Role In Neuroblastoma Tumorigenesis Research Articles

Inhibition of PIM Kinases Promotes Neuroblastoma Cell Differentiation to a Neuronal Phenotype

BackgroundNeuroblastoma arises from aberrancies in neural stem cell differentiation. PIM kinases contribute to cancer formation, but their precise role in neuroblastoma tumorigenesis is poorly understood. In the current study, we evaluated the effects of PIM kinase inhibition on neuroblastoma differentiation. MethodsVersteeg database query assessed the correlation between PIM gene expression and the expression of neuronal stemness markers and relapse free survival. PIM kinases were inhibited with AZD1208. Viability, proliferation, motility were measured in established neuroblastoma cells lines and high-risk neuroblastoma patient-derived xenografts (PDXs). qPCR and flow cytometry detected changes in neuronal stemness marker expression after AZD1208 treatment. ResultsDatabase query showed increased levels of PIM1, PIM2, or PIM3 gene expression were associated with higher risk of recurrent or progressive neuroblastoma. Increased levels of PIM1 were associated with lower relapse free survival rates. Higher levels of PIM1 correlated with lower levels of neuronal stemness markers OCT4, NANOG, and SOX2. Treatment with AZD1208 resulted in increased expression of neuronal stemness markers. ConclusionsInhibition of PIM kinases differentiated neuroblastoma cancer cells toward a neuronal phenotype. Differentiation is a key component of preventing neuroblastoma relapse or recurrence and PIM kinase inhibition provides a potential new therapeutic strategy for this disease.

The RUNX Family Defines Trk Phenotype and Aggressiveness of Human Neuroblastoma through Regulation of p53 and MYCN.

The Runt-related transcription factor (RUNX) family, which is essential for the differentiation of cells of neural crest origin, also plays a potential role in neuroblastoma tumorigenesis. Consecutive studies in various tumor types have demonstrated that the RUNX family can play either pro-tumorigenic or anti-tumorigenic roles in a context-dependent manner, including in response to chemotherapeutic agents. However, in primary neuroblastomas, RUNX3 acts as a tumor-suppressor, whereas RUNX1 bifunctionally regulates cell proliferation according to the characterized genetic and epigenetic backgrounds, including MYCN oncogenesis. In this review, we first highlight the current knowledge regarding the mechanism through which the RUNX family regulates the neurotrophin receptors known as the tropomyosin-related kinase (Trk) family, which are significantly associated with neuroblastoma aggressiveness. We then focus on the possible involvement of the RUNX family in functional alterations of the p53 family members that execute either tumor-suppressive or dominant-negative functions in neuroblastoma tumorigenesis. By examining the tripartite relationship between the RUNX, Trk, and p53 families, in addition to the oncogene MYCN, we endeavor to elucidate the possible contribution of the RUNX family to neuroblastoma tumorigenesis for a better understanding of potential future molecular-based therapies.

Serine Hydroxyl-Methyltransferase 2 Inhibition Induces Differentiation and Impairs Tumorigenesis in High-Risk Neuroblastoma Cells

INTRODUCTION: High-risk neuroblastoma (NB) is associated with SHMT2 (serine hydroxyl-methyltransferase 2) expression, which is upregulated in several cancers and is associated with tumor aggressiveness. We have previously shown that SHMT2 silencing impairs cellular proliferation, migration, and colony formation, and treatment with a SHMT2 inhibitor induces differentiation in vitro. The stem cell marker genes CD133, CKIT, and NOTCH1 have been shown to downregulate differentiation in NB. We hypothesize that treatment with SHIN2, a novel SHMT2 inhibitor, will induce differentiation and impair tumorigenesis via downregulation of CD133, CKIT, and NOTCH1. METHODS: Human NB cell lines, BE(2)-C and SK-N-SH, were used for the study. Cells were treated with SHIN2 for 24 hours and quantitative reverse transcription polymerase chain reaction and immunoblotting were performed. Cellular proliferation was assessed using CCK-8 assays at 24 and 72 hours after SHIN2 treatment. Wound healing assays were performed to assess cellular migration at 24 hours after SHIN2 treatment. RESULTS: SHIN2 decreased CD133, CKIT, and NOTCH1 mRNA and protein expression by 1.2- to 2.5-fold and 1.1- to 1.4-fold, respectively, in both cell lines. SHIN2 significantly impaired cellular proliferation in both cell lines at 24 and 72 hours. SHIN2 treatment significantly increased wound gap and closure rate in both cell lines (Figs. 1A–1C).Figure 1CONCLUSION: SHMT2 plays an important role in NB tumorigenesis. Inhibition of SHMT2 with SHIN2 impairs cellular proliferation and cellular migration, and it induces differentiation via downregulation of stem cell genes CD133, CKIT, and NOTCH1, in vitro. The use of SHIN2 may serve as a novel therapeutic strategy in high-risk NB.

