The Tangential Dialogue Between Science and Medicine: A Case in Point.

The road between a hypothesis about a disease or condition and its cure or palliation is never simply linear. There are many tantalizing tangents to be chased and many seemingly obvious truths with countless exceptions; this is usually a feature, not a bug, as they say in computer programming. In the tangents and exceptions are clues and alternative roads to science and medicine that can provide cures and palliative measures, sometimes for diseases or conditions other than the one being studied. The narrative that follows uses the author's scientific experience in childhood nervous system cancer to illustrate the importance of a robust, bidirectional interaction between the laboratory bench and the clinic bedside in the quest for solutions to problems of health, longevity, and quality of life.

Developing the next-generation cancer research workforce in the National Institutes of Health Intramural Research Program.

Although the National Institutes of Health is renowned for being the largest funder of biomedical research in the world, the research and associated career development programs on its own campuses are relatively unknown. These intramural programs provide many outstanding and programmatically unique opportunities for research-intensive careers and training in cancer biology, prevention, diagnosis, and therapeutics. Their complementary foci, structures, and review mechanisms make the extramural and intramural cancer research contributions of the National Institutes of Health the perfect partners in the quest to rid the world of cancer as we know it.

Identification of a Vitamin-D Receptor Antagonist, MeTC7, which Inhibits the Growth of Xenograft and Transgenic Tumors In Vivo.

Vitamin-D receptor (VDR) mRNA is overexpressed in neuroblastoma and carcinomas of lung, pancreas, and ovaries and predicts poor prognoses. VDR antagonists may be able to inhibit tumors that overexpress VDR. However, the current antagonists are arduous to synthesize and are only partial antagonists, limiting their use. Here, we show that the VDR antagonist MeTC7 (5), which can be synthesized from 7-dehydrocholesterol (6) in two steps, inhibits VDR selectively, suppresses the viability of cancer cell-lines, and reduces the growth of the spontaneous transgenic TH-MYCN neuroblastoma and xenografts in vivo. The VDR selectivity of 5 against RXRα and PPAR-γ was confirmed, and docking studies using VDR-LBD indicated that 5 induces major changes in the binding motifs, which potentially result in VDR antagonistic effects. These data highlight the therapeutic benefits of targeting VDR for the treatment of malignancies and demonstrate the creation of selective VDR antagonists that are easy to synthesize.

PRMT1 promotes neuroblastoma cell survival through ATF5.

Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of cancers, making PRMTs potential therapeutic targets. But it remains not well understood how PRMTs impact specific oncogenic pathways. We previously identified PRMTs as important regulators of cell growth in neuroblastoma, a deadly childhood tumor of the sympathetic nervous system. Here, we demonstrate a critical role for PRMT1 in neuroblastoma cell survival. PRMT1 depletion decreased the ability of murine neuroblastoma sphere cells to grow and form spheres, and suppressed proliferation and induced apoptosis of human neuroblastoma cells. Mechanistic studies reveal the prosurvival factor, activating transcription factor 5 (ATF5) as a downstream effector of PRMT1-mediated survival signaling. Furthermore, a diamidine class of PRMT1 inhibitors exhibited anti-neuroblastoma efficacy both in vitro and in vivo. Importantly, overexpression of ATF5 rescued cell apoptosis triggered by PRMT1 inhibition genetically or pharmacologically. Taken together, our findings shed new insights into PRMT1 signaling pathway, and provide evidence for PRMT1 as an actionable therapeutic target in neuroblastoma.

Using Chemistry to Target Neuroblastoma.

Neuroblastoma is a cancer of the neural crest almost exclusively seen in childhood. While children with single, small primary tumors are often cured with surgery alone, the 65% of children with neuroblastoma whose disease has metastasized have less than a 50% chance of surviving five years after diagnosis. Innovative pharmacological strategies are critically needed for these children. Efforts to identify novel targets that afford ablation of neuroblastoma with minimal toxicity to normal tissues are underway. Developing approaches to neuroblastoma include those that target the catecholamine transporter, ubiquitin E3 ligase, the ganglioside GD2, the retinoic acid receptor, the protein kinases ALK and Aurora, and protein arginine N-methyltransferases. Here, as examples of the use of chemistry to combat neuroblastoma, we describe targeting of the protein arginine N-methyltransferases and their role in prolonging the half-life of the neuroblastoma oncoprotein N-Myc, redox signaling in neuroblastoma, and developmentally regulated proteins expressed in primitive neuroblastoma cells but not in mature neural crest elements.

