Differentiation Of Neuroblastoma Cells Research Articles

ZMYND8 Is a Regulator of Sonic Hedgehog Signaling in ATRA-Mediated Differentiation of Neuroblastoma Cells.

Zinc Finger MYND (Myeloid, Nervy, and DEAF-1) type containing 8 (ZMYND8) is a crucial epigenetic regulator that plays a multifaceted role in governing a spectrum of vital cellular processes, encompassing proliferation, apoptosis, migration, tumor suppression, and differentiation. It has emerged as a key player in neuronal differentiation by orchestrating the expression of neuronal lineage-committed genes. The present study uncovers the role of ZMYND8 in regulating the Sonic Hedgehog (SHH) signaling axis, which is crucial for neuronal differentiation. Genetic deletion of ZMYND8 leads to a significant reduction in SHH pathway genes, GLI1, and PTCH1 expression during all-trans-retinoic acid (ATRA)-induced differentiation. ZMYND8 and RNA pol II S5P are found to co-occupy the GLI1 and PTCH1 gene promoters, positively impacting their gene transcription upon ATRA treatment. Interestingly, ZMYND8 is found to counteract the inhibitory effects of Cyclopamine that block the upstream SHH pathway protein SMO, resulting in enhanced neurite formation in neuroblastoma cells following their treatment with ATRA. These results indicate that ZMYND8 is an epigenetic regulator of the SHH signaling pathway and has tremendous therapeutic potential in ATRA-mediated differentiation of neuroblastoma.

Lipid levels correlate with neuronal and dopaminergic markers during the differentiation of SH-SY5Y cells

Parkinson's Disease (PD) is characterised by the loss of dopaminergic neurons and the deposition of protein inclusions called Lewy Bodies (LBs). LBs are heterogeneous structures composed of protein and lipid molecules and their main constituent is the presynaptic protein α-synuclein. SH-SY5Y cells are neuroblastoma cells commonly used to model PD because they express dopaminergic markers and α-synuclein and they can be differentiated into neuronal cells using established protocols. Despite increasing evidence pointing towards a role of lipids in PD, limited knowledge is available on the lipidome of undifferentiated and differentiated SH-SY5Y cells. Using a combination of lipidomics, proteomics, morphological and electrophysiological measurements, we identified specific lipids, including sphingolipids, whose levels are affected by the differentiation of SH-SY5Y neuroblastoma cells and found that the levels of these lipids correlate with those of neuronal and dopaminergic markers. These results provide a quantitative characterisation of the changes in lipidome associated with the differentiation of SH-SY5Y cells into more neuronal and dopaminergic-like phenotype and serve as a basis for further characterisation of lipid disruptions in association with PD and its risk factors in this dopaminergic-like neuronal cell model.

Effects of Anionic Liposome Delivery of All-Trans-Retinoic Acid on Neuroblastoma Cell Differentiation.

All-trans-retinoic acid (ATRA) has long been known to affect cell growth and differentiation. To improve ATRA's therapeutic efficacy and pharmacodynamics, several delivery systems have been used. In this study, free ATRA and anionic-liposome-encapsulated ATRA were compared for their effects on SK-N-SH human neuroblastoma cell growth and differentiation. Anionic liposomes made of L-α-phosphatidylcholine (PC) and L-α-phosphatidic acid (PA), empty (PC-PA) and loaded with ATRA (PC-PA-ATRA), were characterized by dynamic light scattering (DLS) and electrophoretic mobility measurements, and drug entrapment efficiency (EE%) was measured to evaluate the applicability of the new colloidal formulation. The results of brightfield microscopy and cell growth curves indicated that ATRA, whether free or encapsulated, reduced growth and induced differentiation, resulting in SK-N-SH cells changing from epithelioid to neuronal-like morphologies, and producing a significant increase in neurite growth. To further characterize the neuro-differentiation of SK-N-SH cells, the expression of βIII-Tubulin and synaptophysin and mitochondria localization were analyzed via immunofluorescence. Increased expression of neuronal markers and a peculiar localization of mitochondria in the neuritic extensions were apparent both in ATRA- and PC-PA-ATRA-differentiated cells. As a whole, our results strongly indicate that ATRA treatment, by any means, can induce the differentiation of parent SK-N-SH, and they highlight that its encapsulation in anionic liposomes increases its differentiation ability in terms of the percentage of neurite-bearing cells. Interestingly, our data also suggest an unexpected differentiation capability of anionic liposomes per se. This work highlights the importance of developing and carefully testing novel delivery nanocarriers, which are a necessary first "step" in the development of new therapeutic settings.