Abstract 1678: Single-cell landscape of the TH-MYCN neuroblastoma mouse model

Abstract Background and aims: Neuroblastoma (NB) is the most common and deadly malignancy of infancy. It is an embryonal neuroendocrine tumor arising from the neural crest lineage. Amplification of MYCN is a frequent genetic abnormality, found in approximately 20% of NB tumors, and represents a critical stratifying prognostic marker. Given that MYCN plays a key role in NB tumorigenesis and aggressiveness, the TH-MYCN transgenic mouse model is widely used in NB research. This model is characterized by the overexpression of MYCN in neuroectodermal cells, driven by tyrosine hydroxylase (TH) promoter. This overexpression gives rise to tumors exclusively in the sympathoadrenal system, reflecting human NB features. We aimed to explore the transcriptional landscape of TH-MYCN tumors by single-cell RNA sequencing (scRNA-seq). Methods: Tumors from three homozygous and two hemizygous TH-MYCN mice were dissociated into single cells, profiled by scRNA-seq (Chromium 3’ v2 kit, 10x Genomics), sequenced on the NextSeq platform (Illumina). A total of 41666 single cells were analyzed using pagoda2 and conos. TH-MYCN tumor single-cell transciptomes were further aligned with previously published transcriptomes from the adrenal gland of three mice at embryonic day (E)13.5. To evaluate malignant tumor cells, copy number variants (CNV) were analyzed using inferCNV. Immunohistochemistry was used to validate the bioinformatically predicted cell types. Results: scRNA-seq of tumors from TH-MYCN mice revealed 16 major cell populations, spanning stromal, immune and tumor compartments. MYCN+ tumor cells predominantly resemble sympathetic neuroblasts (Isl1, Stmn1, Nefl, Nefm), while MYCN+ chromaffin-like tumor cells (Pnmt, Cdkn1c, Th, Dbh) were rare. Immunofluorescence stainings confirmed that tumors predominantly consist of proliferating sympathetic neuroblasts. Tumor cell nests were surrounded by supportive stromal and immune cells. The adrenergic tumor cell composition observed in TH-MYCN tumors is highly reminiscent of human NB. Interestingly, we discovered a cluster of immature adrenergic tumor cells aligning with normal fetal cells transitioning from bridge to chromaffin state (characterized by Sox11, Atrx, Xist, Ptprs, Tcf4). Distinct genomic aberrations, particularly unique chromosomal deletions, were found in the immature adrenergic tumor cells relative to their sympathoblast and chromaffin tumor cell counterparts. Conclusions: Examining the cellular composition of TH-MYCN tumors at a transcriptional level gives a detailed view of tumor cell heterogeneity at an unprecedented resolution. This comprehensive atlas of TH-MYCN tumors provides a resource for unmasking novel therapeutic targets. Citation Format: Bethel Tesfai Embaie, Adele Alchahin, Thale Kristin Olsen, Hirak Sarkar, Shenglin Mei, Ninib Baryawno. Single-cell landscape of the TH-MYCN neuroblastoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1678.

Induction of serine hydroxymethyltransferase 2 promotes tumorigenesis and metastasis in neuroblastoma.

