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IC50 for resistant cell lines as compared to IC50 of parental cell line

Supplemental Figure Legends

SN22 and SN38 pharmacokinetics

Supp Figure S1 Figure S1. SRB assay assessing efficacy of free SN-38 and SN38-TOA NP. A, Growth inhibition by SN38-TOA NPs is statistically significant for 4 nM at 72-h (p=0.0002), and 2 nM and 4 nM at 96-h (p0.0001) and 96-h (p>0.0001), and 1 nM at 96-h (p=0.0007). FD = free drug. NP = nanoparticles.

Purpose: Currently, Experimental Design: SN38, the active metabolite of irinotecan (CPT-11), is a potent therapeutic agent that is readily encapsulated in polymeric nanoparticles. Tocopherol oxyacetate (TOA) is a hydrophobic mitocan that was linked to SN38 to significantly increase hydrophobicity and enhance nanoparticle retention. We treated neuroblastomas with SN38-TOA nanoparticles and compared the efficacy with the parent prodrug CPT-11 using a mouse xenograft model.Results: Nanoparticle treatment induced prolonged event-free survival (EFS) in most mice, compared with CPT-11. This was shown for both SH-SY5Y and IMR-32 neuroblastoma xenografts. Enhanced efficacy was likely due to increased and sustained drug levels of SN38 in the tumor compared with conventional CPT-11 delivery. Interestingly, when recurrent CPT-11–treated tumors were re-treated with SN38-TOA nanoparticles, the tumors transformed from undifferentiated neuroblastomas to maturing ganglioneuroblastomas. Furthermore, these tumors were infiltrated with Schwann cells of mouse origin, which may have contributed to the differentiated histology.Conclusions: Nanoparticle delivery of SN38-TOA produced increased drug delivery and prolonged EFS compared to conventional delivery of CPT-11. Also, lower total dose and drug entrapment in nanoparticles during circulation should decrease toxicity. We propose that nanoparticle-based delivery of a rationally designed prodrug is an attractive approach to enhance chemotherapeutic efficacy in pediatric and adult tumors. Clin Cancer Res; 24(11); 2585–93. ©2018 AACR.

Blood counts

Liver function tests

Camptothecins are potent topoisomerase I inhibitors used to treat high-risk pediatric solid tumors, but they often show poor efficacy due to intrinsic or acquired chemoresistance. Here, we developed a multivalent, polymer-based prodrug of a structurally optimized camptothecin (SN22) designed to overcome key chemoresistance mechanisms. The ability of SN22 vs. SN38 (the active form of irinotecan/CPT-11) to overcome efflux pump-driven drug resistance was tested. Tumor uptake and biodistribution of SN22 as a polymer-based prodrug (PEG-[SN22]4) compared with SN38 was determined. The therapeutic efficacy of PEG-[SN22]4 to CPT-11 was compared in: (i) spontaneous neuroblastomas (NB) in transgenic TH-MYCN mice; (ii) orthotopic xenografts of a drug-resistant NB line SK-N-BE(2)C (mutated TP53); (iii) flank xenografts of a drug-resistant NB-PDX; and (iv) xenografts of Ewing sarcoma and rhabdomyosarcoma. Unlike SN38, SN22 inhibited NB cell growth regardless of ABCG2 expression levels. SN22 prodrug delivery resulted in sustained intratumoral drug concentrations, dramatically higher than those of SN38 at all time points. CPT-11/SN38 treatment had only marginal effects on tumors in transgenic mice, but PEG-[SN22]4 treatment caused complete tumor regression lasting over 6 months (tumor free at necropsy). PEG-[SN22]4 also markedly extended survival of mice with drug-resistant, orthotopic NB and it caused long-term (6+ months) remissions in 80% to 100% of NB and sarcoma xenografts. SN22 administered as a multivalent polymeric prodrug resulted in increased and protracted tumor drug exposure compared with CPT-11, leading to long-term “cures” in NB models of intrinsic or acquired drug resistance, and models of high-risk sarcomas, warranting its further development for clinical trials.Significance:SN22 is an effective and curative multivalent macromolecular agent in multiple solid tumor mouse models, overcoming common mechanisms of drug resistance with the potential to elicit fewer toxicities than most cancer therapeutics.

Camptothecins are potent topoisomerase I inhibitors used to treat high-risk pediatric solid tumors, but they often show poor efficacy due to intrinsic or acquired chemoresistance. Here, we developed a multivalent, polymer-based prodrug of a structurally optimized camptothecin (SN22) designed to overcome key chemoresistance mechanisms. The ability of SN22 vs. SN38 (the active form of irinotecan/CPT-11) to overcome efflux pump-driven drug resistance was tested. Tumor uptake and biodistribution of SN22 as a polymer-based prodrug (PEG-[SN22]4) compared with SN38 was determined. The therapeutic efficacy of PEG-[SN22]4 to CPT-11 was compared in: (i) spontaneous neuroblastomas (NB) in transgenic TH-MYCN mice; (ii) orthotopic xenografts of a drug-resistant NB line SK-N-BE(2)C (mutated TP53); (iii) flank xenografts of a drug-resistant NB-PDX; and (iv) xenografts of Ewing sarcoma and rhabdomyosarcoma. Unlike SN38, SN22 inhibited NB cell growth regardless of ABCG2 expression levels. SN22 prodrug delivery resulted in sustained intratumoral drug concentrations, dramatically higher than those of SN38 at all time points. CPT-11/SN38 treatment had only marginal effects on tumors in transgenic mice, but PEG-[SN22]4 treatment caused complete tumor regression lasting over 6 months (tumor free at necropsy). PEG-[SN22]4 also markedly extended survival of mice with drug-resistant, orthotopic NB and it caused long-term (6+ months) remissions in 80% to 100% of NB and sarcoma xenografts. SN22 administered as a multivalent polymeric prodrug resulted in increased and protracted tumor drug exposure compared with CPT-11, leading to long-term “cures” in NB models of intrinsic or acquired drug resistance, and models of high-risk sarcomas, warranting its further development for clinical trials. Significance: SN22 is an effective and curative multivalent macromolecular agent in multiple solid tumor mouse models, overcoming common mechanisms of drug resistance with the potential to elicit fewer toxicities than most cancer therapeutics.

