Low number of neurosecretory vesicles in neuroblastoma impairs massive catecholamine release and prevents hypertension.

Introduction: Neuroblastoma (NB) is a pediatric cancer of the developing sympathetic nervous system. It produces and releases metanephrines, which are used as biomarkers for diagnosis in plasma and urine. However, plasma catecholamine concentrations remain generally normal in children with NB. Thus, unlike pheochromocytoma and paraganglioma (PHEO/PGL), two other non-epithelial neuroendocrine tumors, hypertension is not part of the usual clinical picture of patients with NB. This suggests that the mode of production and secretion of catecholamines and metanephrines in NB is different from that in PHEO/PGL, but little is known about these discrepancies. Here we aim to provide a detailed comparison of the biosynthesis, metabolism and storage of catecholamines and metanephrines between patients with NB and PHEO. Method: Catecholamines and metanephrines were quantified in NB and PHEO/PGL patients from plasma and tumor tissues by ultra-high pressure liquid chromatography tandem mass spectrometry. Electron microscopy was used to quantify neurosecretory vesicles within cells derived from PHEO tumor biopsies, NB-PDX and NB cell lines. Chromaffin markers were detected by qPCR, IHC and/or immunoblotting. Results: Plasma levels of metanephrines were comparable between NB and PHEO patients, while catecholamines were 3.5-fold lower in NB vs PHEO affected individuals. However, we observed that intratumoral concentrations of metanephrines and catecholamines measured in NB were several orders of magnitude lower than in PHEO. Cellular and molecular analyses revealed that NB cell lines, primary cells dissociated from human tumor biopsies as well as cells from patient-derived xenograft tumors (NB-PDX) stored a very low amount of intracellular catecholamines, and contained only rare neurosecretory vesicles relative to PHEO cells. In addition, primary NB expressed reduced levels of numerous chromaffin markers, as compared to PHEO/PGL, except catechol O-methyltransferase and monoamine oxidase A. Furthermore, functional assays through induction of chromaffin differentiation of the IMR32 NB cell line with Bt2cAMP led to an increase of neurosecretory vesicles able to secrete catecholamines after KCl or nicotine stimulation. Conclusion: The low amount of neurosecretory vesicles in NB cytoplasm prevents catecholamine storage and lead to their rapid transformation by catechol O-methyltransferase into metanephrines that diffuse in blood. Hence, in contrast to PHEO/PGL, catecholamines are not secreted massively in the blood, which explains why systemic hypertension is not observed in most patients with NB.

TWIST1 expression is associated with high-risk neuroblastoma and promotes primary and metastatic tumor growth.

The embryonic transcription factors TWIST1/2 are frequently overexpressed in cancer, acting as multifunctional oncogenes. Here we investigate their role in neuroblastoma (NB), a heterogeneous childhood malignancy ranging from spontaneous regression to dismal outcomes despite multimodal therapy. We first reveal the association of TWIST1 expression with poor survival and metastasis in primary NB, while TWIST2 correlates with good prognosis. Secondly, suppression of TWIST1 by CRISPR/Cas9 results in a reduction of tumor growth and metastasis colonization in immunocompromised mice. Moreover, TWIST1 knockout tumors display a less aggressive cellular morphology and a reduced disruption of the extracellular matrix (ECM) reticulin network. Additionally, we identify a TWIST1-mediated transcriptional program associated with dismal outcome in NB and involved in the control of pathways mainly linked to the signaling, migration, adhesion, the organization of the ECM, and the tumor cells versus tumor stroma crosstalk. Taken together, our findings confirm TWIST1 as promising therapeutic target in NB.

Frequency and Prognostic Impact of ALK Amplifications and Mutations in the European Neuroblastoma Study Group (SIOPEN) High-Risk Neuroblastoma Trial (HR-NBL1).

