A nationwide phase II study of delayed local treatment for children with high-risk neuroblastoma: The Japan Children's Cancer Group Neuroblastoma Committee Trial JN-H-11.

PURPOSE: Survival rates of patients with high-risk neuroblastoma are unacceptable. A time-intensified treatment strategy with delayed local treatment to control systemic diseases has been developed in Japan. We conducted a nationwide, prospective, single-arm clinical trial with delayed local treatment. This study evaluated the safety and efficacy of delayed surgery to increase treatment intensity. PATIENTS AND METHODS: Seventy-five patients with high-risk neuroblastoma were enrolled in this study between May 2011 and September 2015. Delayed local treatment consisted of five courses of induction chemotherapy (cisplatin, pirarubicin, vincristine, and cyclophosphamide) and myeloablative high-dose chemotherapy (melphalan, etoposide, and carboplatin), followed by local tumor extirpation with surgery and irradiation. The primary endpoint was progression-free survival (PFS). The secondary endpoints were overall survival (OS), response rate, adverse events, and surgical complications. RESULTS: Seventy-five patients were enrolled, and 64 were evaluable (stage 3, n = 8; stage 4, n = 56). The estimated 3-year PFS and OS rates (95% confidence interval [CI]) were 44.4% [31.8%-56.3%] and 80.7% [68.5%-88.5%], resspectively. The response rate of INRC after completion of the treatment protocol was 66% (42/64; 95% CI: 53%-77%; 23 CR [complete response], 10 VGPR [very good partial response], and nine PR [partial response]). None of the patients died during the protocol treatment or within 30 days of completion. Grade 4 adverse effects, excluding hematological adverse effects, occurred in 48% of patients [31/64; 95% CI: 36%-61%]. Major Surgical complications were observed in 25% of patients [13/51; 95% CI: 14%-40%]. CONCLUSION: This study indicates that delayed local treatment is feasible and shows promising efficacy, suggesting that this treatment should be considered further in a comparative study of high-risk neuroblastoma.

Polycomb group protein BMI1 protects neuroblastoma cells against DNA damage-induced apoptotic cell death.

The overexpression of BMI1, a polycomb protein, correlates with cancer development and aggressiveness. We previously reported that MYCN-induced BMI1 positively regulated neuroblastoma (NB) cell proliferation via the transcriptional inhibition of tumor suppressors in NB cells. To assess the potential of BMI1 as a new target for NB therapy, we examined the effects of reductions in BMI1 on NB cells. BMI1 knockdown (KD) in NB cells significantly induced their differentiation for up to 7 days. BMI1 depletion significantly induced apoptotic NB cell death for up to 14 days along with the activation of p53, increases in p73, and induction of p53 family downstream molecules and pathways, even in p53 mutant cells. BMI1 depletion in vivo markedly suppressed NB xenograft tumor growth. BMI1 reductions activated ATM and increased γ-H2AX in NB cells. These DNA damage signals and apoptotic cell death were not canceled by the transduction of the polycomb group molecules EZH2 and RING1B. Furthermore, EZH2 and RING1B KD did not induce apoptotic NB cell death to the same extent as BMI1 KD. Collectively, these results suggest the potential of BMI1 as a target of molecular therapy for NB and confirmed, for the first time, the shared role of PcG proteins in the DNA damage response of NB cells.

Development of an osteosarcoma model with MYCN amplification and TP53 mutation in hiPS cell-derived neural crest cells.

