Biological Implications and Functional Significance of Transglutaminase Type 2 in Nervous System Tumors.

Transglutaminase type 2 (TG2) is the most ubiquitously expressed member of the transglutaminase family. TG2 catalyzes the transamidation reaction leading to several protein post-translational modifications and it is also implicated in signal transduction thanks to its GTP binding/hydrolyzing activity. In the nervous system, TG2 regulates multiple physiological processes, such as development, neuronal cell death and differentiation, and synaptic plasticity. Given its different enzymatic activities, aberrant expression or activity of TG2 can contribute to tumorigenesis, including in peripheral and central nervous system tumors. Indeed, TG2 dysregulation has been reported in meningiomas, medulloblastomas, neuroblastomas, glioblastomas, and other adult-type diffuse gliomas. The aim of this review is to provide an overview of the biological and functional relevance of TG2 in the pathogenesis of nervous system tumors, highlighting its involvement in survival, tumor inflammation, differentiation, and in the resistance to standard therapies.

Exploring Lin28 proteins: Unravelling structure and functions with emphasis on nervous system malignancies.

Cancer and stem cells share many characteristics related to self-renewal and differentiation. Both cell types express the same critical proteins that govern cellular stemness, which provide cancer cells with the growth and survival benefits of stem cells. LIN28 is an example of one such protein. LIN28 includes two main isoforms, LIN28A and LIN28B, with diverse physiological functions from tissue development to control of pluripotency. In addition to their physiological roles, LIN28A and LIN28B affect the progression of several cancers by regulating multiple cancer hallmarks. Altered expression levels of LIN28A and LIN28B have been proposed as diagnostic and/or prognostic markers for various malignancies. This review discusses the structure and modes of action of the different LIN28 proteins and examines their roles in regulating cancer hallmarks with a focus on malignancies of the nervous system. This review also highlights some gaps in the field that require further exploration to assess the potential of targeting LIN28 proteins for controlling cancer.

Neurons as stromal drivers of nervous system cancer formation and progression.

Similar to their pivotal roles in nervous system development, neurons have emerged as critical regulators of cancer initiation, maintenance, and progression. Focusing on nervous system tumors, we describe the normal relationships between neurons and other cell types relevant to normal nerve function, and discuss how disruptions of these interactions promote tumor evolution, focusing on electrical (gap junctions) and chemical (synaptic) coupling, as well as the establishment of new paracrine relationships. We also review how neuron-tumor communication contributes to some of the complications of cancer, including neuropathy, chemobrain, seizures, and pain. Finally, we consider the implications of cancer neuroscience in establishing risk for tumor penetrance and in the design of future anti-tumoral treatments.

Nuclear receptor NR5A2 negatively regulates cell proliferation and tumor growth in nervous system malignancies.

Nervous system malignancies are characterized by rapid progression and poor survival rates. These clinical observations underscore the need for novel therapeutic insights and pharmacological targets. To this end, here, we identify the orphan nuclear receptor NR5A2/LRH1 as a negative regulator of cancer cell proliferation and promising pharmacological target for nervous system-related tumors. In particular, clinical data from publicly available databases suggest that high expression levels of NR5A2 are associated with favorable prognosis in patients with glioblastoma and neuroblastoma tumors. Consistently, we experimentally show that NR5A2 is sufficient to strongly suppress proliferation of both human and mouse glioblastoma and neuroblastoma cells without inducing apoptosis. Moreover, short hairpin RNA-mediated knockdown of the basal expression levels of NR5A2 in glioblastoma cells promotes their cell cycle progression. The antiproliferative effect of NR5A2 is mediated by the transcriptional induction of negative regulators of the cell cycle, CDKN1A (encoding for p21cip1), CDKN1B (encoding for p27kip1) and Prox1 Interestingly, two well-established agonists of NR5A2, dilauroyl phosphatidylcholine (DLPC) and diundecanoyl phosphatidylcholine, are able to mimic the antiproliferative action of NR5A2 in human glioblastoma cells via the induction of the same critical genes. Most importantly, treatment with DLPC inhibits glioblastoma tumor growth in vivo in heterotopic and orthotopic xenograft mouse models. These data indicate a tumor suppressor role of NR5A2 in the nervous system and render this nuclear receptor a potential pharmacological target for the treatment of nervous tissue-related tumors.