PURPOSE: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through activating point mutations or genomic amplification. We studied ALK genetic alterations in high-risk (HR) patients on the HR-NBL1/SIOPEN trial to determine their frequency, correlation with clinical parameters, and prognostic impact. MATERIALS AND METHODS: Diagnostic tumor samples were available from 1,092 HR-NBL1/SIOPEN patients to determine ALK amplification status (n = 330), ALK mutational profile (n = 191), or both (n = 571). RESULTS: Genomic ALK amplification (ALKa) was detected in 4.5% of cases (41 out of 901), all except one with MYCN amplification (MNA). ALKa was associated with a significantly poorer overall survival (OS) (5-year OS: ALKa [n = 41] 28% [95% CI, 15 to 42]; no-ALKa [n = 860] 51% [95% CI, 47 to 54], [P < .001]), particularly in cases with metastatic disease. ALK mutations (ALKm) were detected at a clonal level (> 20% mutated allele fraction) in 10% of cases (76 out of 762) and at a subclonal level (mutated allele fraction 0.1%-20%) in 3.9% of patients (30 out of 762), with a strong correlation between the presence of ALKm and MNA (P < .001). Among 571 cases with known ALKa and ALKm status, a statistically significant difference in OS was observed between cases with ALKa or clonal ALKm versus subclonal ALKm or no ALK alterations (5-year OS: ALKa [n = 19], 26% [95% CI, 10 to 47], clonal ALKm [n = 65] 33% [95% CI, 21 to 44], subclonal ALKm (n = 22) 48% [95% CI, 26 to 67], and no alteration [n = 465], 51% [95% CI, 46 to 55], respectively; P = .001). Importantly, in a multivariate model, involvement of more than one metastatic compartment (hazard ratio [HR], 2.87; P < .001), ALKa (HR, 2.38; P = .004), and clonal ALKm (HR, 1.77; P = .001) were independent predictors of poor outcome. CONCLUSION: Genetic alterations of ALK (clonal mutations and amplifications) in HR-NB are independent predictors of poorer survival. These data provide a rationale for integration of ALK inhibitors in upfront treatment of HR-NB with ALK alterations.

The noradrenergic profile of plasma metanephrine in neuroblastoma patients is reproduced in xenograft mice models and arise from PNMT downregulation.

Metanephrines (MNs; normetanephrine (NMN), metanephrine (MN) and methoxytyramine (MT)) detected in urine or plasma represent the best biomarker for neuroblastoma (NB) diagnosis, however the metabolism of both catecholamine (CAT) and MNs remains enigmatic in NB. Using patient-derived xenograft (PDX) models derived from primary NB cells, we observed that the plasma levels of MNs in NB-PDX-bearing mice were comparable as in patients. Interestingly, murine plasma displayed an elevated fraction of glucuronidated forms of MNs relative to human plasma where sulfonated forms prevail. In tumors, the concentration ranges of MNs and CAT and the expression levels of the main genes involved in catecholamine metabolism were similar between NB-PDX and human NB tissues. Likewise, plasma and intratumoral profiles of individual MNs, with increased levels of MT and NMN relative to MN, were also conserved in mouse models as in patients. We further demonstrated the downregulation of the Phenylethanolamine N-Methyltransferase gene in NB biopsies and in NB-PDX explaining this biochemical phenotype, and giving a rational to the low levels of epinephrine and MN measured in NB affected patients. Thus, our subcutaneous murine NB-PDX models not only reproduce the phenotype of primary NB tumors, but also the metabolism of catecholamine as observed in patients. This may potentially open new avenues in preclinical studies for the follow up of novel therapeutic options for NB through the quantification of plasma MNs.

Expression of the Neuroblastoma-Associated ALK-F1174L Activating Mutation During Embryogenesis Impairs the Differentiation of Neural Crest Progenitors in Sympathetic Ganglia.

