Sunitinib Suppress Neuroblastoma Growth through Degradation of MYCN and Inhibition of Angiogenesis

<div><p>Neuroblastoma, a tumor of the peripheral sympathetic nervous system, is the most common and deadly extracranial tumor of childhood. The majority of high-risk neuroblastoma exhibit amplification of the MYCN proto-oncogene and increased neoangiogenesis. Both MYCN protein stabilization and angiogenesis are regulated by signaling through receptor tyrosine kinases (RTKs). Therefore, inhibitors of RTKs have a potential as a treatment option for high-risk neuroblastoma. We used receptor tyrosine kinase antibody arrays to profile the activity of membrane-bound RTKs in neuroblastoma and found the multi-RTK inhibitor sunitinib to tailor the activation of RTKs in neuroblastoma cells. Sunitinib inhibited several RTKs and demonstrated potent antitumor activity on neuroblastoma cells, through induction of apoptosis and cell cycle arrest. Treatment with sunitinib decreased MYCN protein levels by inhibition of PI3K/AKT signaling and GSK3β. This effect correlates with a decrease in VEGF secretion in neuroblastoma cells with MYCN amplification. Sunitinib significantly inhibited the growth of established, subcutaneous MYCN-amplified neuroblastoma xenografts in nude mice and demonstrated an anti-angiogenic effect in vivo with a reduction of tumor vasculature and a decrease of MYCN expression. These results suggest that sunitinib should be tested as a treatment option for high risk neuroblastoma patients.</p></div

Sunitinib promotes MYCN protein degradation and inhibit VEGF secretion in neuroblastoma.

<p>(<b>A</b>) Effect of sunitinib on GSK3β phosphorylation and on MYCN total protein in MYCN amplified NB cell lines. (*p≤0.05; ** p≤0.01). (<b>B</b>) Effect of sunitinib on MYCN mRNA evaluated by real time PCR after 72 h of treatment with the drug and represented as relative mRNA level ± SEM (n≥3). (<b>C</b>) ELISA analysis of VEGF secreted to cell culture medium by SK-N-BE(2) cell line after 72 hours of treatment with sunitinib (5 µM), rapamycin (20 nM) or combinations of sunitinib (5 µM) with rapamycin (20 nM) or PD98059 (20 µM). Bars are means ± SEM of three experiments (*p≤0.05 vs. untreated control; †p≤0.05 vs. rapamycin single treatment).</p

Sunitinib impair neuroblastoma growth and potentiates the cytotoxic effects of chemotherapeutic drugs <i>in vitro</i>.

<p>(<b>A</b>) SH-SY5Y, SK-N-BE and SK-N-AS neuroblastoma cells were treated with increasing concentrations of sunitinib for 72 h. The breast epithelial cell line, MCF10, was used to evaluate the therapeutic index of this drug. MTT metabolization was measured by MTT assays and colorimetric evaluation. Percentages compare to control ± SEM are represented. (<b>B</b>) NB cell lines were treated with a combination of sunitinib with increasing concentrations of cisplatin (0,05–10 mg/ml) or doxorubicin (0,02–1 µM) for 72 h. MTT metabolization was measured by MTT assays and the combination index (CI) for each combination was calculated using Calcusyn Software and represented graphically. CI<1, CI = 1 and CI>1 indicates synergism, additive effect, and antagonism, respectively. All experiments were performed at least in triplicate. (<b>C</b>) Synergistic effect of sunitinib with PD98059 on neuroblastoma cell lines. MTT metabolization was measured by MTT assays and the combination index (CI) calculated using Calcusyn Software.</p

Profiling RTK phosphorylation depicts sunitinib as a potential drug for RTK inhibition in neuroblastoma.

<p>(<b>A</b>) Representative nitrocellulose membranes showing the RTKs activation profile of SH-SY5Y, SK-N-BE(2) and IMR-32 NB cell lines before and after treatment with 1 µM dose of sunitinib for 72 h. (<b>B</b>) Sunitinib effect on its principal targets represented graphically as fold of inhibition respect to control. (<b>C</b>) Effect of sunitinib on the phosphorylation of other RTKs (fold change vs. control). (<b>D</b>) Effect of 72 h treatment with sunitinib on SH-SY5Y, SK N BE(2), SK-N-AS and IMR-32 NB cell lines analyzed by Western blot with pSer⁴⁷³Akt, Akt, pThr²⁰²/Thr²⁰⁴ Erk1/2 and Erk1/2 antibodies. β-actin was used as a loading control. (<b>E</b>) Sunitinib induces the phosphorylation of Erk1/2 from 24 hours of sunitinib treatment. All experiments were performed in triplicate.</p

Sunitinib suppress growth of established neuroblastoma xenografts <i>in vivo</i>.

<p>(<b>A</b>) Representative athymic nude mice subcutaneously injected with SH-SY5Y (upper panel) or SK-N-BE(2) (lower panel) cells and treated daily with sunitinib. Tumor samples were evaluated for tumor necrosis by H-E staining. (<b>B</b>) Representation of tumor volume from xenografted nude mice. Sunitinib treated groups showed a significant decrease in tumor growth compared with vehicle treated groups. Tumor dimensions were measured every day. Represented data are means ±SEM (n≥3) (*p≤0.05). (<b>C</b>) Tumor samples were analyzed for microvessel density by von Willebrand factor staining. Positive staining was quantified for each cell line and condition and represented as means ±SEM (n = 3) (*p≤0.05). (<b>D</b>) Immunohistochemical analysis of MYCN protein in SK-N-BE(2) xenografts. Positive staining was quantified and represented as ±SEM (n = 3) (*p≤0.01).</p

Sunitinib inhibit proliferation and induce apotosis of neuroblastoma cells.

<p>(<b>A</b>) Changes in cell cycle were evaluated after treatment with 1 and 5 µM of sunitinib using propidium iodide staining and FACS analysis. The percentage of apoptotic cells in subG₀ regions are represented in plots for each condition. (<b>B</b>) Graphical representation of the percentage of cycling cells (S phase + G₂M phase). Percentage ± SEM are represented (n≥3) (*p≤0.05; **p≤0.01). (<b>C</b>) FACS analysis of a representative example of BrdU+7AAD co-staining after sunitinib treatment. S phase percentage is shown for each experimental condition.</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

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

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

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