Purpose In a previous study, we demonstrated that the combination of fenretinide with lenalidomide, administered by a novel nanomicellar formulation (FLM), provided a strong antitumor effect in a neuroblastoma TrkB-expressing tumor. In this study, we tested the nanomicellar combination in an MYCN amplified neuroblastoma xenograft to assess its efficacy in different tumor genotypes and evaluate the interactions of the nanomicelles with the tumor cells. Experimental design FLM was administered to mice bearing human NLF xenografts to evaluate its efficacy in comparison with the nanomicelles containing fenretinide alone (FM). Confocal laser-scanning fluorescence microscopy images of the NLF cells treated with FLM and FM allowed us to estimate the nanomicelle ability to transport the encapsulated drugs inside the tumor cells. Flow cytometric analysis of the cells from treated tumors was performed to assess the effect of treatment on GD2 expression and NK cell infiltration. Results FLM and FM decreased the growth of NLF xenografts at comparable extents during the treatment period. Afterwards, FLM induced a progressive tumor regression without regrowth, while FM treatment was followed by regrowth within 15-20 days after the end of treatment. Both FLM and FM were able to penetrate the tumor cells transporting the encapsulated drugs. FLM transported higher amount of fenretinide inside the cells. Also, FLM treatment strongly increased GD2 expression in treated tumors and slightly decreased the NK infiltration compared to FM. Conclusion FLM treatment induced a superior antitumor response than FM in NLF xenografts, presumably due to the combined effects of fenretinide cytotoxicity and lenalidomide antiangiogenic activity. The ability of FLM to penetrate tumor cells, transporting the encapsulated drugs, substantially improved the therapeutic efficiency of this system. Moreover, the enhancement of GD2 expression in FLM treated tumors offers the possibility to further increase the antitumor effect by the use of anti-GD2 CAR-T cells and anti-GD2 antibodies in combination with FLM in multimodal therapies.

High-risk solid tumors continue to pose a tremendous therapeutic challenge due to multidrug resistance. Biological mechanisms driving chemoresistance in high-risk primary and recurrent disease are distinct: in newly diagnosed patients, non-response to therapy is often associated with a higher level of tumor "stemness" paralleled by overexpression of the ABCG2 drug efflux pump, whereas in tumors relapsing after non-curative therapy, poor drug sensitivity is most commonly linked to the dysfunction of the tumor suppressor protein, p53. In this study, we used preclinical models of aggressive neuroblastoma featuring these characteristic mechanisms of primary and acquired drug resistance to experimentally evaluate a macromolecular prodrug of a structurally enhanced camptothecin analog, SN22, resisting ABCG2-mediated export, and glucuronidation. Together with extended tumor exposure to therapeutically effective drug levels via reversible conjugation to Pluronic F-108 (PF108), these features translated into rapid tumor regression and long-term survival in models of both ABCG2-overexpressing and p53-mutant high-risk neuroblastomas, in contrast to a marginal effect of the clinically used camptothecin derivative, irinotecan. Our results demonstrate that pharmacophore enhancement, increased tumor uptake, and optimally stable carrier-drug association integrated into the design of the hydrolytically activatable PF108-[SN22]

Purpose Currently >50% of high-risk neuroblastoma (NB) patients, despite intensive therapy and initial partial or complete response, develop recurrent NB due to the persistence of minimal residual disease (MRD) that is resistant to conventional antitumor drugs. Indeed, their low therapeutic index prevents drug-dose escalation and protracted administration schedules, as would be required for MRD treatment. Thus, more effective and less toxic therapies are urgently needed for the management of MRD. To address this aim, we evaluated a new combination of fenretinide and lenalidomide, both endowed with antitumor activity and low-toxicity profiles. New nanomicelles were prepared as carriers for this combination to maximize bioavailability and accumulation at the tumor site because of the enhanced permeability and retention (EPR) effect. Experimental design New nanomicelles containing the fenretinide-lenalidomide combination (FLnMs) were prepared by a one-step method, providing high drug encapsulation and micelle dimensions suitable for tumor accumulation. Their administration to mice bearing human NB xenografts allowed us to evaluate their efficacy in comparison with the nanomicelles containing fenretinide alone (FnMs). Results Treatment by FLnMs significantly decreased the tumor growth of NB xenografts. FLnMs were more active than FnMs despite comparable fenretinide concentrations in tumors, and lenalidomide alone did not show cytotoxic activity in vitro against NB cells. The tumor mass at the end of treatment with FLnMs was predominantly necrotic, with a decreased Ki-67 proliferation index. Conclusion FLnMs provided superior antitumor efficacy in NB xenografts compared to FnMs. The enhanced efficacy of the combination was likely due to the antiangiogenic effect of lenalidomide added to the cytotoxic effect of fenretinide. This new nanomicellar combination is characterized by a low-toxicity profile and offers a novel therapeutic option for the treatment of high-risk tumors where the persistence of MRD requires repeated administrations of therapeutic agents over long periods of time to avoid recurrent disease.

Neuroblastomas (NBs) have heterogeneous clinical behavior, from spontaneous regression or differentiation to relentless progression. Evidence from our laboratory and others suggests that neurotrophin receptors contribute to these disparate behaviors. Previously, the role of TRK receptors in NB pathogenesis was investigated. In the present study, the expression of RET and its co‑receptors in a panel of NB cell lines was investigated and responses to cognate ligands GDNF, NRTN, and ARTN with GFRα1‑3 co‑receptor expression, respectively were found to be correlated. RET expression was high in NBLS, moderate in SY5Y, low/absent in NBEBc1 and NLF cells. All cell lines expressed at least one of GFRα co‑receptors. In addition, NBLS, SY5Y, NBEBc1 and NLF cells showed different morphological changes in response to ligands. As expected, activation of RET/GFRα3 by ARTN resulted in RET phosphorylation. Interestingly, activation of TrkA by its cognate ligand NGF resulted in RET phosphorylation at Y905, Y1015, and Y1062, and this was inhibited in a dose‑dependent manner by the TRK inhibitor (CEP‑701). Conversely, RET activation by ARTN in NBLS cells led to phosphorylation of TrkA. This suggests a physical association between RET and TRK proteins, and cross‑talk between these two receptor pathways. Finally, RET, GFR and TRK expression in primary tumors was investigated and a significant association between RET, its co‑receptors and TRK expression was demonstrated. Thus, the present data support a complex model of interacting neurotrophin receptor pathways in the regulation of cell growth and differentiation in NBs.