Neuroblastoma (NB) is an embryonal malignancy derived from the abnormal differentiation of the sympathetic nervous system. The Anaplastic Lymphoma Kinase (ALK) gene is frequently altered in NB, through copy number alterations and activating mutations, and represents a predisposition in NB-genesis when mutated. Our previously published data suggested that ALK activating mutations may impair the differentiation potential of neural crest (NC) progenitor cells. Here, we demonstrated that the expression of the endogenous ALK gene starts at E10.5 in the developing sympathetic ganglia (SG). To decipher the impact of deregulated ALK signaling during embryogenesis on the formation and differentiation of sympathetic neuroblasts, Sox10-Cre;LSL-ALK-F1174L embryos were produced to restrict the expression of the human ALK-F1174L transgene to migrating NC cells (NCCs). First, ALK-F1174L mediated an embryonic lethality at mid-gestation and an enlargement of SG with a disorganized architecture in Sox10-Cre;LSL-ALK-F1174L embryos at E10.5 and E11.5. Second, early sympathetic differentiation was severely impaired in Sox10-Cre;LSL-ALK-F1174L embryos. Indeed, their SG displayed a marked increase in the proportion of NCCs and a decrease of sympathetic neuroblasts at both embryonic stages. Third, neuronal and noradrenergic differentiations were blocked in Sox10-Cre;LSL-ALK-F1174L SG, as a reduced proportion of Phox2b+ sympathoblasts expressed ßIII-tubulin and almost none were Tyrosine Hydroxylase (TH) positive. Finally, at E10.5, ALK-F1174L mediated an important increase in the proliferation of Phox2b+ progenitors, affecting the transient cell cycle exit observed in normal SG at this embryonic stage. Altogether, we report for the first time that the expression of the human ALK-F1174L mutation in NCCs during embryonic development profoundly disturbs early sympathetic progenitor differentiation, in addition to increasing their proliferation, both mechanisms being potential crucial events in NB oncogenesis.

Aldehyde dehydrogenase activity plays a Key role in the aggressive phenotype of neuroblastoma.

BACKGROUND: The successful targeting of neuroblastoma (NB) by associating tumor-initiating cells (TICs) is a major challenge in the development of new therapeutic strategies. The subfamily of aldehyde dehydrogenases 1 (ALDH1) isoenzymes, which comprises ALDH1A1, ALDH1A2, and ALDH1A3, is involved in the synthesis of retinoic acid, and has been identified as functional stem cell markers in diverse cancers. By combining serial neurosphere passages with gene expression profiling, we have previously identified ALDH1A2 and ALDH1A3 as potential NB TICs markers in patient-derived xenograft tumors. In this study, we explored the involvement of ALDH1 isoenzymes and the related ALDH activity in NB aggressive properties. METHODS: ALDH activity and ALDH1A1/A2/A3 expression levels were measured using the ALDEFLUOR™ kit, and by real-time PCR, respectively. ALDH activity was inhibited using the specific ALDH inhibitor diethylaminobenzaldehyde (DEAB), and ALDH1A3 gene knock-out was generated through the CRISPR/Cas9 technology. RESULTS: We first confirmed the enrichment of ALDH1A2 and ALDH1A3 mRNA expression in NB cell lines and patient-derived xenograft tumors during neurosphere passages. We found that high ALDH1A1 expression was associated with less aggressive NB tumors and cell lines, and correlated with favorable prognostic factors. In contrast, we observed that ALDH1A3 was more widely expressed in NB cell lines and was associated with poor survival and high-risk prognostic factors. We also identified an important ALDH activity in various NB cell lines and patient-derived xenograft tumors. Specific inhibition of ALDH activity with diethylaminobenzaldehyde (DEAB) resulted in a strong reduction of NB cell clonogenicity, and TIC self-renewal potential, and partially enhanced NB cells sensitivity to 4-hydroxycyclophosphamide. Finally, the specific knock-out of ALDH1A3 via CRISPR/Cas9 gene editing reduced NB cell clonogenicity, and mediated a cell type-dependent inhibition of TIC self-renewal properties. CONCLUSIONS: Together our data uncover the participation of ALDH enzymatic activity in the aggressive properties and 4-hydroxycyclophosphamide resistance of NB, and show that the specific ALDH1A3 isoenzyme increases the aggressive capacities of a subset of NB cells.

The CXCR4/CXCR7/CXCL12 Axis Is Involved in a Secondary but Complex Control of Neuroblastoma Metastatic Cell Homing.