Protein arginine methyltransferase 1 is a novel regulator of MYCN in neuroblastoma.

Amplification or overexpression of MYCN is associated with poor prognosis of human neuroblastoma. We have recently defined a MYCN-dependent transcriptional signature, including protein arginine methyltransferase 1 (PRMT1), which identifies a subgroup of patients with high-risk disease. Here we provide several lines of evidence demonstrating PRMT1 as a novel regulator of MYCN and implicating PRMT1 as a potential therapeutic target in neuroblastoma pathogenesis. First, we observed a strong correlation between MYCN and PRMT1 protein levels in primary neuroblastoma tumors. Second, MYCN physically associates with PRMT1 by direct protein-protein interaction. Third, depletion of PRMT1 through siRNA knockdown reduced neuroblastoma cell viability and MYCN expression. Fourth, we showed that PRMT1 regulates MYCN stability and identified MYCN as a novel substrate of PRMT1. Finally, we demonstrated that mutation of putatively methylated arginine R65 to alanine decreased MYCN stability by altering phosphorylation at residues serine 62 and threonine 58. These results provide mechanistic insights into the modulation of MYCN oncoprotein by PRMT1, and suggest that targeting PRMT1 may have a therapeutic impact on MYCN-driven oncogenesis.

The "neuro" of neuroblastoma: Neuroblastoma as a neurodevelopmental disorder.

Neuroblastoma is a childhood cancer derived from cells of neural crest origin. The hallmarks of its enigmatic character include its propensity for spontaneous regression under some circumstances and its association with paraneoplastic opsoclonus, myoclonus, and ataxia. The neurodevelopmental underpinnings of its origins may provide important clues for development of novel therapeutic and preventive agents for this frequently fatal malignancy and for the associated paraneoplastic syndromes. Ann Neurol 2016;80:13-23.

Cell Line-Dependent Variability of Coordinate Expression of p75NTR and CRABP1 and Modulation of Effects of Fenretinide on Neuroblastoma Cells.

Neuroblastoma is a childhood neural crest tumor. Fenretinide, a retinoic acid analogue, induces accumulation of mitochondrial reactive oxygen species and consequent apoptosis in neuroblastoma cells. The p75 neurotrophin receptor (p75NTR) enhances the antineuroblastoma cell efficacy of fenretinide in vitro. We examined the role of the retinoid binding protein, CRABP1, in p75NTR-mediated potentiation of the efficacy of fenretinide. Knockdown and overexpression, respectively, of either p75NTR or CRABP1 were effected in neuroblastoma cell lines using standard techniques. Expression was determined by qRT-PCR and confirmed at the protein level by Western blot. Metabolic viability was determined by Alamar blue assay. While protein content of CRABP1 correlated roughly with that of p75NTR in the three neuroblastoid or epithelioid human neuroblastoma cell lines studied, manipulation of p75NTR expression resulted in cell line-dependent, variable change in CRABP1 expression. Furthermore, in some cell lines, induced expression of CRABP1 in the absence of p75NTR did not alter cell sensitivity to fenretinide treatment. The effects of manipulation of p75NTR expression on CRABP1 expression and the effects of CRABP1 expression on fenretinide efficacy are therefore neuroblastoma cell line-dependent. Potentiation of the antineuroblastoma cell effects of fenretinide by p75NTR is not mediated solely through CRABP1.

Induction of Expression of p75 Neurotrophin Receptor Intracellular Domain Does Not Induce Expression or Enhance Activity of Mitochondrial Complex II.

