Norcantharidin enhances ABT-263-mediated anticancer activity in neuroblastoma cells by upregulation of Noxa

Introduction. Neuroblastoma is the most common extracranial solid tumor in children and accounts for ~15% of all pediatric cancer deaths. DNA-PK, a key regulator of repair responses to double-strand DNA breakages induced by radiation or induction chemotherapy is used in the treatment of high-risk neuroblastomas. Here, we have demonstrated that high DNA-PK expression is associated with poor overall survival in a patient population, DNA-PK is highly activated in neuroblastoma cell lines, and inhibition of DNA-PK enhances therapeutic effects of DNA damaging therapies. Methods. DNA double-strand break damage was induced with etoposide, a topoisomerase II inhibitor. DNA-PK activity was inhibited with peposertib or DNA-PK siRNA. DNA-PK Ser2056 phosphorylation was analyzed in MYCN amplified (SK-NDZ, BE(2)-C, LAN-1) and MYCN non-amplified (SK-N-AS, SK-N-SH) cell lines. Clonogenic and proliferation assays were used to evaluate the efficacy of peposertib in combination with etoposide in BE-2-C and SK-N-DZ neuroblastoma cell lines. DSB-induced DNA repair was visualized with confocal laser scanning microscopy using γ-H2AX immunofluorescence. Western blot was used to confirm inhibition of DNA-PKcs (pDNA-PKcs S2056) and analyze cleaved PARP expression in combination groups. Results. DNA-PK inhibition resulted in enhancement of etoposide cytotoxic therapy (BE-2-C IC50 ET = 2.77 ± 0.2, ET+Pep 0.76 ± 0.04; SK-N-DZ IC50 ET = 0.55 ± 0.06, IC50 ET+Pep = 0.11 ± 0.02) in both BE-2-C and SK-N-DZ cell lines. Immunofluorescence analysis detected γH2AX foci increase in the etoposide and peposertib combination group. DNA-PK inhibition in combination with etoposide treatment significantly increased PARP cleavage in BE-2-C and SK-N-DZ cell lines. The effect of DNA-PK knockdown on neuroblastoma cell survival was even more pronounced compared to DNA-PK inhibition with peposertib. DNA-PK knockdown by siRNA alone increased PARP cleavage to the levels comparable to 2 µM etoposide treatment. The combination of etoposide and DNA-PK knockdown enhanced PARP cleavage even further in neuroblastoma cells. Conclusions. High DNA repair activity is commonly associated with cancer drug resistance. We have shown that DNA-PK activation is continuously supported in both MYCN amplified and MYCN non-amplified neuroblastoma cell lines. The importance of DNA-PK activity in neuroblastoma cell lines is confirmed with DNA-PK knockdown that leads to apoptosis in MYCN amplifiedneuroblastoma cell lines. In addition, the combination of DNA-PK inhibition or DNA-PK knockdown with DNA-damaging therapies stalled DNA damage response and enhanced neuroblastoma cell apoptosis. These findings demonstrate the significance of high DNA-PK activity in neuroblastoma cells and provide a potent therapeutic strategy to enhance neuroblastoma cell apoptosis with selective DNA-PK inhibition. Citation Format: Mahnaz Norouzi, Beibei Zhu, Tadahide Izumi, Eric J. Rellinger, B. Mark Evers, Piotr G. Rychahou. DNA-PK inhibition to enhance etoposide therapy in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 132.

