nutlin-3a and Neuroblastoma

p53 is one of the main regulators of apoptosis, senescence, cell cycle arrest and DNA repair. The expression, function and stabilization of p53 are governed by a complex network of regulators including p14(ARF) and MDM2. MDM2 is the main negative regulator of p53 activity and stability. Unlike tumours in adults, which tend to overcome p53 regulation by p53 mutations, the paediatric tumours neuroblastoma and sarcoma frequently retain wild type p53. Nevertheless, in childhood cancer the p53 pathway is commonly impaired due to upstream MDM2-p14(ARF)-p53 network aberrations. In contrast, aberrations of the p53 downstream pathway are very rare. In cancer cells with intact p53 downstream function MDM2 inhibition, and subsequent rapid increases in nuclear p53 levels, potently "re-activate" dormant apoptotic pathways and rapidly induce apoptotic cell death. As a result MDM2-p53 interaction inhibitors, including cis-imidazolines analogs (Nutlins), are potentially very effective agents in neuroblastoma and sarcomas. Predictive biomarkers are important as a lack of p53 mutations appears to reliably predict response to these inhibitors. Tumours should be screened for p53 mutations in children considered for MDM2-p53 interaction inhibitors. In addition, it is essential that other predictive biomarkers are investigated. The serum concentration of macrophage inhibitory cytokine- 1 (MIC-1) may be a good pharmacodynamic biomarker based on recent findings. In conclusion, targeting the interaction between p53 and its main negative regulator MDM2 represents a major new therapeutic approach in poor prognosis paediatric malignancies without p53 mutations.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Child; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Humans; Imidazoles; Neuroblastoma; Piperazines; Protein Binding; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Adoptive transfer of engineered NK cells, one of clinical approaches to fight cancer, is gaining great interest in the last decade. However, the development of new strategies is needed to improve clinical efficacy and safety of NK cell-based immunotherapy. NK cell-mediated recognition and lysis of tumor cells are strictly dependent on the expression of ligands for NK cell-activating receptors NKG2D and DNAM-1 on tumor cells. Of note, the PVR/CD155 and Nectin-2/CD112 ligands for DNAM-1 are expressed primarily on solid tumor cells and poorly expressed in normal tissue cells. Here, we generated human NK cells expressing either the full length DNAM-1 receptor or three different DNAM-1-based chimeric receptor that provide the expression of DNAM-1 fused to a costimulatory molecule such as 2B4 and CD3ζ chain. Upon transfection into primary human NK cells isolated from healthy donors, we evaluated the surface expression of DNAM-1 and, as a functional readout, we assessed the extent of degranulation, cytotoxicity and the production of IFNγ and TNFα in response to human leukemic K562 cell line. In addition, we explored the effect of Nutlin-3a, a MDM2-targeting drug able of restoring p53 functions and known to have an immunomodulatory effect, on the degranulation of DNAM-1-engineered NK cells in response to human neuroblastoma (NB) LA-N-5 and SMS-KCNR cell lines. By comparing NK cells transfected with four different plasmid vectors and through blocking experiments, DNAM-1-CD3ζ-engineered NK cells showed the strongest response. Furthermore, both LA-N-5 and SMS-KCNR cells pretreated with Nutlin-3a were significantly more susceptible to DNAM-1-engineered NK cells than NK cells transfected with the empty vector. Our results provide a proof-of-concept suggesting that the combined use of DNAM-1-chimeric receptor-engineered NK cells and Nutlin-3a may represent a novel therapeutic approach for the treatment of solid tumors, such as NB, carrying dysfunctional p53.

Topics: Humans; Imidazoles; Killer Cells, Natural; Ligands; Neuroblastoma; Piperazines; Tumor Suppressor Protein p53

In this study, we explored whether Nutlin-3a, a well-known, nontoxic small-molecule compound antagonizing the inhibitory interaction of MDM2 with the tumor suppressor p53, may restore ligands for natural killer (NK) cell-activating receptors (NK-AR) on neuroblastoma cells to enhance the NK cell-mediated killing. Neuroblastoma cell lines were treated with Nutlin-3a, and the expression of ligands for NKG2D and DNAM-1 NK-ARs and the neuroblastoma susceptibility to NK cells were evaluated. Adoptive transfer of human NK cells in a xenograft neuroblastoma-bearing NSG murine model was assessed. Two data sets of neuroblastoma patients were explored to correlate p53 expression with ligand expression. Luciferase assays and chromatin immunoprecipitation analysis of p53 functional binding on