Zebrafish as a Neuroblastoma Model: Progress Made, Promise for the Future.

For nearly a decade, researchers in the field of pediatric oncology have been using zebrafish as a model for understanding the contributions of genetic alternations to the pathogenesis of neuroblastoma (NB), and exploring the molecular and cellular mechanisms that underlie neuroblastoma initiation and metastasis. In this review, we will enumerate and illustrate the key advantages of using the zebrafish model in NB research, which allows researchers to: monitor tumor development in real-time; robustly manipulate gene expression (either transiently or stably); rapidly evaluate the cooperative interactions of multiple genetic alterations to disease pathogenesis; and provide a highly efficient and low-cost methodology to screen for effective pharmaceutical interventions (both alone and in combination with one another). This review will then list some of the common challenges of using the zebrafish model and provide strategies for overcoming these difficulties. We have also included visual diagram and figures to illustrate the workflow of cancer model development in zebrafish and provide a summary comparison of commonly used animal models in cancer research, as well as key findings of cooperative contributions between MYCN and diverse singling pathways in NB pathogenesis.

The Role of Aryl Hydrocarbon Receptor (AhR) in Brain Tumors.

Primary brain tumors, both malignant and benign, are diagnosed in adults at an incidence rate of approximately 23 people per 100 thousand. The role of AhR in carcinogenesis has been a subject of debate, given that this protein may act as either an oncogenic protein or a tumor suppressor in different cell types and contexts. Lately, there is growing evidence that aryl hydrocarbon receptor (AhR) plays an important part in the development of brain tumors. The role of AhR in brain tumors is complicated, depending on the type of tumor, on ligands that activate AhR, and other features of the pathological process. In this review, we summarize current knowledge about AhR in relation to brain tumors and provide an overview of AhR's potential as a therapeutic target.

Long non-coding RNA PVT1 regulates glioma proliferation, invasion, and aerobic glycolysis via miR-140-5p.

OBJECTIVE: To investigate the regulation of long non-coding RNA plasmacytoma variant translocation 1 (LncRNA PVT1) on proliferation, invasion, and aerobic glycolysis in glioma cells via miR-140-5p. PATIENTS AND METHODS: Sixty patients with glioma treated in our hospital were recruited. The expression of PVT1 in tissues and cells was determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and the effects on the prognosis were observed. Glioma cell lines U87 and T98MG were either stably or transiently transfected with over-expression or inhibition vectors. Cell counting kit-8 (CCK-8), transwell, glucose, and lactate detection were employed to measure cell proliferation, invasion, and aerobic glycolysis after transfection. The correlation between PVT1 and miR-140-5p was determined by Dual-Luciferase reporter assay. RNA pull-down and RNA immunoprecipitation (RIP) test were adopted to indicate the correlation between PVT1 and miR-140-5p. RESULTS: PVT1 was highly expressed and had superior diagnostic value in gliomas, and the high expression of PVT1 resulted in poor prognosis of patients. Over-expressing PVT1 increased cell proliferation, invasion, and aerobic glycolysis, while inhibiting PVT1 yielded opposite outcome. Dual-Luciferase reporter assay confirmed that PVT1 could target miR-140-5p. Functional analysis showed that over-expression of miR-140-5p inhibited proliferation, invasion, and aerobic glycolysis in glioma cells. Rescue experiment found that the inhibitory effect of miR-140-5p could be eliminated by up-regulating PVT1 expression. CONCLUSIONS: PVT1 promotes proliferation, invasion, and aerobic glycolysis in glioma cells by regulating miR-140-5p.

Prediction of neuroblastoma cell response to treatment with natural or synthetic retinoids using selected protein biomarkers.

