A Cre-conditional MYCN-driven neuroblastoma mouse model as an improved tool for preclinical studies.

Neuroblastoma, a childhood cancer that originates from neural crest-derived cells, is the most common deadly solid tumor of infancy. Amplification of the MYCN oncogene, which occurs in approximately 20-25% of human neuroblastomas, is the most prominent genetic marker of high-stage disease. The availability of valid preclinical in vivo models is a prerequisite to develop novel targeted therapies. We here report on the generation of transgenic mice with Cre-conditional induction of MYCN in dopamine ß-hydroxylase-expressing cells, termed LSL-MYCN;Dbh-iCre. These mice develop neuroblastic tumors with an incidence of >75%, regardless of strain background. Molecular profiling of tumors revealed upregulation of the MYCN-dependent miR-17-92 cluster as well as expression of neuroblastoma marker genes, including tyrosine hydroxylase and the neural cell adhesion molecule 1. Gene set enrichment analyses demonstrated significant correlation with MYC-associated expression patterns. Array comparative genome hybridization showed that chromosomal aberrations in LSL-MYCN;Dbh-iCre tumors were syntenic to those observed in human neuroblastomas. Treatment of a cell line established from a tumor derived from a LSL-MYCN;Dbh-iCre mouse with JQ1 or MLN8237 reduced cell viability and demonstrated oncogene addiction to MYCN. Here we report establishment of the first Cre-conditional human MYCN-driven mouse model for neuroblastoma that closely recapitulates the human disease with respect to tumor localization, histology, marker expression and genomic make up. This mouse model is a valuable tool for further functional studies and to assess the effect of targeted therapies.

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

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

RNA interference screening identifies a novel role for autocrine fibroblast growth factor signaling in neuroblastoma chemoresistance.

Chemotherapeutic drug resistance is one of the major causes for treatment failure in high-risk neuroblastoma (NB), the most common extra cranial solid tumor in children. Poor prognosis is typically associated with MYCN amplification. Here, we utilized a loss-of-function kinome-wide RNA interference screen to identify genes that cause cisplatin sensitization. We identified fibroblast growth factor receptor 2 (FGFR2) as an important determinant of cisplatin resistance. Pharmacological inhibition of FGFR2 confirmed the importance of this kinase in NB chemoresistance. Silencing of FGFR2 sensitized NB cells to cisplatin-induced apoptosis, which was regulated by the downregulation of the anti-apoptotic proteins BCL2 and BCLXL. Mechanistically, FGFR2 was shown to activate protein kinase C-δ to induce BCL2 expression. FGFR2, as well as the ligand fibroblast growth factor-2, were consistently expressed in primary NB and NB cell lines, indicating the presence of an autocrine loop. Expression analysis revealed that FGFR2 correlates with MYCN amplification and with advanced stage disease, demonstrating the clinical relevance of FGFR2 in NB. These findings suggest a novel role for FGFR2 in chemoresistance and provide a rational to combine pharmacological inhibitors against FGFR2 with chemotherapeutic agents for the treatment of NB.

Bone morphogenetic protein-7 is a MYC target with prosurvival functions in childhood medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is known that overexpression and/or amplification of the MYC oncogene is associated with poor clinical outcome, but the molecular mechanisms and the MYC downstream effectors in MB remain still elusive. Besides contributing to elucidate how progression of MB takes place, most importantly, the identification of novel MYC-target genes will suggest novel candidates for targeted therapy in MB. A group of 209 MYC-responsive genes was obtained from a complementary DNA microarray analysis of a MB-derived cell line, following MYC overexpression and silencing. Among the MYC-responsive genes, we identified the members of the bone morphogenetic protein (BMP) signaling pathway, which have a crucial role during the development of the cerebellum. In particular, the gene BMP7 was identified as a direct target of MYC. A positive correlation between MYC and BMP7 expression was documented by analyzing two distinct sets of primary MB samples. Functional studies in vitro using a small-molecule inhibitor of the BMP/SMAD signaling pathway reproduced the effect of the small interfering RNA-mediated silencing of BMP7. Both approaches led to a block of proliferation in a panel of MB cells and to inhibition of SMAD phosphorylation. Altogether, our findings indicate that high MYC levels drive BMP7 overexpression, promoting cell survival in MB cells. This observation suggests the potential relevance of targeting the BMP/SMAD pathway as a novel therapeutic approach for the treatment of childhood MB.

