Histone-lysine methyltransferase EHMT2 is involved in proliferation, apoptosis, cell invasion, and DNA methylation of human neuroblastoma cells.

Neuroblastoma (NB), a childhood neoplasm arising from neural crest cells, is characterized by a diversity of clinical behaviors ranging from spontaneous remission to rapid tumor progression and death. In addition to genetic abnormalities, recent studies have indicated that epigenetic aberrations also contribute toward NB pathogenesis. However, the epigenetic mechanisms underlying the pathogenesis of NB are largely unknown. Inhibition of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) was evaluated through the measurement of H3K9Me2 levels. Cell proliferation was examined by cell counting in human NB cell lines (LA1-55n, IMR-5, and NMB). The RNA expression of EHMT2, MYCN, and p21 was measured by real-time PCR. The expression of PCNA, MYCN, p53, cyclinD1, H3, H3K27M2, and H3K9Me2 was examined by western blot analysis. In-vitro invasion and the effects of the EHMT2 inhibitor (BIX-01294) were assessed in the Transwell chamber assay. Caspase 3 and 8 activities were measured using a Caspase-Glo assay kit. The level of overall DNA methylation was measured by liquid chromatography-mass spectroscopy. BIX-01294, a specific inhibitor of EHMT2 (a key enzyme for histone H3 dimethylation at lysine-9), specifically decreases the overall H3K9Me2 level but not H3K27Me2. The inhibition of EHMT2 decreased the proliferation of NB cells and induced apoptosis by increasing caspase 8/caspase 3 activity. BIX-01294 inhibited NB cell mobility and invasion. This was accompanied by a decreased expression of the MYCN oncogene. Inhibition of EHMT2 enhanced a doxorubicin-induced inhibitory effect on cell proliferation. Finally, EHMT2 inhibition modulated overall DNA methylation levels in NB cells. Our results show that histone-lysine methylation is involved in cell proliferation, apoptosis, cell invasion, and overall DNA methylation in human NB cells. Further understanding of this mechanism may provide an insight into the pathogenesis of NB progression and lead to novel treatment strategies.

Valproic acid shows a potent antitumor effect with alteration of DNA methylation in neuroblastoma.

Epigenetic aberrations and a CpG island methylator phenotype are associated with poor outcome in children with neuroblastoma (NB). Previously, we have shown that valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, exerts antitumor effects in an NB xenograft model. However, the underlying antitumor molecular mechanisms are largely unknown. In this study, we examined the role of HDAC in cell proliferation, cell cycle progression, gene expression patterns, and epigenome in NB. Cell proliferation, cell cycle progression, caspase activity, RNA and protein expression, quantitative methylation, and global DNA methylation were examined in NBL-W-N and LA1-55n NB cell lines. Our studies showed that inhibition of HDAC decreased NB proliferation, and induced caspase activity and G1 growth arrest. Expression patterns of cancer-related genes were modulated by VPA. The expression of THBS1, CASP8, SPARC, CDKN1A, HIC1, CDKN1B, and HIN1 was upregulated, and that of MYCN and TIG1 was downregulated. HDAC inhibition decreased methylation levels of THBS1 and RASSF1A promoters. Inhibition of HDAC increased acetylation of histone 4 and overall DNA methylation levels. Our studies showed that inhibition of HDAC blocked cell proliferation and cell cycle progression in relation to alteration in cancer-related genes, increased overall DNA methylation, and decreased methylation of tumor suppressor genes. Further studies examining the antitumor effects of VPA in NB are warranted.

Sorafenib inhibits neuroblastoma cell proliferation and signaling, blocks angiogenesis, and impairs tumor growth.