Neuroblastoma (NB) is one of the most common and deadly childhood solid tumors. These tumors are characterized by clinical heterogeneity, from spontaneous regression to relentless progression, and the Trk family of neurotrophin receptors plays an important role in this heterogeneous behavior. We wanted to determine if entrectinib (RXDX-101, Ignyta, Inc.), an oral Pan-Trk, Alk and Ros1 inhibitor, was effective in our NB model. In vitro effects of entrectinib, either as a single agent or in combination with the chemotherapeutic agents Irinotecan (Irino) and Temozolomide (TMZ), were studied on an SH-SY5Y cell line stably transfected with TrkB. In vivo growth inhibition activity was studied in NB xenografts, again as a single agent or in combination with Irino-TMZ. Entrectinib significantly inhibited the growth of TrkB-expressing NB cells in vitro, and it significantly enhanced the growth inhibition of Irino-TMZ when used in combination. Single agent therapy resulted in significant tumor growth inhibition in animals treated with entrectinib compared to control animals [p < 0.0001 for event-free survival (EFS)]. Addition of entrectinib to Irino-TMZ also significantly improved the EFS of animals compared to vehicle or Irino-TMZ treated animals [p < 0.0001 for combination vs. control, p = 0.0012 for combination vs. Irino-TMZ]. We show that entrectinib inhibits growth of TrkB expressing NB cells in vitro and in vivo, and that it enhances the efficacy of conventional chemotherapy in in vivo models. Our data suggest that entrectinib is a potent Trk inhibitor and should be tested in clinical trials for NBs and other Trk-expressing tumors.

TrkB with its ligand, brain-derived neurotrophic factor (BDNF), are overexpressed in the majority of high-risk neuroblastomas (NB). Entrectinib is a novel pan-TRK, ALK, and ROS1 inhibitor that has shown excellent preclinical efficacy in NB xenograft models, and recently it has entered phase 1 trials in pediatric relapsed/refractory solid tumors. We examined entrectinib-resistant NB cell lines to identify mechanisms of resistance. Entrectinib-resistant cell lines were established from five NB xenografts initially sensitive to entrectinib therapy. Clonal cell lines were established in increasing concentrations of entrectinib and had >10X increase in IC50. Cell lines underwent genomic and proteomic analysis using whole-exome sequencing, RNA-Seq, and proteomic expression profiling with confirmatory RT-PCR and Western blot analysis. There was no evidence of NTRK2 (TrkB) gene mutation in any resistant cell lines. Inhibition of TrkB was maintained in all cell lines at increasing concentrations of entrectinib (target independent). PTEN pathway downregulation and ERK/MAPK pathway upregulation were demonstrated in all resistant cell lines. One of these clones also had increased IGF1R signaling, and two additional clones had increased P75 expression, which likely increased TrkB sensitivity to ligand. In conclusion, NB lines overexpressing TrkB developed resistance to entrectinib by multiple mechanisms, including activation of ERK/MAPK and downregulation of PTEN signaling. Individual cell lines also had IGF1R activation and increased P75 expression, allowing preservation of downstream TrkB signaling in the presence of entrectinib. An understanding of changes in patterns of expression can be used to inform multimodal therapy planning in using entrectinib in phase II/III trial planning.

Purpose: Currently, Experimental Design: SN38, the active metabolite of irinotecan (CPT-11), is a potent therapeutic agent that is readily encapsulated in polymeric nanoparticles. Tocopherol oxyacetate (TOA) is a hydrophobic mitocan that was linked to SN38 to significantly increase hydrophobicity and enhance nanoparticle retention. We treated neuroblastomas with SN38-TOA nanoparticles and compared the efficacy with the parent prodrug CPT-11 using a mouse xenograft model. Results: Nanoparticle treatment induced prolonged event-free survival (EFS) in most mice, compared with CPT-11. This was shown for both SH-SY5Y and IMR-32 neuroblastoma xenografts. Enhanced efficacy was likely due to increased and sustained drug levels of SN38 in the tumor compared with conventional CPT-11 delivery. Interestingly, when recurrent CPT-11–treated tumors were re-treated with SN38-TOA nanoparticles, the tumors transformed from undifferentiated neuroblastomas to maturing ganglioneuroblastomas. Furthermore, these tumors were infiltrated with Schwann cells of mouse origin, which may have contributed to the differentiated histology. Conclusions: Nanoparticle delivery of SN38-TOA produced increased drug delivery and prolonged EFS compared to conventional delivery of CPT-11. Also, lower total dose and drug entrapment in nanoparticles during circulation should decrease toxicity. We propose that nanoparticle-based delivery of a rationally designed prodrug is an attractive approach to enhance chemotherapeutic efficacy in pediatric and adult tumors. Clin Cancer Res; 24(11); 2585–93. ©2018 AACR.

Eukaryotic gene expression is developmentally regulated, in part by chromatin remodelling, and its dysregulation has been linked to cancer. CHD5 (chromodomain helicase DNA-binding protein 5) is a tumour suppressor gene (TSG) that maps to a region of consistent deletion on 1p36.31 in neuroblastomas (NBs) and other tumour types. CHD5 encodes a protein with chromatin remodelling, helicase and DNA-binding motifs that is preferentially expressed in neural and testicular tissues. CHD5 is highly homologous to CHD3 and CHD4, which are the core subunits of nucleosome remodelling and deacetylation (NuRD) complexes. To determine if CHD5 forms a similar complex, we performed studies on nuclear extracts from NBLS, SY5Y (both with endogenous CHD5 expression), NLF (CHD5 null) and NLF cells stably transfected with CHD5 cDNA (wild-type and V5–histidine-tagged). Immunoprecipitation (IP) was performed with either CHD5 antibody or antibody to V5/histidine-tagged protein. We identified NuRD components both by GST–FOG1 (Friend Of GATA1) pull-down and by IP. We also performed MS/MS analysis to confirm the presence of CHD5 or other protein components of the NuRD complex, as well as to identify other novel proteins. CHD5 was clearly associated with all canonical NuRD components, including metastasis-associated protein (MTA)1/2, GATA zinc finger domain containing 2A (GATAD2A), histone deacetylase (HDAC)1/2, retinoblastoma-binding protein (RBBP)4/7 and methyl DNA-binding domain protein (MBD)2/3, as determined by Western blotting and MS/MS. Our data suggest CHD5 forms a NuRD complex similar to CHD4. However, CHD5–NuRD may also have unique protein associations that confer functional specificity and may contribute to normal development and to tumour suppression in NB and other cancers.

Nanomedicine-based strategies have the potential to improve therapeutic performance of a wide range of anticancer agents. However, the successful implementation of nanoparticulate delivery systems requires the development of adequately sized nanocarriers delivering their therapeutic cargo to the target in a protected, pharmacologically active form. The present studies focused on a novel nanocarrier-based formulation strategy for SN-38, a topoisomerase I inhibitor with proven anticancer potential, whose clinical application is compromised by toxicity, poor stability and incompatibility with conventional delivery vehicles. SN-38 encapsulated in biodegradable sub-100 nm sized nanoparticles (NP) in the form of its rapidly activatable prodrug derivative with tocopherol succinate potently inhibited the growth of neuroblastoma cells in a dose- and exposure time-dependent manner, exhibiting a delayed response pattern distinct from that of free SN-38. In a xenograft model of neuroblastoma, prodrug-loaded NP caused rapid regression of established large tumors, significantly delayed tumor regrowth after treatment cessation and markedly extended animal survival. The NP formulation strategy enabled by a reversible chemical modification of the drug molecule offers a viable means for SN-38 delivery achieving sustained intratumoral drug levels and contributing to the potency and extended duration of antitumor activity, both prerequisites for effective treatment of neuroblastoma and other cancers.