High-risk neuroblastoma (NB) remains an extremely difficult subgroup to cure and is associated with MYCN amplification. Serine hydroxymethyltransferase 2 (SHMT2) regulates serine metabolism in a myc-dependent manner; it is upregulated in several cancers and is associated with tumor aggressiveness. Akt-2, an important regulator of MYCN via the PI3K/Akt pathway, induces metastatic potential in NB. The association between SHMT2 and PI3K/Akt in hepatocyte regeneration has been well established but its mechanistic interaction in cancer has yet to be clearly elucidated. Herein, we evaluated the exact role of SHMT2 on the PI3K/Akt pathway, in addition to NB tumorigenesis and metastatic potential in vitro. SHMT2 gene expression and overall survival (OS) were assessed. Two human NB cell lines were examined. SHMT2 silencing and overexpression were performed. The downstream effects were analyzed with immunoblotting, RT-qPCR and functional assays were performed. We found SHMT2 gene expression is associated with decreased OS and MYCN amplification. SHMT2 protein and mRNA expression are increased in MYCN-amplified cells. SHMT2 expression has a direct interaction with Akt-2 and MYCN. Induction of SHMT2 increased cellular proliferation, colony formation and cellular migration and SHMT2 expression was increased in metastatic NB cells. We conclude that SHMT2 regulates N-Myc via phosphorylation of Akt-2 and plays an important role in NB tumorigenesis by contributing to cell growth, migration, colony formation and metastasis in vitro.

Role of Energy Metabolism in the Progression of Neuroblastoma.

Neuroblastoma is a common childhood cancer possessing a significant risk of death. This solid tumor manifests variable clinical behaviors ranging from spontaneous regression to widespread metastatic disease. The lack of promising treatments calls for new research approaches which can enhance the understanding of the molecular background of neuroblastoma. The high proliferation of malignant neuroblastoma cells requires efficient energy metabolism. Thus, we focus our attention on energy pathways and their role in neuroblastoma tumorigenesis. Recent studies suggest that neuroblastoma-driven extracellular vesicles stimulate tumorigenesis inside the recipient cells. Furthermore, proteomic studies have demonstrated extracellular vesicles (EVs) to cargo metabolic enzymes needed to build up a fully operative energy metabolism network. The majority of EV-derived enzymes comes from glycolysis, while other metabolic enzymes have a fatty acid β-oxidation and tricarboxylic acid cycle origin. The previously mentioned glycolysis has been shown to play a primary role in neuroblastoma energy metabolism. Therefore, another way to modify the energy metabolism in neuroblastoma is linked with genetic alterations resulting in the decreased activity of some tricarboxylic acid cycle enzymes and enhanced glycolysis. This metabolic shift enables malignant cells to cope with increasing metabolic stress, nutrition breakdown and an upregulated proliferation ratio.

Common variants upstream of MLF1 at 3q25 and within CPZ at 4p16 associated with neuroblastoma

Neuroblastoma is a cancer of the developing sympathetic nervous system that most commonly presents in young children and accounts for approximately 12% of pediatric oncology deaths. Here, we report on a genome-wide association study (GWAS) in a discovery cohort or 2,101 cases and 4,202 controls of European ancestry. We identify two new association signals at 3q25 and 4p16 that replicated robustly in multiple independent cohorts comprising 1,163 cases and 4,396 controls (3q25: rs6441201 combined P = 1.2x10-11, Odds Ratio 1.23, 95% CI:1.16–1.31; 4p16: rs3796727 combined P = 1.26x10-12, Odds Ratio 1.30, 95% CI: 1.21–1.40). The 4p16 signal maps within the carboxypeptidase Z (CPZ) gene. The 3q25 signal resides within the arginine/serine-rich coiled-coil 1 (RSRC1) gene and upstream of the myeloid leukemia factor 1 (MLF1) gene. Increased expression of MLF1 was observed in neuroblastoma cells homozygous for the rs6441201 risk allele (P = 0.02), and significant growth inhibition was observed upon depletion of MLF1 (P < 0.0001) in neuroblastoma cells. Taken together, we show that common DNA variants within CPZ at 4p16 and upstream of MLF1 at 3q25 influence neuroblastoma susceptibility and MLF1 likely plays an important role in neuroblastoma tumorigenesis.

Growth, progression and chromosome instability of Neuroblastoma: a new scenario of tumorigenesis?