Neuroblastoma (NB) is the most common extracranial solid tumor in infants and young children. NB often metastasizes to lymph nodes and bone marrow. When compared to low or intermediate risk tumors, high risk neuroblastoma (HRNB) is more aggressive with low (40-50%) 5 year survival rates. Furthermore, current intensive treatment methods induce higher toxicity and side effects. In search of identifying less-toxic agents to induce anti-cancer activity in NB cells, our group reported the anti-cancer activity of a non-steroidal anti-inflammatory drug, Clotam (TA) using HRNB cell lines potentially inhibiting the transcription factor, Specificity protein 1 (Sp1) and an inhibitor of apoptosis protein, survivin. Recently, our laboratories tested a copper complex of TA (Cu-TA) using 12 cell lines and found that Cu-TA's IC50 values were 30-80% lower when compared to TA. In this study, we investigated the cytotoxicity of SH-SY 5Y, LA1-55n and SMS-KCNR cells in the presence of vehicle (Dimethyl sulfoxide) or TA or Cu-TA using CellTiter-Glo kit and determined the IC50 values using SigmaPlot software. The effect of TA or Cu-TA on apoptosis and cell cycle phase distribution was evaluated by flow cytometry using Annexin-V and Propidium Iodide kits respectively. The characterization and stability of the Cu-TA was also determined. SH SY5Y and SMS-KCNR cells were treated with IC50 values of TA or Cu-TA and the expression of Sp1 and survivin was measured by qPCR and Western blot analysis. The results showed that while both TA and Cu-TA inhibited NB cell growth, Cu-TA's IC50 values were 60-70% less than TA. The inhibition of NB cells with Cu-TA was accompanied by induced apoptotic cell population and cell cycle arrest at G2M phase. Western blot and qPCR results confirmed that Cu-TA inhibited the expression of both Sp1 and survivin with predominant effect on SMS-KCNR cells. These results demonstrate that Cu-TA is more effective than TA for inhibiting Sp1 and survivin in NB cells, which was consistent with the anti-proliferative activity against NB cells. Since TA and Cu-TA are less toxic than standard chemotherapeutic agents, employing these agents for the treatment of pediatric malignancies could be useful. Eventually, combination of Cu-TA and chemotherapeutic agents will be tested. If Cu-TA sensitizes NB cells to chemotherapy, it will help to address the issues associated with the side effects of chemotherapy.Citation Format: Yazmin Hernandez, Sarah Sarah Grebennikov, Emily Zakhary, Ashni Dudhia, Rajasekhar Maram, Diego Casanova, Umesh T. Sankpal, W. Paul Bowman, Deondra T. Brown, Jaya Chhabra, Alvin A. Holder, Riyaz M. Basha. Antiproliferative effect of copper complex of clotam in neuroblastoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2881.

The TrkA and TrkB tyrosine kinases are members of the neurotrophin receptor family and mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, NGF and BDNF, respectively. Expression levels of TrkA/TrkB are important prognostic factors in a variety of embryonal tumors including neuroblastoma, the most common solid tumor of childhood. Because TrkA/TrkB exhibit a high level of sequence similarity and use overlapping pathways for signal transduction, the existence of specific effector molecules crucial for receptor and cell-type-specific response is likely. To identify these effectors by analyzing biological effects of TrkA and TrkB activation in a defined model, we performed a proteome study using the human neuroblastoma SY5Y cell line stably transfected with the TrkA or TrkB cDNA. The use of the recently introduced DIGE (fluorescence two-dimensional difference gel electrophoresis) system (Amersham Biosciences, Piscataway, NJ) allowed us to monitor differences in protein expression between samples in one gel. Proteomic changes were monitored in a time course of 0, 0.5, 1, 6, and 24 h following receptor activation. Using MALDI mass spectrometry, we identified, respectively, 22 and 9 differentially expressed proteins upon the addition of neurotrophin in SY5Y-TrkB and SY5Y-TrkA cells. Functional assignment revealed that the majority of these proteins are involved in organization and maintenance of cellular structures.

To elucidate the molecular mechanism of norcantharidin (NCTD) in inducing apoptosis of liver cancer cells so as to provide basic rationales for its application in liver cancer treatment. Liver cancer cell lines of SMMC-7721 and BEL-7402 were treated with NCTD. The cell growth inhibition was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, cell death detected by trypan blue exclusion assay and apoptosis examined by Annexin V/PI staining and flow cytometry. The cleavage of caspase-9, -3 and PARP, and the expression of the anti-apoptotic Bcl-2 proteins were analyzed by Western blot. The MTT results showed that, after a treatment with NCTD for 24, 48 and 72 h, the IC50 of NCTD in SMMC-7721 cell line was 12, 6 and 1.6 µg/ml respectively; in BEL-7402, the IC50 10, 4 and 2 µg/ml respectively. Trypan blue exclusion assay showed that NCTD mediated substantial cell death in two cancer cell lines. Apoptosis assay showed that, after a 12 h treatment with 10 µg/ml NCTD, 27% of SMMC-7721 cells were induced to undergo apoptosis, an increment of 20% over the untreated control cells (7%); 30% of BEL-7402 cells became apoptotic, an increment of 22% over the untreated control cells (8%). Western blot analysis showed that NCTD treatment potently induced the activation of caspase-9, -3 and the cleavage of PARP, and markedly down-regulated the expression of Bcl-2, Bcl-X(L) and Mcl-1. NCTD strongly inhibits liver cancer cell growth and potently induces apoptotic cell death in two liver cancer cell lines. The strong anticancer activity of NCTD may be induced through targeting multiple Bcl-2 anti-apoptotic family members.

