Sunitinib suppress neuroblastoma growth through degradation of MYCN and inhibition of angiogenesis.
ABSTRACT: 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 GSK3?. 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.
Project description:Neuroblastoma, a tumor of peripheral neural crest origin, numbers among the most common childhood cancers. Both amplification of the proto-oncogene MYCN and increased neoangiogenesis mark high-risk disease. Because angiogenesis is regulated by phosphatidylinositol 3-kinase (PI3K), we tested a clinical PI3K inhibitor, NVP-BEZ235, in MYCN-dependent neuroblastoma. NVP-BEZ235 decreased angiogenesis and improved survival in both primary human (highly pretreated recurrent MYCN-amplified orthotopic xenograft) and transgenic mouse models for MYCN-driven neuroblastoma. Using both gain- and loss-of-function approaches, we demonstrated that the antiangiogenic efficacy of NVP-BEZ235 depended critically on MYCN in vitro and in vivo. Thus, clinical PI3K/mammalian target of rapamycin inhibitors drove degradation of MYCN in tumor cells, with secondary paracrine blockade of angiogenesis. Our data demonstrated significantly improved survival in treated animals and suggest that NVP-BEZ235 should be tested in children with high-risk, MYCN-amplified neuroblastoma.
Project description:Neuroblastoma is the most common extracranial solid tumor of childhood. This tumor is characterized by poor survival, especially when it features amplification of the MYCN oncogene. The ability for human cancers to propagate is marked by their ability to invade and metastasize to distant sites. Focal adhesion kinase (FAK) is a key tyrosine kinase involved in the survival and metastasis of a number of human tumor types. We have shown that FAK is present in human neuroblastoma and that its expression in neuroblastoma is related to the MYCN oncogene. We have also demonstrated that inhibition of FAK in neuroblastoma leads to decreased tumor cell survival. The current review addresses the relationship between the MYCN oncogene, focal adhesion kinase and neuroblastoma.
Project description:Neuroblastoma is a pediatric tumor of the sympathetic nervous system. MYCN (V-myc myelocytomatosis viral-related oncogene, neuroblastoma derived [avian]) is amplified in 20% of neuroblastomas, and these tumors carry a poor prognosis. However, tumors without MYCN amplification also may have a poor outcome. Here, we identified downstream targets of MYCN by shRNA-mediated silencing MYCN in neuroblastoma cells. From these targets, 157 genes showed an expression profile correlating with MYCN mRNA levels in NB88, a series of 88 neuroblastoma tumors, and therefore represent in vivo relevant MYCN pathway genes. This 157-gene signature identified very poor prognosis tumors in NB88 and independent neuroblastoma cohorts and was more powerful than MYCN amplification or MYCN expression alone. Remarkably, this signature also identified poor outcome of a group of tumors without MYCN amplification. Most of these tumors have low MYCN mRNA levels but high nuclear MYCN protein levels, suggesting stabilization of MYCN at the protein level. One tumor has an MYC amplification and high MYC expression. Chip-on-chip analyses showed that most genes in this signature are directly regulated by MYCN. MYCN induces genes functioning in cell cycle and DNA repair while repressing neuronal differentiation genes. The functional MYCN-157 signature recognizes classical neuroblastoma with MYCN amplification, as well as a newly identified group marked by MYCN protein stabilization.
Project description:Detection of amplification of the MYCN gene is essential for determining optimal treatment and estimating prognosis of patients with neuroblastoma (NB). DNA FISH with neuroblastoma tissues or patient-derived bone marrow cells is the standard clinical practice for the detection of MYCN amplification. As tumor cells may often be unavailable, we developed a method to detect MYCN amplification in the plasma of patients with neuroblastoma. Taking single-copy NAGK DNA as reference, we used real-time quantitative PCR (qPCR) to determine the MYCN/NAGK ratio in the plasma of 115 patients diagnosed with NB. An increased MYCN/NAGK ratio in the plasma was consistent with MYCN amplification as assessed by DNA FISH. The AUC for a MYCN/NAGK ratio equal to 6.965 was 0.943, with 86% sensitivity and 100% specificity. Beyond the threshold of 6.965, the MYCN/NAGK ratio correlated with a heavier tumor burden. Event-free and overall survival of two years were significantly shortened in stage 4 patients with a MYCN/NAGK ratio higher than 6.965. Plasma MYCN/NAGK ratios increased in patients with progressive disease and relapse. Thus, we conclude that the determination of the plasma MYCN/NAGK ratio by qPCR is a noninvasive and reproducible method to measure MYCN amplification in patients with NB.