Fenretinide is a chemotherapeutic agent in clinical trials for the treatment of neuroblastoma, among the most common and most deadly cancers of childhood. Fenretinide induces apoptosis in neuroblastoma cells through accumulation of mitochondrial reactive oxygen species released from Complex II. The neurotrophin receptor, p75NTR, potentiates this effect. The signaling activity of p75NTR is dependent upon its cleavage to its intracellular domain, p75ICD, trafficking of p75ICD to the nucleus, and functioning of p75ICD as a transcription factor. Mitochondrial Complex II comprises 4 subunits, all of which are encoded by nuclear DNA. We therefore hypothesized that the fenretinide-potentiating effects of p75NTR are the result of transcriptional enrichment of Complex II by p75ICD. However, the present studies demonstrate that neither induced expression of p75ICD or its active fragments nor overexpression of p75NTR results in altered expression or activity of Complex II.

EYA1 expression and subcellular localization in neuroblastoma and its association with prognostic markers.

Neuroblastoma, the most frequently occurring extracranial solid tumor of childhood, arises from neural crest-derived cells that are arrested at an early stage of differentiation in the developing sympathetic nervous system. There is an urgent need to identify clinically relevant biomarkers for better prognosis and treatment of this aggressive malignancy. Eyes Absent 1 (EYA1) is an essential transcriptional coactivator for neuronal developmental programs during organogenesis. Whether or not EYA1 is implicated in neuroblastoma and subcellular localization of EYA1 is relevant to clinical behaviour of neuroblastoma is not known. We studied EYA1 expression and subcellular localization by immunohistochemistry in tissue microarrays containing tumor specimens from 98 patients, 66 of which were characterized by known clinical prognostic markers of neuroblastoma. Immunostaining results were evaluated and statistically correlated with the degree of histologic differentiation and with neuroblastoma risk stratification group characteristics, including stage of disease, patient age, tumor histology and mitosis-karyorrhexis index (MKI), respectively. We found that EYA1 levels were significantly higher in neuroblastomas than in ganglioneuromas and ganglioneuroblastomas. EYA1 was more highly expressed in stage 1,2,3 or 4S tumors as compared to stage 4 tumors (P<0.01). Tumors with high levels of nuclear EYA1 were more frequently associated with high nuclear MYCN levels. These results suggest that modulation of expression and intracellular localization of EYA1 in neural crest cells may provide a novel direction for therapeutic strategies.

p75 neurotrophin receptor and fenretinide-induced signaling in neuroblastoma.

PURPOSE: Neuroblastoma is the most common extracranial solid tumor of childhood. The retinoic acid analogue, fenretinide (4-hydroxyphenyl retinamide; 4-HPR), induces apoptosis in neuroblastoma cells in vitro and is currently in clinical trials for children with refractory neuroblastoma. We have previously shown that expression of the p75 neurotrophin receptor (p75NTR) enhances apoptosis induction and mitochondrial accumulation of reactive oxygen species by 4-HPR in neuroblastoma cells. We now examine the signaling events that underlie this effect. METHODS: Systematic examination of pro- and anti-apoptotic signaling effectors was performed by Western blot. Specific inhibitors of JNK phosphorylation and scavengers of mitochondrial reactive oxygen species were used to demonstrate the roles of these phenomena in the enhancement of fenretinide efficacy. RESULTS: The present studies demonstrate that enhancement of 4-HPR-induced apoptosis by p75NTR is dependent upon p38MAPK phosphorylation, JNK phosphorylation, caspase 3 activation, Akt cleavage, and decreased Akt phosphorylation. In addition, treatment with 4-HPR results in upregulation of MKK4 and MEKK1, and phosphorylation of MKK3/6. Efforts to enhance the efficacy of 4-HPR and to identify those tumors most likely to respond to it might exploit these effectors of 4-HPR-induced apoptosis. CONCLUSIONS: Pharmacological agents that enhance MKK4 or MEKK1 expression or JNK expression or phosphorylation may enhance efficacy of 4-HPR in neuroblastomas that do not express high levels of p75NTR.

Kidins220/ARMS is expressed in neuroblastoma tumors and stabilizes neurotrophic signaling in a human neuroblastoma cell line.

