Evaluation of Metabolic and Cardiovascular Risk Measured by Laboratory Biomarkers and Cardiopulmonary Exercise Test in Children and Adolescents Recovered from Brain Tumors: The CARMEP Study.

In recent decades, the improvement of treatments and the adoption of therapeutic protocols of international cooperation has led to an improvement in the survival of children affected by brain tumors. However, in parallel with the increase in survival, long-term side effects related to treatments have been observed over time, including the activation of chronic inflammatory processes and metabolic alterations, which can facilitate the onset of metabolic syndrome and increased cardiovascular risk. The aim of this study was to find possible statistically significant differences in the serum concentrations of early biomarkers of metabolic syndrome and in the results of cardiopulmonary exercise testing between survivors of childhood brain tumors and healthy controls. This is a prospective and observational study conducted on a group of 14 male patients who survived childhood brain tumors compared with the same number of healthy controls. The concentrations of early metabolic syndrome biomarkers [adiponectin, leptin, TNF-α, IL-1, IL-6, IL-10, endothelin-1, apolipoprotein B, and lipoprotein (a)] were measured and a cardiopulmonary exercise test (CPET) was performed. Results: Childhood brain tumor survivors performed worse on average than controls on the CPET. Furthermore, they showed higher endothelin-1 values than controls (p = 0.025). The CPET results showed an inverse correlation with leptin. The differences found highlight the greater cardiovascular risk of brain tumor survivors, and radiotherapy could be implicated in the genesis of this greater cardiovascular risk.

Drug Repurposing in Pediatric Brain Tumors: Posterior Fossa Ependymoma and Diffuse Midline Glioma under the Looking Glass.

Tumors of the Central Nervous System (CNS) represent the leading cause of cancer-related deaths in children. Current treatment options are not curative for most malignant histologies, and intense preclinical and clinical research is needed to develop more effective therapeutic interventions against these tumors, most of which meet the FDA definition for orphan diseases. Increased attention is being paid to the repositioning of already-approved drugs for new anticancer indications as a fast-tracking strategy for identifying new and more effective therapies. Two pediatric CNS tumors, posterior fossa ependymoma (EPN-PF) type A and diffuse midline glioma (DMG) H3K27-altered, share loss of H3K27 trimethylation as a common epigenetic hallmark and display early onset and poor prognosis. These features suggest a potentially common druggable vulnerability. Successful treatment of these CNS tumors raises several challenges due to the location of tumors, chemoresistance, drug blood-brain barrier penetration, and the likelihood of adverse side effects. Recently, increasing evidence demonstrates intense interactions between tumor cell subpopulations and supportive tumor microenvironments (TMEs) including nerve, metabolic, and inflammatory TMEs. These findings suggest the use of drugs, and/or multi-drug combinations, that attack both tumor cells and the TME simultaneously. In this work, we present an overview of the existing evidence concerning the most preclinically validated noncancer drugs with antineoplastic activity. These drugs belong to four pharmacotherapeutic classes: antiparasitic, neuroactive, metabolic, and anti-inflammatory. Preclinical evidence and undergoing clinical trials in patients with brain tumors, with special emphasis on pediatric EPN-PF and DMG, are summarized and critically discussed.

Cell-of-Origin and Genetic, Epigenetic, and Microenvironmental Factors Contribute to the Intra-Tumoral Heterogeneity of Pediatric Intracranial Ependymoma.

Intra-tumoral heterogeneity (ITH) is a complex multifaceted phenomenon that posits major challenges for the clinical management of cancer patients. Genetic, epigenetic, and microenvironmental factors are concurrent drivers of diversity among the distinct populations of cancer cells. ITH may also be installed by cancer stem cells (CSCs), that foster unidirectional hierarchy of cellular phenotypes or, alternatively, shift dynamically between distinct cellular states. Ependymoma (EPN), a molecularly heterogeneous group of tumors, shows a specific spatiotemporal distribution that suggests a link between ependymomagenesis and alterations of the biological processes involved in embryonic brain development. In children, EPN most often arises intra-cranially and is associated with an adverse outcome. Emerging evidence shows that EPN displays large intra-patient heterogeneity. In this review, after touching on EPN inter-tumoral heterogeneity, we focus on the sources of ITH in pediatric intra-cranial EPN in the framework of the CSC paradigm. We also examine how single-cell technology has shed new light on the complexity and developmental origins of EPN and the potential impact that this understanding may have on the therapeutic strategies against this deadly pediatric malignancy.