Neuroblastoma (NB) is one of the most deadly solid tumors of the young child, for which new efficient and targeted therapies are strongly needed. The CXCR4/CXCR7/CXCL12 chemokine axis has been involved in the progression and organ-specific dissemination of various cancers. In NB, CXCR4 expression was shown to be associated to highly aggressive undifferentiated tumors, while CXCR7 expression was detected in more differentiated and mature neuroblastic tumors. As investigated in vivo, using an orthotopic model of tumor cell implantation of chemokine receptor-overexpressing NB cells (IGR-NB8), the CXCR4/CXCR7/CXCL12 axis was shown to regulate NB primary and secondary growth, although without any apparent influence on organ selective metastasis. In the present study, we addressed the selective role of CXCR4 and CXCR7 receptors in the homing phase of metastatic dissemination using an intravenous model of tumor cell implantation. Tail vein injection into NOD-scid-gamma mice of transduced IGR-NB8 cells overexpressing CXCR4, CXCR7, or both receptors revealed that all transduced cell variants preferentially invaded the adrenal gland and typical NB metastatic target organs, such as the liver and the bone marrow. However, CXCR4 expression favored NB cell dissemination to the liver and the lungs, while CXCR7 was able to strongly promote NB cell homing to the adrenal gland and the liver. Finally, coexpression of CXCR4 and CXCR7 receptors significantly and selectively increased NB dissemination toward the bone marrow. In conclusion, CXCR4 and CXCR7 receptors may be involved in a complex and organ-dependent control of NB growth and selective homing, making these receptors and their inhibitors potential new therapeutic targets.

Wild-type ALK and activating ALK-R1275Q and ALK-F1174L mutations upregulate Myc and initiate tumor formation in murine neural crest progenitor cells.

The anaplastic lymphoma kinase (ALK) gene is overexpressed, mutated or amplified in most neuroblastoma (NB), a pediatric neural crest-derived embryonal tumor. The two most frequent mutations, ALK-F1174L and ALK-R1275Q, contribute to NB tumorigenesis in mouse models, and cooperate with MYCN in the oncogenic process. However, the precise role of activating ALK mutations or ALK-wt overexpression in NB tumor initiation needs further clarification. Human ALK-wt, ALK-F1174L, or ALK-R1275Q were stably expressed in murine neural crest progenitor cells (NCPC), MONC-1 or JoMa1, immortalized with v-Myc or Tamoxifen-inducible Myc-ERT, respectively. While orthotopic implantations of MONC- 1 parental cells in nude mice generated various tumor types, such as NB, osteo/ chondrosarcoma, and undifferentiated tumors, due to v-Myc oncogenic activity, MONC-1-ALK-F1174L cells only produced undifferentiated tumors. Furthermore, our data represent the first demonstration of ALK-wt transforming capacity, as ALK-wt expression in JoMa1 cells, likewise ALK-F1174L, or ALK-R1275Q, in absence of exogenous Myc-ERT activity, was sufficient to induce the formation of aggressive and undifferentiated neural crest cell-derived tumors, but not to drive NB development. Interestingly, JoMa1-ALK tumors and their derived cell lines upregulated Myc endogenous expression, resulting from ALK activation, and both ALK and Myc activities were necessary to confer tumorigenic properties on tumor-derived JoMa1 cells in vitro.

Involvement of the CXCR7/CXCR4/CXCL12 axis in the malignant progression of human neuroblastoma.

Neuroblastoma (NB) is a typical childhood and heterogeneous neoplasm for which efficient targeted therapies for high-risk tumors are not yet identified. The chemokine CXCL12, and its receptors CXCR4 and CXCR7 have been involved in tumor progression and dissemination. While CXCR4 expression is associated to undifferentiated tumors and poor prognosis, the role of CXCR7, the recently identified second CXCL12 receptor, has not yet been elucidated in NB. In this report, CXCR7 and CXCL12 expressions were evaluated using a tissue micro-array including 156 primary and 56 metastatic NB tissues. CXCL12 was found to be highly associated to NB vascular and stromal structures. In contrast to CXCR4, CXCR7 expression was low in undifferentiated tumors, while its expression was stronger in matured tissues and specifically associated to differentiated neural tumor cells. As determined by RT-PCR, CXCR7 expression was mainly detected in N-and S-type NB cell lines, and was slightly induced upon NB cell differentiation in vitro. The relative roles of the two CXCL12 receptors were further assessed by overexpressing CXCR7 or CXCR4 receptor alone, or in combination, in the IGR-NB8 and the SH-SY5Y NB cell lines. In vitro functional analyses indicated that, in response to their common ligand, both receptors induced activation of ERK1/2 cascade, but not Akt pathway. CXCR7 strongly reduced in vitro growth, in contrast to CXCR4, and impaired CXCR4/CXCL12-mediated chemotaxis. Subcutaneous implantation of CXCR7-expressing NB cells showed that CXCR7 also significantly reduced in vivo growth. Moreover, CXCR7 affected CXCR4-mediated orthotopic growth in a CXCL12-producing environment. In such model, CXCR7, in association with CXCR4, did not induce NB cell metastatic dissemination. In conclusion, the CXCR7 and CXCR4 receptors revealed specific expression patterns and distinct functional roles in NB. Our data suggest that CXCR7 elicits anti-tumorigenic functions, and may act as a regulator of CXCR4/CXCL12-mediated signaling in NB.

