B A Spengler's research while affiliated with Fordham University and other places

Neuroblastoma is a childhood cancer originating from embryonic neural crest cells. Amplification of the proto‑oncogene N-myc, seen in ~30% of neuroblastoma tumors, is a marker for poor prognosis. Recently discovered small regulatory RNAs, microRNAs (miRNAs), are implicated in cancers, including neuroblastoma. miRNAs downregulate the expression of genes by binding to the 3'-untranslated regions (3'-UTRs), thereby inhibiting translation or inducing degradation of cognate mRNAs. Our study sought to identify miRNAs that regulate N-myc expression and thereby malignancy in neuroblastoma. miRNAs whose expression negatively correlates with N-myc expression were identified from a miRNA microarray of 4 N-myc-amplified neuroblastoma cell lines. Three of these miRNAs (miR-17, miR-20a and miR-18a) belong to the miR-17-92 cluster, previously shown to be upregulated by N-myc. qPCR validation of these miRNAs in a larger panel of cell lines revealed that levels of miR-17 were inversely proportional to N-myc mRNA amounts in the N-myc-amplified cell lines. Notably, miR-17 also downregulated N-myc protein synthesis in the N-myc-amplified cells, thereby generating a negative feedback regulatory loop between the proto-oncogene and this miRNA. Moreover, the neuronal-specific RNA-binding protein HuD (ELAVL4), which regulates the processing/stability of N-myc mRNA, competes with miR-17 for a binding site in the 3'-UTR of N-myc. Thus, N-myc levels appear to be modulated by the antagonistic interactions of both miR-17, as a negative regulator, and HuD, as a positive regulator, providing further evidence of the complex cellular control mechanisms of this oncogene in N-myc-amplified neuroblastoma cells.

Background Neuroblastoma (NB) is the most common extracranial solid tumor in children. NB tumors and derived cell lines are phenotypically heterogeneous. Cell lines are classified by phenotype, each having distinct differentiation and tumorigenic properties. The neuroblastic phenotype is tumorigenic, has neuronal features and includes stem cells (I-cells) and neuronal cells (N-cells). The non-neuronal phenotype (S-cell) comprises cells that are non-tumorigenic with features of glial/smooth muscle precursor cells. This study identified miRNAs associated with each distinct cell phenotypes and investigated their role in regulating associated differentiation and tumorigenic properties. Methods A miRNA microarray was performed on the three cell phenotypes and expression verified by qRT-PCR. miRNAs specific for certain cell phenotypes were modulated using miRNA inhibitors or stable transfection. Neuronal differentiation was induced by RA; non-neuronal differentiation by BrdU. Changes in tumorigenicity were assayed by soft agar colony forming ability. N-myc binding to miR-375 promoter was assayed by chromatin-immunoprecipitation. Results Unsupervised hierarchical clustering of miRNA microarray data segregated neuroblastic and non-neuronal cell lines and showed that specific miRNAs define each phenotype. qRT-PCR validation confirmed that increased levels of miR-21, miR-221 and miR-335 are associated with the non-neuronal phenotype, whereas increased levels of miR-124 and miR-375 are exclusive to neuroblastic cells. Downregulation of miR-335 in non-neuronal cells modulates expression levels of HAND1 and JAG1, known modulators of neuronal differentiation. Overexpression of miR-124 in stem cells induces terminal neuronal differentiation with reduced malignancy. Expression of miR-375 is exclusive for N-myc-expressing neuroblastic cells and is regulated by N-myc. Moreover, miR-375 downregulates expression of the neuronal-specific RNA binding protein HuD. Conclusions Thus, miRNAs define distinct NB cell phenotypes. Increased levels of miR-21, miR-221 and miR-335 characterize the non-neuronal, non-malignant phenotype and miR-335 maintains the non-neuronal features possibly by blocking neuronal differentiation. miR-124 induces terminal neuronal differentiation with reduction in malignancy. Data suggest N-myc inhibits neuronal differentiation of neuroblastic cells possibly by upregulating miR-375 which, in turn, suppresses HuD. As tumor differentiation state is highly predictive of patient survival, the involvement of these miRNAs with NB differentiation and tumorigenic state could be exploited in the development of novel therapeutic strategies for this enigmatic childhood cancer.

Cellular heterogeneity is a well-known feature of human neuroblastoma tumors and cell lines. Of the 3 phenotypes (N-, I-, and S-type) isolated and characterized, the I-type cancer stem cell of neuroblastoma is the most malignant. Here, we report that, although wild-type N-Ras protein is expressed at the same level in all 3 neuroblastoma cell phenotypes, activated N-Ras-GTP level is significantly higher in I-type cancer stem cells. When activated N-Ras levels were decreased by transfection of a dominant-negative N-Ras construct, the malignant potential of I-type cancer stem cells decreased significantly. Conversely, when weakly malignant N-type cells were transfected with a constitutively active N-Ras construct, activated N-Ras levels, and malignant potential, were significantly increased. Thus, high levels of N-Ras-GTP are required for the increased malignancy of I-type neuroblastoma cancer stem cells. Moreover, increased activation of N-Ras results from significant down-regulation of neurofibromin (NF1), an important RasGAP. This specific down-regulation is mediated by an ubiquitin-proteasome-dependent pathway. Thus, decreased expression of NF1 in I-type neuroblastoma cancer stem cells causes a high level of activated N-Ras that is, at least in part, responsible for their higher tumorigenic potential.