The BET Inhibitor OTX015 Exhibits In Vitro and In Vivo Antitumor Activity in Pediatric Ependymoma Stem Cell Models.

Childhood ependymomas are heterogenous chemoresistant neoplasms arising from aberrant stem-like cells. Epigenome deregulation plays a pivotal role in ependymoma pathogenesis, suggesting that epigenetic modifiers hold therapeutic promise against this disease. Bromodomain and extraterminal domain (BET) proteins are epigenome readers of acetylated signals in histones and coactivators for oncogenic and stemness-related transcriptional networks, including MYC/MYCN (Proto-Oncogene, BHLH Transcritpion Factor)-regulated genes. We explored BET inhibition as an anticancer strategy in a panel of pediatric patient-derived ependymoma stem cell models by OTX015-mediated suppression of BET/acetylated histone binding. We found that ependymoma tissues and lines express BET proteins and their targets MYC and MYCN. In vitro, OTX015 reduced cell proliferation by inducing G0/G1-phase accumulation and apoptosis at clinically tolerable doses. Mechanistically, inhibitory p21 and p27 increased in a p53-independent manner, whereas the proliferative driver, phospho-signal transducer and activator of transcription 3 (STAT3), decreased. Upregulation of apoptosis-related proteins and survivin downregulation were correlated with cell line drug sensitivity. Minor alterations of MYC/MYCN expression were reported. In vivo, OTX015 significantly improved survival in 2/3 orthotopic ependymoma models. BET proteins represent promising targets for pharmaceutical intervention with OTX015 against ependymoma. The identification of predictive determinants of sensitivity may help identify ependymoma molecular subsets more likely to benefit from BET inhibitor therapies.

CN133, a Novel Brain-Penetrating Histone Deacetylase Inhibitor, Hampers Tumor Growth in Patient-Derived Pediatric Posterior Fossa Ependymoma Models.

Pediatric ependymoma (EPN) is a highly aggressive tumor of the central nervous system that remains incurable in 40% of cases. In children, the majority of cases develop in the posterior fossa and can be classified into two distinct molecular entities: EPN posterior fossa A (PF-EPN-A) and EPN posterior fossa B (PF-EPN-B). Patients with PF-EPN-A have poor outcome and are in demand of new therapies. In general, PF-EPN-A tumors show a balanced chromosome copy number profile and have no recurrent somatic nucleotide variants. However, these tumors present abundant epigenetic deregulations, thereby suggesting that epigenetic therapies could provide new opportunities for PF-EPN-A patients. In vitro epigenetic drug screening of 11 compounds showed that histone deacetylase inhibitors (HDACi) had the highest anti-proliferative activity in two PF-EPN-A patient-derived cell lines. Further screening of 5 new brain-penetrating HDACi showed that CN133 induced apoptosis in vitro, reduced tumor growth in vivo and significantly extended the survival of mice with orthotopically-implanted EPN tumors by modulation of the unfolded protein response, PI3K/Akt/mTOR signaling, and apoptotic pathways among others. In summary, our results provide solid preclinical evidence for the use of CN133 as a new therapeutic agent against PF-EPN-A tumors.

Novel SEC61G-EGFR Fusion Gene in Pediatric Ependymomas Discovered by Clonal Expansion of Stem Cells in Absence of Exogenous Mitogens.