MicroRNA miR-885-5p targets CDK2 and MCM5, activates p53 and inhibits proliferation and survival.

Several microRNA (miRNA) loci are found within genomic regions frequently deleted in primary neuroblastoma, including miR-885-5p at 3p25.3. In this study, we demonstrate that miR-885-5p is downregulated on loss of 3p25.3 region in neuroblastoma. Experimentally enforced miR-885-5p expression in neuroblastoma cell lines inhibits proliferation triggering cell cycle arrest, senescence and/or apoptosis. miR-885-5p leads to the accumulation of p53 protein and activates the p53 pathway, resulting in upregulation of p53 targets. Enforced miR-885-5p expression consistently leads to downregulation of cyclin-dependent kinase (CDK2) and mini-chromosome maintenance protein (MCM5). Both genes are targeted by miR-885-5p via predicted binding sites within the 3'-untranslated regions (UTRs) of CDK2 and MCM5. Transcript profiling after miR-885-5p introduction in neuroblastoma cells reveals alterations in expression of multiple genes, including several p53 target genes and a number of factors involved in p53 pathway activity. Taken together, these data provide evidence that miR-885-5p has a tumor suppressive role in neuroblastoma interfering with cell cycle progression and cell survival.

Comparison of performance of one-color and two-color gene-expression analyses in predicting clinical endpoints of neuroblastoma patients.

Microarray-based prediction of clinical endpoints may be performed using either a one-color approach reflecting mRNA abundance in absolute intensity values or a two-color approach yielding ratios of fluorescent intensities. In this study, as part of the MAQC-II project, we systematically compared the classification performance resulting from one- and two-color gene-expression profiles of 478 neuroblastoma samples. In total, 196 classification models were applied to these measurements to predict four clinical endpoints, and classification performances were compared in terms of accuracy, area under the curve, Matthews correlation coefficient and root mean-squared error. Whereas prediction performance varied with distinct clinical endpoints and classification models, equivalent performance metrics were observed for one- and two-color measurements in both internal and external validation. Furthermore, overlap of selected signature genes correlated inversely with endpoint prediction difficulty. In summary, our data strongly substantiate that the choice of platform is not a primary factor for successful gene expression based-prediction of clinical endpoints.

MYCN/c-MYC-induced microRNAs repress coding gene networks associated with poor outcome in MYCN/c-MYC-activated tumors.

Increased activity of MYC protein-family members is a common feature in many cancers. Using neuroblastoma as a tumor model, we established a microRNA (miRNA) signature for activated MYCN/c-MYC signaling in two independent primary neuroblastoma tumor cohorts and provide evidence that c-MYC and MYCN have overlapping functions. On the basis of an integrated approach including miRNA and messenger RNA (mRNA) gene expression data we show that miRNA activation contributes to widespread mRNA repression, both in c-MYC- and MYCN-activated tumors. c-MYC/MYCN-induced miRNA activation was shown to be dependent on c-MYC/MYCN promoter binding as evidenced by chromatin immunoprecipitation. Finally, we show that pathways, repressed through c-MYC/MYCN miRNA activation, are highly correlated to tumor aggressiveness and are conserved across different tumor entities suggesting that c-MYC/MYCN activate a core set of miRNAs for cooperative repression of common transcriptional programs related to disease aggressiveness. Our results uncover a widespread correlation between miRNA activation and c-MYC/MYCN-mediated coding gene expression modulation and further substantiate the overlapping functions of c-MYC and MYCN in the process of tumorigenesis.

Integrated genomic profiling identifies two distinct molecular subtypes with divergent outcome in neuroblastoma with loss of chromosome 11q.