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; Cell Line, Tumor; Cytotoxicity, Immunologic; Female; Humans; Imidazoles; Killer Cells, Natural; Ligands; Mice; Mice, Inbred NOD; Neuroblastoma; NK Cell Lectin-Like Receptor Subfamily K; Piperazines; Receptors, Natural Killer Cell; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Cell lines established from the TH-MYCN transgenic murine model of neuroblastoma are a valuable preclinical, immunocompetent, syngeneic model of neuroblastoma, for which knowledge of their p53 pathway status is important. In this study, the Trp53 status and functional response to Nutlin-3 and ionising radiation (IR) were determined in 6 adherent TH-MYCN transgenic cell lines using Sanger sequencing, western blot analysis and flow cytometry. Sensitivity to structurally diverse MDM2 inhibitors (Nutlin-3, MI-63, RG7388 and NDD0005) was determined using XTT proliferation assays. In total, 2/6 cell lines were Trp53 homozygous mutant (NHO2A and 844MYCN+/+) and 1/6 (282MYCN+/-) was Trp53 heterozygous mutant. For 1/6 cell lines (NHO2A), DNA from the corresponding primary tumour was found to be Trp53 wt. In all cases, the presence of a mutation was consistent with aberrant p53 signalling in response to Nutlin-3 and IR. In comparison to TP53 wt human neuroblastoma cells, Trp53 wt murine control and TH-MYCN cell lines were significantly less sensitive to growth inhibition mediated by MI-63 and RG7388. These murine Trp53 wt and mutant TH-MYCN cell lines are useful syngeneic, immunocompetent neuroblastoma models, the former to test p53-dependent therapies in combination with immunotherapies, such as anti-GD2, and the latter as models of chemoresistant relapsed neuroblastoma when aberrations in the p53 pathway are more common. The spontaneous development of Trp53 mutations in 3 cell lines from TH-MYCN mice may have arisen from MYCN oncogenic driven and/or ex vivo selection. The identified species-dependent selectivity of MI-63 and RG7388 should be considered when interpreting in vivo toxicity studies of MDM2 inhibitors.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Humans; Imidazoles; Indoles; Mice; Mice, Transgenic; Mutation; N-Myc Proto-Oncogene Protein; Neuroblastoma; para-Aminobenzoates; Piperazines; Proto-Oncogene Proteins c-mdm2; Pyrrolidines; Radiation, Ionizing; Signal Transduction; Spiro Compounds; Tumor Suppressor Protein p53

Neuroblastoma is a malignant embryonal tumor occurring in young children, consisting of undifferentiated neuroectodermal cells derived from the neural crest. Current therapies for high-risk neuroblastoma are insufficient, resulting in high mortality rates and high incidence of relapse. With the intent to find new therapies for neuroblastomas, we investigated the efficacy of low-doses of actinomycin D, which at low concentrations preferentially inhibit RNA polymerase I-dependent rRNA trasncription and therefore, ribosome biogenesis.. Neuroblastoma cell lines with different p53 genetic background were employed to determine the response on cell viability and apoptosis of low-dose of actinomycin D. Subcutaneously-implanted SK-N-JD derived neuroblastoma tumors were used to assess the effect of low-doses of actinomycin D on tumor formation.. Low-dose actinomycin D treatment causes a reduction of cell viability in neuroblastoma cell lines and that this effect is stronger in cells that are wild-type for p53. MYCN overexpression contributes to enhance this effect, confirming the importance of this oncogene in ribosome biogenesis. In the wild-type SK-N-JD cell line, apoptosis was the major mechanism responsible for the reduction in viability and we demonstrate that treatment with the MDM2 inhibitor Nutlin-3, had a similar effect to that of actinomycin D. Apoptosis was also detected in p53(-/-)deficient LA1-55n cells treated with actinomycin D, however, only a small recovery of cell viability was found when apoptosis was inhibited by a pan-caspase inhibitor, suggesting that the treatment could activate an apoptosis-independent cell death pathway in these cells. We also determined whether actinomycin D could increase the efficacy of the histone deacetylase inhibitor, SAHA, which is in being used in neuroblastoma clinical trials. We show that actinomycin D synergizes with SAHA in neuroblastoma cell lines. Moreover, on subcutaneously-implanted neuroblastoma tumors derived from SK-N-JD cells, actinomycin D led to tumor regression, an effect enhanced in combination with SAHA.. The results presented in this work demonstrate that actinomycin D, at low concentrations, inhibits proliferation and induces cell death in vitro, as well as tumor regression in vivo. From this study, we propose that use of ribosome biogenesis inhibitors should be clinically considered as a potential therapy to treat neuroblastomas.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Survival; Dactinomycin; Dose-Response Relationship, Drug; Drug Synergism; Female; Hydroxamic Acids; Imidazoles; Mice; Neuroblastoma; Piperazines; Proto-Oncogene Proteins c-myc; Time Factors; Tumor Suppressor Protein p53; Vorinostat