Functional sphere profiling reveals the complexity of neuroblastoma tumor-initiating cell model.

Neuroblastoma (NB) is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC) model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC) has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically "profile" the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB.

Fenretinide-induced caspase-8 activation and apoptosis in an established model of metastatic neuroblastoma.

BACKGROUND: Resistance of high-risk metastatic neuroblastoma (HR-NB) to high dose chemotherapy (HD-CT) raises a major therapeutic challenge in pediatric oncology. Patients are treated by maintenance CT. For some patients, an adjuvant retinoid therapy is proposed, such as the synthetic retinoid fenretinide (4-HPR), an apoptotic inducer. Recent studies demonstrated that NB metastasis process is enhanced by the loss of caspase-8 involved in the Integrin-Mediated Death (IMD) process. As the role of caspase-8 appears to be critical in preventing metastasis, we aimed at studying the effect of 4-HPR on caspase-8 expression in metastatic neuroblasts. METHODS: We used the human IGR-N-91 MYCN-amplified NB experimental model, able to disseminate in vivo from the primary nude mouse tumor xenograft (PTX) into myocardium (Myoc) and bone marrow (BM) of the animal. NB cell lines, i.e., IGR-N-91 and SH-EP, were treated with various doses of Fenretinide (4-HPR), then cytotoxicity was analyzed by MTS proliferation assay, apoptosis by the propidium staining method, gene or protein expressions by RT-PCR and immunoblotting and caspases activity by colorimetric protease assays. RESULTS: The IGR-N-91 parental cells do not express detectable caspase-8. However the PTX cells established from the primary tumor in the mouse, are caspase-8 positive. In contrast, metastatic BM and Myoc cells show a clear down-regulation of the caspase-8 expression. In parallel, the caspases -3, -9, -10, Bcl-2, or Bax expressions were unchanged. Our data show that in BM, compared to PTX cells, 4-HPR up-regulates caspase-8 expression that parallels a higher sensitivity to apoptotic cell death. Stable caspase-8-silenced SH-EP cells appear more resistant to 4-HPR-induced cell death compared to control SH-EP cells. Moreover, 4-HPR synergizes with drugs since apoptosis is restored in VP16- or TRAIL-resistant-BM cells. These results demonstrate that 4-HPR in up-regulating caspase-8 expression, restores and induces apoptotic cell death in metastatic neuroblasts through caspase-8 activation. CONCLUSION: This study provides basic clues for using fenretinide in clinical treatment of HR-NB patients. Moreover, since 4-HPR induces cell death in caspase-8 negative NB, it also challenges the concept of including 4-HPR in the induction of CT of these patients.

Complex molecular mechanisms cooperate to mediate histone deacetylase inhibitors anti-tumour activity in neuroblastoma cells.