The basis for molecular and cellular heterogeneity in ependymomas of the central nervous system is not understood. This study suggests a basis for this phenomenon in the selection for mitogen-independent (MI) stem-like cells with impaired proliferation but increased intracranial tumorigenicity. MI ependymoma cell lines created by selection for EGF/FGF2-independent proliferation exhibited constitutive activation of EGFR, AKT, and STAT3 and sensitization to the antiproliferative effects of EGFR tyrosine kinase inhibitors (TKI). One highly tumorigenic MI line harbored membrane-bound, constitutively active, truncated EGFR. Two EGFR mutants (ΔN566 and ΔN599) were identified as products of intrachromosomal rearrangements fusing the 3' coding portion of the EGFR gene to the 5'-UTR of the SEC61G, yielding products lacking the entire extracellular ligand-binding domain of the receptor while retaining the transmembrane and tyrosine kinase domains. EGFR TKI efficiently targeted ΔN566/ΔN599-mutant-mediated signaling and prolonged the survival of mice bearing intracranial xenografts of MI cells harboring these mutations. RT-PCR sequencing of 16 childhood ependymoma samples identified SEC61G-EGFR chimeric mRNAs in one infratentorial ependymoma WHO III, arguing that this fusion occurs in a small proportion of these tumors. Our findings demonstrate how in vitro culture selections applied to genetically heterogeneous tumors can help identify focal mutations that are potentially pharmaceutically actionable in rare cancers. Cancer Res; 77(21); 5860-72. ©2017 AACR.

Ependymoma stem cells are highly sensitive to temozolomide in vitro and in orthotopic models.

BACKGROUND: Ependymoma management remains challenging because of the inherent chemoresistance of this tumor. To determine whether ependymoma stem cells (SCs) might contribute to therapy resistance, we investigated the sensitivity of ependymoma SCs to temozolomide and etoposide. METHODS: The efficacies of the two DNA damaging agents were explored in two ependymoma SC lines in vitro and in vivo models. RESULTS: Ependymoma SC lines were highly sensitive to temozolomide and etoposide in vitro, but only temozolomide impaired tumor-initiation properties. Consistently, temozolomide but not etoposide showed significant antitumoral activity on ependymoma SC-driven subcutaneous and orthotopic xenografts by reducing the mitotic fraction. In vitro temozolomide at the EC50 (10 µM) induced accumulation of cells in the G2/M phase that was unexpectedly accompanied by downregulation of p27 and p21 without modulation of full-length p53 (FLp53). Differentiation-committed ependymoma SCs acquired resistance to temozolomide. Inhibition of proliferation was partly due to apoptosis, that occurred earlier in differentiated cells as compared to neurospheres. The activation of apoptosis correlated with an increase in p53ß/γ isoforms without modulation of FLp53 under both serum-free and differentiation-promoting media. Incubation of cells in both conditions with temozolomide resulted in increased glioneuronal differentiation exhibiting elevated glial fibrillary acidic protein, galactosylceramidase, and ßIII-tubulin expression compared to untreated controls. O(6)-methylguanine DNA methyltransferase (MGMT) transcript levels were very low in SCs, and were increased by treatment and, epigenetically, by differentiation through MGMT promoter unmethylation. CONCLUSION: Ependymoma growth might be impaired by temozolomide through preferential depletion of a less differentiated, more tumorigenic, MGMT-negative cell population with stem-like properties.

Epigenetic Silencing of DKK3 in medulloblastoma.

Medulloblastoma (MB) is a malignant pediatric brain tumor arising in the cerebellum consisting of four distinct subgroups: WNT, SHH, Group 3 and Group 4, which exhibit different molecular phenotypes. We studied the expression of Dickkopf (DKK) 1-4 family genes, inhibitors of the Wnt signaling cascade, in MB by screening 355 expression profiles derived from four independent datasets. Upregulation of DKK1, DKK2 and DKK4 mRNA was observed in the WNT subgroup, whereas DKK3 was downregulated in 80% MBs across subgroups with respect to the normal cerebellum (p < 0.001). Since copy number aberrations targeting the DKK3 locus (11p15.3) are rare events, we hypothesized that epigenetic factors could play a role in DKK3 regulation. Accordingly, we studied 77 miRNAs predicting to repress DKK3; however, no significant inverse correlation between miRNA/mRNA expression was observed. Moreover, the low methylation levels in the DKK3 promoters (median: 3%, 5% and 5% for promoter 1, 2 and 3, respectively) excluded the downregulation of gene expression by methylation. On the other hand, the treatment of MB cells with Trichostatin A (TSA), a potent inhibitor of histone deacetylases (HDAC), was able to restore both DKK3 mRNA and protein. In conclusion, DKK3 downregulation across all MB subgroups may be due to epigenetic mechanisms, in particular, through chromatin condensation.