Mesenchymal stem cell- or osteoblast-derived osteosarcoma is the most common malignant bone tumor. Its highly metastatic malignant phenotypes, which are often associated with a poor prognosis, have been correlated with the modulation of TP53- and cell-cycle-related pathways. MYC, which regulates the transcription of cell-cycle modulating genes, is used as a representative prognostic marker for osteosarcoma. Another member of the MYC oncoprotein family, MYCN, is highly expressed in a subset of osteosarcoma, however its roles in osteosarcoma have not been fully elucidated. Here, we attempted to create an in vitro tumorigenesis model using hiPSC-derived neural crest cells, which are precursors of mesenchymal stem cells, by overexpressing MYCN on a heterozygous TP53 hotspot mutation (c.733G>A; p.G245S) background. MYCN-expressing TP53 mutated transformed clones were isolated by soft agar colony formation, and administered subcutaneously into the periadrenal adipose tissue of immunodeficient mice, resulting in the development of chondroblastic osteosarcoma. MYCN suppression decreased the proliferation of MYCN-induced osteosarcoma cells, suggesting MYCN as a potential target for a subset of osteosarcoma treatment. Further, comprehensive analysis of gene expression and exome sequencing of MYCN-induced clones indicated osteosarcoma-specific molecular features, such as the activation of TGF-ß signaling and DNA copy number amplification of GLI1. The model of MYCN-expressing chondroblastic osteosarcoma was developed from hiPSC-derived neural crest cells, providing a useful tool for the development of new tumor models using hiPSC-derived progenitor cells with gene modifications and in vitro transformation.

ATM depletion induces proteasomal degradation of FANCD2 and sensitizes neuroblastoma cells to PARP inhibitors.

BACKGROUND: Genomic alterations, including loss of function in chromosome band 11q22-23, are frequently observed in neuroblastoma, which is the most common extracranial childhood tumour. In neuroblastoma, ATM, a DNA damage response-associated gene located on 11q22-23, has been linked to tumorigenicity. Genetic changes in ATM are heterozygous in most tumours. However, it is unclear how ATM is associated with tumorigenesis and cancer aggressiveness. METHODS: To elucidate its molecular mechanism of action, we established ATM-inactivated NGP and CHP-134 neuroblastoma cell lines using CRISPR/Cas9 genome editing. The knock out cells were rigorously characterized by analyzing proliferation, colony forming abilities and responses to PARP inhibitor (Olaparib). Western blot analyses were performed to detect different protein expression related to DNA repair pathway. ShRNA lentiviral vectors were used to knockdown ATM expression in SK-N-AS and SK-N-SH neuroblastoma cell lines. ATM knock out cells were stably transfected with FANCD2 expression plasmid to over-expressed the FANCD2. Moreover, knock out cells were treated with proteasome inhibitor MG132 to determine the protein stability of FANCD2. FANCD2, RAD51 and γH2AX protein expressions were determined by Immunofluorescence microscopy. RESULTS: Haploinsufficient ATM resulted in increased proliferation (p < 0.01) and cell survival following PARP inhibitor (olaparib) treatment. However, complete ATM knockout decreased proliferation (p < 0.01) and promoted cell susceptibility to olaparib (p < 0.01). Complete loss of ATM suppressed the expression of DNA repair-associated molecules FANCD2 and RAD51 and induced DNA damage in neuroblastoma cells. A marked downregulation of FANCD2 expression was also observed in shRNA-mediated ATM-knockdown neuroblastoma cells. Inhibitor experiments demonstrated that the degradation of FANCD2 was regulated at the protein level through the ubiquitin-proteasome pathway. Reintroduction of FANCD2 expression is sufficient to reverse decreased proliferation mediated by ATM depletion. CONCLUSIONS: Our study revealed the molecular mechanism underlying ATM heterozygosity in neuroblastomas and elucidated that ATM inactivation enhances the susceptibility of neuroblastoma cells to olaparib treatment. These findings might be useful in the treatment of high-risk NB patients showing ATM zygosity and aggressive cancer progression in future.