Resistance formation after initial therapy response (acquired resistance) is common in high-risk neuroblastoma patients. YM155 is a drug candidate that was introduced as a survivin suppressant. This mechanism was later challenged, and DNA damage induction and Mcl-1 depletion were suggested instead. Here we investigated the efficacy and mechanism of action of YM155 in neuroblastoma cells with acquired drug resistance. The efficacy of YM155 was determined in neuroblastoma cell lines and their sublines with acquired resistance to clinically relevant drugs. Survivin levels, Mcl-1 levels, and DNA damage formation were determined in response to YM155. RNAi-mediated depletion of survivin, Mcl-1, and p53 was performed to investigate their roles during YM155 treatment. Clinical YM155 concentrations affected the viability of drug-resistant neuroblastoma cells through survivin depletion and p53 activation. MDM2 inhibitor-induced p53 activation further enhanced YM155 activity. Loss of p53 function generally affected anti-neuroblastoma approaches targeting survivin. Upregulation of ABCB1 (causes YM155 efflux) and downregulation of SLC35F2 (causes YM155 uptake) mediated YM155-specific resistance. YM155-adapted cells displayed increased ABCB1 levels, decreased SLC35F2 levels, and a p53 mutation. YM155-adapted neuroblastoma cells were also characterized by decreased sensitivity to RNAi-mediated survivin depletion, further confirming survivin as a critical YM155 target in neuroblastoma. In conclusion, YM155 targets survivin in neuroblastoma. Furthermore, survivin is a promising therapeutic target for p53 wild-type neuroblastomas after resistance acquisition (neuroblastomas are rarely p53-mutated), potentially in combination with p53 activators. In addition, we show that the adaptation of cancer cells to molecular-targeted anticancer drugs is an effective strategy to elucidate a drug's mechanism of action.

Topics: ATP Binding Cassette Transporter, Subfamily B; Cell Line, Tumor; Cell Survival; DNA Damage; Drug Resistance, Neoplasm; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Kinetics; Membrane Transport Proteins; Mutation; Naphthoquinones; Neuroblastoma; Piperazines; Proto-Oncogene Proteins c-mdm2; RNA, Small Interfering; Survivin; Tumor Suppressor Protein p53

Flubendazole was shown to exert anti-leukaemia and anti-myeloma activity through inhibition of microtubule function. Here, flubendazole was tested for its effects on the viability of in total 461 cancer cell lines. Neuroblastoma was identified as highly flubendazole-sensitive cancer entity in a screen of 321 cell lines from 26 cancer entities. Flubendazole also reduced the viability of five primary neuroblastoma samples in nanomolar concentrations thought to be achievable in humans and inhibited vessel formation and neuroblastoma tumour growth in the chick chorioallantoic membrane assay. Resistance acquisition is a major problem in high-risk neuroblastoma. 119 cell lines from a panel of 140 neuroblastoma cell lines with acquired resistance to various anti-cancer drugs were sensitive to flubendazole in nanomolar concentrations. Tubulin-binding agent-resistant cell lines displayed the highest flubendazole IC50 and IC90 values but differences between drug classes did not reach statistical significance. Flubendazole induced p53-mediated apoptosis. The siRNA-mediated depletion of the p53 targets p21, BAX, or PUMA reduced the neuroblastoma cell sensitivity to flubendazole with PUMA depletion resulting in the most pronounced effects. The MDM2 inhibitor and p53 activator nutlin-3 increased flubendazole efficacy while RNAi-mediated p53-depletion reduced its activity. In conclusion, flubendazole represents a potential treatment option for neuroblastoma including therapy-refractory cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Cell Line, Tumor; Chickens; Cyclin-Dependent Kinase Inhibitor p21; Humans; Imidazoles; Mebendazole; Neuroblastoma; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; RNA Interference; RNA, Small Interfering; Signal Transduction; Tumor Suppressor Protein p53

Restoration of the antitumor activity of p53 could offer a promising approach for the treatment of neuroblastoma. MicroRNAs (miRNAs) are important mediators of p53 activity, but their role in the p53 response has not yet been comprehensively addressed in neuroblastoma. Therefore, we set out to characterize alterations in miRNA expression that are induced by p53 activation in neuroblastoma cells. Genome-wide miRNA expression analysis showed that miR-34a-5p, miR-182-5p, miR-203a, miR-222-3p, and miR-432-5p are upregulated following nutlin-3 treatment in a p53 dependent manner. The function of miR-182-5p, miR-203a, miR-222-3p, and miR-432-5p was analyzed by ectopic overexpression of miRNA mimics. We observed that these p53-regulated miRNAs inhibit the proliferation of neuroblastoma cells to varying degrees, with the most profound growth inhibition recorded for miR-182-5p. Overexpression of miR-182-5p promoted apoptosis in some neuroblastoma cell lines and induced neuronal differentiation of NGP cells. Using Chromatin Immunoprecipitation-qPCR (ChIP-qPCR), we did not observe direct binding of p53 to MIR182, MIR203, MIR222, and MIR432 in neuroblastoma cells. Taken together, our findings yield new insights in the network of p53-regulated miRNAs in neuroblastoma.