Neuroblastoma (NB) is the most common and deadly solid tumor in children. The majority of NB patients have advanced stage disease with poor prognosis, so more effective, less toxic therapy is needed. We developed a novel nanocarrier-based strategy for tumor-targeted delivery of a prodrug of SN38, the active metabolite of irinotecan. We formulated ultrasmall-sized ( < 100 nm) biodegradable poly(lactide)-poly(ethylene glycol) based nanoparticles (NPs) containing SN38 conjugated to tocopherol succinate (SN38-TS). Alternative dosing schedules of SN38-TS NPs were compared to irinotecan. Comparison of SN38-TS NPs (2 doses) with irinotecan (20 doses) showed equivalent efficacy but no cures. Comparison of SN38-TS NPs (8, 8, and 16 doses, respectively) to irinotecan (40 doses) showed that all SN38-TS NP regimens were far superior to irinotecan, and “cures” were obtained in all NP arms. SN38-TS NP delivery resulted in 200x the amount of SN38 in NB tumors at 4 hr post-treatment, compared to SN38 detected for the irinotecan arm; no toxicity was seen with NPs. We conclude that this SN38-TS NP formulation improved delivery, retention, and efficacy, without causing systemic toxicity.

CHD5 was first identified because of its location on 1p36 in a region of frequent deletion in neuroblastomas. CHD5 (chromodomain–helicase–DNA–binding-5) is the fifth member of a family of chromatin remodeling proteins, and it probably functions by forming a nucleosome remodeling and deacetylation (NuRD) complex that regulates transcription of particular genes. CHD5 is preferentially expressed in the nervous system and testis. On the basis of its position, pattern of expression, and function in neuroblastoma cells and xenografts, CHD5 was identified as a tumor suppressor gene (TSG). Evidence soon emerged that CHD5 also functioned as a TSG in gliomas and a variety of other tumor types, including breast, colon, lung, ovary, and prostate cancers. Although one copy of CHD5 is deleted frequently, inactivating mutations of the remaining allele are rare. However, DNA methylation of the CHD5 promoter is found frequently, and this epigenetic mechanism leads to biallelic inactivation. Furthermore, low CHD5 expression is strongly associated with unfavorable clinical and biologic features as well as outcome in neuroblastomas and many other tumor types. Thus, based on its likely involvement as a TSG in neuroblastomas, gliomas, and many common adult tumors, CHD5 may play an important developmental role in many other tissues besides the nervous system and testis. Cancer Res; 74(3); 652–8. ©2014 AACR.

Purpose: Neuroblastomas (NBs) have genomic, biological, and clinical heterogeneity. High-risk NBs are characterized by several genomic changes, including MYCN amplification and 1p36 deletion. We identified the chromatin-remodeling gene CHD5 as a tumor suppressor gene that maps to 1p36.31. Low or absent CHD5 expression is associated with a 1p36 deletion and an unfavorable outcome, but the mechanisms of CHD5 inactivation in NBs are unknown. Experimental Design: We examined (i) the CHD5 sequence in 188 high-risk NBs investigated through the TARGET initiative, (ii) the methylation status of the CHD5 promoter in 108 NBs with or without 1p36 deletion and/or MYCN amplification, and (iii) mRNA expression of CHD5 and MYCN in 814 representative NBs using TaqMan low-density array microfluidic cards. Results: We found no examples of somatically acquired CHD5 mutations, even in cases with 1p36 deletion, indicating that homozygous genomic inactivation is rare. Methylation of the CHD5 promoter was common in the high-risk tumors, and it was generally associated with both 1p deletion and MYCN amplification. High CHD5 expression was a powerful predictor of favorable outcome, and it showed prognostic value even in multivariable analysis after adjusting for MYCN amplification, 1p36 deletion, and/or 11q deletion. Conclusions: We conclude that (i) somatically acquired CHD5 mutations are rare in primary NBs, so inactivation probably occurs by deletion and epigenetic silencing; (ii) CHD5 expression and promoter methylation are associated with MYCN amplification, suggesting a possible interaction between these 2 genes; and (iii) high CHD5 expression is strongly correlated with favorable clinical/biological features and outcome. Clin Cancer Res; 18(6); 1588–97. ©2012 AACR.

Background Neuroblastomas (NBs) are characterized by clinical heterogeneity, from spontaneous regression to relentless progression. The pattern of NTRK family gene expression contributes to these disparate behaviors. TrkA/NTRK1 is expressed in favorable NBs that regress or differentiate, whereas TrkB/NTRK2 and its ligand brain-derived neurotrophic factor (BDNF) are co-expressed in unfavorable NBs, representing an autocrine survival pathway. We determined the significance of NTRK family gene expression in a large, representative set of primary NBs. Patients and Methods We analyzed the expression of the following genes in 814 NBs using quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR): NTRK1, NTRK2, NTRK3, P75/NGFR, nerve growth factor (NGF), BDNF, IGFR1, and EGFR. Expression (high vs. low) was dichotomized by median expression value and compared to clinical and biological variables as well as outcome. Results High NTRK1 expression was strongly correlated with favorable age, stage, MYCN status, histology, ploidy, risk group, and outcome (P

Background: Neuroblastoma (NB) ranks as the most common solid tumor in infants and is responsible for 15% of all childhood cancer deaths. Chemotherapeutics used as a first-line treatment in high-risk NB patients are inherently toxic and ultimately ineffective. High-risk NB patients have Citation Format: David Guerrero, Ivan S. Alferiev, Ferro Nguyen, Peng Guan, Venkatadri Kolla, Danielle Soberman, Ilia Fishbein, Robert J. Levy, Garrett M. Brodeur, Michael Chorny. Nanocarriers with a camptothecin-mitocan co-drug suppress tumor growth and extend survival in models of chemosensitive and chemoresistant neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A119.