Project description:The mechanisms causing persistence of embryonal cells that later give rise to tumors is unknown. One tumorigenic factor in the embryonal childhood tumor neuroblastoma is the MYCN protooncogene. Here we show that normal mice developed neuroblast hyperplasia in paravertebral ganglia at birth that completely regressed by 2 weeks of age. In contrast, ganglia from MYCN transgenic (TH-MYCN) mice demonstrated a marked increase in neuroblast hyperplasia and MycN expression during week 1. Regression of neuroblast hyperplasia was then delayed and incomplete before neuroblastoma tumor formation at 6 and 13 weeks in homo- and hemizygote mice, respectively. Paravertebral neuronal cells cultured from perinatal TH-MYCN mice exhibited 3- to 10-fold resistance to nerve growth factor (NGF) withdrawal, compared with normal mice. Both low- and high-affinity NGF receptors were expressed in perinatal neuroblast hyperplasia but not in neuroblastoma tumor tissue. MYCN transgene amplification was present at low levels in perinatal neuroblast hyperplasia from both homo- and hemizygote TH-MYCN mice. However, only in hemizygous mice did tumor formation correlate with a stepwise increase in the frequency of MYCN amplification. These data suggest that inappropriate perinatal MycN expression in paravertebral ganglia cells from TH-MYCN mice initiated tumorigenesis by altering the physiologic process of neural crest cell deletion. Persisting embryonal neural crest cells underwent further changes, such as MYCN amplification and repression of NGF receptor expression, during tumor progression. Our studies provide a model for studying perinatal factors influencing embryonal tumor initiation.
Project description:Amplification of MYCN occurs commonly in neuroblastoma. We report that phosphatidylinositol 3-kinase (PI3K) inhibition in murine neuroblastoma (driven by a tyrosine hydroxylase-MYCN transgene) led to decreased tumor mass and decreased levels of Mycn protein without affecting levels of MYCN mRNA. Consistent with these observations, PI3K inhibition in MYCN-amplified human neuroblastoma cell lines resulted in decreased levels of Mycn protein without affecting levels of MYCN mRNA and caused decreased proliferation and increased apoptosis. To clarify the importance of Mycn as a target of broad-spectrum PI3K inhibitors, we transduced wild-type N-myc and N-myc mutants lacking glycogen synthase kinase 3beta phosphorylation sites into human neuroblastoma cells with no endogenous expression of myc. In contrast to wild-type N-myc, the phosphorylation-defective mutant proteins were stabilized and were resistant to the antiproliferative effects of PI3K inhibition. Our results show the importance of Mycn as a therapeutic target in established tumors in vivo, offer a mechanistic rationale to test PI3K inhibitors in MYCN-amplified neuroblastoma, and represent a therapeutic approach applicable to a broad range of cancers in which transcription factors are stabilized through a PI3K-dependent mechanism.