Although the administration of retinoids represents an important part of treatment for children suffering from high-risk neuroblastomas, approximately 50% of these patients do not respond to this therapy or develop resistance to retinoids during treatment. Our study focused on the comparative analysis of the expression of five genes and corresponding proteins (DDX39A, HMGA1, HMGA2, HOXC9 and PBX1) that have recently been discussed as possible predictive biomarkers of clinical response to retinoid differentiation therapy. Expression of these five candidate biomarkers was evaluated at both the mRNA and protein level in the same subset of 8 neuroblastoma cell lines after treatment with natural or synthetic retinoids. We found that the cell lines that were HMGA2-positive and/or HOXC9-negative have a reduced sensitivity to retinoids. Furthermore, the experiments revealed that the retinoid-sensitive cell lines showed a uniform pattern of change after treatment with both natural and sensitive retinoids: increased DDX39A and decreased PBX1 protein levels. Our results showed that in NBL cells, these putative protein biomarkers are associated with sensitivity or resistance to retinoids, and their endogenous or induced expression can distinguish between these two phenotypes.

Synergistic drug combination GC7/DFMO suppresses hypusine/spermidine-dependent eIF5A activation and induces apoptotic cell death in neuroblastoma.

The eukaryotic initiation factor 5A (eIF5A), which contributes to several crucial processes during protein translation, is the only protein that requires activation by a unique post-translational hypusine modification. eIF5A hypusination controls cell proliferation and has been linked to cancer. eIF5A hypusination requires the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase and uniquely depends on the polyamine (PA) spermidine as the sole substrate. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in PA biosynthesis. Both ODC and PAs control cell proliferation and are frequently dysregulated in cancer. Since only spermidine can activate eIF5A, we chose the hypusine-PA nexus as a rational target to identify new drug combinations with synergistic antiproliferative effects. We show that elevated mRNA levels of the two target enzymes DHPS and ODC correlate with poor prognosis in a large cohort of neuroblastoma (NB) tumors. The DHPS inhibitor GC7 (N1-guanyl-1,7-diaminoheptane) and the ODC inhibitor α-difluoromethylornithine (DFMO) are target-specific and in combination induced synergistic effects in NB at concentrations that were not individually cytotoxic. Strikingly, while each drug alone at higher concentrations is known to induce p21/Rb- or p27/Rb-mediated G1 cell cycle arrest, we found that the drug combination induced caspase 3/7/9, but not caspase 8-mediated apoptosis, in NB cells. Hypusinated eIF5A levels and intracellular spermidine levels correlated directly with drug treatments, signifying specific drug targeting effects. This two-pronged GC7/DFMO combination approach specifically inhibits both spermidine biosynthesis and post-translational, spermidine-dependent hypusine-eIF5A activation, offering an exciting clue for improved NB drug therapy.

Targeting effect of microRNA on CD133 and its impact analysis on proliferation and invasion of glioma cells.

MiR-200b, a member of the microRNA-200 family, has been identified to be capable of suppressing glioma cell growth through targeting CREB1 or CD133. However, whether miR-200b affects the biological behavior (proliferation, invasion, and migration) of glioma cells is poorly understood. The aim of this study was to evaluate the effect of miR-200b on the biological behavior of glioma cells in vitro. MiRNA-200b mimics, miRNA-200b inhibitor, and mimic control were transfected into conventionally cultured glioma U251 cells, followed by measuring the expression of miR-200b and CD133 in transfected cells by RT-PCR; effect of miR-200b on CD133 mRNA 3'-UTR luciferase activity by luciferase reporter assay; proliferation activity of transfected U251 cells by MTT method; and changes in U251 cell invasion and migration by Transwell method after transfection. Compared to that in the miRNA-200b inhibitor, mimic control, and blank control groups, miRNA-200b expression was significantly increased and CD133 mRNA expression was significantly decreased in the mimic miRNA-200b group in a time-dependent manner (P < 0.05). Meanwhile, dual luciferase reporter assay showed that miR-200b could inhibit CD133 activity through binding to the 3'-UTR of CD133 mRNA (P < 0.05). Furthermore, the proliferation activity and invasion and migration abilities of U251 cells transfected with miRNA-200b mimic were significantly decreased (P < 0.05). In conclusion, overexpression of miR-200b inhibited the proliferation, invasion, and migration of glioma cells possibly through targeting CD133.

Therapeutic targeting of the MYC signal by inhibition of histone chaperone FACT in neuroblastoma.

Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. We used a MYC target gene signature that predicts poor neuroblastoma prognosis to identify the histone chaperone FACT (facilitates chromatin transcription) as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small-molecule curaxin compound CBL0137 markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with standard chemotherapy by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells and suggesting a treatment strategy for MYCN-driven neuroblastoma.

Integrin α4 Enhances Metastasis and May Be Associated with Poor Prognosis in MYCN-low Neuroblastoma.

High-risk neuroblastoma is associated with an overall survival rate of 30-50%. Neuroblastoma-expressed cell adhesion receptors of the integrin family impact cell adhesion, migration, proliferation and survival. Integrin α4 is essential for neural crest cell motility during development, is highly expressed on leukocytes, and is critical for transendothelial migration. Thus, cancer cells that express this receptor may exhibit increased metastatic potential. We show that α4 expression in human and murine neuroblastoma cell lines selectively enhances in vitro interaction with the alternatively spliced connecting segment 1 of fibronectin, as well as vascular cell adhesion molecule-1 and increases migration. Integrin α4 expression enhanced experimental metastasis in a syngeneic tumor model, reconstituting a pattern of organ involvement similar to that seen in patients. Accordingly, antagonism of integrin α4 blocked metastasis, suggesting adhesive function of the integrin is required. However, adhesive function was not sufficient, as mutants of integrin α4 that conserved the matrix-adhesive and promigratory function in vitro were compromised in their metastatic capacity in vivo. Clinically, integrin α4 is more frequently expressed in non-MYNC amplified tumors, and is selectively associated with poor prognosis in this subset of disease. These results reveal an unexpected role for integrin α4 in neuroblastoma dissemination and identify α4 as a potential prognostic indicator and therapeutic target.

NRSF: an angel or a devil in neurogenesis and neurological diseases.

The neuron-restrictive silencer factor (NRSF) a transcriptional regulator that function as a hub that coordinately regulates multiple aspects of neurogenesis, orchestrates neural differentiation, and preserves the unique neural phenotype. NRSF also acts as an oncogene in neural tumorigenesis, although its effect differs depending on the cell type and tissues. Intriguingly, far more than above functions, potential roles for NRSF and its target genes have also been implicated in the pathogenesis and therapeutic mechanism of neurodegenerative diseases. NRSF acts as a flexible and complicated regulator in nervous system, from transcriptional repressor to activator or modulator, and plays a part in neuronal survival or neuronal death. Here, we present the mechanisms proposed to account for the multiple roles of NRSF in neurogenesis and neurological diseases and discuss the therapeutic perspective of recent advances. The mechanisms underlying this duality of NRSF are helpful to understanding the physiological and pathological conditions of neurons and provide new therapeutic approaches to neurological disorders and diseases.

p19-INK4d inhibits neuroblastoma cell growth, induces differentiation and is hypermethylated and downregulated in MYCN-amplified neuroblastomas.

Uncontrolled cell cycle entry, resulting from deregulated CDK-RB1-E2F pathway activity, is a crucial determinant of neuroblastoma cell malignancy. Here we identify neuroblastoma-suppressive functions of the p19-INK4d CDK inhibitor and uncover mechanisms of its repression in high-risk neuroblastomas. Reduced p19-INK4d expression was associated with poor event-free and overall survival and neuroblastoma risk factors including amplified MYCN in a set of 478 primary neuroblastomas. High MYCN expression repressed p19-INK4d mRNA and protein levels in different neuroblastoma cell models with conditional MYCN expression. MassARRAY and 450K methylation analyses of 105 primary neuroblastomas uncovered a differentially methylated region within p19-INK4d. Hypermethylation of this region was associated with reduced p19-INK4d expression. In accordance, p19-INK4d expression was activated upon treatment with the demethylating agent, 2'-deoxy-5-azacytidine, in neuroblastoma cell lines. Ectopic p19-INK4d expression decreased viability, clonogenicity and the capacity for anchorage-independent growth of neuroblastoma cells, and shifted the cell cycle towards the G1/0 phase. p19-INK4d also induced neurite-like processes and markers of neuronal differentiation. Moreover, neuroblastoma cell differentiation, induced by all-trans retinoic acid or NGF-NTRK1-signaling, activated p19-INK4d expression. Our findings pinpoint p19-INK4d as a neuroblastoma suppressor and provide evidence for MYCN-mediated repression and for epigenetic silencing of p19-INK4d by DNA hypermethylation in high-risk neuroblastomas.