The purpose of this study was to determine the expression and cellular functions of the epithelial NADPH oxidase DUOX1 during alveolar type II cell development. When human fetal lung cells (gestational age 11–22 wk) were cultured to confluency on permeable filters, exposure of cells to a hormone mixture (dexamethasone, 8-Br-cAMP, and IBMX, together referred to as DCI) resulted in differentiation of cells into a mature type II phenotype as assessed by expression of lamellar bodies, surfactant proteins, and transepithelial electrical parameters. After 6 days in culture in presence of DCI, transepithelial resistance (2,616 ± 529 Ω·cm2) and potential (−8.5 ± 0.6 mV) indicated epithelial polarization. At the same time, treatment with DCI significantly increased the mRNA expression of DUOX1 (∼21-fold), its maturation factor DUOXA1 (∼12-fold), as well as DUOX protein (∼12-fold), which was localized near the apical cell pole in confluent cultures. For comparison, in fetal lung specimens, DUOX protein was not detectable at up to 27 wk of gestational age but was strongly upregulated after 32 wk. Function of DUOX1 was assessed by measuring H2O2 and acid production. Rates of H2O2 production were increased by DCI treatment and blocked by small interfering RNA directed against DUOX1 or by diphenylene iodonium. DCI-treated cultures also showed increased intracellular acid production and acid release into the mucosal medium, and acid production was largely blocked by knockdown of DUOX1 mRNA. These data establish the regulated expression of DUOX1 during alveolar maturation, and indicate DUOX1 in alveolar H2O2 and acid secretion by differentiated type II cells.

Background Chromodomain-helicase DNA binding protein 5 (CHD5) is an important tumor suppressor gene deleted from 1p36.31 in neuroblastomas (NBs). High CHD5 expression is associated with a favorable prognosis, but deletion or low expression is frequent in high-risk tumors. We explored the role of CHD5 expression in the neuronal differentiation of NB cell lines. Methods NB cell lines SH-SY5Y (SY5Y), NGP, SK-N-DZ, IMR5, LAN5, SK-N-FI, NB69 and SH-EP were treated with 1–10 μM 13-cis-retinoic acid (13cRA) for 3–12 days. qRT-PCR and Western blot analyses were performed to measure mRNA and protein expression levels, respectively. Morphological differences were examined by both phase contrast and immunofluorescence studies. Results Treatment of SY5Y cells with 13cRA caused upregulation of CHD5 expression in a time- and dose-dependent manner (1, 5, or 10 μM for 7 or 12 days) and also induced neuronal differentiation. Furthermore, both NGP and SK-N-DZ cells showed CHD5 upregulation and neuronal differentiation after 13cRA treatment. In contrast, 13cRA treatment of IMR5, LAN5, or SK-N-FI induced neither CHD5 expression nor neuronal differentiation. NB69 cells showed two different morphologies (neuronal and substrate adherent) after 12 days treatment with 10 μM of 13cRA. CHD5 expression was high in the neuronal cells, but low/absent in the flat, substrate adherent cells. Finally, NGF treatment caused upregulation of CHD5 expression and neuronal differentiation in SY5Y cells transfected to express TrkA (SY5Y-TrkA) but not in TrkA-null parental SY5Y cells, and both changes were blocked by a pan-TRK inhibitor. Conclusions Treatment with 13cRA induces neuronal differentiation only in NB cells that upregulate CHD5. In addition, NGF induced CHD5 upregulation and neuronal differentiation only in TrkA expressing cells. Together, these results suggest that CHD5 is downstream of TrkA, and CHD5 expression may be crucial for neuronal differentiation induced by either 13cRA or TrkA/NGF signaling. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0425-y) contains supplementary material, which is available to authorized users.

Effect of 13cRA on cell proliferation by SRB assay. SY5Y and NB69 cells were plated on 96 well plates and treated with or without 10Â ÎźM retinoic acid. Plates were harvested after 2, 5, and 7Â days of 13cRA treatment. Cell viability was analyzed by an SRB assay measuring optical density (OD). Cell lines treated with 13cRA showed lower OD, indicating reduced cell number compared to untreated control cells. (DOC 46 kb)

(A) Neurite inhibition by CEP-701 in SY5Y-TrkA cells. Representative phase contrast images of SY5Y-TrkA cells depicting extension of neurites in response to 13cRA treatment after 4 days, and subsequent neurite inhibition following 100 nM CEP-701 treatment (μm-micrometer) (B) Statistical analysis of neurite outgrowth following 13cRA as well as CEP-701 treatment of SY5Y-TrkA cells. Length of neurites (μm-micrometer) from three to five representative fields of 13cRA and CEP-701 treated cells were measured in μm and compared with neurites from untreated cells. P-values were calculated by one-way ANOVA using Prism followed by Tukey’s post-test. (DOC 148 kb)

(A) Detection of neurites in NB69 cells in response to retinoic acid. Representative phase contrast images of NB69 cells showing long neurites upon 13cRA treatment for 6 and 10 days. (B) Statistical analysis of various neurites upon 13cRA treatment in NB69 cells. Length of neurites from various selected fields were measured in μm and compared with neurites from untreated cells. Neurites were measured (μm-micrometer) from at least three independent experiments and p-values were calculated by one-way ANOVA using Prism followed by Tukey’s post-test. (DOC 296 kb)

(A) Neurite formation in SY5Y cells in response to 13cRA. Representative phase contrast images depicting extension of neurites in response to 13cRA treatment after 6 days and 10 days. Long neurites were observed when cells were treated with 13cRA. (B) Statistical analysis of neurite outgrowth following 13cRA treatment in SY5Y cells. Length of neurites from three to five representative fields of 13cRA treated cells were measured in μm and compared with neurites from untreated cells. Neurites were measured (μm-micrometer) from at least three independent experiments, and p-values were calculated by one-way ANOVA using Prism followed by Tukey’s post-test. (DOC 208 kb)

Hop is an unusual homeodomain protein that was first identified in the developing heart where it functions downstream of Nkx2.5 to modulate cardiac gene expression. Hop functions through interactions with histone deacetylase (HDAC) 2 to mediate repression of cardiac-specific genes, and recent studies show that HDAC activity and HDAC2 expression are decreased in people with chronic obstructive pulmonary disease. Here, we show that Hop is expressed in airway epithelium coincident with HDAC2, and expression is induced by the combination of dexamethasone and cAMP in parallel with induction of surfactant protein gene expression. Hop functions in the developing pulmonary airway, acting downstream of Nkx2.1 and GATA6, to negatively regulate surfactant protein expression. Loss of Hop expression in vivo results in defective type 2 pneumocyte development with increased surfactant production and disrupted alveolar formation. Thus Hop represents a novel regulator of pulmonary maturation that is induced by glucocorticoids to mediate functionally important HDAC-dependent negative feedback regulation.