BACKGROUND: More effective therapy for children with high-risk neuroblastoma is desperately needed. Preclinical studies have shown that neuroblastoma tumor growth can be inhibited by agents that block angiogenesis. We hypothesized that drugs which target both neuroblastoma cells and tumor angiogenesis would have potent anti-tumor activity. In this study we tested the effects of sorafenib, a multi-kinase inhibitor, on neuroblastoma cell proliferation and signaling, and in mice with subcutaneous human neuroblastoma xenografts or orthotopic adrenal tumors. PROCEDURE: Mice with subcutaneous neuroblastoma xenografts or orthotopic adrenal tumors were treated with sorafenib, and tumor growth rates were measured. Blood vessel architecture and vascular density were evaluated histologically in treated and control neuroblastoma tumors. The in vitro effects of sorafenib on neuroblastoma proliferation, cell cycle, and signaling were also evaluated. RESULTS: Sorafenib inhibited tumor growth in mice with subcutaneous and orthotopic adrenal tumors. Decreased numbers of cycling neuroblastoma cells and tumor blood vessels were seen in treated versus control tumors, and the blood vessels in the treated tumors had more normal architecture. Sorafenib treatment also decreased neuroblastoma cell proliferation, attenuated ERK signaling, and enhanced G(1) /G(0) cell cycle arrest in vitro. CONCLUSIONS: Our results demonstrate that sorafenib inhibits the growth of neuroblastoma tumors by targeting both neuroblastoma cells and tumor blood vessels. Single agent sorafenib should be evaluated in future phase II neuroblastoma studies.

MYCN and HIF-1 directly regulate TET1 expression to control 5-hmC gains and enhance neuroblastoma cell migration in hypoxia.

Ten-Eleven-Translocation 5-methylcytosine dioxygenases 1-3 (TET1-3) convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC), using oxygen as a co-substrate. Contrary to expectations, hypoxia induces 5-hmC gains in MYCN-amplified neuroblastoma (NB) cells via upregulation of TET1. Here, we show that MYCN directly controls TET1 expression in normoxia, and in hypoxia, HIF-1 augments TET1 expression and TET1 protein stability. Through gene-editing, we identify two MYCN and HIF-1 binding sites within TET1 that regulate gene expression. Bioinformatic analyses of 5-hmC distribution and RNA-sequencing data from hypoxic cells implicate hypoxia-regulated genes important for cell migration, including CXCR4. We show that hypoxic cells lacking the two MYCN/HIF-1 binding sites within TET1 migrate slower than controls. Treatment of MYCN-amplified NB cells with a CXCR4 antagonist results in slower migration under hypoxic conditions, suggesting that inclusion of a CXCR4 antagonist into NB treatment regimens could be beneficial for children with MYCN-amplified NBs.

In MYCN-amplified neuroblastoma cell lines, MYCN directly controls TET1 expression in normoxia.In MYCN-amplified neuroblastoma cell lines exposed to hypoxia, HIF-1 augments TET1 expression and TET1 protein stability.Hypoxic MYCN-amplified neuroblastoma cell lines have increased cell migration, mediated by genes including CXCR4 that gain 5-hydroxymethylcytosine density.Treatment of MYCN-amplified NB cells with a CXCR4 antagonist slows hypoxia-associated migration, suggesting a CXCR4 antagonist could be beneficial in treatment regimens for children with MYCN-amplified neuroblastomas.

The quinoxaline anti-tumor agent (R+)XK469 inhibits neuroblastoma tumor growth.

The quinoxaline anti-tumor agent (R+)XK469 mediates its effects by topoisomerase IIB inhibition. This report describes a 14-year old with relapsed neuroblastoma who experienced disease stabilization for 14 months while receiving (R+)XK469 monotherapy. Due to this favorable response, laboratory studies were undertaken to determine efficacy in the preclinical setting. (R+)XK469 inhibited proliferation, caused G(2) cell cycle arrest of neuroblastoma cells in vitro, and inhibited growth of neuroblastoma xenograft tumors. These preclinical results, coupled with the favorable clinical response, demonstrate that (R+)XK469 and similar anti-tumor agents may be effective in the treatment of high-risk neuroblastoma and warrant further testing.

Anti-angiogenic SPARC peptides inhibit progression of neuroblastoma tumors.