BACKGROUND: Neurotrophic signaling is an important factor in the survival of developing neurons, and the expression of neurotrophic receptors correlates with prognosis in neuroblastoma. Kinase D-interacting substrate of 220 kDa (Kidins220) associates with neurotrophic receptors and stabilizes them, but the expression and function of Kidins220 in neuroblastoma are unknown. METHODS: We study Kidins220 expression in human neuroblastoma cell lines and tumor samples by western blotting and microarray analyses. We determine the functional consequences of downregulation of Kidins220 for response of cell lines to oxidative stress, chemotherapeutic treatment, and neurotrophins using small interfering RNA silencing and by measuring cell survival, signaling, and migration. RESULTS: Kidins220 is expressed in all neuroblastoma tumors and cell lines studied. Downregulation of Kidins220 leads to attenuation of nerve growth factor (NGF)-induced, but not brain-derived neurotrophic factor (BDNF)-induced, MAPK signaling. However, downregulation of Kidins220 does not alter the response to chemotherapeutic drugs or oxidative stress or affect cellular motility. CONCLUSION: Kidins220 is expressed in neuroblastoma tumors and stabilizes NGF-induced, but not BDNF-induced, survival signaling in neuroblastoma cell lines.

Aiming at neuroblastoma and hitting other worthy targets.

Neuroblastoma is, at once, the most common and deadly extracranial solid tumor of childhood. Efforts aimed at targeting the neural characteristics of these tumors have taught us much about neural crest cell biology, apoptosis induction in the nervous system, and neurotrophin receptor signaling and intracellular processing. But neuroblastoma remains a formidable enemy to the oncologist and an enigmatic target to the neuroscientist.

Life at the interface: adults with "pediatric" disorders of the nervous system.

The increasing longevity of patients with congenital and developmental disorders of the nervous system reflects the palliative and social success of pediatrics in the past 2 decades. This success has resulted in an increasing number of adult patients with residua or sequelae of childhood disease and/or its treatment. It is critically important that residencies and subspecialty fellowships train a cadre of physicians to prepare patients and families for the transition of children with special health care needs to adulthood and to attend to their unique medical, psychological, and social concerns. Health services and education research must better define the needs of this growing population and the best ways to educate their physicians and families and empower them to become as independent as their fullest potential allows.

Kidins220/ARMS depletion is associated with the neural-to Schwann-like transition in a human neuroblastoma cell line model.

Peripheral neuroblastic tumors exist as a heterogeneous mixture of neuroblastic (N-type) cells and Schwannian stromal (S-type) cells. These stromal cells not only represent a differentiated and less aggressive fraction of the tumor, but also have properties that can influence the further differentiation of nearby malignant cells. In vitro neuroblastoma cultures exhibit similar heterogeneity with N-type and S-type cells representing the neuroblastic and stromal portions of the tumor, respectively, in behavior, morphology, and molecular expression patterns. In this study, we deplete kinase D-interacting substrate of 220kD (Kidins220) with an shRNA construct and thereby cause morphologic transition of the human SH-SY5Y neuroblastoma cell line from N-type to S-type. The resulting cells have similar morphology and expression profile to SH-EP1 cells, a native S-type cell line from the same parent cell line, and to SH-SY5Y cells treated with BrdU, a treatment that induces S-type morphology. Specifically, both Kidins220-deficient SH-SY5Y cells and native SH-EP1 cells demonstrate down-regulation of the genes DCX and STMN2, markers for the neuronal lineage. We further show that Kidins220, DCX and STMN2 are co-down-regulated in cells of S-type morphology generated by methods other than Kidins220 depletion. Finally, we report that the association of low Kidins220 expression with S-type morphology and low DCX and STMN2 expression is demonstrated in spontaneously occurring human peripheral neuroblastic tumors. We propose that Kidins220 is critical in N- to S-type transition of neural crest tumor cells.

p75NTR: an enhancer of fenretinide toxicity in neuroblastoma.