Comparative epigenomics by machine learning approach for neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the second most common pediatric solid tumor. Because the number of genetic mutations found in tumors are small, even in some patients with unfavorable NB, epigenetic variation is expected to play an important role in NB progression. DNA methylation is a major epigenetic mechanism, and its relationship with NB prognosis has been a concern. One limitation with the analysis of variation in DNA methylation is the lack of a suitable analytical model. Therefore, in this study, we performed a random forest (RF) analysis of the DNA methylome data of NB from multiple databases. RESULTS: RF is a popular machine learning model owing to its simplicity, intuitiveness, and computational cost. RF analysis identified novel intermediate-risk patient groups with characteristic DNA methylation patterns within the low-risk group. Feature selection analysis based on probe annotation revealed that enhancer-annotated regions had strong predictive power, particularly for MYCN-amplified NBs. We developed a gene-based analytical model to identify candidate genes related to disease progression, such as PRDM8 and FAM13A-AS1. RF analysis revealed sufficient predictive power compared to other machine learning models. CONCLUSIONS: RF is a useful tool for DNA methylome analysis in cancer epigenetic studies, and has potential to identify a novel cancer-related genes.

FGFR2 loss sensitizes MYCN-amplified neuroblastoma CHP134 cells to CHK1 inhibitor-induced apoptosis.

Checkpoint kinase 1 (CHK1) plays a key role in genome surveillance and integrity throughout the cell cycle. Selective inhibitors of CHK1 (CHK1i) are undergoing clinical evaluation for various human malignancies, including neuroblastoma. In this study, one CHK1i-sensitive neuroblastoma cell line, CHP134, was investigated, which characteristically carries MYCN amplification and a chromosome deletion within the 10q region. Among several cancer-related genes in the chromosome 10q region, mRNA expression of fibroblast growth factor receptor 2 (FGFR2) was altered in CHP134 cells and associated with an unfavorable prognosis of patients with neuroblastoma. Induced expression of FGFR2 in CHP134 cells reactivated downstream MEK/ERK signaling and resulted in cells resistant to CHK1i-mediated cell growth inhibition. Consistently, the MEK1/2 inhibitor, trametinib, potentiated CHK1 inhibitor-mediated cell death in these cells. These results suggested that FGFR2 loss might be prone to highly effective CHK1i treatment. In conclusion, extreme cellular dependency of ERK activation may imply a possible application for the MEK1/2 inhibitor, either as a single inhibitor or in combination with CHK1i in MYCN-amplified neuroblastomas.

Polycomb EZH1 regulates cell cycle/5-fluorouracil sensitivity of neuroblastoma cells in concert with MYCN.

In the present study, we found that EZH1 depletion in MYCN-amplified neuroblastoma cells resulted in significant cell death as well as xenograft inhibition. EZH1 depletion decreased the level of H3K27me1; the interaction and protein stabilization of MYCN and EZH1 appear to play roles in epigenetic transcriptional regulation. Transcriptome analysis of EZH1-depleted cells resulted in downregulation of the cell cycle progression-related pathway. In particular, Gene Set Enrichment Analysis revealed downregulation of reactome E2F-mediated regulation of DNA replication along with key genes of this process, TYMS, POLA2, and CCNA1. TYMS and POLA2 were transcriptionally activated by MYCN and EZH1-related epigenetic modification. Treatment with the EZH1/2 inhibitor UNC1999 also induced cell death, decreased H3K27 methylation, and reduced the levels of TYMS in neuroblastoma cells. Previous reports indicated neuroblastoma cells are resistant to 5-fluorouracil (5-FU) and TYMS (encoding thymidylate synthetase) has been considered the primary site of action for folate analogues. Intriguingly, UNC1999 treatment significantly sensitized MYCN-amplified neuroblastoma cells to 5-FU treatment, suggesting that EZH inhibition could be an effective strategy for development of a new epigenetic treatment for neuroblastoma.

Infantile Stage M Neuroblastoma With 11q Deletion, Mimicking Stage MS.

A 4-month-old boy with abdominal distension was diagnosed with adrenal neuroblastoma with numerous metastases to the liver and nodules in the skin and muscles. Marked hepatomegaly spontaneously regressed with decreasing tumor marker levels, and the final diagnosis was stage M based on radiologic findings confirming metastasis to the pancreas. The neuroblastoma did not have the MYCN amplification but had an 11q aberration. Chemotherapy was initiated at age 6 months with a successful response. Our case reflects the heterogenous clinical behavior of neuroblastoma and highlights the challenging issue of the difference between stage M and stage MS neuroblastoma in infants.