Topics: Cell Line, Tumor; Cell Proliferation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome-Wide Association Study; Humans; Imidazoles; MicroRNAs; Neuroblastoma; Piperazines; Promoter Regions, Genetic; Protein Binding; Tumor Suppressor Protein p53

A frequent mechanism of acquired multidrug resistance in human cancers is overexpression of ATP-binding cassette transporters such as the Multi-Drug Resistance Protein 1 (MDR-1). Nutlin-3, an MDM2-p53 antagonist, has previously been reported to be a competitive MDR-1 inhibitor.. This study assessed whether the structurally diverse MDM2-p53 antagonists, MI-63, NDD0005, and RG7388 are also able to modulate MDR-1 function, particularly in p53 mutant neuroblastoma cells, using XTT-based cell viability assays, western blotting, and liquid chromatography-mass spectrometry analysis.. Verapamil and the MDM2-p53 antagonists potentiated vincristine-mediated growth inhibition in a concentration-dependent manner when used in combination with high MDR-1-expressing p53 mutant neuroblastoma cell lines at concentrations that did not affect the viability of cells when given alone. Liquid chromatography-mass spectrometry analyses showed that verapamil, Nutlin-3, MI-63 and NDD0005, but not RG7388, led to increased intracellular levels of vincristine in high MDR-1-expressing cell lines.. These results show that in addition to Nutlin-3, other structurally unrelated MDM2-p53 antagonists can also act as MDR-1 inhibitors and reverse MDR-1-mediated multidrug resistance in neuroblastoma cell lines in a p53-independent manner. These findings are important for future clinical trial design with MDM2-p53 antagonists when used in combination with agents that are MDR-1 substrates.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Cisplatin; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Humans; Imidazoles; Indoles; Inhibitory Concentration 50; Neuroblastoma; para-Aminobenzoates; Piperazines; Proto-Oncogene Proteins c-mdm2; Pyrrolidines; Spiro Compounds; Tumor Suppressor Protein p53; Verapamil; Vincristine

Aurora kinase inhibitors displayed activity in pre-clinical neuroblastoma models. Here, we studied the effects of the pan-aurora kinase inhibitor tozasertib (VX680, MK-0457) and the aurora kinase inhibitor alisertib (MLN8237) that shows some specificity for aurora kinase A over aurora kinase B in a panel of neuroblastoma cell lines with acquired drug resistance. Both compounds displayed anti-neuroblastoma activity in the nanomolar range. The anti-neuroblastoma mechanism included inhibition of aurora kinase signalling as indicated by decreased phosphorylation of the aurora kinase substrate histone H3, cell cycle inhibition in G2/M phase, and induction of apoptosis. The activity of alisertib but not of tozasertib was affected by ABCB1 expression. Aurora kinase inhibitors induced a p53 response and their activity was enhanced in combination with the MDM2 inhibitor and p53 activator nutlin-3 in p53 wild-type cells. In conclusion, aurora kinases are potential drug targets in therapy-refractory neuroblastoma, in particular for the vast majority of p53 wild-type cases.

Topics: Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; Aurora Kinase A; Aurora Kinase B; Azepines; Brain Neoplasms; Cell Cycle Checkpoints; Cell Proliferation; Drug Resistance, Neoplasm; Drug Synergism; Gene Expression Regulation, Neoplastic; Histones; Humans; Imidazoles; Neuroblastoma; Phosphorylation; Piperazines; Primary Cell Culture; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-mdm2; Pyrimidines; Signal Transduction; Tumor Suppressor Protein p53

Chemotherapy induces apoptosis and tumor regression primarily through activation of p53-mediated transcription. Neuroblastoma is a p53 wild type malignancy at diagnosis and repression of p53 signaling plays an important role in its pathogenesis. Recently developed small molecule inhibitors of the MDM2-p53 interaction are able to overcome this repression and potently activate p53 dependent apoptosis in malignancies with intact p53 downstream signaling. We used the small molecule MDM2 inhibitor, Nutlin-3a, to determine the p53 drug response signature in neuroblastoma cells. In addition to p53 mediated apoptotic signatures, GSEA and pathway analysis identified a set of p53-repressed genes that were reciprocally over-expressed in neuroblastoma patients with the worst overall outcome in multiple clinical cohorts. Multifactorial regression analysis identified a subset of four genes (CHAF1A, RRM2, MCM3, and MCM6) whose expression together strongly predicted overall and event-free survival (p<0.0001). The expression of these four genes was then validated by quantitative PCR in a large independent clinical cohort. Our findings further support the concept that oncogene-driven transcriptional networks opposing p53 activation are essential for the aggressive behavior and poor response to therapy of high-risk neuroblastoma.

Topics: Apoptosis; Cell Line, Tumor; HCT116 Cells; Humans; Imidazoles; Neuroblastoma; Oligonucleotide Array Sequence Analysis; Piperazines; Prognosis; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3(r)RITA(10 μM) to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells.