Mature alveolar type II cells that produce pulmonary surfactant are essential for adaptation to extrauterine life. We profiled gene expression in human fetal lung epithelial cells cultured in serum-free medium containing dexamethasone and cyclic AMP, a treatment that induces differentiation of type II cells. Microarray analysis identified 388 genes that were induced > 1.5-fold by 72 h of hormone treatment. Induced genes represented all categories of molecular function and subcellular location, with increased frequency in the categories of ionic channel, cell adhesion, surface film, lysosome, extracellular matrix, and basement membrane. In time-course experiments, self-organizing map analysis identified a cluster of 17 genes that were slowly but highly induced (5- to approximately 190-fold) and represented four functional categories: surfactant-related (SFTPC, SFTPA, PGC, SFTPB, LAMP3, LPL), regulatory (WIF2, IGF2, IL1RL1, NR4A2, HIF3A), metabolic (MAOA, ADH1B, SEPP1), and transport (SCNN1A, CLDN18, AQP4). Induction of both mRNA and protein for these genes, which included nine newly identified regulated genes, was confirmed, and cellular localization was determined in both fetal and postnatal tissue. Induction of lysosomal-associated membrane protein 3 required both hormones, and expression was localized to limiting membranes of lamellar bodies. Hormone-induced differentiation of human type II cells is associated with genome-wide increased expression of genes with diverse functions.

Neuroblastoma (NB) is the most common and deadly solid tumor in children. Despite recent improvements, the long-term outlook for high-risk NB is still50%. Further, there is considerable short- and long-term toxicity. More effective, less toxic therapy is needed, and the development of targeted therapies offers great promise.Relevant literature was reviewed to identify current and future therapeutic targets that are critical to malignant transformation and progression of NB. The potential or actual NB therapeutic targets are classified into four categories: i) genes activated by amplification, mutation, translocation or autocrine overexpression; ii) genes inactivated by deletion, mutation or epigenetic silencing; iii) membrane-associated genes expressed on most NBs but few other tissues; or iv) common target genes relevant to NB as well as other tumors.Therapeutic approaches have been developed to some of these targets, but many remain untargeted at the present time. It is unlikely that single targeted agents will be sufficient for long-term cure, at least for high-risk NBs. The challenge will be how to integrate targeted agents with each other and with conventional therapy to enhance their efficacy, while simultaneously reducing systemic toxicity.

Nuclear factor-kappaB (NF-kappaB) is a ubiquitous transcription factor that regulates the expression of multiple inflammatory and immune response genes and plays a critical role in host defense and in chronic inflammatory diseases. The mineralocorticoid receptor (MR) belongs to the steroid/thyroid hormone receptor super-family of ligand-induced transcription factors. We demonstrate a dose-dependent, mutual transcriptional antagonism between NF-kappaB and MR in transient transfection experiments. We also show that the antagonism is limited to the p65 subunit of NF-kappaB heterodimer but not p50. Transient cotransfection experiments with MR deletion constructs reveal the necessity of various N-terminal MR domains for this phenomenon. Inhibition of NF-kappaB by IkappaB relieves the repression of NF-kappaB function by MR. These data suggest that the p65 subunit of NF-kappaB interacts with MR indirectly and transrepresses MR activation.

MB78 is a virulent phage of Salmonella typhimurium that possesses a number of interesting features, making it a suitable organism to study the regulation of gene expression. A detailed physical map of this phage genome has been constructed and is being extensively studied at the molecular level. Here, we demonstrate the expression of two late proteins of bacteriophage MB78 derived from the same gene as a result of possible ribosomal frameshifting. In vitro transcription-translation yields a major protein that migrates as 28 kDa, whereas in vivo expression using pET expression vectors yields two equally expressed proteins of molecular sizes 28 and 26 kDa. A putative slippery sequence TTTAAAG and a pseudoknot structure, two essential cis elements required for the classical ribosomal frameshifting, are identified in the reading frame. Mutations created at the slippery sequence resulted in a single 28 kDa protein and completely abolished the expression of 26 kDa protein. Thus, we have produced the first evidence that ribosomal frameshifting occurs in bacteriophage MB78 of Salmonella typhimurium.

Bacteriophage MB78, a virulent phage of Salmonella typhimurium isolated in our laboratory. It is different from the well-known temperate phage P22 and 9NA. A detailed physical map has been constructed. To understand more about the physiology and genetics of this interesting phage it has become necessary to fragment the phage genome, clone the fragments and analyze in depth. A number of promoters of bacteriophage MB78 have been cloned and characterized recently. As a part of this program, in this investigation, we report cloning, sequencing and expression and promoter analysis of the ClaI G fragment. We identified the expressed protein as phage structural. Phage structural proteins play a vital role in forming the core head of the phage particle.

The mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) are members of the steroid/thyroid hormone receptor superfamily of ligand inducible transcription factors and have been shown to bind the glucocorticoid response element (GRE). Sodium-potassium ATPase (Na/K ATPase) is a major target of mineralocorticoids. Both aldosterone and glucocorticoids activate the human Na/K ATPase α1 subunit and β1 subunit genes transcriptionally. However, the mechanisms of corticosteroid regulation of mammalian Na/K ATPase subunit gene expression are not known. In this investigation, we report for the first time that cell lines (T-84 and 293) express endogenous MR by RT-PCR message expression. However, the protein product was not expressed as determined by western blot analyses. In transactivation studies of MR with GRE31, we detected MR expression at low concentrations of aldosterone. We also performed Northern blot and nuclear run-off transcription assays to further confirm that the regulation is transcriptional. We conclude that the transcriptional regulation of the human Na/K ATPase α1 and β1 subunits by aldosterone occurs via the involvement of the MR.

Sodium-potassium ATPase (Na/K ATPase) is a major target of mineralocorticoids. Both aldosterone and glucocorticoids activate the human Na/K ATPase alpha1 and beta1 genes transcriptionally. The mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) have been shown to bind the glucocorticoid response element (GRE); however, a specific element responsible for the activation of the MR is not known. Sequence analysis of the putative regulatory region of the Na/K ATPase alpha1 gene revealed the presence of a hormone response element that allows the MR to interact with it, at least as well as if not better than the GR. This response element is designated MRE/GRE. In this investigation, we demonstrated the MR and GR induced gene expression in COS-1 cells by cotransfecting with respective expression plasmids (RshMR and RshGR) along with a luciferase reporter. The synthetic MRE/GRE linked to a neutral promoter was activated by MR (6-fold); however, the GR induced a lower level of expression (3.8-fold), suggesting that the element may be preferably MR responsive. Mutations in the synthetic MRE/GRE could not induce the expression with MR, whereas GR had a small effect. Electrophoretic mobility shift analyses demonstrated a direct interaction of MR and GR with the MRE/GRE that was supershifted by an antiMR antibody and the complex was partially cleared by an antiGR antibody, respectively, whereas nonimmune serum had no effect. Footprinting analyses of the promoter region showed that a portion of the DNA containing this element is protected by recombinant MR and GR. Thus these data confirm that this MRE/GRE interacts with both MR and GR but interaction with receptors may be more MR-responsive than response elements previously described.