BACKGROUND: Histone deacetylase inhibitors (HDACi) are a new class of promising anti-tumour agent inhibiting cell proliferation and survival in tumour cells with very low toxicity toward normal cells. Neuroblastoma (NB) is the second most common solid tumour in children still associated with poor outcome in higher stages and, thus NB strongly requires novel treatment modalities. RESULTS: We show here that the HDACi Sodium Butyrate (NaB), suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA) strongly reduce NB cells viability. The anti-tumour activity of these HDACi involved the induction of cell cycle arrest in the G2/M phase, followed by the activation of the intrinsic apoptotic pathway, via the activation of the caspases cascade. Moreover, HDACi mediated the activation of the pro-apoptotic proteins Bid and BimEL and the inactivation of the anti-apoptotic proteins XIAP, Bcl-xL, RIP and survivin, that further enhanced the apoptotic signal. Interestingly, the activity of these apoptosis regulators was modulated by several different mechanisms, either by caspases dependent proteolytic cleavage or by degradation via the proteasome pathway. In addition, HDACi strongly impaired the hypoxia-induced secretion of VEGF by NB cells. CONCLUSION: HDACi are therefore interesting new anti-tumour agents for targeting highly malignant tumours such as NB, as these agents display a strong toxicity toward aggressive NB cells and they may possibly reduce angiogenesis by decreasing VEGF production by NB cells.

The chemokine receptor CXCR4 strongly promotes neuroblastoma primary tumour and metastatic growth, but not invasion.

Neuroblastoma (NB) is a heterogeneous, and particularly malignant childhood neoplasm in its higher stages, with a propensity to form metastasis in selected organs, in particular liver and bone marrow, and for which there is still no efficient treatment available beyond surgery. Recent evidence indicates that the CXCR4/CXCL12 chemokine/receptor axis may be involved in promoting NB invasion and metastasis. In this study, we explored the potential role of CXCR4 in the malignant behaviour of NB, using a combination of in vitro functional analyses and in vivo growth and metastasis assessment in an orthotopic NB mouse model. We show here that CXCR4 overexpression in non-metastatic CXCR4-negative NB cells IGR-NB8 and in moderately metastatic, CXCR4 expressing NB cells IGR-N91, strongly increased tumour growth of primary tumours and liver metastases, without altering the frequency or the pattern of metastasis. Moreover shRNA-mediated knock-down experiments confirmed our observations by showing that silencing CXCR4 in NB cells impairs in vitro and almost abrogates in vivo growth. High levels of CXCL12 were detected in the mouse adrenal gland (the primary tumour site), and in the liver suggesting a paracrine effect of host-derived CXCL12 on NB growth. In conclusion, this study reveals a yet unreported NB-specific predominant growth and survival-promoting role of CXCR4, which warrants a critical reconsideration of the role of CXCR4 in the malignant behaviour of NB and other cancers.

Histone deacetylase inhibitors strongly sensitise neuroblastoma cells to TRAIL-induced apoptosis by a caspases-dependent increase of the pro- to anti-apoptotic proteins ratio.

BACKGROUND: Neuroblastoma (NB) is the second most common solid childhood tumour, an aggressive disease for which new therapeutic strategies are strongly needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumour cells, but not in normal tissues and therefore represents a valuable candidate in apoptosis-inducing therapies. Caspase-8 is silenced in a subset of highly malignant NB cells, which results in full TRAIL resistance. In addition, despite constitutive caspase-8 expression, or its possible restoration by different strategies, NB cells remain weakly sensitive to TRAIL indicating a need to develop strategies to sensitise NB cells to TRAIL. Histone deacetylase inhibitors (HDACIs) are a new class of anti-cancer agent inducing apoptosis or cell cycle arrest in tumour cells with very low toxicity toward normal cells. Although HDACIs were recently shown to increase death induced by TRAIL in weakly TRAIL-sensitive tumour cells, the precise involved sensitisation mechanisms have not been fully identified. METHODS: NB cell lines were treated with various doses of HDACIs and TRAIL, then cytotoxicity was analysed by MTS/PMS proliferation assays, apoptosis was measured by the Propidium staining method, caspases activity by colorimetric protease assays, and (in)activation of apoptotic proteins by immunoblotting. RESULTS: Sub-toxic doses of HDACIs strongly sensitised caspase-8 positive NB cell lines to TRAIL induced apoptosis in a caspases dependent manner. Combined treatments increased the activation of caspases and Bid, and the inactivation of the anti-apoptotic proteins XIAP, Bcl-x, RIP, and survivin, thereby increasing the pro- to anti-apoptotic protein ratio. It also enhanced the activation of the mitochondrial pathway. Interestingly, the kinetics of caspases activation and inactivation of anti-apoptotic proteins is accelerated by combined treatment with TRAIL and HDACIs compared to TRAIL alone. In contrast, cell surface expression of TRAIL-receptors or TRAIL is not affected by sub-toxic doses of HDACIs. CONCLUSION: HDACIs were shown to activate the mitochondrial pathway and to sensitise NB cells to TRAIL by enhancing the amplitude of the apoptotic cascade and by restoring an apoptosis-prone ratio of pro- to anti-apoptotic proteins. Combining HDACIs and TRAIL could therefore represent a weakly toxic and promising strategy to target TRAIL-resistant tumours such as neuroblastomas.