Topics: Apoptosis; Cell Line, Tumor; Cisplatin; Cluster Analysis; Drug Resistance, Neoplasm; Furans; Humans; Imidazoles; Mutation; Neuroblastoma; Phenotype; Piperazines; Proto-Oncogene Proteins c-mdm2; Transcriptome; Tumor Suppressor Protein p53; Vincristine

MYCN amplification occurs in about 20-25% of human neuroblastomas and characterizes the majority of the high-risk cases, which display less than 50% prolonged survival rate despite intense multimodal treatment. Somehow paradoxically, MYCN also sensitizes neuroblastoma cells to apoptosis, understanding the molecular mechanisms of which might be relevant for the therapy of MYCN amplified neuroblastoma. We recently reported that the apoptosis-sensitive phenotype induced by MYCN is linked to stabilization of p53 and its proapoptotic kinase HIPK2. In MYCN primed neuroblastoma cells, further activation of both HIPK2 and p53 by Nutlin-3 leads to massive apoptosis in vitro and to tumor shrinkage and impairment of metastasis in xenograft models. Here we report that Galectin-3 impairs MYCN-primed and HIPK2-p53-dependent apoptosis in neuroblastoma cells. Galectin-3 is broadly expressed in human neuroblastoma cell lines and tumors and is repressed by MYCN to induce the apoptosis-sensitive phenotype. Despite its reduced levels, Galectin-3 can still exert residual antiapoptotic effects in MYCN amplified neuroblastoma cells, possibly due to its specific subcellular localization. Importantly, Nutlin-3 represses Galectin-3 expression, and this is required for its potent cell killing effect on MYCN amplified cell lines. Our data further characterize the apoptosis-sensitive phenotype induced by MYCN, expand our understanding of the activity of MDM2-p53 antagonists and highlight Galectin-3 as a potential biomarker for the tailored p53 reactivation therapy in patients with high-risk neuroblastomas.

Topics: Apoptosis; Carrier Proteins; Cell Line, Tumor; Cytoprotection; DNA Damage; Galectin 3; Gene Amplification; Gene Dosage; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Imidazoles; Models, Biological; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Phenotype; Piperazines; Protein Serine-Threonine Kinases; Protein Transport; Tumor Suppressor Protein p53

Suppression of p53 activity is essential for proliferation and survival of tumor cells. A direct p53-activating compound, nutlin-3, was used in this study, together with p53 mutation analysis, to characterize p53 pathway defects in a set of 34 human neuroblastoma cell lines. We identified 9 cell lines (26%) with a p53 loss-of-function mutation, including 6 missense mutations, 1 nonsense mutation, 1 in-frame deletion, and 1 homozygous deletion of the 3' end of the p53 gene. Sensitivity to nutlin-3 was highly predictive of absence of p53 mutation. Signaling pathways downstream of p53 were functionally intact in 23 of 25 cell lines with wild-type p53. Knockdown and overexpression experiments revealed a potentiating effect of p14(ARF) expression on the response of neuroblastoma cells to nutlin-3. Our findings shed light on the spectrum of p53 pathway lesions in neuroblastoma cells, indicate that defects in effector molecules downstream of p53 are remarkably rare in neuroblastoma, and identify p14(ARF) as a determinant of the outcome of the response to MDM2 inhibition. These insights may prove useful for the clinical translation of evolving strategies aimed at p53 reactivation and for the development of new therapeutic approaches.

Topics: Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p16; Genes, p53; Humans; Imidazoles; Mutation; Neuroblastoma; Piperazines; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Tumor Suppressor Protein p53