Anti-tumor activity of the beta-adrenergic receptor antagonist propranolol in neuroblastoma.

Neuroblastoma (NB) is a pediatric tumor of the sympathetic nervous system, which is often associated with elevated catecholamines. More than half of patients with metastatic NB relapse and survival is extremely poor with current therapies. In a high-throughput screen of FDA-approved drugs we identified anti-NB activity for the nonselective ß-adrenergic receptor antagonist propranolol hydrochloride. Propranolol inhibited growth of a panel of fifteen NB cell lines irrespective of MYCN status, and treatment induced apoptosis and decreased proliferation. Activity was dependent on inhibition of the ß2, and not ß1, adrenergic receptor, and treatment resulted in activation of p53 and p73 signaling in vitro. The majority of NB cell lines and primary tumors express ß2 adrenergic receptor and higher mRNA levels correlate with improved patient survival, but expression levels did not correlate with in vitro sensitivity to propranolol. Furthermore, propranolol is synergistic with the topoisomerase I inhibitor SN-38 and propranolol inhibits growth of NB xenografts in vivo at doses similar to those used to treat infants with hemangiomas and hypertension. Taken together, our results suggest that propranolol has activity against NB and thus should be considered in combination treatments for patients with relapsed and refractory NB.

LGI proteins in the nervous system.

The development and function of the vertebrate nervous system depend on specific interactions between different cell types. Two examples of such interactions are synaptic transmission and myelination. LGI1-4 (leucine-rich glioma inactivated proteins) play important roles in these processes. They are secreted proteins consisting of an LRR (leucine-rich repeat) domain and a so-called epilepsy-associated or EPTP (epitempin) domain. Both domains are thought to function in protein-protein interactions. The first LGI gene to be identified, LGI1, was found at a chromosomal translocation breakpoint in a glioma cell line. It was subsequently found mutated in ADLTE (autosomal dominant lateral temporal (lobe) epilepsy) also referred to as ADPEAF (autosomal dominant partial epilepsy with auditory features). LGI1 protein appears to act at synapses and antibodies against LGI1 may cause the autoimmune disorder limbic encephalitis. A similar function in synaptic remodelling has been suggested for LGI2, which is mutated in canine Benign Familial Juvenile Epilepsy. LGI4 is required for proliferation of glia in the peripheral nervous system and binds to a neuronal receptor, ADAM22, to foster ensheathment and myelination of axons by Schwann cells. Thus, LGI proteins play crucial roles in nervous system development and function and their study is highly important, both to understand their biological functions and for their therapeutic potential. Here, we review our current knowledge about this important family of proteins, and the progress made towards understanding their functions.

Hox-C9 activates the intrinsic pathway of apoptosis and is associated with spontaneous regression in neuroblastoma.

Neuroblastoma is an embryonal malignancy of the sympathetic nervous system. Spontaneous regression and differentiation of neuroblastoma is observed in a subset of patients, and has been suggested to represent delayed activation of physiologic molecular programs of fetal neuroblasts. Homeobox genes constitute an important family of transcription factors, which play a fundamental role in morphogenesis and cell differentiation during embryogenesis. In this study, we demonstrate that expression of the majority of the human HOX class I homeobox genes is significantly associated with clinical covariates in neuroblastoma using microarray expression data of 649 primary tumors. Moreover, a HOX gene expression-based classifier predicted neuroblastoma patient outcome independently of age, stage and MYCN amplification status. Among all HOX genes, HOXC9 expression was most prominently associated with favorable prognostic markers. Most notably, elevated HOXC9 expression was significantly associated with spontaneous regression in infant neuroblastoma. Re-expression of HOXC9 in three neuroblastoma cell lines led to a significant reduction in cell viability, and abrogated tumor growth almost completely in neuroblastoma xenografts. Neuroblastoma growth arrest was related to the induction of programmed cell death, as indicated by an increase in the sub-G1 fraction and translocation of phosphatidylserine to the outer membrane. Programmed cell death was associated with the release of cytochrome c from the mitochondria into the cytosol and activation of the intrinsic cascade of caspases, indicating that HOXC9 re-expression triggers the intrinsic apoptotic pathway. Collectively, our results show a strong prognostic impact of HOX gene expression in neuroblastoma, and may point towards a role of Hox-C9 in neuroblastoma spontaneous regression.