BACKGROUND: New, more effective strategies are needed to treat highly aggressive neuroblastoma. Our laboratory has previously shown that full-length Secreted Protein Acidic and Rich in Cysteine (SPARC) and a SPARC peptide corresponding to the follistatin domain of the protein (FS-E) potently block angiogenesis and inhibit the growth of neuroblastoma tumors in preclinical models. Peptide FS-E is structurally complex and difficult to produce, limiting its potential as a therapeutic in the clinic. RESULTS: In this study, we synthesized two smaller and structurally more simple SPARC peptides, FSEN and FSEC, that respectively correspond to the N-and C-terminal loops of peptide FS-E. We show that both peptides FSEN and FSEC have anti-angiogenic activity in vitro and in vivo, although FSEC is more potent. Peptide FSEC also significantly inhibited the growth of neuroblastoma xenografts. Histologic examination demonstrated characteristic features of tumor angiogenesis with structurally abnormal, tortuous blood vessels in control neuroblastoma xenografts. In contrast, the blood vessels observed in tumors, treated with SPARC peptides, were thin walled and structurally more normal. Using a novel method to quantitatively assess blood vessel abnormality we demonstrated that both SPARC peptides induced changes in blood vessel architecture that are consistent with blood vessel normalization. CONCLUSION: Our results demonstrate that SPARC peptide FSEC has potent anti-angiogenic and anti-tumorigenic effects in neuroblastoma. Its simple structure and ease of production indicate that it may have clinical utility in the treatment of high-risk neuroblastoma and other types of pediatric and adult cancers, which depend on angiogenesis.

Epigenetic alterations differ in phenotypically distinct human neuroblastoma cell lines.

BACKGROUND: Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Seven cancer related genes (THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC, and HIC-1) that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype. METHODS: Two NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s) that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around THBS-1, HIN-1, TIG-1 and CASP8 promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the THBS-1 promoter. Luciferase assay was used to determine THBS-1 promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity. RESULTS: Promoter methylation values for THBS-1, HIN-1, TIG-1, and CASP8 were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3) were identified in the THBS-1 promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3) were present in the THBS-1 promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for THBS-1 expression. We also show that 5-Aza-dC treatment of LA1-55n cells alters the DNA methylation status and the histone code in the THBS-1 promoter modifies cell morphology, and inhibits their ability to form colonies in soft agar. CONCLUSION: Our results suggest that epigenetic aberrations contribute to NB phenotype, and that tumorigenic properties can be inhibited by reversing the epigenetic changes with 5-Aza-dC.

5-Hydroxymethylcytosine Profiles in Circulating Cell-Free DNA Associate with Disease Burden in Children with Neuroblastoma.

PURPOSE: 5-Hydroxymethylcytosine (5-hmC) is an epigenetic marker of open chromatin and active gene expression. We profiled 5-hmC with Nano-hmC-Seal technology using 10 ng of plasma-derived cell-free DNA (cfDNA) in blood samples from patients with neuroblastoma to determine its utility as a biomarker. EXPERIMENTAL DESIGN: For the Discovery cohort, 100 5-hmC profiles were generated from 34 well children and 32 patients (27 high-risk, 2 intermediate-risk, and 3 low-risk) at various time points during the course of their disease. An independent Validation cohort encompassed 5-hmC cfDNA profiles (n = 29) generated from 21 patients (20 high-risk and 1 intermediate-risk). Metastatic burden was classified as high, moderate, low, or none per Curie metaiodobenzylguanidine scores and percentage of tumor cells in bone marrow. Genes with differential 5-hmC levels between samples according to metastatic burden were identified using DESeq2. RESULTS: Hierarchical clustering using 5-hmC levels of 347 genes identified from the Discovery cohort defined four clusters of samples that were confirmed in the Validation cohort and corresponded to high, high-moderate, moderate, and low/no metastatic burden. Samples from patients with increased metastatic burden had increased 5-hmC deposition on genes in neuronal stem cell maintenance and epigenetic regulatory pathways. Further, 5-hmC cfDNA profiles generated with 1,242 neuronal pathway genes were associated with subsequent relapse in the cluster of patients with predominantly low or no metastatic burden (sensitivity 65%, specificity 75.6%). CONCLUSIONS: cfDNA 5-hmC profiles in children with neuroblastoma correlate with metastatic burden and warrants development as a biomarker of treatment response and outcome.