Molecular cytogenetic characterization of doxorubicin-resistant neuroblastoma cell lines: evidence that acquired multidrug resistance results from a unique large amplification of the 7q21 region.

Neuroblastoma is a heterogeneous neural crest-derived embryonic childhood neoplasm that is the second most common solid tumor found in children. Despite recent advances in combined therapy, the overall survival of patients with high-stage disease has not improved in the last decades. Treatment failure is in part attributed to multidrug resistance. To address the mechanisms involved in the development of multidrug resistance, we have generated two doxorubicin-resistant neuroblastoma cell lines (IGRN-91R and LAN-1R). These cells were shown to overexpress the MDR1 gene coding for the P-glycoprotein and were resistant to other MDR1- and non-MDR1-substrate drugs. Indeed, the MDR1 inhibitor verapamil only partially restored sensitivity to drugs, confirming that P-glycoprotein-mediated drug efflux was not responsible for 100% resistance. High-resolution and array-based comparative genomic hybridization analyses revealed the presence of an amplicon in the 7q21 region as the unique genomic alteration common to both doxorubicin-resistant cell lines. In addition to the MDR1 locus, this large amplified region is likely to harbor additional genes potentially involved in the development of drug resistance. This study represents the first molecular cytogenetic and genomic approach to identifying genomic regions involved in the multidrug-resistant phenotype of neuroblastoma. These results could lead to the identification of relevant target genes for the development of new therapeutic modalities.

In vivo echographic evidence of tumoral vascularization and microenvironment interactions in metastatic orthotopic human neuroblastoma xenografts.

Human neuroblastoma (NB) is the second most frequent solid tumor of childhood and represents a highly heterogeneous disease at clinical and biologic levels. Little progress has been made to improve the poor prognosis of patients with high-stage NB. Tumor progression and metastatic dissemination still represent major obstacles to the successful treatment of advanced stage disease. In order to develop and evaluate new, targeted, therapeutic strategies, fully defined and biologically relevant in vivo models of NB are strongly needed. We have developed an orthotopic model of metastatic human NB in the nude mouse, using 2 well-characterized NB cell lines. Tumor growth, vascular properties and metastatic patterns were investigated using a sensitive and newly developed in vivo echographic technology in addition to immunohistochemistry and PCR analyses. Results show that implantation of low numbers of NB cells directly into the adrenal gland of nude mice resulted in rapid and homogeneous tumor growth without tumor morbidity. Nude mice were shown to rapidly develop highly vascularized adrenal tumors that selectively metastasized to the liver and bone marrow. In addition, the newly formed mouse vessels in orthotopic but not in heterotopic tumors, were found to express the highly angiogenic alphavbeta3 integrin marker, indicating the development of a truly malignant neovasculature in orthotopic conditions only. This observation confirms the impact of the regional microenvironment on tumor biology and suggests the existence of cross-talk with the tumor cells. In conclusion, such model faithfully reproduces the growth, vascular and metastatic patterns as observed in patients. It therefore represents a powerful and biologically relevant tool to improve our understanding of the biology of NB and to develop and assess new antiangiogenic and metastasis-targeted therapies.

The S box of major histocompatibility complex class II promoters is a key determinant for recruitment of the transcriptional co-activator CIITA.