Abstract 2852: Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells

Abstract Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system derived from migratory neural crest cells that have committed to become sympathetic neuroblasts, but their definitive differentiation is blocked. Neuroblastoma cell identity depends on the adrenergic core regulatory circuit (CRC) of transcription factors that collaborate with MYCN to drive cell proliferation and the oncogenic gene expression program. Retinoic acid is used as differentiation therapy for patients with high-risk neuroblastoma, with the goal to block proliferation and drive sympathetic neuronal differentiation. We recently showed that retinoic acid re-organizes the enhancer landscape of neuroblastoma and promotes a new retino-sympathetic cell state characterized by MYCN downregulation, proliferative arrest, and sympathetic neuronal differentiation. However, the effects of retinoic acid on epigenetic rewiring of the adrenergic CRC and suppression of MYCN expression are reversible in vitro and in vivo, leading to tumor re-growth when the retinoic acid is withdrawn. Therefore, we sought to determine if retinoid-induced differentiation can be forced to progress to irreversible neuronal maturation by the addition of inhibitors of epigenetic modifying enzymes. Here, we conducted a drug screen of epigenetic modifying drugs alone and in combination with retinoic acid to determine their effects on neuroblastoma cell growth and differentiation. In this screen, we found that an inhibitor of the histone H3K23 acetyltransferases KAT6A/B, PF-9363, synergistically inhibits neuroblastoma cell growth in combination with retinoic acid. Moreover, retinoid-resistant neuroblastoma cell lines can be sensitized to retinoic acid when combined with a KAT6 inhibitor. Importantly, this combination treatment renders the differentiated cell state irreversible, such that it is maintained after retinoic acid is withdrawn, with continued suppression of the adrenergic CRC and MYCN expression. In conclusion, the retino-sympathetic differentiated cell state induced by retinoic acid in neuroblastoma becomes irreversible when the cells are also treated with an inhibitor of the KAT6A/B histone H3K23 acetyltransferases, implicating the essential role of these enzymes in restoring the proliferative immature progenitor phenotype in adrenergic neuroblastoma cells. Citation Format: Nina Weichert-Leahey, Alla Berezovskaya, Mark Zimmerman, Brian J. Abraham, Adam W. Durbin, A Thomas Look. Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2852.

A Potential Prognostic Gene Signature Associated with p53-Dependent NTRK1 Activation and Increased Survival of Neuroblastoma Patients.

Neuroblastoma is the most common extracranial solid tumour in children, comprising close to 10% of childhood cancer-related deaths. We have demonstrated that activation of NTRK1 by TP53 repression of PTPN6 expression is significantly associated with favourable survival in neuroblastoma. The molecular mechanisms by which this activation elicits cell molecular changes need to be determined. This is critical to identify dependable biomarkers for the early detection and prognosis of tumours, and for the development of personalised treatment. In this investigation we have identified and validated a gene signature for the prognosis of neuroblastoma using genes differentially expressed upon activation of the NTRK1-PTPN6-TP53 module. A random survival forest model was used to construct a gene signature, which was then assessed across validation datasets using Kaplan-Meier analysis and ROC curves. The analysis demonstrated that high BASP1, CD9, DLG2, FNBP1, FRMD3, IL11RA, ISGF10, IQCE, KCNQ3, and TOX2, and low BSG/CD147, CCDC125, GABRB3, GNB2L1/RACK1 HAPLN4, HEBP2, and HSD17B12 expression was significantly associated with favourable patient event-free survival (EFS). The gene signature was associated with favourable tumour histology and NTRK1-PTPN6-TP53 module activation. Importantly, all genes were significantly associated with favourable EFS in an independent manner. Six of the signature genes, BSG/CD147, GNB2L1/RACK1, TXNDC5, FNPB1, B3GAT1, and IGSF10, play a role in cell differentiation. Our findings strongly suggest that the identified gene signature is a potential prognostic biomarker and therapeutic target for neuroblastoma patients and that it is associated with neuroblastoma cell differentiation through the activation of the NTRK1-PTPN6-TP53 module.