RRS1 plays an important role in regulating ribosome biogenesis. Recently, RRS1 has emerged as an oncoprotein involved in tumorigenicity of some cancers. However its role in neuroblastoma remains unknown. RRS1 expression was detected in pediatric neuroblastoma patients' tissues and cell lines. The effects of RRS1 knockdown on proliferation, apoptosis, and cell cycle were evaluated in neuroblastoma cell lines. RRS1-related survival pathway was analyzed by co-immunoprecipitation (Co-IP), mass spectrometry, reverse transcription-quantitative real-time PCR (RT-qPCR), and western blot. Protein-protein interaction (PPI) network was constructed using Cytoscape software and the STRING databases. Increased RRS1 level was found in neuroblastoma cases (35.6%) and cell lines. High RRS1 expression levels were associated with poor prognosis. RRS1 knockdown inhibited cell proliferation, induced apoptosis, and caused cell cycle arrest in SK-N-AS and SH-SY5Y cells. Co-IP and mass spectrometry analysis showed that RRS1 affects PI3K/Akt and nuclear factor κB (NF-κB) pathways. RT-qPCR and western blot results revealed that RRS1 knockdown inhibited the PI3K/Akt/NF-κB pathway through dephosphorylation of key proteins. In PPI network, AKT, PI3K, and P65 connected RRS1 with differentially expressed proteins more closely. This study suggests RRS1 knockdown may inhibit neuroblastoma cell proliferation by the PI3K/Akt/NF-κB pathway. Therefore, RRS1 may be a potential target for neuroblastoma treatment. RRS1 is involved in the progression of neuroblastoma. Knockdown of RRS1 contributes to inhibit the survival of neuroblastoma cells. RRS1 is associated with the PI3K/Akt/NF-κB signaling pathway in neuroblastoma cells. RRS1 may be a promising target for neuroblastoma therapy.

In this study we aimed to (1). screen phenothiazines for cytotoxic activity in glioma, neuroblastoma, and primary mouse brain tissue; and (2). determine the mechanism of the cytotoxic effect (apoptosis, necrosis) and the roles of calmodulin inhibition and sigma receptor modulation. Rat glioma (C6) and human neuroblastoma (SHSY-5Y) cell lines were treated with different phenothiazines. All agents induced a dose-dependent decrease in viability and proliferation, with the highest activity elicited by thioridazine. Sensitivity to thioridazine of glioma and neuroblastoma cells was significantly higher (p < 0.05) than that of primary mouse brain culture (IC50 11.2 and 15.1 microM vs 41.3 microM, respectively). The N-mustard fluphenazine induced significantly lower cytotoxicity in glioma cells, compared to fluphenazine. The sigma receptor selective ligand (+)-SK&F10047 increased viability slightly while combined with fluphenazine; SK&F10047 did not alter fluphenazine activity. Flow cytometry of propidium iodide (PI)-stained glioma cells treated with thioridazine, fluphenazine, or perphenazine (6-50 microM) resulted in a concentration-dependent increase of fragmented DNA up to 94% vs 3% in controls by all agents. Thioridazine (12.5 microM)-treated glioma cells costained with PI and Hoechst 33342 revealed a red fluorescence of fragmented nuclei in treated cells and a blue fluorescence of intact control nuclei. After 4-h exposure to thioridazine (25 and 50 microM), a 25- to 30-fold increase in caspase-3 activity in neuroblastoma cells was noted. Overall, the marked apoptotic effect of phenothiazines in brain-derived cancer cells, and the low sensitivity of primary brain tissue suggest the potential use of selected agents as therapeutic modalities in brain cancer.