Imbalances in chromosome 11q occur in approximately 30% of primary neuroblastoma and are associated with poor outcome. It has been suggested that 11q loss constitutes a distinct clinico-genetic neuroblastoma subgroup by affecting expression levels of corresponding genes. This study analysed the relationship of 11q loss, clinical phenotype and global transcriptomic profiles in four clinico-genetic subgroups (11q alteration/favourable outcome, n=7; 11q alteration/unfavourable outcome, n=14; no 11q alteration/favourable outcome, n=81; no 11q alteration/unfavourable outcome, n=8; tumours with MYCN amplification and/or 1p loss were excluded). Unsupervised and supervised comparisons of gene expression profiles consistently showed significantly different mRNA patterns between favourable and unfavourable neuroblastomas, both in the subgroups with and without 11q loss. In contrast, favourable tumours with and without 11q loss showed highly similar transcriptomic profiles. Disproportionate downregulation of 11q genes was observed only in unfavourable tumours with 11q loss. The diverging molecular profiles were neither caused by considerable differences in the size of the deleted regions nor by differential methylation patterns of 11q genes. Together, this study shows that neuroblastoma with 11q loss comprises two biological subgroups that differ both in their clinical phenotype and gene expression patterns, indicating that 11q loss is not a primary determinant of neuroblastoma tumour behaviour.

Risk estimation of neuroblastoma patients using molecular markers.

The pediatric tumor neuroblastoma is a heterogeneous disease: Patients' clinical courses can range from spontaneous regression to fatal progression of the disease. Accordingly, treatment protocols vary from "wait and see" approaches to intensive multimodal therapies. Accurate risk estimation of the patients is therefore mandatory to choose the most adequate therapy. Current trials stratify by a limited number of clinical variables, such as stage of the disease and age of the patient at diagnosis, as well as molecular markers, such as amplification of the oncogene MYCN and loss of the short arm of chromosome 1. However, misclassifications of patients still occur, and thus, a precise prediction of the clinical courses remains a challenge of neuroblastoma research. In recent years, genomic alterations and gene expression profiles of this neoplasm have been characterized thoroughly. It has been shown that the diverse clinical phenotypes are reflected by both specific cytogenetic aberrations and distinct gene expression patterns. Moreover, a variety of DNA copy number changes and gene expression-based classifiers have been described that could predict the outcome of neuroblastoma patients more precisely than established prognostic variables. In this review, the recent advances in the detection and evaluation of molecular prognostic markers for neuroblastoma patients are summarized, and their current and potential contribution to risk stratification systems is discussed.

Expression of the tumour suppressor gene CADM1 is associated with favourable outcome and inhibits cell survival in neuroblastoma.

Cell adhesion molecule 1 (CADM1) is a putative tumour suppressor gene, which is downregulated in many solid tumours. In neuroblastoma, loss of CADM1 expression has recently been found in disseminated tumours with adverse outcome, prompting us to investigate its role in neuroblastoma tumour progression. Oligonucleotide-microarray analysis of 251 neuroblastoma specimens demonstrated that CADM1 downregulation is associated with unfavourable prognostic markers like disseminated stage 4, age >18 months, MYCN amplification and chromosome 11q alterations (P<0.001 each). Furthermore, low CADM1 expression was significantly correlated with unfavourable gene expression-based classification (P<0.001) and adverse patient outcome (P<0.001). Bisulphite sequencing and genetic analysis of 18 primary neuroblastomas suggested that neither haploinsufficiency nor hypermethylation is regularly involved in CADM1 gene silencing in neuroblastoma, which is in contrast to results obtained in other malignancies. In addition, no mutations disrupting the CADM1 reading frame were found in 25 primary neuroblastomas. Over-expression of CADM1 in neuroblastoma cells resulted in significant reduction of proliferation, viability and colony formation in soft agar. Collectively, our results suggest that downregulation of CADM1 tumour suppressor gene expression is a critical event in neuroblastoma pathogenesis resulting in tumour progression and unfavourable patient outcome.

Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks.