Aagab is required for zebrafish larval development by regulating neural activity

Clathrin-mediated endocytosis has been implicated in various physiological processes, including nutrient uptake, signal transduction, synaptic vesicle recycling, maintenance of cell polarity, and antigen presentation. Despite prior knowledge of its importance as a key regulator in promoting clathrin-mediated endocytosis, the physiological function of α- and γ-adaptin binding protein (aagab) remains elusive. In this study, we investigate the biological function of aagab during zebrafish development. We establish a loss-of-function mutant of the aagab gene in zebrafish, revealing impaired swimming and early larval mortality. Given the high expression level of the aagab gene in the brain, we probe into its physiological role in the nervous system. aagab mutants display subdued calcium responses and local field potential in the optic tectal neurons, aligning with reduced neurotransmitter release (e.g., norepinephrine) in the tectal neuropil of aagab mutants. Overexpressing aagab message RNA (mRNA) or nervous stimulant treatment in mutants restores neurotransmitter release, calcium responses, swimming ability, and survival. Furthermore, our observations show delayed release of FM 1–43 in AAGAB knockdown differentiated neuroblastoma cells, pointing towards a probable link to defective clathrin-mediated synaptic vesicle recycling. In conclusion, our study underscores the significance of Aagab in neurobiology and suggests its potential impacts in neurological disorders.

Identification of CDKN3 as a Key Gene that Regulates Neuroblastoma Cell Differentiation.

We conducted a high-content screening (HCS) in neuroblastoma BE(2)-C cells to identify cell cycle regulators that control cell differentiation using a library of siRNAs against cell cycle-regulatory genes. We discovered that knocking down expression of cyclin dependent kinase inhibitor 3 (CDKN3) showed the most potent effect in inducing neurite outgrowth, the morphological cell differentiation marker of neuroblastoma cells. We then demonstrated that CDKN3 knockdown increased expression of neuroblastoma molecular differentiation markers, neuron specific enolase (NSE), βIII-tubulin and growth associated protein 43 (GAP43). We further showed that CDKN3 knockdown reduced expression of cell proliferation markers Ki67 and proliferating cell nuclear antigen (PCNA), and reduced colony formation of neuroblastoma cells. More importantly, we observed a correlation of high tumor CDKN3 mRNA levels with poor patient survival in the investigation of public neuroblastoma patient datasets. In exploring the mechanisms that regulate CDKN3 expression, we found that multiple strong differentiation-inducing molecules, including miR-506-3p and retinoic acid, down-regulated CDKN3 expression. In addition, we found that N-Myc promoted CDKN3 expression at the transcriptional level by directly binding to the CDKN3 promoter. Furthermore, we found that CDKN3 and two additional differentiation-regulating cell cycle proteins identified in our HCS, CDC6 and CDK4, form an interactive network to promote expression of each other. In summary, we for the first time discovered the function of CDKN3 in regulating neuroblastoma cell differentiation and characterized the transcriptional regulation of CDKN3 expression by N-Myc in neuroblastoma cells. Our findings support that CDKN3 plays a role in modulating neuroblastoma cell differentiation and that overexpression of CDKN3 may contribute to neuroblastoma progression.

Nordihydroguaiaretic Acid Affects Undifferentiated and Differentiated Neuroblastoma Cells Differently through Mechanisms that Impact on Cell Viability.

We aimed to investigate the mechanisms involved in the neurotoxic effects of NDGA on differentiated and undifferentiated human neuroblastoma cells (MSN), assessing cell viability, changes in the actin cytoskeleton, cell migration and the expression of the 5-LOX enzyme and the inhibitor of cell cycle progression p21WAF1/CIP1. High expression and activity of the lipoxygenase enzyme (LOX) have been detected in several tumors, including neuroblastoma samples, suggesting the use of LOX inhibitors as potential therapy molecules. Among these, the natural compound nordihydroguaiaretic acid (NDGA) has been extensively tested as an antiproliferative drug against diverse types of cancer cells. In this study, we analyzed the toxic effect of NDGA on neuroblastoma cells at a dose that did not affect cell survival when they differentiated to a neuron-like phenotype and the potential mechanisms involved in the anticancer properties. We exposed human neuroblastoma cells (MSN) to different concentrations of NDGA before and after a differentiation protocol with retinoic acid and nerve growth factor and analyzed cell viability, cell migration, actin cytoskeleton morphology and the levels of the cell cycle inhibitor p21WAF1/CIP1 and 5-LOX. We found that differentiated human neuroblastoma cells are more resistant to NDGA than undifferentiated cells. The toxic effects of NDGA were accompanied by reduced cell migration, changes in actin cytoskeleton morphology, induction of p21WAF1/CIP1 and decreased levels of the 5- LOX enzyme. This study provides new evidence regarding the potential use of NDGA to induce cell death in human neuroblastoma.