[Introduction] BMI1 is a polycomb protein and its overexpression has been correlated with cancer development and aggressiveness. We previously reported that v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN)-induced BMI1 positively regulated neuroblastoma (NB) cell proliferation via the transcriptional suppression of tumor suppressors in NB cells (ONCOGENE, 2010). The role of BMI1 in DNA damage repair pathways has recently been highlighted. The ubiquitination of H2AK119 is also up-regulated locally at DNA damage sites, namely those of double-strand breaks and UV-damaged lesions. BMI1 and other PcG proteins make foci at DNA damage sites and are involved in DNA damage repair pathways; however, the exact role of PcG proteins in DNA damage repair pathways remains to be elucidated. [Results and Discussion] In order to evaluate the potential of BMI1 as a new target for NB therapy, we herein examined the effects of BMI1 reductions on NB cell differentiation and apoptotic NB cell death. BMI1 knockdown (KD) up to 7 days in NB cells significantly induced their differentiation. BMI1 depletion up to 14 days significantly induced apoptotic NB cell death along with the activation of p53, increases in p73, and induction of p53 family downstream molecules. BMI1 depletion in vivo markedly suppressed NB xenograft tumor growth. A pathological analysis using the TUNEL assay indicated the induction of apoptotic cell death. BMI1 reductions activated ATM and increased γ-H2AX in NB cells. The recently identified polycomb protein L3MBTL2 was clearly decreased in BMI1 KD NB cells. BMI1 KD-induced L3MBTL2 reductions occurred at the protein level. BMI1 interacted with Ring1b, but not with L3MBTL2. Furthermore, L3MBTL2 KD by siRNA effectively accelerated apoptosis induced by the BMI1 inhibitor, PTC-209, related to apoptotic cell death accompanied by Jun/EGR-1/p53 pathway activation in NB cells. In neuronal cells, NGF depletion induced JUN pathway activation and apoptotic cell death. Furthermore, JUN modulates myeloma cell apoptosis by interacting with EGR-1, which down-regulates Survivin and triggers caspase signaling; JUN up-regulates EGR-1 expression by binding its promoter, which suggests that EGR-1 is the direct downstream target gene of the JUN transcription factor. In solid tumors, EGR-1 regulates radiation-induced cell death via apoptotic pathways including p53-dependent apoptosis. These results clarified, for the first time, the cooperation between PcG BMI1 and L3MBTL2 in DNA damage responses. Therefore, BMI1 and L3MBTL2 appear to be promising targets in new therapies for NB tumors. Citation Format: Nobuhiro Akita, Hisanori Takenobu, Hiroshi Chikaraishi, Miki Ohira, Takehiko Kamijo. Polycomb group protein BMI1 protects neuroblastoma cells from DNA damage-induced apoptotic death in cooperation with L3MBTL2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2427.

Background: Gain of 17q is the most powerful genetic predictor of adverse clinical outcome in neuroblastoma (NB). 17q+ correlated with poor survival in our population-based material where we found aberrations of chromosome 17 in 85% of primary neuroblastoma tumors. Specifically, gain of WIP1 at 17q23 is frequently detected in NB. WIP1 is a serine/threonine phosphatase encoded by the gene PPM1D, member of the PP2C family known to be regulators of DNA repair pathways. Methods: A formazan-based spectrophotometric method was used to measure cell viability in several NB cell lines treated with different WIP1 inhibitors. Comparative genomic hybridization (CGH) was used to analyze primary NB tumors and cell lines. NB cells were examined for WIP1 expression using immunoblotting, immunohistochemistry and qPCR. Stable WIP1 knockdown NB cells were generated by shRNA transfections. Clonogenicity of WIP1 knockdown were examined by clonogenic assay. In vivo, WIP1 knocked- and non-silenced cells were injected in nude NMRI mice followed by daily evaluation of tumor formation. Results: Detailed CGH analysis revealed that genomic gain of WIP1 is evident with at least one extra copy in all tumor samples showing 17q gain. Results from our NB cell lines demonstrate 17q aberrations in all samples. These findings were corroborated by expression pattern of WIP1 mRNA and different immunostaining techniques. In a wide range of NB cell lines we found that all WIP1 inhibitors had good cytotoxic effect at higher concentrations (50µM) while some showed cytotoxic effects at lower concentrations. Transfection experiments with shRNA against WIP1 showed decreased cell viability, proliferation and colony formation in NB cells. Tumor xenograft development after WIP1 downregulation was significantly delayed with time to tumor development (0.10 mL) more than doubled compared to animals injected with cells transfected with the scrambled construct (15 days median, vs. 33 days). Conclusions: Our data suggest that WIP1 expression have significant oncogenic function in neuroblastoma development. Our results showed that WIP1 is present in at least one extra genomic copy in the majority of primary NB. WIP1 knocked cells exhibited decreased cell growth and clonogenic capacity compared to non-silenced cells. In addition, WIP1 inhibition could be a potential target in treating high-risk neuroblastoma but more studies investigating the effects of WIP1 inhibition in neuroblastoma are needed to evaluate its clinical significance. Citation Format: Jelena Milosevic, Malin Wickström, Diana Treis, Hjalmar Ståhlberg Nordegren, Lotta Elfman, Ninib Baryawno, Susanne Fransson, Baldur Sveinbjörnsson, Tommy Martinsson, John Inge Johnsen, Per Kogner. PPM1D/WIP1, a potential target in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3971. doi:10.1158/1538-7445.AM2014-3971