Project description:Neuroblastoma derived from primitive sympathetic neural precursors is a common type of solid tumor in infants. MYCN proto?oncogene bHLH transcription factor (MYCN) amplification and 1p36 deletion are important factors associated with the poor prognosis of neuroblastoma. Expression levels of MYCN and c?MYB proto?oncogene transcription factor (c?myb) decline during the differentiation of neuroblastoma cells; E2F transcription factor 1 (E2F1) activates the MYCN promoter. However, the underlying mechanism of MYCN overexpression and amplification requires further investigation. In the present study, potential c?Myb target genes, and the effect of c?myb RNA interference (RNAi) on MYCN expression and amplification were investigated in MYCN?amplified neuroblastoma cell lines. The mRNA expression levels and MYCN gene copy number in five neuroblastoma cell lines were determined by quantitative polymerase chain reaction. In addition, variations in potential target gene expression and MYCN gene copy number between pre? and post?c?myb RNAi treatment groups in MYCN?amplified Kelly, IMR32, SIMA and MHH?NB?11 cell lines, normalized to those of non?MYCN?amplified SH?SY5Y, were examined. To determine the associations between gene expression levels and chromosomal aberrations, MYCN amplification and 1p36 alterations in interphases/metaphases were analyzed using fluorescence in situ hybridization. Statistical analyses revealed correlations between 1p36 alterations and the expression of c?myb, MYB proto?oncogene like 2 (B?myb) and cyclin dependent kinase inhibitor 1A (p21). Additionally, the results of the present study also demonstrated that c?myb may be associated with E2F1 and L3MBTL1 histone methyl?lysine binding protein (L3MBTL1) expression, and that E2F1 may contribute to MYCN, B?myb, p21 and chromatin licensing and DNA replication factor 1 (hCdt1) expression, but to the repression of geminin (GMNN). On c?myb RNAi treatment, L3MBTL1 expression was silenced, while GMNN was upregulated, indicating G2/M arrest. In addition, MYCN gene copy number increased following treatment with c?myb RNAi. Notably, the present study also reported a 43.545% sequence identity between upstream of MYCN and Drosophila melanogaster amplification control element 3, suggesting that expression and/or amplification mechanisms of developmentally?regulated genes may be evolutionarily conserved. In conclusion, c?myb may be associated with regulating MYCN expression and amplification. c?myb, B?myb and p21 may also serve a role against chromosome 1p aberrations. Together, it was concluded that MYCN gene is amplified during S phase, potentially via a replication?based mechanism.
Project description:Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX-histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma.
Project description:MYCN amplification occurs in 20% of neuroblastomas and is strongly related to poor clinical outcome. We have identified folate-mediated one-carbon metabolism as highly upregulated in neuroblastoma tumors with MYCN amplification and have validated this finding experimentally by showing that MYCN amplified neuroblastoma cell lines have a higher requirement for folate and are significantly more sensitive to the antifolate methotrexate than cell lines without MYCN amplification. We have demonstrated that methotrexate uptake in neuroblastoma cells is mediated principally by the reduced folate carrier (RFC; SLC19A1), that SLC19A1 and MYCN expression are highly correlated in both patient tumors and cell lines, and that SLC19A1 is a direct transcriptional target of N-Myc. Finally, we assessed the relationship between SLC19A1 expression and patient survival in two independent primary tumor cohorts and found that SLC19A1 expression was associated with increased risk of relapse or death, and that SLC19A1 expression retained prognostic significance independent of age, disease stage and MYCN amplification. This study adds upregulation of folate-mediated one-carbon metabolism to the known consequences of MYCN amplification, and suggests that this pathway might be targeted in poor outcome tumors with MYCN amplification and high SLC19A1 expression.
Project description:The MYC oncoproteins are thought to stimulate tumor cell growth and proliferation through amplification of gene transcription, a mechanism that has thwarted most efforts to inhibit MYC function as potential cancer therapy. Using a covalent inhibitor of cyclin-dependent kinase 7 (CDK7) to disrupt the transcription of amplified MYCN in neuroblastoma cells, we demonstrate downregulation of the oncoprotein with consequent massive suppression of MYCN-driven global transcriptional amplification. This response translated to significant tumor regression in a mouse model of high-risk neuroblastoma, without the introduction of systemic toxicity. The striking treatment selectivity of MYCN-overexpressing cells correlated with preferential downregulation of super-enhancer-associated genes, including MYCN and other known oncogenic drivers in neuroblastoma. These results indicate that CDK7 inhibition, by selectively targeting the mechanisms that promote global transcriptional amplification in tumor cells, may be useful therapy for cancers that are driven by MYC family oncoproteins.