Neuroblastoma is the most common pediatric abdominal tumor and principally a p53 wild-type, highly vascular, aggressive tumor, with limited response to anti-VEGF therapies alone. MDM2 is a key inhibitor of p53 and a positive activator of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) activity with an important role in neuroblastoma pathogenesis. We hypothesized that concurrent inhibition of both MDM2 and VEGF signaling would have cooperative anti-tumor effects, potentiating anti-angiogenic strategies for neuroblastoma and other p53 wild-type tumors. We orthotopically implanted SH-SY5Y neuroblastoma cells into nude mice (n = 40) and treated as follows: control, bevacizumab, Nutlin-3a, combination of bevacizumab plus Nutlin-3a. Expression of HIF-1α and VEGF were measured by qPCR, Western blot, and ELISA. Tumor apoptosis was measured by immunohistochemistry and caspase assay. Angiogenesis was evaluated by immunohistochemistry for vascular markers (CD-31, type-IV collagen, αSMA). Both angiogenesis and metastatic burden were digitally quantified. In vitro, Nutlin-3a suppresses HIF-1α expression with subsequent downregulation of VEGF. Bevacizumab plus Nutlin-3a leads to significant suppression of tumor growth compared to control (P < 0.01) or either agent alone. Combination treated xenograft tumors display a marked decrease in endothelial cells (P < 0.0001), perivascular basement membrane (P < 0.04), and vascular mural cells (P < 0.004). Nutlin-3a alone and in combination with bevacizumab leads to significant tumor apoptosis (P < 0.0001 for both) and significant decrease in incidence of metastasis (P < 0.05) and metastatic burden (P < 0.03). Bevacizumab plus Nutlin-3a cooperatively inhibits tumor growth and angiogenesis in neuroblastoma in vivo with dramatic effects on tumor vascularity. Concomitantly targeting VEGF and p53 pathways potently suppresses tumor growth, and these results support further clinical development of this approach.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal, Humanized; Bevacizumab; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Imidazoles; Mice; Mice, Nude; Neoplasm Metastasis; Neovascularization, Pathologic; Neuroblastoma; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Murine double minute 2 (MDM2) negatively regulates the activity of the tumor suppressor protein p53. Nutlin-3 is a MDM2 inhibitor under preclinical investigation as nongenotoxic activator of the p53 pathway for cancer therapy. Here, nutlin-3 was evaluated for its activity alone or in combination with established chemotherapeutic drugs for antitumor action in chemosensitive and chemoresistant neuroblastoma and rhabdomyosarcoma cell lines. Effects of nutlin-3 single treatment were much more pronounced in p53 wild-type cell lines (IC(50)s <3 micromol/L) than in p53-mutated cell lines (IC(50)s >17 micromol/L). In sharp contrast to the expectations, nutlin-3 concentrations that did not affect viability of p53-mutated cell lines strongly increased the efficacy of vincristine in p53-mutated, P-glycoprotein (P-gp)-overexpressing cell lines (decrease in IC(50)s 92- to 3,434-fold). Similar results were obtained for other P-gp substrates. Moreover, nutlin-3 reduced efflux of rhodamine 123 and other fluorescence dyes that are effluxed by P-gp. Investigation of Madin-Darby canine kidney (MDCK) II cells stably transfected with plasmids encoding for P-gp (MDCKII MDR1) or multidrug resistance protein 1 (MRP-1, MDCKII MRP1) revealed that nutlin-3 not only interferes with P-gp but also affects MRP-1-mediated efflux. Kinetic studies and investigation of P-gp-ATPase activity showed that nutlin-3 is likely to act as a P-gp transport substrate. Examination of the nutlin-3 enantiomers nutlin-3a and nutlin-3b revealed that, in contrast to MDM2-inhibitory activity that is limited to nutlin-3a, both enantiomers similarly interfere with P-gp-mediated drug efflux. In conclusion, nutlin-3-induced inhibition of P-gp and MRP-1 was discovered as a novel anticancer mechanism of the substance in this report.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Humans; Imidazoles; Mice; Mutation; Neuroblastoma; Piperazines; Proto-Oncogene Proteins c-mdm2; Rhabdomyosarcoma, Alveolar; Rhodamine 123; Stereoisomerism; Tumor Suppressor Protein p53; Vincristine

Neuroblastoma (NB) is a primitive neuroectodermal tumor and the second most common solid tumor in children. NB exhibits heterogeneous behavior and spontaneous regression can occur in patients under 12 months of age. Response to treatment is both age- and stage-specific; however, patients over 1 year of age are generally considered high risk. NB tumors from these patients are often characterized by alterations in p53 expression and murine double minute (MDM2) activity with concomitant resistance to chemotherapy. We evaluated the ability of nutlin-3 to sensitize a p53-null and doxorubicin-resistant NB cell line, LA155N, to doxorubicin. Nutlin-3 treatment upregulated TAp73 and E2F1 protein levels. It potentiated the ability of doxorubicin to block cell proliferation and activate apoptosis and TAp73 knockdown resulted in a reduction of this sensitization. Additionally, PUMA expression was induced by the combination treatment, but reduced by knockdown of either TAp73 or E2F1. We conclude that, following nutlin-3 treatment, TAp73 and E2F1 are released from MDM2 and activated by doxorubicin to induce PUMA and apoptosis. This study addresses p53-independent mechanisms of nutlin-3 action in chemoresistant NB, especially in combination with chemotherapeutics. We believe that this model has strong clinical relevance for chemoresistant and p53 dysfunctional NB.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Proliferation; DNA-Binding Proteins; Doxorubicin; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Drug Synergism; E2F1 Transcription Factor; Gene Expression Regulation, Neoplastic; Genes, p53; Humans; Imidazoles; Models, Biological; Neuroblastoma; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Cells, Cultured; Tumor Protein p73; Tumor Suppressor Proteins