BACKGROUND: Retinoic acid (RA) is a mainstay of neuroblastoma treatment as it promotes tumor differentiation. However, the mechanism underlying RA-mediated neuronal differentiation is not well understood. The purpose of this study is to better define the molecular targets through which RA promotes differentiation in neuroblastoma cells. The RE-1 Silencing Transcription Factor (REST) is a repressor of neuronal genes. It is expressed in embryonic stem cells and neural progenitors, and is downregulated during neurogenesis. REST is overexpressed in many neuroblastoma tumor samples and cell lines, and its expression is associated with an undifferentiated phenotype. Since REST is critical for neuronal differentiation, we hypothesized that RA promotes differentiation in neuroblastoma cell lines by downregulating REST expression. METHODS AND RESULTS: Quantitative reverse transcription polymerase chain reaction, immunofluorescence assay and western blotting revealed REST expression to be elevated in a number of human neuroblastoma tumors and cell lines. Treatment with RA promoted neuronal differentiation in some but not all cell lines. In RA-responsive cells, induction of neuronal differentiation was associated with a decline in REST protein levels and upregulation of REST-target gene expression. However, REST protein levels were elevated upon RA treatment in resistant cells, REST transcript was upregulated in both RA-sensitive and resistant cells as expected. These results suggested that RA downregulated REST through post-transcriptional mechanisms, which was confirmed by measurement of REST protein levels in the presence of cycloheximide. Treatment with MG-132 blocked RA-mediated degradation of REST, indicating the involvement of the proteasome. The E3-ligase, SCFβ-TRCP, is known to be involved in REST ubiquitination and degradation. Low levels SCFβ-TRCP were associated with elevated REST protein in a number of human neuroblastoma tumors and cell lines. We observed that SCFβ-TRCP transcript and protein were elevated upon RA-treatment in responsive cell lines but not in RA-resistant cells with high REST levels. Ectopic expression of SCFβ-TRCP promoted REST ubiquitination and degradation in RA-resistant cells and formed a complex with ubiquitinated REST as determined by immunoprecipitation and co-immunoprecipitation experiments. Since these events were associated with induction of neuronal differentiation, our findings suggest that a failure to degrade REST blocks neuronal differentiation upon RA treatment. CONCLUSION: Our study indicates that deranged proteasomal machinery may contribute to maintenance of REST expression and the blockade of neuronal differentiation in neuroblastoma cells. Agents with potential to upregulate SCFβ-TRCP expression may have therapeutic value in patients that fail to respond to RA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2025. doi:10.1158/1538-7445.AM2011-2025

Backgroundc-Met is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), and both c-Met and its ligand are expressed in a variety of tissues. C-Met/HGF/SF signaling is essential for normal embryogenesis, organogenesis, and tissue regeneration. Abnormal c-Met/HGF/SF signaling has been demonstrated in different tumors and linked to aggressive and metastatic tumor phenotypes. In vitro and in vivo studies have demonstrated inhibition of c-Met/HGF/SF signaling by the small-molecule inhibitor PHA665752. This study investigated c-Met and HGF expression in two neuroblastoma (NBL) cell lines and tumor tissue from patients with NBL, as well as the effects of PHA665752 on growth and motility of NBL cell lines. The effect of the tumor suppressor protein PTEN on migration and proliferation of tumor cells treated with PHA665752 was also evaluated.MethodsExpression of c-Met and HGF in NBL cell lines SH-EP and SH-SY5Y and primary tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. The effect of PHA665752 on c-Met/HGF signaling involved in NBL cell proliferation and migration was evaluated in c-Met-positive cells and c-Met-transfected cells. The transwell chemotaxis assay and the MTT assay were used to measure migration and proliferation/cell-survival of tumor cells, respectively. The PPAR-γ agonist rosiglitazone was used to assess the effect of PTEN on PHA665752-induced inhibition of NBL cell proliferation/cell-survival and migrationResultsHigh c-Met expression was detected in SH-EP cells and primary tumors from patients with advanced-stage disease. C-Met/HGF signaling induced both migration and proliferation of SH-EP cells. Migration and proliferation/cell-survival were inhibited by PHA665752 in a dose-dependent manner. We also found that induced overexpression of PTEN following treatment with rosiglitazone significantly enhanced the inhibitory effect of PHA665752 on NBL-cell migration and proliferation.Conclusionc-Met is highly expressed in most tumors from patients with advanced-stage, metastatic NBL. Furthermore, using the NBL cell line SH-EP as a model, PHA665752 was shown to inhibit cMet/HGF/SF signaling in vitro, suggesting c-Met inhibitors may have efficacy for blocking local progression and/or metastatic spread of c-Met-positive NBL in vivo. These are novel findings for this disease and suggest that further studies of agents targeting the c-Met/HGF axis in NBL are warranted