Identification of ALK germline mutation (3605delG) in pediatric anaplastic medulloblastoma.

The anaplastic lymphoma kinase (ALK) gene has been found either rearranged or mutated in several neoplasms such as anaplastic large-cell lymphoma, non-small-cell lung cancer, neuroblastoma and anaplastic thyroid cancer. Medulloblastoma (MB) is an embryonic pediatric cancer arising from nervous system, a tissue in which ALK is expressed during embryonic development. We performed an ALK mutation screening in 52 MBs and we found a novel heterozygous germline deletion of a single base in exon 23 (3605delG) in a case with marked anaplasia. This G deletion results in a frameshift mutation producing a premature stop codon in exon 25 of ALK tyrosine kinase domain. We also screened three human MB cell lines without finding any mutation of ALK gene. Quantitative expression analysis of 16 out of 52 samples showed overexpression of ALK mRNA in three MBs. In the present study, we report the first mutation of ALK found in MB. Moreover, a deletion of ALK gene producing a stop codon has not been detected in human tumors up to now. Further investigations are now required to elucidate whether the truncated form of ALK may have a role in signal transduction.

Effects of epidermal growth factor receptor blockade on ependymoma stem cells in vitro and in orthotopic mouse models.

Some lines of evidence suggest that tumors, including ependymoma, might arise from a subpopulation of cells, termed cancer stem cells (CSCs), with self-renewal and tumor-initiation properties. Given the strict dependence of CSCs on epidermal growth factor (EGF) through EGF receptor (EGFR), we investigated the effects of EGFR inhibitors in ependymoma-stem cells (SCs) in vitro and in orthotopic mouse models. We established two ependymoma-SC lines from two recurrent pediatric ependymoma. Both lines expressed markers of radial glia--the candidate SCs of ependymoma--and showed renewal ability, multipotency, and tumorigenicity after orthotopic implantation, despite markedly different expression of CD133 (94 vs. 6%). High phosphorylated-EGFR/EGFR ratio was detected, which decreased after differentiation. EGFR inhibitors (gefitinib and AEE788) reduced clonogenicity, proliferation and survival of ependymoma-SC lines dose-dependently, and blocked EGF-induced activation of EGFR, Akt and extracellular signal-regulated kinase 1/2. Overall, AEE788 was more effective than gefitinib. EGFR blockade as well as differentiation strongly reduced CD133 expression. However, ex vivo treatment with AEE788 did not impair orthotopic tumor engraftment, whereas ex vivo differentiation did, suggesting that CD133 does not absolutely segregate for tumorigenicity in ependymoma-SCs. Orally administered AEE788 prolonged survival of mice bearing ependymoma-SC-driven orthotopic xenografts from 56 to 63 days, close to statistical significance (log-rank p=0.06). Our study describes for the first time EGFR signaling in ependymoma-SCs and the effects of EGFR blockade in complementary in vitro and in vivo systems. The experimental models we developed can be used to further investigate the activity of EGFR inhibitors or other antineoplastic agents in this tumor.

Chromosome 9q and 16q loss identified by genome-wide pooled-analysis are associated with tumor aggressiveness in patients with classic medulloblastoma.