Tightly regulated expression of major histocompatibility complex (MHC) class II genes is critical for the immune system. A conserved regulatory module consisting of four cis-acting elements, the W, X, X2 and Y boxes, controls transcription of MHC class II genes. The X, X2, and Y boxes are bound, respectively, by RFX, CREB, and NF-Y to form a MHC class II-specific enhanceosome complex. The latter constitutes a landing pad for recruitment of the transcriptional co-activator CIITA. In contrast to the well defined roles of the X, X2, and Y boxes, the role of the W region has remained controversial. In vitro binding studies have suggested that it might contain a second RFX-binding site. We demonstrate here by means of promoter pull-down assays that the most conserved subsequence within the W region, called the S box, is a critical determinant for tethering of CIITA to the enhanceosome complex. Binding of CIITA to the enhanceosome requires both integrity of the S box and a remarkably stringent spacing between the S and X boxes. Even a 1-2-base pair change in the native S-X distance is detrimental for CIITA recruitment and promoter function. In contrast to current models, binding of RFX to a putative duplicated binding site in the W box is thus not required for either CIITA recruitment or promoter activity. This paves the way for the identification of novel factors mediating the contribution of the S box to the activation of MHC class II promoters.

Drug-mediated sensitization to TRAIL-induced apoptosis in caspase-8-complemented neuroblastoma cells proceeds via activation of intrinsic and extrinsic pathways and caspase-dependent cleavage of XIAP, Bcl-xL and RIP.

Neuroblastoma (NB) is a childhood neoplasm which heterogeneous behavior can be explained by differential regulation of apoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces rapid apoptosis in most tumor cells and thus represents a promising anticancer agent. We have reported silencing of caspase-8 expression in highly malignant NB cells as a possible mechanism of resistance to TRAIL-induced apoptosis. To explore the particular contribution of caspase-8 in such resistance, retroviral-mediated stable caspase-8 expression was induced in the IGR-N91 cells. As a result, sensitivity to TRAIL was fully restored in the caspase-8-complemented cells. TRAIL-induced cell death could be further enhanced by cotreatment of IGR-N91-C8 and SH-EP cells with cycloheximide or subtoxic concentrations of chemotherapeutic drugs in a caspase-dependent manner. Sensitization to TRAIL involved enhanced death receptor DR5 expression, activation of Bid and the complete caspases cascade. Interestingly, combined treatments also enhanced the cleavage-mediated inactivation of antiapoptotic molecules, XIAP, Bcl-x(L) and RIP. Our results show that restoration of active caspase-8 expression in a caspase-8-deficient NB cell line is necessary and sufficient to fully restore TRAIL sensitivity. Moreover, the synergistic effect of drugs and TRAIL results from activation of the caspase cascade via a mitochondrial pathway-mediated amplification loop and from the inactivation of apoptosis inhibitors.

Restoration of TRAIL-induced apoptosis in a caspase-8-deficient neuroblastoma cell line by stable re-expression of caspase-8.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumor cells, a process sometimes potentiated by chemotherapeutic drugs or cycloheximide (CHX). Childhood neuroblastoma (NB) is a clinically and biologically heterogeneous neoplasm whose behavior can be explained by differential regulation of apoptosis. The non-invasive S-type NB cell lines are sensitive to TRAIL, whereas the invasive N-type NB cell lines are resistant. We have reported the silencing of caspase-8 expression in N-type cells as a possible mechanism of death receptor-mediated resistance to apoptosis in NB. The recently observed deregulation of caspase-10 in these cells prompted us to investigate the particular contribution of caspase-8 silencing in the resistance to TRAIL in N-type cells. Stable caspase-8 expression was therefore restored in the IGR-N91 cell line by retroviral infection. The IGR-N91-C8 cells became sensitive to TRAIL-mediated apoptosis, whereas the control vector-infected IGR-N91-M cells remained resistant. Interestingly, the apoptotic response to TRAIL was enhanced by co-treatment of SH-EP and IGR-N91-C8 cells with CHX or with sub-toxic concentration of doxorubicin (DOX) in a caspase-dependent manner, as cells could be protected from death by specific caspase-8 or pan-caspase inhibitors. CHX or DOX was shown to enhance TRAIL-induced caspase-8 activation and loss of mitochondrial transmembrane potential. In conclusion, restoration of active caspase-8 expression in caspase-8- and caspase-10-deficient IGR-N-91 cell line is necessary and sufficient to fully restore TRAIL-mediated cell death. Moreover, DOX and CHX were able to sensitize NB cell lines to TRAIL-induced apoptosis in a caspase-8-dependent manner by engaging death receptor and mitochondrial signaling pathways.