BackgroundNeuroblastoma is a pediatric cancer with a low survival rate of patients with metastatic stage 4 disease. Tumor aggressiveness and progression have been associated with structural copy number variations (CNVs) that are observed in malignant cells. In contrast, localized Neuroblastomas, which are associated with a low number of structural CNVs but frequent numerical CNVs, are less aggressive, and patients have good outcomes. Finally, whole-genome and whole-exome sequencing of Neuroblastoma tissues have shown few damaging mutations in these tumors.ConclusionsIn the present report it is proposed that chromosome instability (CIN) plays a major role in Neuroblastoma tumorigenesis and that CIN is already present in the early phases of tumor development. High CIN can promote several types of chromosomal damage including chromothripsis, gene deletion, amplification and rearrangements, which deregulate gene expression. Indeed, gene rearrangements have been reported as a new scenario in the development of Neuroblastoma, which supports the hypothesis that CIN is an early step preliminary to the late catastrophic events leading to tumor development.

A combined gene expression and functional study reveals the crosstalk between N-Myc and differentiation-inducing microRNAs in neuroblastoma cells.

MYCN amplification is the most common genetic alteration in neuroblastoma and plays a critical role in neuroblastoma tumorigenesis. MYCN regulates neuroblastoma cell differentiation, which is one of the mechanisms underlying its oncogenic function. We recently identified a group of differentiation-inducing microRNAs. Given the demonstrated inter-regulation between MYCN and microRNAs, we speculated that MYCN and the differentiation-inducing microRNAs might form an interaction network to control the differentiation of neuroblastoma cells. In this study, we found that eight of the thirteen differentiation-inducing microRNAs, miR-506-3p, miR-124-3p, miR-449a, miR-34a-5p, miR-449b-5p, miR-103a-3p, miR-2110 and miR-34b-5p, inhibit N-Myc expression by either directly targeting the MYCN 3′UTR or through indirect regulations. Further investigation showed that both MYCN-dependent and MYCN-independent pathways play roles in mediating the differentiation-inducing function of miR-506-3p and miR-449a, two microRNAs that dramatically down-regulate MYCN expression. On the other hand, we found that N-Myc inhibits the expression of multiple differentiation-inducing microRNAs, suggesting that these miRNAs play a role in mediating the function of MYCN. In examining the published dataset collected from clinical neuroblastoma specimens, we found that expressions of two miRNAs, miR-137 and miR-2110, were significantly anti-correlated with MYCN mRNA levels, suggesting their interactions with MYCN play a clinically-relevant role in maintaining the MYCN and miRNA expression levels in neuroblastoma. Our findings altogether suggest that MYCN and differentiation-inducing miRNAs form an interaction network that play an important role in neuroblastoma tumorigenesis through regulating cell differentiation.

Abstract 3283: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis

Abstract The formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor with pattern recognition properties and is mainly expressed by myeloid cells. It is involved in a broad range of host defense mechanisms and a variety of host-derived agonists of FPR1 have been identified, including formyl peptides released from disrupted mitochondria of necrotic cells. In the present study, we demonstrate the expression of FPR1 in 7 different neuroblastoma cell lines and in primary tumors. Furthermore, FPR1 is expressed at increased levels in high stage tumors. Addition of the FPR1 agonist N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) to neuroblastoma cells in vitro caused enhanced proliferative activity, increase of intracellular calcium response and activation of STAT3 and MAPK/ERK signal transduction pathways. All these signal transduction events were abrogated by the use of Cyclosporin H, a specific FPR1 antagonist. To assess the significance of this receptor in vivo, we developed a set of neuroblastoma cell clones with different expression levels of FPR1. Xenograft models showed that cells with overexpression of the receptor developed tumors significantly faster compared to control group. Our findings so far suggest that FPR1 may play a significant role in neuroblastoma tumorigenesis and that therapeutic intervention of the FPR1 pathway may be an important clinical strategy in neuroblastoma therapy. Citation Format: Igor Snapkov, Carl Otto Öqvist, Yngve Anton Figenschau, Per Kogner, John Inge Johnsen, Baldur Sveinbjørnsson. The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3283. doi:10.1158/1538-7445.AM2015-3283

Abstract 1946: Activating transcription factor 5 (ATF5) in highly expressed in Stage 4, MYCN-amplified neuroblastoma