Summary BacteriophageMB78,avirulentphageof Salmonellatyphimu-rium ,doesnotallowotherphages,suchasP22and9NA,togrowinitspresence.Adetailedphysicalmapofthisphagehasbeenconstructedinourlaboratory.Inanongoingefforttounderstandthegeneticsofthisinterestingphage,variousgeneswerecharac-terized.Here,wereportcloning,sequencing,andexpressionoftwolateproteins,codedina Sal I– Hind IIIfragment(SH9),byusingtheminicellexpressionsystem.Further,weperformedakineticstudyofphageproteinsbyinfectingthehostLT2cellsandcomparedtheproteinsproduced,withproteinsobtainedbytheminicellexpres-sionsystem.Bothsetsofproteinsrunexactlyparallelandmigratedas14-and15-kDaproteinsonapolyacrylamidegel.Thesynthesisofthesetwoproteinsstarted15minafterinfectionwithMB78andwasprominentafter45min.Oneoftheproteinsexhibited57%homologytothestructuralproteinofmycobacteriophageL5. IUBMB Life ,48:493–497,1999Keywords Bacteriophage MB78; minicell expression; phageproteins. INTRODUCTION BacteriophageMB78isavirulentphageof Salmonellaty-phimurium

The mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) belong to the steroid/thyroid hormone superfamily of ligand-induced transcription factors. Both activate the human Na/K ATPase alpha1 and beta1 genes transcriptionally. To assess the role of the transcription factor Sp1 and the nuclear factor I (NF-I), in MR- and GR-mediated gene expression using the human Na/K ATPase beta1 full-length promoter, we have examined the functions of Sp-I and NF-I functions in two different cell lines, COS-1 and T-84. By transient transfections we have shown that Sp-I significantly enhances MR and GR expression, whereas NF-I had negligible effect. We propose that the transcriptional enhancement could be through a direct interaction physically between MR or GR with Sp1 that allows other factors to bind the responsive element resulting in synergistic upregulation of transcription.

We have previously demonstrated that up-regulation of STAT1 protein by all-trans-retinoic acid (RA) in interferon (IFN)-unresponsive cells permits growth inhibition by IFNs. Here, we show that the promoter of STAT1 directly responds to retinoic acid treatment. Sequence and functional analysis of the murine STAT1 promoter have identified a direct repeat motif that serves as a retinoic acid response element. Mutagenesis of this element resulted in a loss of response to RA. This element is activated by RA receptors alpha, beta, and gamma. In vivo, RA receptor beta and retinoid X receptor alpha preferentially interacted with this element. Thus, these data define a molecular basis for the synergy between IFNs and retinoids in tumor growth inhibition.

The molecular basis for the enhanced growth inhibition of MCF-7 human breast cancer xenografts by a combination of human interferon-β (IFN-β) and tamoxifen was investigated. Treatment of MCF-7, MDA-MB-231, and BT-20 cells with the combination of IFN-β and tamoxifen resulted in enhanced antiproliferative effects in vitro. Treatment with the combination of IFN-β and tamoxifen enhanced the expression of several IFN-β-inducible genes in human breast carcinoma cell lines relative to levels induced by IFN-β alone. Tamoxifen alone did not induce transcription of IFN-stimulated genes (ISGs). Augmentation of ISG expression by the combination of IFN-β and tamoxifen was noted in breast tumor cell lines irrespective of their functional estrogen receptor (ER) status or their dependence on estradiol for growth, suggesting that upregulation of ISGs was independent of ER status. Enhancement of IFN-stimulated gene expression by tamoxifen occurred at the transcripti onal level. Expression of transfected reporter genes under the control of IFN-α/β regulated promoters was also enhanced in IFN-β and tamoxifen-treated cells. Similarly, transcriptional induction of chimeric reporter plasmids driven by an IFN-γ inducible promoter (GAS; IFN-γ activated site) was also enhanced by the combination of IFN-γ and tamoxifen. In tamoxifen treated cells, IFN-β and IFN-γ readily activated transcription factors ISGF-3 and GAF, respectively. Therefore, augmentation of ISG expression by tamoxifen is an early event in the antitumoral activity of this drug combination. (Mol Cell Biochem 167: 169-177, 1997)

Interferons (IFN) and retinoids failed to inhibit the growth of a number of breast tumor cell lines. However, a combination of these two biological response modifiers significantly suppressed the cell growth at pharmacologically achievable doses. The molecular basis for such enhancement was investigated in MCF-7, a breast tumor cell line resistant to growth inhibition by IFN-β. Pretreatment of cells with retinoic acid (RA) for 16 h followed by IFN-β, but not the converse, induced cytotoxic effects in the cells. Continuous presence of RA was not necessary, although it enhanced the degree of cell death when present. Further analyses revealed that IFN-β failed to activate IFN-stimulated gene transcription. However, IFN-β strongly up-regulated the gene expression in RA-pretreated cells. Both IFN-β- and IFN-γ-inducible gene expression were enhanced via a modulation of the transcriptional factor IFN-stimulated gene factors-3 and GAF binding to respective cognate regulatory elements. STAT1 was undetectable in these cells prior to RA treatment. RA increased the levels of this crucial regulator, thereby restoring IFN responses. Thus, RA augmentation of STAT1 may be an early step in the cooperative anti-tumor effects of IFN and RA.

Review on MYCN (v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)), with data on DNA, on the protein encoded, and where the gene is implicated.

Review on CHD5 (chromodomain helicase DNA binding protein 5), with data on DNA, on the protein encoded, and where the gene is implicated.