Dyngo-4a Induces Neuroblastoma Cell Differentiation Through The AKT and ERK1/2 Pathway.

The aim of the study is to check whether dyngo-4a can inhibit neuroblastoma (NB) proliferation and induce NB cell differentiation Background: Dynamin plays a role in regulating neurotransmission, signaling pathways, nutrient uptake, and pathogen infection, enhancing cell proliferation, tumor invasion, and metastasis. Studies have reported that dyngo-4a, a dynamin inhibitor, can be used to identify potential biomarkers and promising novel therapeutic targets for cancer treatment. To our knowledge, no published reports are showing that dynamin inhibitors can reduce NB cell proliferation and induce differentiation. In this study, we report that dyngo-4a can inhibit NB proliferation and induce NB cell differentiation. In this study, mouse neuroblastoma (Neuro-2a) cells were cultured in the presence or absence of dyngo-4a or retinoic acid (RA), or in the presence of both dyngo-4a and RA, or in the presence of sequential administration of dyngo-4a and RA to compare the effects on the inhibition of cell proliferation and effects on neuroblastoma cell differentiation induction. The neural cell markers, Nestin and Tuj 1 (Neuron-specific class III beta-tubulin), were used to demonstrate that the differentiated cells have neuronal cell features. The phosphorylation of Protein Kinase B (AKT), extracellular signalregulated kinases1/2 (ERK1/2), and epidermal growth factor receptor (EGFR) were determined to examine the potential mechanisms of induced differentiation. Dyngo-4a or RA or dyngo-4a with subsequent RA administration induced Neuro-2a cell differentiation. However, RA with subsequent dyngo-4a administration results in almost total death of the Neuro-2a cells. The differentiation rate induced by dyngo-4a was significantly higher than the rate by RA treatment (72.5 ± 1.4% vs. 52.9 ± 3.1% with neuron features, P<0.05; 39.0 ± 0.8% vs. 29.9 ± 1.8% for axons under light microscopy, p<0.05). The differentiation rate of cells treated with dyngo-4a first, followed by RA, was greater than when they were added together (74.8 ± 3.8% vs. 10.6 ± 3.6%; 45.5 ± 1.6% vs. 12.4 ± 0.6%, p<0.01). Co-administration of dyngo-4a and RA at the same time diminished differentiation efficacy significantly. Dyngo-4a induced Neuro-2a cell differentiation and increased Tuj-1 positive staining by the 6th day post- treatment. Dyngo-4a also inhibited Neuro-2a cell proliferation in a dose-dependent manner. Regarding the mechanism, dyngo-4a treatment showed a significant increase in p-AKT and p-ERK1/2 but not in p-EGFR. At a level comparable to RA, dynamin inhibition with dyngo-4a lowers proliferation and causes differentiation of Neuro-2a mouse NB cells in vitro. The AKT pathway is activated by dynago- 4a, which results in differentiation. The combination of RA with dynago-4a reduces the efficiency of differentiation. The application of dynago-4a followed by RA, on the other hand, enhances the differentiating effect, implying alternative mechanistic roles in the process.

Differentiated neuroblastoma cells remain epigenetically poised for de-differentiation to an immature state.

Neuroblastoma is the most common extracranial solid tumor of childhood and accounts for a significant share of childhood cancer deaths. Prior studies utilizing RNA sequencing of bulk tumor populations showed two predominant cell states characterized by high and low expression of neuronal genes. Although cells respond to treatment by altering their gene expression, it is unclear whether this reflects shifting balances of distinct subpopulations or plasticity of individual cells. Using mouse and human neuroblastoma cell lines lacking MYCN amplification, we show that the antigen CD49b (also known as ITGA2) distinguishes these subpopulations. CD49b expression marked proliferative cells with an immature gene expression program, whereas CD49b-negative cells expressed differentiated neuronal marker genes and were non-cycling. Sorted populations spontaneously switched between CD49b expression states in culture, and CD49b-negative cells could generate rapidly growing, CD49b-positive tumors in mice. Although treatment with the chemotherapy drug doxorubicin selectively killed CD49b-positive cells in culture, the CD49b-positive population recovered when treatment was withdrawn. We profiled histone 3 (H3) lysine 27 acetylation (H3K27ac) to identify enhancers and super enhancers that were specifically active in each population and found that CD49b-negative cells maintained the priming H3 lysine 4 methylation (H3K4me1) mark at elements that were active in cells with high expression of CD49b. Improper maintenance of primed enhancer elements might thus underlie cellular plasticity in neuroblastoma, representing potential therapeutic targets for this lethal tumor.