Presence of cancer-associated fibroblasts inversely correlates with Schwannian stroma in neuroblastoma tumors.

Stromal cells have a central function in the regulation of tumor angiogenesis. Recent studies have shown that stromal myofibroblasts (cancer-associated fibroblasts) actively promote tumor growth and enhance tumor angiogenesis in many types of adult carcinomas. To evaluate the function cancer-associated fibroblasts have in neuroblastoma angiogenesis and investigate their relationship to stromal Schwann cells, we quantified cancer-associated fibroblasts in 60 primary neuroblastoma tumors and in a novel neuroblastoma xenograft model in which murine Schwann cells were induced to infiltrate into the tumor stroma. Tumor sections were examined for presence of microvascular proliferation, a hallmark of tumor angiogenesis. Cancer-associated fibroblasts were characterized by positive immunostaining for alpha-smooth muscle actin (alpha-SMA) and were distinguished from pericytes by staining negatively for high-molecular-weight caldesmon. alpha-SMA-positive cells were quantified and their number was defined as high when >1.0% of the area was positive. Associations between high cancer-associated fibroblast number, microvascular proliferation and established prognosticators were analyzed. High numbers of cancer-associated fibroblasts were associated with Schwannian stroma-poor histopathology and microvascular proliferation. Thirty-seven (80%) of the 46 Schwannian stroma-poor tumors had high numbers of cancer-associated fibroblasts in the tumor stroma compared to only 2 (14%) of the 14 Schwannian stroma-rich/dominant tumors (P<0.001). Thirty-three (89%) of 37 tumors with microvascular proliferation had high numbers of cancer-associated fibroblasts compared to 9 (40%) of 22 tumors without microvascular proliferation (P<0.001). In the xenografts with infiltrating Schwann cells (n=10), the number of cancer-associated fibroblasts per mm(2) was approximately sevenfold less than in the control xenografts without stromal Schwann cells (n=9) (mean of 51+/-30 vs 368+/-105, respectively; P<0.001). Thus, cancer-associated fibroblasts were inversely associated with presence of Schwann cells, suggesting that Schwann cells may prevent the activation of fibroblasts. A deeper understanding of the function cancer-associated fibroblasts have in neuroblastoma angiogenesis may guide future development of stroma-directed therapeutic strategies.

Thrombospondin-1 peptide ABT-510 combined with valproic acid is an effective antiangiogenesis strategy in neuroblastoma.

In the pediatric cancer neuroblastoma, clinically aggressive disease is associated with increased levels of angiogenesis stimulators and high vascular index. We and others have hypothesized that blocking angiogenesis may be effective treatment for this pediatric malignancy. However, little is known about the efficacy of antiangiogenic agents in pediatric malignancies. Recently, promising results have been reported in an adult phase I study of ABT-510, a peptide derivative of the natural angiogenic inhibitor thrombospondin-1. Histone deacetylase inhibitors, such as valproic acid (VPA), have also been shown to have antiangiogenic activity in several cancer models. In this study, we evaluated the effects of ABT-510 and VPA on neuroblastoma tumor growth and angiogenesis. Although only VPA was capable of blocking the proliferation of neuroblastoma cells and inducing neuroblastoma cell apoptosis in vitro, treatment with VPA or ABT-510 alone significantly suppressed the growth of neuroblastoma xenografts established from two different MYCN-amplified cell lines. Combination therapy more effectively inhibited the growth of small neuroblastoma xenografts than single-agent treatment, and in animals with large xenografts, total cessation of tumor growth was achieved with this treatment approach. The microvascular density was significantly reduced in the xenografts treated with combination therapy compared with controls or tumors treated with single agents. In addition, the number of structurally abnormal vessels was reduced, suggesting that these agents may "normalize" the tumor vasculature. Our results indicate that ABT-510 combined with VPA may be an effective antiangiogenic treatment strategy for children with high-risk neuroblastoma.