Alveolar type II cells have multiple functions, including surfactant production and fluid clearance, which are critical for lung function. Differentiation of type II cells occurs in cultured fetal lung epithelial cells treated with dexamethasone plus cAMP and isobutylmethylxanthine (DCI) and involves increased expression of 388 genes. In this study, type II cells of human adult lung were isolated at approximately 95% purity, and gene expression was determined (Affymetrix) before and after culturing 5 days on collagen-coated dishes with or without DCI for the final 3 days. In freshly isolated cells, highly expressed genes included SFTPA/B/C, SCGB1A, IL8, CXCL2, and SFN in addition to ubiquitously expressed genes. Transcript abundance was correlated between fetal and adult cells (r = 0.88), with a subset of 187 genes primarily related to inflammation and immunity that were expressed10-fold higher in adult cells. During control culture, expression increased for 8.1% of expressed genes and decreased for approximately 4% including 118 immune response and 10 surfactant-related genes. DCI treatment promoted lamellar body production and increased expression of approximately 3% of probed genes byor =1.5-fold; 40% of these were also induced in fetal cells. Highly induced genes (or =10-fold) included PGC, ZBTB16, DUOX1, PLUNC, CIT, and CRTAC1. Twenty-five induced genes, including six genes related to surfactant (SFTPA/B/C, PGC, CEBPD, and ADFP), also had decreased expression during control culture and thus are candidates for hormonal regulation in vivo. Our results further define the adult human type II cell molecular phenotype and demonstrate that a subset of genes remains hormone responsive in cultured adult cells.

Purpose: Neuroblastoma (NB) is the most common and deadly solid tumor in childhood. The majority of NB patients have advanced stage disease and a poor prognosis, so more effective, less toxic therapy is needed. We wanted to determine if more targeted delivery of chemotherapeutic agents would improve efficacy and reduce systemic toxicity. We developed a novel nanocarrier-based strategy for tumor-targeted delivery of a prodrug of SN38, the active metabolite of irinotecan. Experimental Design: We formulated ultrasmall-sized ( Results: All SN38-TS NP regimens were far superior to irinotecan, and “cures” were obtained in all NP arms (no cures with irinotecan). SN38-TS NP delivery resulted in 200x the amount of SN38 in NB tumors at 4 hr post-treatment, and 25% of the 4-hr SN38 level remained at 72 hr post-treatment, compared to no SN38 detected at 24 hr post-treatment for the irinotecan arm; no toxicity was seen with NPs. Conclusions: We conclude that this SN38-TS NP formulation improved delivery, retention, and efficacy, without causing systemic toxicity. Citation Format: Radhika Iyer, Jamie L. Croucher, Michael Chorny, Jennifer L. Mangino, Ivan S. Alferiev, Robert J. Levy, Venkatadri Kolla, Garrett M. Brodeur. Nanoparticle delivery of an SN38 conjugate is more effective than Irinotecan in a mouse model of Neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5537. doi:10.1158/1538-7445.AM2015-5537

Background: Neuroblastoma (NB) is a tumor of the sympathetic nervous system that is the most common extracranial solid tumor of childhood. NBs show remarkable clinical heterogeneity, and we, along with others, have identified different patterns of genomic change that underlie these diverse clinical behaviors. We first identified 1p deletion as a characteristic change in advanced stage NBs. CHD5, a tumor suppressor gene, was first identified because of its location on 1p36. However, mutation of the remaining allele of CHD5 is rare in these tumors. Therefore, it is likely that epigenetic mechanisms play important roles in CHD5 downregulation. MicroRNAs (miRNAs) are small RNAs that bind to the 3′ UTR to mediate downregulation of target gene expression by translational repression or cleavage of the target gene message. Dysregulation of CHD5 via miR-211 has been reported in colon cancer. In order to understand the role of miRNA regulation of CHD5 in NBs, we wanted to assess the function of all miRNAs that are predicted to target CHD5. Method/approach: We used TargetScan, miRanda, MirTarget2 and other prediction algorithms to identify miRNAs that were predicted to bind to the CHD5-3′UTR. We identified 18 miRNAs that were predicted by 2 or more programs: miR-204-5p, -211, -216b, -17, -19ab, -20ab, -93, -106ab, -130ab, -301ab, -454, -519d, -3666. We used a renilla-luciferase reporter plasmid that contains 103 bp of the 3′UTR of CHD5 targeted by all miRNAs except miR-204, -211, - 219b, and -3666. We also created control plasmids with no CHD5 3′UTR insert and a mutated CHD5 3′UTR with mutated miRNA binding sites. Allstar siRNA served as an additional negative control. We performed transient transfections in two NB cell lines, NLF (a MYCN amplified cell line) and SY5Y (a MYCN nonamplified cell line), with the reporter plasmid and miRNA mimic. We measured luciferase (internal control) and renilla (reporter) activity. Results: Our results from the luciferase reporter assay indicate that four miRNAs are strong candidates for CHD5 downregulation in NB: miR-17, -93, -20b, and miR-106b. Each exhibited a significant decrease of fluorescence activity in cells transfected with CHD5 3′UTR in comparison to cells transfected with either of the control plasmids. Interestingly, MYCN upregulates three of the four miRNA candidates: miR-17, -93, & -20b. We further confirmed consistent downregulation of the CHD5 by all four miRNAs. Conclusion: CHD5 expression is downregulated by miR-17, -93, -2b, and 106b. MYCN amplification and overexpression leads to upregulation of miR-17, -93, and -20b, and this may lead to down regulation of CHD5. Citation Format: Koumudi Naraparaju, Venkatadri Kolla, Tiangang Zhuang, Mayumi Higashi, Gerd A. Blobel, Garrett M. Brodeur. The role of microRNAs in the epigenetic silencing of CHD5, a tumor suppressor in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 191. doi:10.1158/1538-7445.AM2015-191

Thyroid transcription factor-1 (TTF-1, product of the Nkx2.1 gene) is essential for branching morphogenesis of the lung and enhances expression of surfactant proteins by alveolar type II cells. We investigated expression of two TTF-1 mRNA transcripts, generated by alternative start sites and coding for 42- and 46-kD protein isoforms in the mouse, during hormone-induced differentiation of human fetal lung type II cells in culture. Transcript for 42-kD TTF-1 was 20-fold more abundant than TTF-1(46) mRNA by RT-PCR. Only 42-kD protein was detected in lung cells, and its content increased during in vivo development and in response to in vitro glucocorticoid plus cAMP treatment. To examine TTF-1 target proteins, recombinant, phosphorylated TTF-1(42) was expressed in nuclei of cells by adenovirus transduction. By microarray analysis, 14 genes were comparably induced by recombinant TTF-1 (rTTF-1) and hormone treatment, and 9 additional hormone-responsive genes, including surfactant proteins-A/B/C, were partially induced by rTTF-1. The most highly (approximately 10-fold) TTF-1-induced genes were DC-LAMP (LAMP3) and CEACAM6 with induction confirmed by Western analysis and immunostaining. Treatment of cells with hormones plus small inhibitory RNA directed toward TTF-1 reduced TTF-1 content by approximately 50% and inhibited hormone induction of the 23 genes induced by rTTF-1. In addition, knockdown of TTF-1 inhibited 72 of 274 other genes induced by hormones. We conclude that 42-kD TTF-1 is required for induction of a subset of regulated genes during type II cell differentiation.