Identification of miRNAs that regulate neuroblast growth and differentiation could be important in understanding the onset and progression of the childhood cancer neuroblastoma, and in developing new therapeutics to fight this often fatal disease. Neuroblastoma tumors and derived cell lines are heterogeneous, with three basic phenotypes ‐ neuroblastic (N), substrate‐adherent (S), and cancer stem cells (I). These types differ significantly in their morphology, biochemistry and tumorigenic potential. Five miRNAs differentially expressed with respect to cell phenotype were identified from a miRNA microarray, validated by RT‐PCR, and quantified by real‐time RT‐PCR taqman assays in 14 different cell lines (6 N‐, 5 I‐ and 3 S‐types). Out of these miRNAs, three are highly expressed in non‐tumorigenic S‐type cell lines compared to I‐ and N‐type cell lines: miR‐21 (10‐ fold, p<0.05), miR‐221 (18‐fold, p<0.05) and miR‐335 (136‐fold, p<0.05). Moreover, miRNA expression increased significantly with BrdU‐induced I‐ to S‐type differentiation of BE(2)‐C. By contrast, miR‐124a levels are 10‐fold higher in N‐type cells compared to both I‐ and S‐type cells and are elevated 2‐fold (p<0.05) following retinoic acid‐induced neuronal differentiation of I‐type BE(2)‐C. Transient transfection of miR‐124a into I‐cells induced a neuronal‐like phenotype with increased neurite growth, supporting its active role in neuronal differentiation. Another neuronal‐specific miRNA, miR‐375, expressed in N‐ and I‐ but not in S‐type cells, significantly decreased with induced differentiation from an I‐ to S‐type phenotype. A second factor affecting survival of neuroblastoma patients is overexpression/amplification of the protooncogene N‐myc. Thus, the microarray was sorted for miRNAs whose levels correlated directly or inversely with N‐myc gene copy number. Candidate miRNAs were quantified by real‐time RT‐PCR taqman assays in the same 14 cell lines (8 N‐myc amplified and 6 non‐amplified). Three members of the miR‐17‐92 cluster (miR‐17‐5p, miR‐18a and miR‐20a) are highly expressed in N‐myc‐amplified cell lines compared to non‐amplified cells (p<0.05). Moreover, siRNA‐induced reduction of N‐myc protein (2.5‐fold) down‐regulated expression levels of all three miRNAs by 70% (p<0.05), suggesting N‐myc up‐regulates the expression of the miR‐17‐92 cluster. A screen of potential targets of the miR‐17‐92 cluster members revealed, interestingly, that N‐myc mRNA itself is a predicted target of miR‐17‐5p. Down‐regulation of this miRNA (95%) by transient transfection with specific inhibitors increased N‐myc mRNA 2‐fold (p<0.05) and protein 1.5‐fold (p<0.05), confirming and extending results of Cloonan et al (2008). In N‐myc amplified cell lines, N‐myc mRNA levels negatively correlate with levels of miR‐17‐5p. Together these data reveal a possible negative feedback loop between N‐myc and miR‐17‐5p. In conclusion, miRNAs that are differentially expressed with respect to both phenotype and N‐myc amplification status may significantly contribute to the progression and aggressiveness of neuroblastoma. Citation Information: Cancer Res 2009;69(23 Suppl):B76.

Human neuroblastoma is an embryonic cancer of the neural crest. Cellular heterogeneity is a characteristic feature of both tumors and derived cell lines. Recent studies have revealed that both cell lines and tumors contain cancer stem cells. In culture, these cells are self-renewing, multipotent, and highly malignant; in tumors their frequency correlates with a worse prognosis. Their identification and characterization should now permit a targeted approach to more effective treatment of this often fatal childhood cancer.

In human neuroblastoma tumors, amplification of the N-myc proto-oncogene and loss of all or part of the short arm of chromosome #1 are both associated with a poor prognosis. Accruing evidence indicates that it is the absence of one allele of the HuD (ELAVL4) gene, encoding the neuronal-specific RNA-binding protein HuD and localized to 1p34, that is linked to amplification. In 12 human neuroblastoma cell lines, N-myc amplification correlates with loss of one HuD allele and decreased HuD expression. Transfection experiments demonstrate that modulating HuD expression affects N-myc gene copy number as well as expression. Introduction of a sense HuD construct into two N-myc amplified cell lines considerably increases N-myc expression whereas gene copy number decreases. Conversely, expression of antisense HuD in N-myc nonamplified SH-SY5Y cells reduces HuD and N-myc mRNA levels even as cells show amplification of the N-myc gene. Thus, N-myc gene copy number is modulated by alteration of HuD expression. We propose that haploinsufficiency of HuD due to chromosome #1p deletion in neuroblastoma selects for cells that amplify N-myc genes. Application of these findings could lead to more effective therapies in the treatment of those patients with the worst prognosis.