The purpose of this study was to develop a method of classifying cancers to specific diagnostic categories based on their gene expression signatures using artificial neural networks (ANNs). We trained the ANNs using the small, round blue-cell tumors (SRBCTs) as a model. These cancers belong to four distinct diagnostic categories and often present diagnostic dilemmas in clinical practice. The ANNs correctly classified all samples and identified the genes most relevant to the classification. Expression of several of these genes has been reported in SRBCTs, but most have not been associated with these cancers. To test the ability of the trained ANN models to recognize SRBCTs, we analyzed additional blinded samples that were not previously used for the training procedure, and correctly classified them in all cases. This study demonstrates the potential applications of these methods for tumor diagnosis and the identification of candidate targets for therapy.

Heterozygous mutations in ANKH, the human ortholog of the mouse progressive ankylosis gene, result in craniometaphyseal dysplasia.

Craniometaphyseal dysplasia (CMD) is a bone dysplasia characterized by overgrowth and sclerosis of the craniofacial bones and abnormal modeling of the metaphyses of the tubular bones. Hyperostosis and sclerosis of the skull may lead to cranial nerve compressions resulting in hearing loss and facial palsy. An autosomal dominant form of the disorder (MIM 123000) was linked to chromosome 5p15.2-p14.1 (ref. 3) within a region harboring the human homolog (ANKH) of the mouse progressive ankylosis (ank) gene. The ANK protein spans the outer cell membrane and shuttles inorganic pyrophosphate (PPi), a major inhibitor of physiologic and pathologic calcification, bone mineralization and bone resorption. Here we carry out mutation analysis of ANKH, revealing six different mutations in eight of nine families. The mutations predict single amino acid substitutions, deletions or insertions. Using a helix prediction program, we propose for the ANK molecule 12 membrane-spanning helices with an alternate inside/out orientation and a central channel permitting the passage of PPi. The mutations occur at highly conserved amino acid residues presumed to be located in the cytosolic portion of the protein. Our results link the PPi channel ANK with bone formation and remodeling.

A new and accurate prediction model for growth response to growth hormone treatment in children with growth hormone deficiency.

OBJECTIVE: To identify parameters which predict individual growth response to recombinant human GH (rhGH) therapy and to combine these parameters in a prediction model. DESIGN: Fifty-eight prepubertal patients with GH deficiency (17 females) participated in this prospective multicenter trial with 1 year of follow-up. METHODS: Auxological measurements, parameters of GH status and markers of bone metabolism were measured at baseline and at 1, 3 and 6 months after the start of rhGH treatment. Correlations with height velocity during the first 12 months of treatment (HV+12) were calculated. Prediction models were derived by multiple regression analysis. RESULTS: The model which best predicted HV+12 combined the following parameters: pretreatment bone age retardation as a fraction of chronological age, pretreatment serum levels of IGF-I, urinary levels of deoxypyridinoline (a marker of bone resorption) after 1 month of treatment and height velocity after 3 months of treatment. This model explained 89% of the variation in HV+12 with a standard deviation of the residuals of 0.93 cm/year. Defining successful rhGH therapy as a doubling of pretreatment height velocity, the model had a specificity of 90% and a sensitivity of 100% in predicting therapeutic success. CONCLUSIONS: This model is an accurate and practicable tool to predict growth response in GH-deficient children. It may help to optimize rhGH therapy by individual dose adjustment and contribute to improved overall outcomes.

Serum leptin is suppressed by growth hormone therapy in growth hormone-deficient children.

Leptin is a hormone which is exclusively synthesized and secreted by adipocytes. As of yet, little is known about the complex interplay of hormones in the modulation of circulating leptin levels. To investigate the effect of growth hormone (GH) therapy on leptin, leptin serum concentrations were measured by a specific radioimmunoassay in 29 children with GH deficiency (21 boys, 8 girls; age range 3-14 years) before and after 1, 3 and 6 months of treatment with recombinant human GH. At baseline, serum leptin levels were identical to those of healthy children. Serum leptin correlated with body mass index (BMI; r=0.60, p < 0.001) and weight (r=0.48, p=0.004), but not with height, age, insulinlike growth factor 1, or insulinlike growth factor-binding protein 3, and there was no sex difference (p > 0.05). After 1 month of treatment, the leptin levels had decreased to 73+/-(SEM) 13% of individual pretreatment levels (p=0.002) and remained constant thereafter. While the correlation between leptin, BMI, and weight persisted throughout the study period, the changes in leptin concentrations during treatment were not associated with changes in BMI, weight, height, insulinlike growth factor 1, and insulinlike growth factor binding protein 3. In conclusion, this preliminary study demonstrates that serum leptin decreases during GH treatment in children with GH deficiency.