OBJECTIVE: Neuroblastoma is a common, frequently fatal, neural crest tumor of childhood. Chemotherapy-resistant neuroblastoma cells typically have Schwann cell-like ("S-type") morphology and express the p75 neurotrophin receptor (p75NTR). p75NTR has been previously shown to modulate the redox state of neural crest tumor cells. We, therefore, hypothesized that p75NTR expression level would influence the effects of the redox-active chemotherapeutic drug fenretinide on neuroblastoma cells. METHODS: Transfection and lentiviral transduction were used to manipulate p75NTR expression in these cell lines. Sensitivity to fenretinide was determined by concentration- and time-cell survival studies. Apoptosis incidence was determined by morphological assessment and examination of cleavage of poly-ADP ribose polymerase and caspase-3. Generation and subcellular localization of reactive oxygen species were quantified using species- and site-specific stains and by examining the effects of site-selective antioxidants on cell survival after fenretinide treatment. Studies of mitochondrial electron transport employed specific inhibitors of individual proteins in the electron transport chain. RESULTS: Knockdown of p75NTR attenuates fenretinide-induced accumulation of mitochondrial superoxide and apoptosis. Overexpression of p75NTR has the opposite effects. Pretreatment of cells with 2-thenoyltrifluoroacetone or dehydroascorbic acid uniquely prevents mitochondrial superoxide accumulation and cell death after fenretinide treatment, indicating that mitochondrial complex II is the likely site of fenretinide-induced superoxide generation and p75NTR-induced potentiation of these phenomena. CONCLUSION: Modification of expression of p75NTR in a particular neuroblastoma cell line modifies its susceptibility to fenretinide. Enhancers of p75NTR expression or signaling could be potential drugs for use as adjuncts to chemotherapy of neural tumors.

Chopper is prodeath regardless of the effect of p75ICD on sensitivity to oxidative stress.

BACKGROUND: The intracellular domain (ICD) of the neurotrophin receptor, p75NTR, exhibits variably pro- and antiapoptotic activity and has been implicated in neurodegenerative and neurodestructive disease. The molecular determinants of these cellular effects are not completely understood. The "Chopper" domain of p75ICD has been shown to be proapoptotic in in vitro systems in which p75ICD is proapoptotic. The effects of Chopper in systems in which p75ICD is antiapoptotic and, therefore, whether or not Chopper accounts for the variability of the cellular effects of p75ICD are not known. We therefore examined the effects of deletion of Chopper on the effects of p75ICD on in vitro cell culture systems in which p75ICD is pro- or antiapoptotic, respectively. RESULTS: In HN33.11 murine neuroblastoma-hippocampal neuron hybrid cells, p75ICD is antiapoptotic. In NIH 3T3 cells, p75ICD is proapoptotic. In both cell lines deletion of the Chopper domain from p75ICD decreases the incidence of apoptosis resulting from oxidative stress. Thus, irrespective of the nature of the effects of p75ICD on the cell, its Chopper domain is proapoptotic. CONCLUSIONS: Expression of p75ICD can enhance or attenuate oxidative induction of apoptosis. Variability of the effects of p75ICD is not related to variability of the effects of its Chopper domain.

Pharmacologic management of high-risk neuroblastoma in children.

Neuroblastoma is the most common extracranial solid tumor of childhood. It accounts for 15% of pediatric cancer deaths. Children with high-risk disease have a 3-year event-free survival rate of only 20%. Chemotherapy is the mainstay of treatment in children with advanced neuroblastoma. The aim of this article was to review and critically evaluate the pharmacotherapy of neuroblastoma, using peer reviewed and review literature from 2000-11. All peer reviewed, published human subject studies of therapy for neuroblastoma in children were included. Animal model and in vitro studies were included only if they added to the understanding of the mechanism of a proposed or existing human neuroblastoma therapy. Current therapeutic options for neuroblastoma involve insufficient differentiation of normal from neoplastic tissue. Critically needed new approaches will increasingly exploit targeting of therapy for unique characteristics of the neuroblastoma cell. Pharmacotherapy for neuroblastoma still suffers from an inadequate therapeutic window. Enhancement of toxicity for tumor and safety for normal tissues will entail innovation in targeting neuroblastoma cells and rescuing or protecting normal tissue elements.