Correction to: Results of a phase II trial for high-risk neuroblastoma treatment protocol JN-H-07: a report from the Japan Childhood Cancer Group Neuroblastoma Committee (JNBSG).

In the October 2018 issue of International Journal of Clinical Oncology.

TAp63 represses transcription of MYCN/NCYM gene and its high levels of expression are associated with favorable outcome in neuroblastoma.

TAp63 is an isoform of p63 gene, a p53 family gene that suppresses tumorigenesis via transcriptional regulation. TAp63 represses transcription of MYC oncogene in glioblastomas; however, its role in another MYC family gene, MYCN, has remained elusive. In this study, we showed that TAp63 repressed transcription of the MYCN gene in human cancer cells. Overexpression of TAp63 in HeLa cells suppressed MYCN expression, whereas knockdown of TAp63 had the opposite effect. By binding to exon 1 of MYCN gene, TAp63 suppressed the promoter activities of MYCN and its cis-antisense gene, NCYM. Other p53 family members, p53 and TAp73, showed lesser ability to suppress MYCN/NCYM promoter activities compared with that of TAp63. All-trans-retinoic acid (ATRA) treatment of MYCN/NCYM-amplified neuroblastoma CHP134 cells induced TAp63 and reduced p53 expressions, accompanied by downregulation of MYCN/NCYM expressions. Meanwhile, TAp63 knockdown inhibited ATRA-induced repression of NCYM gene expression. Blocking the p53 family binding sites by CRISPR-dCas9 system in CHP134 cells induced MYCN/NCYM expression and promoted apoptotic cell death. Expression levels of TAp63 mRNA inversely correlated with those of MYCN/NCYM expression in primary neuroblastomas, which was associated with a favorable prognosis. Collectively, TAp63 repressed MYCN/NCYM bidirectional transcription, contributing to the suppression of neuroblastoma growth.

PPP3CB contributes to poor prognosis through activating nuclear factor of activated T-cells signaling in neuroblastoma.

We previously identified a gain-of-function mutation in PPP3CB in a neuroblastoma (NB) with MYCN amplification. Here we investigated the functional and clinical role of PPP3CB in NB. High PPP3CB expression was an independent indicator predicting poor prognosis of NB. Overexpression of wildtype or mutated PPP3CB (PPP3CBmut) promoted cell growth, but PPP3CB knockdown decreased cell growth in NB cells. Forced expressions of PPP3CB and PPP3CBmut activated NFAT2 and NFAT4 transcription factors and inhibited GSK3ß activity, resulting in the increase in the expressions of c-Myc, MYCN, and ß-catenin, which were downregulated in response to PPP3CB knockdown. Treatment with calcineurin inhibitor cyclosporin A (CsA) or FK506 suppressed cell proliferation and induced apoptotic cell death in both MYCN-amplified and MYCN-non-amplified NB cell lines. Expression of PPP3CB protein was decreased in response to two calcineurin inhibitors. c-Myc, MYCN, and ß-catenin were downregulated at the mRNA and protein levels in CsA or FK506-treated NB cells. Our data indicate that elevated expression of PPP3CB and the expression of its constitutively active mutant contribute to the aggressive behavior of NB tumors and therefore suggest that inhibition of calcineurin activity might have therapeutic potential for high-risk NB.

Results of a phase II trial for high-risk neuroblastoma treatment protocol JN-H-07: a report from the Japan Childhood Cancer Group Neuroblastoma Committee (JNBSG).