Medulloblastoma (MB) is one of the most aggressive pediatric brain tumor. We report genome-wide pooled-analysis of classic MB variant of patients over 3 years of age at diagnosis. We combined array comparative genomic hybridization (aCGH) results from experimental analysis (31 cases) with two public databases (55 cases) in a final evaluation of 86 MBs. The most common chromosome structural aberrations were gains of 17q (45.3%), 1q (22.1%), and losses of 8p (15.1%), 10q (19.8%), 17p (37.2%), and 16q (16.3%). Isochromome (17q) was observed in 29.1% MBs. A significant association between poor patients survival and losses of 9q (p < 0.0023), 10q (p < 0.012), and 16q (p < 0.036) was observed. Univariate analysis showed association of 9q loss (p < 0.008) and 16q loss (p = 0.05) with adverse overall survival (OS). Chromosome 6 monosomy was a protective event although statistically borderline (p = 0.066). After adjusting for confounding factors, a poor OS was found for patients whose tumor has 9q loss [hazard ratio (HR) = 3.97; p < 0.006) or 16q loss (HR = 2.41; p = 0.038). Our results highlight the importance of genomic studies in different MB histological variants and indicate a genotype-phenotype correlation.

Dual Inhibitor AEE788 Reduces Tumor Growth in Preclinical Models of Medulloblastoma.

Medulloblastoma is the most frequent malignant pediatric brain tumor with a dismal prognosis in 30% of cases. We examined the activity of AEE788, a dual inhibitor of human epidermal receptor (HER) 1/2 and vascular endothelial growth factor receptor (VEGFR) 1/2, in medulloblastoma preclinical models. Established lines (Daoy and D283), chemoresistant (Daoy(Pt)), and ectopically HER2-overexpressing (Daoy(HER2)) cells expressed diverse levels of total and activated AEE788 target receptors. In vitro, AEE788 inhibited cell proliferation (IC(50) from 1.7 to 3.8 µM) and prevented epidermal growth factor- and neuregulin-induced HER1, HER2, and HER3 activation. Inhibition of Akt paralleled that of HER receptors. In vivo, AEE788 growth inhibited Daoy, Daoy(Pt), and Daoy(HER2) xenografts by 51%, 45%, and 72%, respectively. Immunohistochemical analysis of mock- and HER2-transfected xenografts revealed that the latter showed, along with high HER2 expression, high VEGFR2 staining in tumor and endothelial cells and increased expression of the endothelial marker CD31. AEE788 reduced the activation of target receptors and angiogenesis. In 21 primary medulloblastoma, HER2 expression significantly correlated (P < .01) with VEGFR2 (r = 0.56) and VEGF (r = 0.61). In conclusion, AEE788 shows similar growth-suppressive activities in chemosensitive and chemoresistant medulloblastoma cells in vitro and in vivo. Ectopic HER2 overexpression sensitizes cells to AEE788 in vivo, but not in vitro, possibly through host-mediated processes. Together with the experimental data, the finding that HER2 positively correlates with VEGFR2 and VEGF in human medulloblastoma specimens indicates HER2-overexpressing medulloblastoma as the subset that most likely might benefit from AEE788 treatment.

Antitumor activity and pharmacokinetics of oral gimatecan on pediatric cancer xenografts.

PURPOSE: This study compared the antitumor activity and the pharmacological profile of gimatecan given orally and irinotecan (CPT-11) on pediatric tumor xenografts. EXPERIMENTAL DESIGN: Gimatecan was tested in two neuroblastoma cell lines (SK-N-DZ and SK-N-(BE)2c) and on TE-671 rhabdomyosarcoma cells using two different schedules. We characterized its pharmacokinetic profile in nude mice bearing human SK-N-DZ and TE-671 cell lines. RESULTS: Gimatecan appears to have high plasma disposition. The drug was present in plasma almost completely as the intact lactone form and showed substantial activity in all tumor models. Prolonged daily treatment with low doses of gimatecan produced significant tumor regression in all tumor xenografts. CONCLUSION: The antitumor activity and the promising pharmacological profile indicate gimatecan as an excellent candidate for clinical treatment of pediatric tumors.