MYCN gene amplification is a negative prognostic indicator in neuroblastoma and high level MycN expression in Stage IV neuroblastoma is generally a hallmark of poor patient outcome. However, high level expression of the MycN protein in neuroblastoma cells lacking MYCN amplification suppresses growth and drives apoptosis; this, in part, explains the absence of clinical observations of high level MycN in neuroblastoma lacking MYCN amplification. In the current study, we found that combination treatment with nutlin-3 and doxorubicin upregulated MycN expression in non-MYCN-amplified neuroblastoma cells at both the protein and mRNA levels. The induced expression of MycN in non-MYCN-amplified cells inhibited cell proliferation and increased apoptosis. MycN induction also upregulated p53, p21 and Bax protein levels, as well as mRNA levels for the positive neuroblastoma prognostic factors CD44 and EFNB3. Blocking MycN reversed these effects. These results were corroborated by findings using a MycN-inducible system in SHEP cells, another MYCN non-amplified neuroblastoma cell line. Our results indicate that doxorubicin/nutlin-3 combination treatment both induces expression of MycN in a non-MYCN-amplified background and sensitizes neuroblastoma cells to chemotherapy. These findings support the idea that induction of MycN in non-MYCN-amplified cells drives neuroblastoma cells toward apoptosis and suggest that combination nutlin-3/doxorubicin treatment may be clinically important.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Doxorubicin; Ephrin-B3; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Imidazoles; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Piperazines; Prognosis; RNA, Messenger; Tumor Suppressor Protein p53

Restoring p53 function by antagonizing its interaction with the negative regulator MDM2 is an appealing nongenotoxic approach to treating tumors with wild-type p53. Mutational inactivation of p53 is rare in neuroblastoma tumors at diagnosis and occurs in only a subset of multidrug-resistant neuroblastomas.. The antiproliferative and cytotoxic effect of nutlin-3, a small-molecule MDM2 antagonist, was examined in chemosensitive (UKF-NB-3) and matched chemoresistant neuroblastoma cells with wild-type p53 (UKF-NB-3(r)DOX20) or with mutant p53 (UKF-NB-3(r)VCR10). Activation of the p53 pathway was assessed by expression analysis of p53 target genes, flow cytometric cell cycle analysis, and apoptosis assays. Mice with established chemoresistant tumor xenografts were treated orally with nutlin-3 or vehicle control (n = 5-10 mice per group) and were used to evaluate effects on tumor growth, p53 pathway activity, and metastatic tumor burden. All statistical tests were two-sided.. Nutlin-3 induced a similar activation of the p53 pathway in UKF-NB-3 and UKF-NB-3(r)DOX20 cells, as evidenced by increased expression of p53 target genes, G1 cell cycle arrest, and induction of apoptosis. No such response was observed in UKF-NB-3(r)VCR10 cells with mutant p53. Oral administration of nutlin-3 to UKF-NB-3(r)DOX20 xenograft-bearing mice led to inhibition of primary tumor growth (mean tumor volume after 3 weeks of treatment, nutlin-3- vs vehicle-treated mice: 772 vs 1661 mm3, difference = 890 mm3, 95% confidence interval = 469 to 1311 mm3, P < .001), p53 pathway activation, and reduction in the extent of metastatic disease. The growth of UKF-NB-3(r)VCR10 xenografts was unaffected by nutlin-3.. Nutlin-3 activates the p53 pathway and suppresses tumor growth in this model system of chemoresistant neuroblastoma, provided that wild-type p53 is present.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Caspase 3; Caspase 7; Cell Cycle; Cell Line, Tumor; Diploidy; DNA Fragmentation; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Immunoblotting; Immunohistochemistry; Mice; Mice, Nude; Mutation; Neuroblastoma; Piperazines; Proto-Oncogene Proteins c-mdm2; Reverse Transcriptase Polymerase Chain Reaction; Transplantation, Heterologous; Tumor Suppressor Protein p53

Topics: Animals; Antineoplastic Agents; Cyclin-Dependent Kinase Inhibitor p16; Humans; Imidazoles; Mice; Mice, Transgenic; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Piperazines; Proto-Oncogene Proteins c-mdm2; Transcriptional Activation; Tumor Suppressor Protein p53

Neuroblastoma (NB) is the most common solid malignancy in childhood and its prognosis is still generally poor. In contrast to many other cancers, mutations of the p53 tumor suppressor are rare. Instead, significant cytosolic sequestration of wtp53 is one of several mechanisms that attenuate p53 function in this cancer. Here, we report that aberrant p53 hyperubiquitylation contributes to p53 cytoplasmic sequestration in NB. NB lines constitutively harbor an elevated portion of wtp53 as stable ubiquitylated species confined to the cytoplasm. p53 hyperubiquitylation is not due to dysregulation by Hdm2 or proteasomal dysfunction. Instead, the defect lies in p53 regulation by HAUSP, a major p53-deubiquitylating enzyme. In contrast to non-NB cancer cells with nuclear p53 and normal ubiquitylation, p53 from NB cells shows impaired HAUSP interaction. Conversely, interference with p53 hyperubiquitylation in NB cells by Nutlin 3a or by a C-terminal p53 peptide (aa 305-393) results in p53 relocalization from the cytoplasm to the nucleus, and in case of Nutlin, in reactivation of p53's transcriptional and apoptotic functions. Moreover, nutlin and camptothecin act synergistically in inducing NB cell apoptosis. Hence, this study strengthens the rationale for targeting p53 deubiquitylation by drugs like Nutlin as a promising new strategy in NB therapy.