Abstract Background: Activating transcription factor 5 (ATF5), a member of the ATF/CREB family transcription factor, has been shown to regulate the differentiation of neuroprogenitor cells into mature neurons. ATF5 has also been implicated in progression of glioblastoma multiforme. We have recently identified ATF5 as a potential synthetic lethal gene to MYCN-amplified neuroblastoma (NB) through a whole genome shRNA screen. In the present study we investigate the role of ATF5 in NB tumorigenesis. Methods: We analyzed the human NB patient data GEO (GSE45480, Kocak et al.) to investigate and correlate the expression of ATF5 with different stages of NB. Immunohistochemical staining for ATF5 was performed on primary human NB tumors. Quantitative RT-PCR and western blot for ATF5 were conducted on a panel of NB cell lines. Immunocytochemistry was performed to determine the intercellular localization of ATF5. ATF5 was silenced by a tetracycline inducible (tet-on) shRNA construct in BE(2)-C and SK-N-DZ cells. The cells were subjected to cell viability assay and soft agar colony formation assay to assess the function of ATF5. Results: Analysis of NB patient data GEO (GSE45480) from Kocak et al., demonstrated that ATF5 is highly expressed in Stage 4 MYCN-amplified tumors compared to Stage 4, MYCN-non-amplified tumors (P = 2.56e-10) or Stage 1 tumors (P = 7.30e-18). ATF5 is also highly expressed in Stage 4, MYCN-non-amplified tumors vs. Stage 1 tumors (P = 2.8e-7). High expression of ATF5 strongly correlated with poor survival of NB patients (P = 2.88e-6, NCI Oncogenomics, Obertheur data set). Quantitative PCR detected ATF5 transcript in all eight NB cell lines tested. Western blot analyses demonstrated high expression of ATF5 in the MYCN-amplified neuroblastoma cell lines BE(2)-C, SK-N-DZ, and IMR32. Nuclear ATF5 staining was detected in all three cell lines as well as in Stage 4 NB tumors. Silencing of ATF5 reduced BE(2)-C cell viability to 30%, 48 hr after the addition of doxycycline. Silencing of ATF5 significantly reduced the colony formation of BE(2)-C and SK-N-DZ cells in soft agar (p&amp;lt;0.05) Conclusions: Our study demonstrates that ATF5 is highly expressed in Stage 4 MYCN-amplified tumors, and that silencing of ATF5 inhibits proliferation. These results suggest that ATF5 has a role in NB tumorigenesis and it represents a potential therapeutic target in MYCN-amplified neuroblastoma. Citation Format: Debarshi Banerjee, Shuobo Zhang, Gonzalo Lopez, Andrea Califano, Angela Kadenhe-Chiweshe, Darrell Yamashiro. Activating transcription factor 5 (ATF5) in highly expressed in Stage 4, MYCN-amplified neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1946. doi:10.1158/1538-7445.AM2015-1946

Deoxyhypusine synthase (DHPS) inhibitor GC7 induces p21/Rb-mediated inhibition of tumor cell growth and DHPS expression correlates with poor prognosis in neuroblastoma patients.

Neuroblastoma (NB) is an aggressive pediatric malignancy that typically occurs in infants and children under the age of 5 years. High-stage tumors relapse frequently even after aggressive multimodal treatment, resulting in therapy resistance and eventually in patient death. Clearly, new biologically-targeted drugs are needed that more efficiently suppress tumor growth and prevent relapse. We and others previously showed that polyamines such as spermidine play an essential role in NB tumorigenesis and that DFMO, an inhibitor of the central polyamine synthesis gene ODC, is effective in vitro and in vivo, prompting its evaluation in NB clinical trials. However, the specific molecular actions of polyamines remain poorly defined. Spermidine and deoxyhypusine synthase (DHPS) are essential components in the hypusination-driven post-translational activation of eukaryotic initiation factor 5A (eIF5A). We assessed the role of DHPS in NB and the impact of its inhibition by N(1)-guanyl-1,7-diaminoheptane (GC7) on tumor cell growth using cell proliferation assays, Western blot, immunofluorescence microscopy, and Affymetrix micro-array mRNA expression analyses in NB tumor samples. We found that GC7 inhibits NB cell proliferation in a dose-dependent manner, through induction of the cell cycle inhibitor p21 and reduction of total and phosphorylated Rb proteins. Strikingly, high DHPS mRNA expression correlated significantly with unfavorable clinical parameters, including poor patient survival, in a cohort of 88 NB tumors (all P < 0.04). These results suggest that spermidine and DHPS are key contributing factors in NB tumor proliferation through regulation of the p21/Rb signaling axis.