Cellular heterogeneity is a hallmark of human neuroblastoma tumors and cell lines. Within a single neuroblastoma are cells from distinct neural crest lineages whose relative abundance is significant for prognosis. We postulate that a self-renewing multipotent tumor stem cell, which gives rise to diverse cell lineages, is the malignant progenitor of this cancer. To test this hypothesis, we have established 22 cloned, phenotypically homogeneous populations of the three major cell types from 17 neuroblastoma cell lines. In vitro, malignant neuroblastoma stem cells, termed I-type (intermediate type), have distinct morphologic, biochemical, differentiative, and tumorigenic properties. I-type cells express features of both neuroblastic (N) cells (scant cytoplasm, neuritic processes, neurofilaments, pseudoganglia, and granin and neurotransmitter enzyme expression) and substrate-adherent (S) cells (extensive cytoplasm and vimentin and CD44 expression). Moreover, they show bidirectional differentiation to either N or S cells when induced by specific agents. I-type cells are significantly more malignant than N- or S-type cells, with four- to five-fold greater plating efficiencies in soft agar and six-fold higher tumorigenicity in athymic mice. Differences in malignant potential are unrelated to N-myc amplification/overexpression or the ability to digest and migrate through the extracellular matrix. Immunocytochemical analyses of a small series of tumors reveal that frequency of cells coexpressing N and S cell markers correlates with poor prognosis. Thus, I-type stem cells may be instrumental in the genesis and growth of tumors in the patient. Their unique biology deserves attention and further investigation.

Neuroblastomas, tumors of the sympathetic nervous system, account for 7-10% of the cancers of childhood. Genetic studies have shown, and this study has confirmed, that neuroblastomas are very heterogeneous; no single genetic change common to all neuroblastomas has yet been identified. One genetic aberration found frequently in this pediatric tumor is MYCN gene amplification. Recently we identified a new subset of tumors showing MYCN gain (small increases in gene number arising from unbalanced translocation). To investigate whether gain precedes amplification or is an independent event, we surveyed 200 primary tumors for MYCN copy number with fluorescence in situ hybridization; 152 of 200 (76%) were MYCN single-copy tumors, whereas 48 of 200 (24%) tumors harbored MYCN abnormalities: 36 of the 48 (75%) had MYCN amplification and 12 (25%) had MYCN gain. Among the 36 with MYCN amplified gene, we found four that also showed gain. In three tumors exhibiting simultaneous gain and amplification, these two events were detected in neighboring cells. In the fourth case we detected only MYCN gain in metastatic neuroblasts in the bone marrow, but both MYCN amplification and gain in the primary tumor. The detailed study of these four cases suggests that there may be several different mechanisms leading to increase in MYCN copy number. Further studies in other human malignancies are necessary to determine whether simultaneous gain and amplification are specific to neuroblastoma or constitute a general mechanism by which tumor cells can acquire selective growth advantage.

Examining the methylation patterns of gene promoters has become a powerful tool in the effort to identify and distinguish different tumor subtypes (1,2). Typically associated with tumor suppressor genes, epigenetic silencing of gene expression has been seen in several different cancers. Aberrant hypermethyl- ation has been recorded for genes that function in various aspects of cancer biology, including cell cycle regulation, tumor sup- pression, apoptosis, and metastasis. The article by Alaminos et al. (3) in this issue is a clear extension of this previous research. DNA methylation silences gene expression through the ad- dition of methyl groups to cytosine residues of CpG-rich islands present in the promoter region of genes. Whereas housekeeping genes possess CpG regions that are typically unmethylated and therefore transcriptionally active in all cell types, tissue-specific genes are unmethylated in cells actively expressing the genes and are normally methylated in all other differentiated tissues. Patterns of silencing are specified during development; methyl- ation is essential for normal imprinting, X-chromosome inacti- vation, and differential gene expression during embryonic growth. Changes in DNA methylation patterns have recently been associated with specific cancers, and CpG island hyper- methylation profiles for primary tumors have become accepted as a common means to distinguish human cancer subtypes (1,2). Alaminos et al. (3) initially chose 45 candidate genes repre- sentative of important cellular processes and compared their methylation states in 10 human neuroblastoma cell lines and clones by using methylation-specific polymerase chain reaction. The cell lines comprise homogeneous populations of the various cell types—that is, S-type (substrate adherent), N-type (neuro- blastic), and I-type (stem) cells— derived from tumors and were used to determine whether specific differences in the methyl- ation profiles of the candidate genes were associated with cell phenotype. I-type cells can differentiate into either neuroblasts