Topics: Active Transport, Cell Nucleus; Camptothecin; Cell Compartmentation; Cell Line, Tumor; Cytoplasm; Drug Synergism; Enzyme Inhibitors; Humans; Imidazoles; Neuroblastoma; Neurons; Peptide Fragments; Piperazines; Tumor Suppressor Protein p53; Ubiquitin; Ubiquitin Thiolesterase; Ubiquitin-Specific Peptidase 7

In this study, we have analyzed the consequences, on several neuroblastoma cell lines, of combined treatments with (R)-roscovitine (CYC202, Seliciclib), a CDK inhibitory drug, and nutlin-3, a p53 activating drug. Both compounds were found to synergize, causing significant levels of apoptosis in cultured cells when combined at sublethal concentrations. In SH-SY5Y cells, Bcl-XL protein overexpression protected from apoptosis induced by either nutlin-3 alone or the (R)-roscovitine plus nutlin-3 association but failed to prevent apoptosis triggered by (R)-roscovitine alone. Moreover, Western blot studies showed that (R)-roscovitine increased nutlin-3-mediated p53 stabilization. Therefore, we conclude the contribution of (R)-roscovitine to the synergism is basically the sensitization of SH-SY5Y cells to the action of nutlin-3 on p53. The relevance of this pharmacological synergism with respect to the treatment of neuroblastoma is discussed.

Topics: Antineoplastic Agents; Apoptosis; bcl-X Protein; Caspases; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Drug Synergism; Enzyme Inhibitors; Humans; Imidazoles; L-Lactate Dehydrogenase; Neuroblastoma; Peptide Hydrolases; Piperazines; Proto-Oncogene Proteins c-mdm2; Purines; Roscovitine; Time Factors; Transfection; Tumor Suppressor Protein p53

Novel therapeutic approaches are urgently needed for high-stage neuroblastoma, a major therapeutic challenge in pediatric oncology. The majority of neuroblastoma tumors are p53 wild type with intact downstream p53 signaling pathways. We hypothesize that stabilization of p53 would sensitize this aggressive tumor to genotoxic chemotherapy via inhibition of MDM2, the primary negative upstream regulator of p53. We used pharmacologic inhibition of the MDM2-p53 interaction with the small-molecule inhibitor Nutlin and studied the subsequent response to chemotherapy in neuroblastoma cell lines. We did 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and terminal deoxynucleotidyl transferase assays to measure proliferation and apoptosis in several cell lines (IMR32, MYCN3, and JF) treated with combinations of cisplatin, etoposide, and Nutlin. We found consistent and robust decreases in proliferation and increases in apoptosis with the addition of Nutlin 3a to etoposide or cisplatin in all cell lines tested and no response to the inactive Nutlin 3b enantiomer. We also show a rapid and robust accumulation of p53 protein by Western blot in these cells within 1 to 2 hours of treatment. We conclude that MDM2 inhibition dramatically enhances the activity of genotoxic drugs in neuroblastoma and should be considered as an adjuvant to chemotherapy for this aggressive pediatric cancer and for possibly other p53 wild-type solid tumors.

Topics: Animals; Antineoplastic Agents; Apoptosis; Drug Synergism; HCT116 Cells; Humans; Imidazoles; Mice; Neuroblastoma; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Circumvention of the p53 tumor suppressor barrier in neuroblastoma is rarely caused by TP53 mutation but might arise from inappropriately increased activity of its principal negative regulator MDM2. We show here that targeted disruption of the p53-MDM2 interaction by the small-molecule MDM2 antagonist nutlin-3 stabilizes p53 and selectively activates the p53 pathway in neuroblastoma cells with wild-type p53, resulting in a pronounced antiproliferative and cytotoxic effect through induction of G(1) cell cycle arrest and apoptosis. A nutlin-3 response was observed regardless of MYCN amplification status. Remarkably, surviving SK-N-SH cells adopted a senescence-like phenotype, whereas CLB-GA and NGP cells underwent neuronal differentiation. p53 dependence of these alternative outcomes of nutlin-3 treatment was evidenced by abrogation of the effects when p53 was knocked down by lentiviral-mediated short hairpin RNA interference. The diversity of cellular responses reveals pleiotropic mechanisms of nutlins to disable neuroblastoma cells and exemplifies the feasibility of exploiting, by a single targeted intervention, the multiplicity of anticancer activities exerted by a key tumor suppressor as p53. The observed treatment effects without the need of imposing a genotoxic burden suggest that selective MDM2 antagonists might be beneficial for treatment of neuroblastoma patients with and without MYCN amplification.

Topics: Apoptosis; Cell Differentiation; Cell Line, Tumor; Cellular Senescence; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; G1 Phase; Gene Dosage; Gene Targeting; Genes, myc; Genes, p53; Genetic Vectors; Humans; Imidazoles; Neoplasm Proteins; Neuroblastoma; Piperazines; Proto-Oncogene Proteins c-mdm2; RNA, Small Interfering; Tumor Suppressor Protein p53