Abstract 3976: Expression of formyl peptide receptor 1 (FPR1) in neuroblastoma: Implications in tumorigenesis

Abstract The formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor with pattern recognition properties. It is mainly expressed by myeloid cells and is involved in a broad range of host defense mechanisms. However, a variety of host-derived agonists of FPR1 have been identified, including formyl peptides released from disrupted mitochondria of necrotic cells. In the present study, we demonstrate that FPR1 is expressed in neuroblastoma cell lines and primary tumors and high expression is correlated with poor overall survival. Addition of the FPR1 agonist N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) to neuroblastoma cells in vitro causes enhanced proliferative activity, increase of intracellular calcium response and activation of STAT3 and MAPK/Erk signaling pathways. Currently, clonal neuroblastoma cell line with inducible activity of FPR1 gene is under testing in a mouse model to assess the significance of this receptor in vivo. Our findings so far suggest that FPR1 may play a significant role in neuroblastoma tumorigenesis and that therapeutic intervention of the FPR1 pathway may become an important clinical strategy in neuroblastoma therapy. Citation Format: Igor Snapkov, Per Kogner, John-Inge Johnsen, Baldur Sveinbjørnsson. Expression of formyl peptide receptor 1 (FPR1) in neuroblastoma: Implications in tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3976. doi:10.1158/1538-7445.AM2014-3976

Abstract 3323: PRAF2 regulates Rab5-dependent endocytic trafficking of EGFR in neuroblastoma

Abstract Receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), mediate extracellular signaling that activates downstream cell proliferation, differentiation, and migration. Through ligand stimulation, clathrin-mediated endocytosis regulates the EGFR signal transduction pathway. Prenylated Rab acceptor 1 domain family, membrane 2 (PRAF2) is a novel 19-kDa protein with a prenylated Rab acceptor 1 (PRA1) motif and four transmembrane-spanning domains. Similar to PRAF1 and PRAF3, PRAF2 belongs to the PRAF family of vesicle transport-associated proteins. Our lab identified PRAF2 as a candidate prognostic marker of neuroblastic tumors. Using RNA interference, we showed that PRAF2 plays an essential role in neuroblastoma tumorigenesis and metastasis. In the present study, we examined the function of PRAF2 in EGFR endocytic trafficking. We generated stable PRAF2-knockdown (SK-shPRAF2) and PRAF2-overexpressing (SK-PRAF2) neuroblastoma SK-N-SH cell lines and stimulated both cell lines with EGF (10 ng/ml) for various time periods in order to determine the role of PRAF2 in EGFR endocytic trafficking. As judged by immunofluorescence microscopy, we detected larger Rab5-positive punctuate structures in PRAF2-overexpressing SK-PRAF2 cells compared to PRAF2-knockdown SK-shPRAF2 cells or control cells, after 15 min EGF stimulation. After EGF treatment, PRAF2 clearly colocalized with Rab5-positive punctae in SK-PRAF2 cells, suggesting that PRAF2 interacts with Rab5, a critical regulator for receptor endocytosis and early endosome fusion. Moreover, using a receptor internalization assay, we observed a delay in the internalization of EGFR in EGF-treated SK-shPRAF2 cells, in contrast to SK-PRAF2 and control cells. This is in support of our immunofluorescence microscopy results in which Rab5-postive punctae distribution is observed mostly along the membrane of the EGF-treated SK-shPRAF2 cells. Notably, the level of phosphorylation of Akt (Ser473) transiently elevated in EGF-treated SK-PRAF2 cells after 15 min and diminished thereafter, suggesting that overexpression of PRAF2 accelerates the EGFR internalization and degradation. Based on our findings, we propose that PRAF2 affects EGFR endocytosis by regulating early endosome trafficking and Akt activation in neuroblastoma. Citation Format: Lisette P. Yco, Andre S. Bachmann. PRAF2 regulates Rab5-dependent endocytic trafficking of EGFR in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3323. doi:10.1158/1538-7445.AM2014-3323