Antitumor effect in medulloblastoma cells by gefitinib: Ectopic HER2 overexpression enhances gefitinib effects in vivo.

The effects of the epidermal growth factor receptor (EGFR) inhibitor gefitinib on cell growth and signaling were evaluated in three medulloblastoma (MB) cell lines (D283, D341, Daoy), one supratentorial primitive neuroectodermal tumor cell line (PFSK), and four MB primary cultures. Cell lines showed diverse expression of EGFR and human epidermal receptor 2 (HER2), with high levels of constitutively activated HER2 in the HER2-overexpressing D341 and D283 cells. Gefitinib sensitivity varied across lines and was not related to expression of HER receptors or receptor baseline activation. Gefitinib induced G(0)/G(1) arrest in all lines, whereas apoptosis was dose-dependently induced only in D283 and D341 cells. The molecular response to gefitinib was investigated in Daoy and D341 lines, which showed a higher (half-maximal inhibitory concentration [IC(50)], 3.8 microM) and lower (IC(50), 6.6 microM) sensitivity to the agent, respectively. Gefitinib inhibited constitutive and EGF-triggered EGFR phosphorylation in both lines but was ineffective in constitutive activation of HER2 in D341 cells. Phosphorylated AKT inhibition paralleled that of phosphorylated EGFR, suggesting the presence of an autocrine gefitinib-sensitive EGFR/AKT pathway. On the whole, EGF-dependent signaling was less responsive to ligand stimulation and gefitinib inhibition in D341 cells, which correlated with the lower sensitivity to gefitinib's antiproliferative effect of this line. In vivo, the growth of D341 and Daoy xenografts treated with gefitinib at 150 mg/kg per day was inhibited by approximately 50%. Ectopically overexpressed HER2 in Daoy cells significantly increased sensitivity to gefitinib's antitumor effects in vivo (tumor volume inhibition = 78%). Our data indicate that gefitinib might be a molecularly targeted agent for the treatment of MB.

Chemoresistant tumor cell lines display altered epidermal growth factor receptor and HER3 signaling and enhanced sensitivity to gefitinib.

Deregulated signaling through the epidermal growth factor receptor (EGFR) is involved in chemoresistance. To identify the molecular determinants of sensitivity to the EGFR inhibitor gefitinib (Iressa, ZD1839) in chemoresistance, we compared the response of matched chemosensitive and chemoresistant glioma and ovarian cancer cell lines. We found that chemoresistant cell lines were 2- to 3-fold more sensitive to gefitinib growth-inhibitory effects, because of decreased proliferation rather than survival. Sensitivity to gefitinib correlated with overexpression and constitutive phosphorylation of HER2 and HER3, but not EGFR, altered HER ligand expression, and enhanced activation of EGF-triggered EGFR pathway. No activating mutations were found in EGFR. Gefitinib fully inhibited EGF-induced and constitutive Akt activation only in chemoresistant cells. In parallel, gefitinib downregulated constitutively phosphorylated HER2 and HER3, and activated GSK3beta with a concomitant degradation of cyclin D1. Ectopically overexpressed HER2 on its own was insufficient to sensitize chemonaive cells to gefitinib. pHER3 coimmunoprecipitated with p85-PI3K in chemoresistant cells and gefitinib dissociated these complexes. siRNA-mediated inhibition of HER3 decreased constitutive activation of Akt and sensitivity to gefitinib in chemoresistant cells. Our study indicates that in chemoresistant cells gefitinib inhibits both an enhanced EGF-triggered pathway and a constitutive HER3-mediated Akt activation, indicating that inhibition of HER3 together with that of EGFR could be relevant in chemorefractory tumors. Furthermore, in combination experiments gefitinib enhanced the effects of coadministered drugs more in chemoresistant than chemosensitive ovarian cancer cells. Combined treatment might be therapeutically beneficial in chemoresistant tumors from ovary and likely from other tissues.

Increased sensitivity to the platelet-derived growth factor (PDGF) receptor inhibitor STI571 in chemoresistant glioma cells is associated with enhanced PDGF-BB-mediated signaling and STI571-induced Akt inactivation.