Optimization of 3D Synthetic Scaffolds for Neuronal Tissue Engineering Applications.

The increasing prevalence of neurodegenerative diseases has spurred researchers to develop advanced 3D models that accurately mimic neural tissues. Hydrogels stand out as ideal candidates as their properties closely resemble those of the extracellular matrix. A critical challenge in this regard is to comprehend the influence of the scaffold's mechanical properties on cell growth and differentiation, thus enabling targeted modifications. In light of this, a synthesis and comprehensive analysis of acrylamide-based hydrogels incorporating a peptide has been conducted. Adequate cell adhesion and development is achieved due to their bioactive nature and specific interactions with cellular receptors. The integration of a precisely controlled physicochemical hydrogel matrix and inclusion of the arginine-glycine-aspartic acid peptide sequence has endowed this system with an optimal structure, thus providing a unique ability to interact effectively with biomolecules. The analysis fully examined essential properties governing cell behavior, including pore size, mechanical characteristics, and swelling ability. Cell-viability experiments were performed to assess the hydrogel's biocompatibility, while the incorporation of grow factors aimed to promote the differentiation of neuroblastoma cells. The results underscore the hydrogel's ability to stimulate cell viability and differentiation in the presence of the peptide within the matrix.

Alpha-secretase dependent nuclear localization of the amyloid-β precursor protein-binding protein Fe65 promotes DNA repair

Fe65 is a brain enriched adaptor protein involved in various cellular processes, including actin cytoskeleton regulation, DNA repair and transcription. A well-studied interacting partner of Fe65 is the transmembrane amyloid-β precursor protein (APP), which can undergo regulated intramembrane proteolysis (RIP). Following β- and γ-secretase-mediated RIP, the released APP intracellular domain (AICD) together with Fe65 can translocate to the nucleus and regulate transcription. In this study, we investigated if Fe65 nuclear localization can also be regulated by different α-secretases, also known to participate in RIP of APP and other transmembrane proteins. We found that in both Phorbol 12-myristate 13-acetate and all-trans retinoic acid differentiated neuroblastoma cells a strong negative impact on Fe65 nuclear localization, equal to the effect observed upon γ-secretase inhibition, could be detected following inhibition of all three (ADAM9, ADAM10 and ADAM17) α-secretases. Moreover, using the comet assay and analysis of Fe65 dependent DNA repair associated posttranslational modifications of histones, we could show that inhibition of α-secretase-mediated Fe65 nuclear translocation resulted in impaired capacity of the cells to repair DNA damage. Taken together this suggests that α-secretase processing of APP and/or other Fe65 interacting transmembrane proteins play an important role in regulating Fe65 nuclear translocation and DNA repair.

Cell Cycle Reactivation, at the Start of Neurodegeneration, Induced by Forskolin and Aniline in Differentiated Neuroblastoma Cells.

A characteristic hallmark of Alzheimer's disease (AD) is the intracellular accumulation of hyperphosphorylated tau protein, a phenomenon that appears to have associations with oxidative stress, double-stranded DNA breakage, and the de-condensation of heterochromatin. Re-entry into the cell division cycle appears to be involved in the onset of this neurodegenerative process. Indeed, the cell cycle cannot proceed regularly in the differentiated neurons leading to cell death. Here, we induced cell cycle reactivation in neuronal-like cells, obtained by neuroblastoma cells treated with retinoic acid, by exposure to forskolin or aniline. These compounds determine tau hyperphosphorylation or oxidative stress, respectively, resulting in the appearance of features resembling the start of neuronal degeneration typical of AD, such as tau hyperphosphorylation and re-entry into the cell cycle. Indeed, we detected an increased transcriptional level of cyclins and the appearance of a high number of mitotic cells. We also observed a delay in the initiation of the cell cycle when forskolin was co-administered with pituitary adenylate cyclase-activating polypeptide (PACAP). This delay was not observed when PACAP was co-administered with aniline. Our data demonstrate the relevance of tau hyperphosphorylation in initiating an ectopic cell cycle in differentiated neuronal cells, a condition that can lead to neurodegeneration. Moreover, we highlight the utility of neuroblastoma cell lines as an in vitro cellular model to test the possible neuroprotective effects of natural molecules.