BACKGROUND: The Japanese Children's Cancer Group (JCCG) Neuroblastoma Committee (JNBSG) conducted a phase II clinical trial for high-risk neuroblastoma treatment. We report the result of the protocol treatment and associated genomic aberration studies. METHODS: JN-H-07 was a single-arm, late phase II trial for high-risk neuroblastoma treatment with open enrollment from June 2007 to February 2009. Eligible patients underwent five courses of induction chemotherapy followed by high-dose chemotherapy with hematopoietic stem cell rescue. Surgery for the primary tumor was scheduled after three or four courses of induction chemotherapy. Radiotherapy was administered to the primary tumor site and to any bone metastases present at the end of induction chemotherapy. RESULTS: The estimated 3-year progression-free and overall survival rates of the 50 patients enrolled were 36.5 ± 7.0 and 69.5 ± 6.6%, respectively. High-dose chemotherapy caused severe toxicity including three treatment-related deaths. In response to this, the high-dose chemotherapy regimen was modified during the trial by infusing melphalan before administering carboplatin and etoposide. The modified high-dose chemotherapy regimen was less toxic. Univariate analysis revealed that patients younger than 547 days and patients whose tumor showed a whole chromosomal gains / losses pattern had a significantly poor prognosis. Notably, the progression-free survival of cases with MYCN amplification were not inferior to those without MYCN amplification. CONCLUSIONS: The outcome of patients treated with the JN-H-07 protocol showed improvement over the results reported by previous studies conducted in Japan. Molecular and genetic profiling may enable a more precise stratification of the high-risk cohort.

Recent trends in microRNA research into breast cancer with particular focus on the associations between microRNAs and intrinsic subtypes.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate the function of target genes at the post-transcriptional phase. miRNAs are considered to have roles in the development, progression and metastasis of cancer. Recent studies have indicated that particular miRNA signatures are correlated with tumor aggressiveness, response to drug therapy and patient outcome in breast cancer. On the other hand, in routine clinical practice, the treatment regimens for breast cancer are determined based on the intrinsic subtype of the primary tumor. Previous studies have shown that miRNA expression profiles of each intrinsic subtypes of breast cancer differ. In hormone receptor-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer, miRNA expressions are found to be correlated with endocrine therapy resistance, progesterone receptor expression and heat shock protein activity. Some miRNAs are associated with resistance to HER2-targeted therapy and HER3 expression in HER2-positive breast cancer. In triple-negative breast cancer, miRNA expressions are found to be associated with BRCA mutations, immune system, epithelial-mesenchymal transition, cancer stem cell properties and androgen receptor expression. As it has been clarified that the expression levels and functions of miRNA differ among the various subtypes of breast cancer, and it is necessary to take account of the characteristics of each breast cancer subtype during research into the roles of miRNA in breast cancer. In addition, the discovery of the roles played by miRNAs in breast cancer might provide new opportunities for the development of novel strategies for diagnosing and treating breast cancer.

Identification of novel mutations in Japanese ovarian clear cell carcinoma patients using optimized targeted NGS for clinical diagnosis.

OBJECTIVE: Ovarian clear cell carcinoma (OCCC) is an aggressive ovarian cancer with a higher frequency in Japan and often becomes chemorefractory disease. Reliable genetic diagnosis is essential to affirm the success of precision medicine for OCCC treatment. The aim of this study is, therefore, to identify novel mutations in OCCCs and develop a feasible clinical next generation sequencing (NGS) approach using formalin-fixed paraffin-embedded (FFPE) rather than preferable but not always available fresh frozen (FF) samples. METHODS: We optimized and evaluated exome analyses of 409 cancer-related genes using FFPE and FF DNA and analyzed NGS data to identify somatic mutations in Japanese OCCCs. RESULTS: Sufficient and good quality DNAs from FFPE samples were extracted from 18 (FIGO Stage I: 12) out of 29 pairs of matched normal and OCCC for NGS (63%). The fine quality of extracted DNAs depended on the length of storage period (<2years storage). We also identified 45 somatic mutations in 34 genes including unreported variants from those FFPE DNA, in which somatic mutations in the PIK3CA gene was the most common (28%) as previously reported. Seven genes (PIK3CA, ARID1A, CTNNB1, CSMD3, LPHN3, LRP1B, and TP53) were mutated in at least two independent OCCCs. FF samples from 3 out of those 18 OCCCs were available and 13 out of 14 FFPE somatic mutations were confirmed. CONCLUSIONS: We successfully identified novel genetic alterations in Japanese OCCCs and demonstrated a feasible clinical diagnostic procedure using targeted NGS for OCCC FFPE samples.