The surface membrane antigens of 7 neuroblastoma and 91 leukemia-lymphoma cell lines were studied with the use of a total of 36 murine monoclonal antibodies (MoAb) primarily developed against hematopoietic cells and 2 MoAb developed against human fetal brain. Five of the MoAb against hematopoietic cells (BA-1, BA-2, DU-ALL-1, J-5, and BA-3) consistently bound to common acute lymphoblastic leukemia cell lines, and 2 others (MCS-2 and OKM-1) reacted uniformly with acute myeloblastic-acute monoblastic leukemia cell lines. However, these 7 MoAb also reacted with 1-7 neuroblastoma cell lines. All the human neuroblastoma cell lines bound MoAb BA-2 and DU-ALL-1. Six of the 7 lines reacted with BA-1. Only 1 neuroblastoma cell line (SJ-N-CG) gave positive staining with J-5 and BA-3, and another line (SK-N-AS) bound MoAb MCS-2 and OKM-1. Anti-fetal brain MoAb (UJ-13A and UJ-127-11) were highly positive for all the neuroblastoma cell lines. By contrast, 4 of 43 leukemia-lymphoma cell lines tested bound these anti-fetal brain antibodies. Both B3/25 and OKT-9, anti-transferrin receptor antibodies, reacted with all of the hematopoietic and neuroblastoma cell lines. These results demonstrate that neuroblastoma and hematopoietic cell lines possess common antigenic determinants despite their different embryologic origins. The neuroblastoma cell lines may be classified into subgroups on the basis of phenotype profiles determined by the MoAb. MoAb may be useful in characterization and classification of neuroblastoma cells, as has already proved to be the case for cells of the hematopoietic lineages.

Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.

Background: In neuroblastoma (NB) 17q+ is the most powerful genetic predictor of adverse clinical outcome. We found aberrations of chromosome 17 in 85% in a national study of NB with 17q+ in 46% and whole 17 gain in 39%. Gain of 17q correlated with poor survival in our population-based material. In NB gain of wild-type p53-induced phosphatase 1 (Wip1) at 17q23 is frequently detected. Wip1 is a serine/threonine phosphatase and was described as a gatekeeper in the Mdm2-p53 regulatory loop by promoting Mdm2-mediated proteolysis of p53. Methods: Comparative genomic hybridization (CGH) was used to analyze primary NB tumors and cell lines. NB cells were examined for Wip1 expression using immunoblotting, immunohistochemistry and qPCR. Stable Wip1 knockdown NB cells were generated by shRNA transfections. H2AX phosphorylation and clonogenicity of Wip1 knockdown were examined using flow cytometry and clonogenic assay, respectively. In vivo, Wip1 knocked- and non-silenced cells were injected in nude NMRI mice. Tumor formation was followed by daily evaluation by digital caliper. Results: Detailed CGH analysis revealed that Wip1 is present in at least one extra genomic copy in all 63 tumor samples containing 17q gain. Results from our wide range of NB cell lines demonstrate 17q aberrations in all samples. These findings have been confirmed by analyzing the expression pattern of Wip1 using different immunostaining techniques. Moreover, mRNA levels of Wip1 correlate to Wip1 protein expression. Transfection experiments with shRNA against Wip1 showed decreased cell viability, proliferation and colony formation in NB cells. In addition, substantial increase of gamma-H2AX expression after Wip1 knockdown compared to the non-silencing transfection was observed. Tumor xenograft development was significantly delayed showing median tumor development (0.10 mL) to be more than doubled (15 days median, vs. 33 days) after Wip1 downregulation compared to animals injected with cells transfected with the scrambled construct. Conclusions: Wip1 downregulation inhibits tumor development. Our results showed that Wip1 is present in at least one extra genomic copy in the majority of primary NB. We identified NB cell lines expressing high, moderate and low levels of Wip1 where Wip1 knocked cells exhibited decreased cell growth and clonogenic capacity compared to non-silenced cells. Moreover, knockdown of Wip1 induced phosphorylation of histone H2AX, indicating a significant role of Wip1 in the DNA damage response of these tumors. Our data suggest that Wip1 expression have significant oncogenic function in neuroblastoma development. Citation Format: Jelena Milosevic, Malin Wickström, Lotta Elfman, Ninib Baryawno, Baldur Sveinbjörnsson, Tommy Martinsson, John Inge Johnsen, Per Kogner. The potential oncogenic significance of Wip1 in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3817. doi:10.1158/1538-7445.AM2013-3817