Maternal Embryonic Leucine Zipper Kinase (MELK), a Potential Therapeutic Target for Neuroblastoma.

Maternal embryonic leucine zipper kinase (MELK) activates pathways that mediate aggressive tumor growth and therapy resistance in many types of adult cancers. Pharmacologic and genomic inhibition of MELK impairs tumor growth and increases sensitivity to radiation and chemotherapy. On the basis of these promising preclinical studies, early-phase adult clinical trials testing the MELK inhibitor OTS167 are ongoing. To investigate whether MELK is also a therapeutic target in neuroblastoma, we analyzed MELK expression in primary tumors and cell lines, and examined the effects of OTS167 on neuroblastoma growth. In primary tumors, high levels of MELK were associated with advanced stage disease and inferior survival. Higher levels of MELK were also detected in tumorigenic versus nontumorigenic neuroblastoma cell lines, and cells with higher levels of MELK expression were more sensitive to OTS167 than low-MELK expressing cells. OTS167 suppressed the growth of neuroblastoma xenografts, and in a preclinical model of minimal residual disease, survival was prolonged with MELK inhibition. OTS167 treatment downregulated MELK and its target enhancer of zeste homolog 2 (EZH2), a component of the polycomb repressive complex 2 (PRC2) that is known to modulate the DNA damage response. We also show that OTS167 reduced the formation of collapsed replication forks induced by camptothecin or radiation. Taken together, our results indicate that MELK indirectly mediates efficient processing of replication-associated DNA lesions in neuroblastoma, and that OTS167 sensitizes cells to DNA-damaging agents by abrogating this process. Further studies evaluating the activity of combination treatment regimens with OTS167 in neuroblastoma are warranted.

5-Hydroxymethylcytosine Profiles Are Prognostic of Outcome in Neuroblastoma and Reveal Transcriptional Networks That Correlate With Tumor Phenotype.

PURPOSE: Whole-genome profiles of the epigenetic modification 5-hydroxymethylcytosine (5-hmC) are robust diagnostic biomarkers in adult patients with cancer. We investigated if 5-hmC profiles would serve as novel prognostic markers in neuroblastoma, a clinically heterogeneous pediatric cancer. Because this DNA modification facilitates active gene expression, we hypothesized that 5-hmC profiles would identify transcriptomic networks driving the clinical behavior of neuroblastoma. PATIENTS AND METHODS: Nano-hmC-Seal sequencing was performed on DNA from Discovery (n = 51), Validation (n = 38), and Children's Oncology Group (n = 20) cohorts of neuroblastoma tumors. RNA was isolated from 48 tumors for RNA sequencing. Genes with differential 5-hmC or expression between clusters were identified using DESeq2. A 5-hmC model predicting outcome in high-risk patients was established using linear discriminant analysis. RESULTS: Comparison of low- versus high-risk tumors in the Discovery cohort revealed 577 genes with differential 5-hmC. Hierarchical clustering of tumors from the Discovery and Validation cohorts using these genes identified two main clusters highly associated with established prognostic markers, clinical risk group, and outcome. Genes with increased 5-hmC and expression in the favorable cluster were enriched for pathways of neuronal differentiation and KRAS activation, whereas genes involved in inflammation and the PRC2 complex were identified in the unfavorable cluster. The linear discriminant analysis model trained on high-risk Discovery cohort tumors was prognostic of outcome when applied to high-risk tumors from the Validation and Children's Oncology Group cohorts (hazard ratio, 3.8). CONCLUSION: 5-hmC profiles may be optimal DNA-based biomarkers in neuroblastoma. Analysis of transcriptional networks regulated by these epigenomic modifications may lead to a deeper understanding of drivers of neuroblastoma phenotype.

Prominent microvascular proliferation in clinically aggressive neuroblastoma.