PRAF2 stimulates cell proliferation and migration and predicts poor prognosis in neuroblastoma

Prenylated Rab acceptor 1 domain family, member 2 (PRAF2) is a novel 19-kDa protein with four transmembrane-spanning domains that belongs to the PRAF protein family. Neuroblastoma (NB) is the most common malignant extracranial solid tumor of childhood that originates in primitive cells of the developing sympathetic nervous system. We investigated the correlation of PRAF2 mRNA expression to NB clinical and genetic parameters using Affymetrix expression analysis of a series of 88 NB tumors and examined the functional role of PRAF2 in an NB cell line using RNA interference. We found that high PRAF2 expression is significantly correlated to several unfavorable NB characteristics: MYCN amplification, high age at diagnosis, poor outcome and high INSS stage. The shRNA-mediated PRAF2 downregulation in the SK-N-SH NB cell line resulted in decreased cellular proliferation, migration and matrix-attachment. These findings were confirmed in NB patient tumor samples, where high PRAF2 expression is significantly correlated to bone and bone marrow metastasis, the main cause of death in NB patients. The present study shows that PRAF2 plays an essential role in NB tumorigenesis and metastasis.

Time-Lapse Imaging of Neuroblastoma Cells to Determine Cell Fate upon Gene Knockdown

Neuroblastoma is the most common extra-cranial solid tumor of early childhood. Standard therapies are not effective in case of poor prognosis and chemotherapy resistance. To improve drug therapy, it is imperative to discover new targets that play a substantial role in tumorigenesis of neuroblastoma. The mitotic machinery is an attractive target for therapeutic interventions and inhibitors can be developed to target mitotic entry, spindle apparatus, spindle activation checkpoint, and mitotic exit. We present an elaborate analysis pipeline to determine cancer specific therapeutic targets by first performing a focused gene expression analysis to select genes followed by a gene knockdown screening assay of live cells. We interrogated gene expression studies of neuroblastoma tumors and selected 240 genes relevant for tumorigenesis and cell cycle. With these genes we performed time-lapse screening of gene knockdowns in neuroblastoma cells. We classified cellular phenotypes and used the temporal context of the perturbation effect to determine the sequence of events, particularly the mitotic entry preceding cell death. Based upon this phenotype kinetics from the gene knockdown screening, we inferred dynamic gene functions in mitosis and cell proliferation. We identified six genes (DLGAP5, DSCC1, SMO, SNRPD1, SSBP1, and UBE2C) with a vital role in mitosis and these are promising therapeutic targets for neuroblastoma. Images and movies of every time point of all screened genes are available at https://ichip.bioquant.uni-heidelberg.de.

Intersectin 1 is required for Neuroblastoma tumorigenesis

Neuroblastoma (NB) is a malignant tumor deriving from the neural crest that is responsible for 13% of cancer‐related deaths in children. Phosphoinositide 3‐kinases (PI3Ks) play a central role in NB tumorigenesis. PI3Ks activate AKT, which regulates survival and stabilizes the MYCN oncogene. Both MYCN and AKT are markers for poor prognosis in NBs. Inhibiting PI3Ks with pan‐inhibitors blocks malignant progression in vivo. These inhibitors have high toxicity that limits their use in NB treatment. Though there are three classes of PI3Ks, only Class I has been implicated in NB. Class 2 beta PI3K (PI3KC2β) is overexpressed in cancer and involved in adherent and anchorage‐independent growth of cancer cells. We found that the scaffold protein intersectin 1 (ITSN1) interacts with PI3KC2β and regulates its activation and Akt activation in mouse NB lines. Thus, we examined the role of the ITSN1‐PI3KC2β pathway in human NB tumorigenesis. We found that stable silencing of ITSN1 in human NB cells attenuates their growth both in soft agar assays in vitro and in xenograft tumor assays in vivo. Furthermore, PI3KC2β overexpression restores anchorage‐independent growth of ITSN1‐depleted NB cells suggesting that PI3KC2β mediates ITSN1's tumorigenic phenotype. This study reveals a new pathway, i.e., ITSN1‐ PI3KC2β is involved in regulating the pathogenesis of NBs.