Palbociclib releases the latent differentiation capacity of neuroblastoma cells

Neuroblastoma is the most common extracranial solid tumor in infants, arising from developmentally stalled neural crest-derived cells. Driving tumor differentiation is a promising therapeutic approach for this devastating disease. Here, we show that the CDK4/6 inhibitor palbociclib not only inhibits proliferation but induces extensive neuronal differentiation of adrenergic neuroblastoma cells. Palbociclib-mediated differentiation is manifested by extensive phenotypic and transcriptional changes accompanied by the establishment of an epigenetic program driving expression of mature neuronal features. Invivo palbociclib significantly inhibits tumor growth in mouse neuroblastoma models. Furthermore, dual treatment with retinoic acid resets the oncogenic adrenergic core regulatory circuit of neuroblastoma cells, further suppresses proliferation, and can enhance differentiation, altering gene expression in ways that significantly correlate with improved patient survival. We therefore identify palbociclib as a therapeutic approach to dramatically enhance neuroblastoma differentiation efficacy that could be used in combination with retinoic acid to improve patient outcomes.

Directed Neuronal Differentiation of SH-SY5Y Human Neuroblastoma Cells on 2D Matrices Containing Recombinant Spidroins Modified with Cell Adhesion Peptides

Directed Neuronal Differentiation of SH-SY5Y Human Neuroblastoma Cells on 2D Matrices Containing Recombinant Spidroins Modified with Cell Adhesion Peptides

Discovery of 5-sulfonyltetrazoles as neuroblastoma differentiation agents

Previously, we developed an innovative high-content screening (HCS) approach to quantify neuroblastoma cell differentiation based on neurite outgrowth, a morphological differentiation marker of neuroblastoma cells. Here, we report the utilization of this platform to identify 1-methyl-5-(ethylsulfonyl)-1H-tetrazole (3a) as a new neuroblastoma differentiation agent using the ChemBridge DiversetTM commercial synthetic small molecule compound library. We show that this activity can be extended to a group of analogues, which can be accessed via a short two-step synthetic sequence. A new analogue, 5-(allylsulfonyl)-1-methyl-1H-tetrazole (3c) was identified in this synthetic effort as a compound that has even more pronounced differentiation and cytotoxic activities than the original hit compound 3a.

In Vivo Identification of H3K9me2/H3K79me3 as an Epigenetic Barrier to Carcinogenesis.

The highly dynamic nature of chromatin's structure, due to the epigenetic alterations of histones and DNA, controls cellular plasticity and allows the rewiring of the epigenetic landscape required for either cell differentiation or cell (re)programming. To dissect the epigenetic switch enabling the programming of a cancer cell, we carried out wide genome analysis of Histone 3 (H3) modifications during osteogenic differentiation of SH-SY5Y neuroblastoma cells. The most significant modifications concerned H3K27me2/3, H3K9me2, H3K79me1/2, and H3K4me1 that specify the process of healthy adult stem cell differentiation. Next, we translated these findings in vivo, assessing H3K27, H3K9, and H3K79 methylation states in biopsies derived from patients affected by basalioma, head and neck carcinoma, and bladder tumors. Interestingly, we found a drastic decrease in H3K9me2 and H3K79me3 in cancer specimens with respect to their healthy counterparts and also a positive correlation between these two epigenetic flags in all three tumors. Therefore, we suggest that elevated global levels of H3K9me2 and H3K79me3, present in normal differentiated cells but lost in malignancy, may reflect an important epigenetic barrier to tumorigenesis. This suggestion is further corroborated, at least in part, by the deranged expression of the most relevant H3 modifier enzymes, as revealed by bioinformatic analysis. Overall, our study indicates that the simultaneous occurrence of H3K9me2 and H3K79me3 is fundamental to ensure the integrity of differentiated tissues and, thus, their combined evaluation may represent a novel diagnostic marker and potential therapeutic target.

Targeting TRIM24 promotes neuroblastoma differentiation and decreases tumorigenicity via LSD1/CoREST complex.