PURPOSE: Tumor vasculature is disorganized and glomeruloid microvascular proliferation (MVP) has been identified as a poor prognosticator in some adult cancers. To determine the clinical significance of MVP, including glomeruloid MVP in neuroblastoma, we initially examined vessel architecture in tumor sections from 51 children diagnosed at Children's Memorial Hospital (CMH) and subsequently evaluated 154 neuroblastoma tumors on a tissue microarray constructed at Children's Hospital of Philadelphia (CHOP). EXPERIMENTAL DESIGN: H&E sections were examined for the presence of structurally abnormal vessels and further characterized by immunostaining for CD31 and von Willebrand factor to highlight endothelial cells and alpha-smooth muscle actin for pericytes. Tumors with thickened walls containing a complete layer of hypertrophic endothelial cells plus additional layers of vascular mural cells were classified as MVP positive. Associations between MVP and established clinicopathologic features and outcome were assessed. RESULTS: In both series, MVP was significantly associated with Schwannian stroma-poor histology (CMH, P = 0.008; CHOP, P < 0.001) and decreased survival probability (CMH, P = 0.017; CHOP, P = 0.014). In the CHOP series, MVP was associated with high-risk group classification (P < 0.001), although this association was not seen in the smaller CMH cohort. CONCLUSIONS: The association between MVP and poor outcome provides further support for the concept that angiogenesis plays an important role in determining the biological behavior of neuroblastoma tumors. Our results also indicate that angiogenesis is regulated differently in Schwannian stroma-rich versus stroma-poor neuroblastoma tumors. Further studies investigating the activity of angiogenic inhibitors in children with clinically aggressive stroma-poor neuroblastoma are warranted.

Methylation of CASP8, DCR2, and HIN-1 in neuroblastoma is associated with poor outcome.

PURPOSE: Epigenetic aberrations have been shown to play an important role in the pathogenesis of most cancers. To investigate the clinical significance of epigenetic changes in neuroblastoma, we evaluated the relationship between clinicopathologic variables and the pattern of gene methylation in neuroblastoma cell lines and tumors. EXPERIMENTAL DESIGN: Methylation-specific PCR was used to evaluate the gene methylation status of 19 genes in 14 neuroblastoma cell lines and 8 genes in 70 primary neuroblastoma tumors. Associations between gene methylation, established prognostic factors, and outcome were evaluated. Log-rank tests were used to identify the number of methylated genes that was most predictive of overall survival. RESULTS: Epigenetic changes were detected in the neuroblastoma cell lines and primary tumors, although the pattern of methylation varied. Eight of the 19 genes analyzed were methylated in >70% of the cell lines. Epigenetic changes of four genes were detected in only small numbers of cell lines. None of the cell lines had methylation of the other seven genes analyzed. In primary neuroblastoma tumors, high-risk disease and poor outcome were associated with methylation of DCR2, CASP8, and HIN-1 individually. Although methylation of the other five individual genes was not predictive of poor outcome, a trend toward decreased survival was seen in patients with a methylation phenotype, defined as > or =4 methylated genes (P = 0.055). CONCLUSION: Our study indicates that clinically aggressive neuroblastoma tumors have aberrant methylation of multiple genes and provides a rationale for exploring treatment strategies that include demethylating agents.

Neuroblastoma angiogenesis is inhibited with a folded synthetic molecule corresponding to the epidermal growth factor-like module of the follistatin domain of SPARC.