Activation of sonic hedgehog signaling pathway in S-type neuroblastoma cell lines

The effects of Sonic hedgehog (Shh) signaling pathway activation on S-type neuroblastoma (NB) cell lines and its role in NB tumorigenesis were investigated. Immunohistochemistry was used to detect the expression of Shh pathway components-Patched1 (PTCH1) and Gli1 in 40 human primary NB samples. Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Gli1 in three kinds of S-type NB cell lines (SK-N-AS, SK-N-SH and SHEP1), respectively. Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway. S-type NB cell lines were treated with different concentrations of Shh or/and cyclopamine for different durations. Cell viability was measured by using MTT method. Apoptosis rate and cell cycle were assayed by flow cytometry. The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice. High-level expression of PTCH1 and Gli1 was detected in both NB samples and S-type NB cell lines. Cyclopamine decreased the survival rate of the three cell lines while Shh increased it, and the inhibition effects of cyclopamine could be partially reversed by shh pre-treatment. Cyclopamine induced the cell apoptosis and the cell cycle arrest in G0/G1 phase, while Shh induced the reverse effects and could partially prevent effects of cyclopamine. Cyclopamine could also inhibit the growth of NB in vivo. Our studies revealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle, suggesting a new therapeutic approach to NB.

Expression and Methylation Pattern of p16 in Neuroblastoma Tumorigenesis

Understanding migration, population and differentiation of primordial neural crest cells will help in evolving biology of neuroblastoma. P16 is a tumour suppressor gene contributing in cell cycle arrest as cyclin dependent kinase inhibitor. Methylation is an important mechanism for silencing tumor suppressor genes. The aim of this study was to evaluate the role of p16 and its methylation pattern in neuroblastoma tumorigenesis. This study included 23 cases (11 male; 12 female) and 31 samples from archival paraffin embedded tissues. P16 was studied in 5 samples of normal adrenal medullar tissue, 5 samples of adrenal tissue including blastic rests, 5 samples of neuroblastoma in situ tissue and in 8 samples of neuroblastoma tissues primary and after chemotherapy in each group. The adrenal gland tissues were obtained from paediatric autopsy cases. Expression of p16 was searched by immunohistochemistry. Methylation specific PCR was used to detect the methylation rate of p16. The age range of autopsy cases was between 20 weeks of foetal age and 36 months of infant age. The mean age of neuroblastoma cases was 45 months. P16 expression was positive in normal adrenal tissues, in one of 5 samples of adrenal blastic rest tissue and in all of samples of after chemotherapy; while no expression was observed in neuroblastoma and neuroblastoma in situ tissues. P16 methylation was observed in samples of neuroblastoma in situ and primary neuroblastoma tissues. Our results suggest that p16 and its methylation seems to play role in neuroblastoma tumorigenesis and in the migration, population and differentiation of primordial neural crest cells. Inhibitors of DNA methylation may provide a useful tool for restoring p16 activity in neuroblastoma treatment.

Gene expression profiling of 1p35-36 genes in neuroblastoma.

Deletion of the chromosome 1p36 region is a frequent abnormality in neuroblastoma. To gain further insights into the role of this alteration in oncogenesis, we have constructed a specific cDNA microarray representing most known genes and ESTs from the 1p35-36 region and analysed the expression profiles of 15 neuroblastoma cell lines and 28 neuroblastoma tumours. Hierarchical clustering using expression levels of 320 cDNAs from 1p35-36 separated localized or 4S cases without 1p deletion from advanced stages and cell lines. Supervised learning classification enabled to predict reliably the status of chromosome 1p according to its expression profile. Around 15% of the genes or ESTs presented a significantly decreased expression in samples with 1p deletion as compared to 1p-normal samples suggesting that 1p deletion results in a gene dosage effect on a subset of genes critical for the development of 1p-deleted neuroblastoma. Several genes presumed to have functions in neural differentiation (CDC42, VAMP3, CLSTN1), signal transduction in neural cells (GNB1) and cell cycle regulation (STMN1, RPA2, RBAF600, FBXO6, MAD2L2) exhibited a decreased expression in samples presenting 1p deletion. The identification of such genes provides baseline information for further studies to elucidate how these genes could individually or collectively play a critical role in neuroblastoma tumorigenesis.