High-risk neuroblastoma (NB) still has an unfavorable prognosis and inducing NB differentiation is a potential strategy in clinical treatment, yet underlying mechanisms are still elusive. Here we identify TRIM24 as an important regulator of NB differentiation. Multiple datasets and clinical specimens were analyzed to define the role of TRIM24 in NB. The effects of TRIM24 on differentiation and growth of NB were determined by cell morphology, spheres formation, soft agar assay, and subcutaneous xenograft in nude mice. RNA-Seq and qRT-PCR were used to identify genes and pathways involved. Mass spectrometry and co-immunoprecipitation were used to explore the interaction of proteins. Trim24 is highly expressed in spontaneous NB in TH-MYCN transgenic mice and clinical NB specimens. It is associated with poor NB differentiation and unfavorable prognostic. Knockout of TRIM24 in neuroblastoma cells promotes cell differentiation, reduces cell stemness, and inhibits colony formation in soft agar and subcutaneous xenograft tumor growth in nude mice. Mechanistically, TRIM24 knockout alters genes and pathways related to neural differentiation and development by suppressing LSD1/CoREST complex formation. Besides, TRIM24 knockout activates the retinoic acid pathway. Targeting TRIM24 in combination with retinoic acid (RA) synergistically promotes NB cell differentiation and inhibits cell viability. Our findings demonstrate that TRIM24 is critical for NB differentiation and suggest that TRIM24 is a promising therapeutic target in combination with RA in NB differentiation therapy.

A Glimpse into the Genome-wide DNA Methylation Changes in 6-hydroxydopamine-induced In Vitro Model of Parkinson's Disease.

A cell-based model of Parkinson's disease (PD) is a well-established in vitro experimental prototype to investigate the disease mechanism and therapeutic approach for a potential anti-PD drug. The SH-SY5Y human neuroblastoma cells and 6-OHDA combo is one of the many neurotoxininduced neuronal cell models employed in numerous neuroscience-related research for discovering neuroprotective drug compounds. Emerging studies have reported a significant correlation between PD and epigenetic alterations, particularly DNA methylation. However, the DNA methylation changes of PD-related CpG sites on the 6-OHDA-induced toxicity on human neuronal cells have not yet been reported. We performed a genome-wide association study (GWAS) using Infinium Epic beadchip array surveying 850000 CpG sites in differentiated human neuroblastoma cells exposed to 6-OHDA. We identified 236 differentially methylated probes (DMPs) or 163 differentially methylated regions (DMRs) in 6-OHDA treated differentiated neuroblastoma cells than the untreated reference group with p<0.01, Δbeta cut-off of 0.1. Among 236 DMPs, hypermethylated DMPs are 110 (47%), whereas 126 (53%) are hypomethylated. Our bioinformatic analysis revealed 3 DMRs that are significantly hypermethylated and associated with neurological disorders, namely AKT1, ITPR1 and GNG7. This preliminary study demonstrates the methylation status of PD-related CpGs in the 6-OHDA-induced toxicity in the differentiated neuroblastoma cells model.

Chlorogenic Acid Induced Neuroblastoma Cells Differentiation via the ACAT1-TPK1-PDH Pathway.

Chlorogenic acid (CHA) has been shown to have substantial biological activities, including anti-inflammatory, antioxidant, and antitumor effects. However, the pharmacological role of CHA in neuroblastoma has not yet been assessed. Neuroblastoma is a type of cancer that develops in undifferentiated sympathetic ganglion cells. This study aims to assess the antitumor activity of CHA against neuroblastoma and reveal its mechanism of action in cell differentiation. Be(2)-M17 and SH-SY5Y neuroblastoma cells were used to confirm the differentiation phenotype. Subcutaneous and orthotopic xenograft mouse models were also used to evaluate the antitumor activity of CHA. Seahorse assays and metabolomic analyses were further performed to investigate the roles of CHA and its target ACAT1 in mitochondrial metabolism. CHA induced the differentiation of Be(2)-M17 and SH-SY5Y neuroblastoma cells in vivo and in vitro. The knockdown of mitochondrial ACAT1, which was inhibited by CHA, also resulted in differentiation characteristics in vivo and in vitro. A metabolomic analysis revealed that thiamine metabolism was involved in the differentiation of neuroblastoma cells. These results provide evidence that CHA shows good antitumor activity against neuroblastoma via the induction of differentiation, by which the ACAT1-TPK1-PDH pathway is involved. CHA is a potential drug candidate for neuroblastoma therapy.