a few numerical aberrations. TG8 shown here display whole chromosome gain of mouse chromosome 3 and 11 (indicated by arrows).</p

Summary of genomic aberrations in ten different TH-<i>MYCN</i> tumors.

<p>Abbreviations:</p>a<p>homozygous;</p>b<p>hemizygous;</p>c<p>whole chromosome gain;</p>d<p>whole chromosome loss.</p