Cytostasis and morphological changes induced by mifepristone in human metastatic cancer cells involve cytoskeletal filamentous actin reorganization and impairment of cell adhesion dynamics.

BACKGROUND: Changes in cell shape and plasticity in cytoskeletal dynamics are critically involved in cell adhesion, migration, invasion and the overall process of metastasis. Previous work in our laboratory demonstrated that the synthetic steroid mifepristone inhibited the growth of highly metastatic cancer cells, while simultaneously causing striking changes in cellular morphology. Here we assessed whether such morphological alterations developed in response to cytostatic concentrations of mifepristone are reversible or permanent, involve rearrangement of cytoskeletal proteins, and/or affect the adhesive capacity of the cells. METHODS: Cancer cell lines of the ovary (SKOV-3), breast (MDA-MB-231), prostate (LNCaP), and nervous system (U87MG) were exposed to cytostatic concentrations of mifepristone and studied by phase-contrast microscopy. The transient or permanent nature of the cytostasis and morphological changes caused by mifepristone was assessed, as well as the rearrangement of cytoskeletal proteins. De-adhesion and adhesion assays were utilized to determine if mifepristone-arrested and morphologically dysregulated cells had abnormal de-adhesion/adhesion dynamics when compared to vehicle-treated controls. RESULTS: Mifepristone-treated cells displayed a long, thin, spindle-like shape with boundaries resembling those of loosely adhered cells. Growth arrest and morphology changes caused by mifepristone were reversible in SKOV-3, MDA-MB-231 and U87MG, but not in LNCaP cells that instead became senescent. All cancer cell types exposed to mifepristone displayed greatly increased actin ruffling in association with accelerated de-adhesion from the culture plate, and delayed adhesion capacity to various extracellular matrix components. CONCLUSIONS: Cytostatic concentrations of mifepristone induced alterations in the cellular structure of a panel of aggressive, highly metastatic cancer cells of different tissues of origin. Such changes were associated with re-distribution of actin fibers that mainly form non-adhesive membrane ruffles, leading to dysregulated cellular adhesion capacity.

Expression of WW domain-containing oxidoreductase WOX1 in human nervous system tumors.

BACKGROUND AND OBJECTIVES: We aimed to evaluate the expression levels of the tumor suppressor WOX1 in nervous system tumors and its co-expression with p53 and neurofibromatosis type 2/merlin (NF2) tumor suppressor gene products. METHODS: Immunohistochemistry, western blotting and in situ hybridization were used for WOX1 protein and WWOX mRNA expression. Immunofluorescence and electron microscopical immunohistochemistry were performed for colocalization of gene products. RESULTS: WOX1 expression is low in normal cortical neurons, mainly on the axon fibers, whereas there is moderate to high immunoreactivity in the cytosol and nuclei of certain tumor cells. In the microcystic (WHO grade I) and malignant (WHO grade III) meningiomas, WOX1 expression is intense, but various in transitional (WHO grade I) and atypical (WHO grade II) subtypes. WOX1 levels are moderate to high in the menigiotheliomatous area, but relatively low in the fibroblastic area. WOX1 and NF2/merlin, but not p53, colocalized in certain tumor cells, primarily at the borders of nuclei. Schwannoma and astrocytoma specimens stained moderately to strongly positive for the WOX1 protein. Interestingly, the expression of WOX1, NF2/merlin and mutant p53 is intense in high grade glioblastoma, but WOX1 expression is low in metastatic carcinoma or adenocarcinoma. CONCLUSIONS: The expression of WOX1 on different types of nervous system tumors, including primary and metastatic tumors, is differential.