Secreted protein acidic and rich in cysteine (SPARC) is a multifunctional matricellular glycoprotein. In vitro, SPARC inhibits the proliferation and migration of endothelial cells stimulated by growth factors and induces endothelial cell apoptosis. We previously showed that SPARC also inhibits angiogenesis in vivo and impairs the growth of the pediatric tumor neuroblastoma (NB). SPARC comprises three domains that are independently folded by a complex pattern of disulfide bonds and have a high degree of structural conservation. In this study, separate modules of the SPARC domains were synthesized as cysteine-linked peptides and tested for their ability to inhibit angiogenesis. Peptide FS-E, representing the epidermal growth factor (EGF)-like module of the follistatin (FS) domain, did not cause endothelial cell apoptosis but strongly inhibited basic fibroblast growth factor (bFGF)-induced endothelial cell migration with an ED(50) = 10 pmol/L. In vivo, peptide FS-E blocked bFGF-stimulated angiogenesis and neovascularization induced by NB cells. The EGF-like conformation was essential for peptide FS-E function because reduction of its two disulfide bonds completely abrogated peptide activity. Peptides FS-K and EC-N, corresponding to part of the Kazal module of the FS domain and the conserved alpha-helix in the extracellular calcium-binding domain, respectively, had minimal to no inhibitory activity. Our data show that the EGF-like module of the SPARC FS domain is angiosuppressive, and its structural conformation is critical for antiangiogenic activity.

Methylation-associated silencing of the thrombospondin-1 gene in human neuroblastoma.

Tumor angiogenesis, a major requirement for tumor outgrowth and metastasis, is regulated by pro- and antiangiogenic factors. Methylation-associated inactivation of the angiogenesis inhibitor thrombospondin-1 (TSP-1) has been observed recently in some adult tumors. To investigate the role of TSP-1 in pediatric cancer, we examined its pattern of expression and mechanisms of regulation in neuroblastoma (NB). TSP-1 was silenced in a subset of undifferentiated, advanced-stage tumors and NB cell lines. In contrast, most localized tumors expressed this angiogenesis inhibitor, and a significant correlation between morphological evidence of neuroblast differentiation and TSP-1 expression was observed. Luciferase assays demonstrated the presence of nuclear factors required for TSP-1 transcription in both TSP-1-positive and -negative cell lines, but no correlation between TSP-1 promoter activity and the level of TSP-1 mRNA expression was seen. Our studies indicate that the transcriptional silencing of TSP-1 was caused by methylation. TSP-1 promoter methylation was detected in all of the NB cell lines lacking TSP-1 mRNA and in 37% of the NB clinical tumors analyzed. Furthermore, treatment with the demethylating agent, 5-Aza-2'-deoxycytidine (5-Aza-dC), restored TSP-1 expression in NB cell lines. Disrupting methylation with 5-Aza-dC also led to significant inhibition of NB in vivo and re-expression of TSP-1 in a subset of NB xenografts. These results suggest that 5-Aza-dC inhibits NB growth by augmenting the expression of TSP-1 along with other genes that suppress tumor growth. Demethylating agents may prove to be effective candidates for the treatment of children with NB.

SPARC is a key Schwannian-derived inhibitor controlling neuroblastoma tumor angiogenesis.

Neuroblastoma (NB), a common pediatric neoplasm, consists of two main cell populations: neuroblastic/ganglionic cells and Schwann cells. NB tumors with abundant Schwannian stroma display a more benign clinical behavior than stroma-poor tumors. Recent studies suggest that Schwann cells influence NB tumor growth via secreted factors that induce differentiation, suppress proliferation, and inhibit angiogenesis. Two angiogenesis inhibitors, pigment epithelium-derived factor and tissue inhibitor of metalloproteinase-2, have been detected in Schwann cell secretions. Here, we isolated another Schwann cell-derived secreted inhibitor of angiogenesis, a 43-kDa protein identified as SPARC (secreted protein acidic and rich in cysteine), an extracellular matrix protein. We found SPARC to be critical for the antiangiogenic phenotype of cultured Schwann cells. We also show that purified SPARC potently inhibits angiogenesis and significantly impairs NB tumor growth in vivo. SPARC may be an effective candidate for the treatment of children with clinically aggressive, Schwannian stroma-poor NB tumors.

Methylation-associated silencing of the heat shock protein 47 gene in human neuroblastoma.