NCYM, a Cis-antisense gene of MYCN, encodes a de novo evolved protein that inhibits GSK3ß resulting in the stabilization of MYCN in human neuroblastomas.

The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3ß, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3ß, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3ß inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3ß activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.

Transcriptional regulation of BMCC1 mediated by E2F1 in neuroblastoma cells.

BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1)/PRUNE2 is highly expressed in patients with favorable neuroblastoma (NB), encoding a multifunctional scaffold protein that modulates several signaling networks including RhoA and AKT pathways. Accumulating evidence suggests that BMCC1 acts as a tumor-suppressor. In this study, we addressed molecular mechanism underlying transcriptional regulation of BMCC1 in NBs. We found that transcription factor E2F1 was recruited to E2F-binding site in the promoter region of BMCC1 gene. Indeed, overexpression of E2F1 resulted in an increase in the expression level of BMCC1 in NB cell lines. On the other hand, knockdown of E2F1 in NB cells yielded down-regulation of BMCC1. Also, we showed that BMCC1 and E2F1 were simultaneously induced at G1 to S phase transition. Therefore, we conclude that E2F1 directly facilitated BMCC1 transcription. Taking together, these results suggest that BMCC1 induced by E2F1 acts as a tumor suppressor through its pro-apoptotic function, resulted in favorable prognosis of NB.

Correlation between the International Neuroblastoma Pathology Classification and genomic signature in neuroblastoma.

The International Neuroblastoma Pathology Classification (INPC) has a prognostic impact that distinguishes two categories of neuroblastoma: favorable histology (FH) and unfavorable histology (UH). We analyzed 92 cases of neuroblastoma with the INPC evaluation and genomic grouping to investigate the correlation between the INPC and genomic signature, together with their prognostic significance. The correlation of UH tumor and partial gains and/or losses (GGP), as well as the correlation of FH tumor and whole gains and/or losses (GGW), was statistically significant. Both UH and GGP were late-onset (median age at diagnosis was 36 and 48 months, respectively) and had poor prognosis (overall survival rate [OS], 43.1% and 42.4%, respectively). In contrast, both FH and GGW were early-onset (median age at diagnosis, 4 and 9.5 months, respectively) and had favorable prognosis (OS, 88.6% and 87.1%, respectively). Unfavorable histology and GGP had significantly inferior OS compared to FH and GGW. Overall survival was not significantly different among the genomic groups in FH; however, it was inferior in UH with GGP. In UH with a single copy MYCN, genomic subgroups GGP2s (both 1p and 11q losses) and GGP3s (partial 11q loss but not 1p loss) indicated significantly poor prognosis compared to GGP4s (no partial 1p and 11q loss). As INPC and MYCN amplification were found to be the most powerful prognostic biological factors, they should be included with genomic grouping as treatment stratification for patients with UH and single copy of MYCN.

Functional interplay between MYCN, NCYM, and OCT4 promotes aggressiveness of human neuroblastomas.