Apoptosis in atypical fibroxanthoma and pleomorphic malignant fibrous histiocytoma.

Explanations for the disparate behavior of atypical fibroxanthoma (AFX) as compared with pleomorphic malignant fibrous histiocytoma (MFH) have included the proposition that the former is a pseudosarcoma. Nonetheless, these tumors are now widely regarded as the same process, but with AFX behaving benignly by virtue of its superficial location. However, a neoplasm's metastastatic potential has been proposed to be related to apoptosis. Therefore, the aim of the present study was to examine apoptotic counts, in conjunction with two important regulators of apoptosis: p53 and bcl-2, to determine if a distinction exists that may account for the different outcomes of these lesions. There was no significant statistical difference between eight AFX and nine pleomorphic MFH in terms of apoptotic behavior, proliferative indexes, p53 protein expression, or presence of bcl-2 product. Therefore, our results further support the contention that AFX should be regarded as a form of pleomorphic MFH, which demonstrates low malignant potential by virtue of its location in readily accessible sites.

Interleukin 10 inhibits cytokine production of human AML cells.

BACKGROUND: Unlike normal hematopoietic cells leukemic blasts from patients with AML constitutively express cytokines like IL1, GM-CSF and TNF alpha and it has been suggested that these cytokines may regulate growth and differentiation of the malignant cells. IL10 inhibits cytokine production of activated macrophages and T-helper 1 cells. We analysed whether IL10 can also suppress cytokine production and may inhibit growth of AML cells. MATERIALS AND METHODS: AML blasts of 18 patients were purified by immunomagnetic separation and cultured in serum-free medium in the presence of cytokines. The production of cytokines was analysed by ELISA, DNA synthesis by 3H-thymidine incorporation and mRNA expression of cytokine genes by semiquantitative RT-PCR. RESULTS: Our results confirm previous results that AML blasts produce a variety of cytokines such as GM-CSF, IL1 alpha, IL1 beta, IL6 and TNF alpha. AML cells were induced to proliferation by G-CSF, GM-CSF, IL3, IL1 beta and SCF to a different extent. In contrast, IL10 significantly inhibited the cytokine production at the mRNA and protein level and spontaneous thymidine uptake in a dose-dependent way. This inhibition could be abrogated by IL10 specific antibodies. CONCLUSION: These observations suggest an inhibitory effect of IL10 on the proliferation of cultured AML blasts most likely through suppression of endogenous cytokines.

[Study on the effect of formaldehyde on the immunological reactivity and morphology of hepatitis B-antigen (author's transl)]. / Untersuchungen über die Wirkung von Formaldehyd auf die immunologische Reaktivität und die Morphologie des Hepatitis B-Antigens.

Treatment of purified HB-antigen with formaldehyde solution changes its immunological reactivity and its morphology. A decrease in antigen activity by formaldehyde treatment could be demonstrated by radial immunodiffusion and by radioimmunoassay (Fig. 1). Electron microscopic studies revealed a segmentation of tubular HBAg particles into small round forms (Fig. 3) and a shift of the ratio tubular forms: spherical forms in favour of the spherical particles (Table 1). These findings support the hypothesis that 20 nm particles originate from a division of tubular forms. Round forms became smaller after formaldehyde treatment (Fig. 4). The treatment with formaldehyde changed neither the density of HBsAg testes in CsCl gradients, nor the immunologic identity tested in immunodiffusion tests (Fig. 2). All prescribed effects were observed only in the high concentrations of formalin (10 and 1%), whereas in the concentration of 0.1%, which is usually used for inactivations in the development of vaccines, no immunological and morphological changes of the HBAg could be seen.