Hypermethylation of gene promoter CpG islands is a frequent mechanism for gene inactivation in a variety of human cancers, including neuroblastoma (NB). We demonstrated recently that treatment with the demethylating agent 5'-aza-2'-deoxycytidine (5-Aza-dC) significantly inhibited NB growth in vivo. In an effort to identify the genes and biological pathways that are responsible for the impaired NB tumor growth observed after treatment with 5-Aza-dC, we performed genome-wide gene expression analysis of control and treated NBL-W-S NB cells. We found >or=3-fold changes in expression of 44 genes that play roles in angiogenesis, apoptosis, cell adhesion, transcriptional regulation, and signal transduction. The gene encoding heat shock protein 47 (Hsp47), a collagen-specific molecular chaperon, was up-regulated >80-fold after 5-Aza-dC treatment. Expression studies confirmed that Hsp47 is silenced in a subset of NB cell lines and tumors. We also show that silencing of Hsp47 in NB cells is associated with aberrant methylation of promoter CpG islands and that Hsp47 expression can be restored after treatment with 5-Aza-dC. A strong correlation between Hsp47 and collagen type I and IV expression was seen in NB cells. Interestingly, tumorigenicity was inversely correlated with the level of collagen expression in NB cell lines, and higher levels of collagen were detected in mature NB tumors that are associated with favorable outcome compared with undifferentiated, advanced-stage NBs. Our studies support a role for Hsp47 in the regulation of collagen type I and IV production in NB cells and suggest that the level of collagen expression may influence NB tumor phenotype.

Secreted protein acidic and rich in cysteine (SPARC) induces lipotoxicity in neuroblastoma by regulating transport of albumin complexed with fatty acids.

SPARC is a matrix protein that mediates interactions between cells and the microenvironment. In cancer, SPARC may either promote or inhibit tumor growth depending upon the tumor type. In neuroblastoma, SPARC is expressed in the stromal Schwannian cells and functions as a tumor suppressor. Here, we developed a novel in vivo model of stroma-rich neuroblastoma using non-tumorigenic SHEP cells with modulated levels of SPARC, mixed with tumorigenic KCNR cells. Tumors with stroma-derived SPARC displayed suppressed growth, inhibited angiogenesis and increased lipid accumulation. Based on the described chaperone function of SPARC, we hypothesized that SPARC binds albumin complexed with fatty acids and transports them to tumors. We show that SPARC binds albumin with Kd=18.9±2.3 uM, and enhances endothelial cell internalization and transendothelial transport of albumin in vitro. We also demonstrate that lipids induce toxicity in neuroblastoma cells and show that lipotoxicity is increased when cells are cultured in hypoxic conditions. Studies investigating the therapeutic potential of SPARC are warranted.

Integrative genomics reveals hypoxia inducible genes that are associated with a poor prognosis in neuroblastoma patients.

Neuroblastoma is notable for its broad spectrum of clinical behavior ranging from spontaneous regression to rapidly progressive disease. Hypoxia is well known to confer a more aggressive phenotype in neuroblastoma. We analyzed transcriptome data from diagnostic neuroblastoma tumors and hypoxic neuroblastoma cell lines to identify genes whose expression levels correlate with poor patient outcome and are involved in the hypoxia response. By integrating a diverse set of transcriptome datasets, including those from neuroblastoma patients and neuroblastoma derived cell lines, we identified nine genes (SLCO4A1, ENO1, HK2, PGK1, MTFP1, HILPDA, VKORC1, TPI1, and HIST1H1C) that are up-regulated in hypoxia and whose expression levels are correlated with poor patient outcome in three independent neuroblastoma cohorts. Analysis of 5-hydroxymethylcytosine and ENCODE data indicate that at least five of these nine genes have an increase in 5-hydroxymethylcytosine and a more open chromatin structure in hypoxia versus normoxia and are putative targets of hypoxia inducible factor (HIF) as they contain HIF binding sites in their regulatory regions. Four of these genes are key components of the glycolytic pathway and another three are directly involved in cellular metabolism. We experimentally validated our computational findings demonstrating that seven of the nine genes are significantly up-regulated in response to hypoxia in the four neuroblastoma cell lines tested. This compact and robustly validated group of genes, is associated with the hypoxia response in aggressive neuroblastoma and may represent a novel target for biomarker and therapeutic development.