Neuroblastoma is a pediatric solid tumor that originates from embryonic neural crest cells. The MYCN gene locus is frequently amplified in unfavorable neuroblastomas, and the gene product promotes the progression of neuroblastomas. However, the molecular mechanisms by which MYCN amplification contributes to stem cell-like states of neuroblastoma remain elusive. In this study, we show that MYCN and its cis-antisense gene, NCYM, form a positive feedback loop with OCT4, a core regulatory gene maintaining a multipotent state of neural stem cells. We previously reported that NCYM is co-amplified with the MYCN gene in primary human neuroblastomas and that the gene product promotes aggressiveness of neuroblastoma by stabilization of MYCN. In 36 MYCN-amplified primary human neuroblastomas, OCT4 mRNA expression was associated with unfavorable prognosis and was correlated with that of NCYM. The OCT4 protein induced both NCYM and MYCN in human neuroblastoma cells, whereas NCYM stabilized MYCN to induce OCT4 and stem cell-related genes, including NANOG, SOX2, and LIN28. In sharp contrast to MYCN, enforced expression of c-MYC did not enhance OCT4 expression in human neuroblastoma cells. All-trans retinoic acid treatment reduced MYCN, NCYM, and OCT4 expression, accompanied by the decreased amount of OCT4 recruited onto the intron 1 region of MYCN. Knockdown of NCYM or OCT4 inhibited formation of spheres of neuroblastoma cells and promoted asymmetric cell division in MYCN-amplified human neuroblastoma cells. These results suggest that the functional interplay between MYCN, NCYM, and OCT4 contributes to aggressiveness of MYCN-amplified human neuroblastomas.

Sendai virus-mediated expression of reprogramming factors promotes plasticity of human neuroblastoma cells.

Neuroblastoma (NB) is the most common extracranial solid tumor that originates from multipotent neural crest cells. NB cell populations that express embryonic stem cell-associated genes have been identified and shown to retain a multipotent phenotype. However, whether somatic reprogramming of NB cells can produce similar stem-cell like populations is unknown. Here, we sought to reprogram NB cell lines using an integration-free Sendai virus vector system. Of four NB cell lines examined, only SH-IN cells formed induced pluripotent stem cell-like colonies (SH-IN 4F colonies) at approximately 6 weeks following transduction. These SH-IN 4F colonies were alkaline phosphatase-positive. Array comparative genomic hybridization analysis indicated identical genomic aberrations in the SH-IN 4F cells as in the parental cells. SH-IN 4F cells had the ability to differentiate into the three embryonic germ layers in vitro, but rather formed NBs in vivo. Furthermore, SH-IN 4F cells exhibited resistance to cisplatin treatment and differentiated into endothelial-like cells expressing CD31 in the presence of vascular endothelial growth factor. These results suggest that SH-IN 4F cells are partially reprogrammed NB cells, and could be a suitable model for investigating the plasticity of aggressive tumors.

Identification of a novel E-box binding pyrrole-imidazole polyamide inhibiting MYC-driven cell proliferation.

The MYC transcription factor plays a crucial role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Due to its oncogenic activities and overexpression in a majority of human cancers, it is an interesting target for novel drug therapies. MYC binding to the E-box (5'-CACGTGT-3') sequence at gene promoters contributes to more than 4000 MYC-dependent transcripts. Owing to its importance in MYC regulation, we designed a novel sequence-specific DNA-binding pyrrole-imidazole (PI) polyamide, Myc-5, that recognizes the E-box consensus sequence. Bioinformatics analysis revealed that the Myc-5 binding sequence appeared in 5'- MYC binding E-box sequences at the eIF4G1, CCND1, and CDK4 gene promoters. Furthermore, ChIP coupled with detection by quantitative PCR indicated that Myc-5 has the ability to inhibit MYC binding at the target gene promoters and thus cause downregulation at the mRNA level and protein expression of its target genes in human Burkitt's lymphoma model cell line, P493.6, carrying an inducible MYC repression system and the K562 (human chronic myelogenous leukemia) cell line. Single i.v. injection of Myc-5 at 7.5 mg/kg dose caused significant tumor growth inhibition in a MYC-dependent tumor xenograft model without evidence of toxicity. We report here a compelling rationale for the identification of a PI polyamide that inhibits a part of E-box-mediated MYC downstream gene expression and is a model for showing that phenotype-associated MYC downstream gene targets consequently inhibit MYC-dependent tumor growth.