Solute Carrier Family 3, Member 2 (SLC3A2 or 4F2hc) is a multifunctional glycoprotein that mediates integrin-dependent signaling, acts as a trafficking chaperone for amino acid transporters, and is involved in polyamine transportation. We identified SLC3A2 as a potential Anaplastic Lymphoma Kinase (ALK) interacting partner in a BioID-proximity labeling screen in neuroblastoma (NB) cells. In this work we show that endogenous SLC3A2 and ALK interact in NB cells and that this SLC3A2:ALK interaction was abrogated upon treatment with the ALK inhibitor lorlatinib. We show here that loss of ALK activity leads to decreased SLC3A2 expression and reduced SLC3A2 protein stability in a panel of NB cell lines, while stimulation of ALK with ALKAL2 ligand resulted in increased SLC3A2 protein levels. We further identified MARCH11, an E3 ligase, as a regulator of SLC3A2 ubiquitination downstream of ALK. Further, knockdown of SLC3A2 resulted in inhibition of NB cell growth. To investigate the therapeutic potential of SLC3A2 targeting, we performed monotreatment of NB cells with AMXT-1501 (a polyamine transport inhibitor), which showed only moderate effects in NB cells. In contrast, a combination lorlatinib/AMXT-1501 treatment resulted in synergistic inhibition of cell growth in ALK-driven NB cell lines. Taken together, our results identify a novel role for the ALK receptor tyrosine kinase (RTK), working in concert with the MARCH11 E3 ligase, in regulating SLC3A2 protein stability and function in NB cells. The synergistic effect of combined ALK and polyamine transport inhibition shows that ALK/MARCH11/SLC3A2 regulation of amino acid transport is important for oncogenic growth and survival in NB cells.

Neuroblastoma (NB) and Ewing sarcoma (ES) are neuroectodermal tumors typical of pediatric age that, despite aggressive treatment, still present a poor prognosis when in advanced stages. Studies indicate that c-KIT and platelet-derived growth factor receptor (PDGFR) play a substantial role in the proliferation and survival of NB and ES cells. Dasatinib, an oral multi-targeted inhibitor of several kinases including BCR-ABL and SRC-family kinases, is also active against c-KIT and PDGFR. Here, we evaluated the effect of dasatinib on the NB cell lines SJ-N-KP, SK-N-BE, AF8 and IMR5, and on the ES lines PDE02, TC106 and 6647. Proliferation and viability assays showed that dasatinib exerts an antiproliferative activity with a peak effect occurring at 24 h. After a 24-h exposure to dasatinib at 100 nM, proliferation was inhibited by 29.4+/-5.7% in SJ-N-KP, 41.3+/-11.7% in IMR5, 35.3+/-7.6% in PDE02 and 14+/-10.6% in 6647. Dasatinib did not induce apoptosis in NB and ES cell lines. A possible antimigratory activity of dasatinib was evaluated by scratch test. Dasatinib at 100 nM inhibited the migration of NB and ES cell lines by a mean of 30.2 and 25.3%, respectively. This activity suggests a possible role of dasatinib in inhibiting metastasis and appears of particular interest, given the association between metastatic disease and poor prognosis in these tumors. In conclusion, the cytostatic and antimigratory activity of dasatinib in NB and ES cell lines and the lack of pro-apoptotic activity suggests a possible use for this compound in the treatment of these tumors as a combination with other cytotoxic therapy.