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 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

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

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

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