The platelet-derived growth factor receptor (PDGFR) is a tyrosine kinase, implicated in the development and progression of different tumors, including gliomas. Chemoresistance is a common feature of malignant gliomas. Since receptor tyrosine kinases contribute to chemoresistance in tumors, we addressed whether PDGFR signaling might confer selective growth advantage to chemoresistant cells. The effects of the PDGFR inhibitor STI571 on proliferation and PDGFR signaling were compared in chemosensitive and cisplatin-selected, chemoresistant sublines derived from glioma and from two other PDGFR-expressing tumors (ovarian carcinoma and neuroblastoma). The chemoresistant glioma U87/Pt cells were twofold more sensitive to STI571 growth-inhibitory effects than the chemosensitive U87 cells, and two- to threefold more sensitive than five unrelated glioma cell lines. The other two paired cell lines were equally responsive. Sensitization of U87/Pt cells correlated with upregulation of the PDGF-B isoform and with PDGF-BB-induced Akt overactivation, which was prevented by STI571. STI571 specifically inhibited PDGF-BB-, but not PDGF-AA- or stem cell factor-mediated signaling. In serum-containing medium, STI571 decreased phospho-Akt in U87/Pt cells, but not in U87, while activating extracellular signal-regulated kinase (Erk) in both. STI571 antiproliferative effects were partially reverted by constitutively active Akt. Cotreatment with inhibitors of phosphatidylinositol 3'-kinase (PI3K) or mitogen-activated protein kinase kinase (MEK) resulted in enhanced growth inhibition in glioma cells. Our results suggest that increased PDGF-BB signaling may sensitize chemoresistant glioma cells to STI571, suggesting a therapeutic potential for STI571 in patients with malignant gliomas refractory to chemotherapy. Simultaneous blockade of PDGFR and PI3K or Erk pathway may enhance therapeutic targeting in gliomas.

Neprilysin participates in skeletal muscle regeneration and is accumulated in abnormal muscle fibres of inclusion body myositis.

Neprilysin (NEP, EP24.11), a metallopeptidase originally shown to modulate signalling events by degrading small regulatory peptides, is also an amyloid-beta- (Abeta) degrading enzyme. We investigated a possible role of NEP in inclusion body myositis (IBM) and other acquired and hereditary muscle disorders and found that in all myopathies NEP expression was directly associated with the degree of muscle fibre regeneration. In IBM muscle, NEP protein was also strongly accumulated in Abeta-bearing abnormal fibres. In vitro, during the experimental differentiation of myoblasts, NEP protein expression was regulated at the post-transcriptional level with a rapid increase in the early stage of myoblast differentiation followed by a gradual reduction thereafter, coincident with the progression of the myogenic programme. Treatment of differentiating muscle cells with the NEP inhibitor dl-3-mercapto-2-benzylpropanoylglycine resulted in impaired differentiation that was mainly associated with an abnormal regulation of Akt activation. Therefore, NEP may play an important role during muscle cell differentiation, possibly through the regulation, either directly or indirectly, of the insulin-like growth factor I-driven myogenic programme. In IBM muscle increased NEP may be instrumental in (i) reducing the Abeta accumulation in vulnerable fibres and (ii) promoting a repair/regenerative attempt of muscle fibres possibly through the modulation of insulin-like growth factor I-dependent pathways.

Antitumor activity of imatinib mesylate in neuroblastoma xenografts.

Imatinib mesylate has antitumor activity in vitro and in vivo against neuroblastoma cell lines and xenografts characterized by a different expression of receptor tyrosine kinases. In this article, we report that imatinib tumor concentration can be independent of the administered dose and does not correlate with the antitumor effect. In xenografts, high-dose administration does not improve imatinib efficacy. In conclusion, there is no clear-cut correlation between the levels of expression for imatinib-responsive targets and the in vitro and in vivo sensitivity. This further suggests that in neuroblastoma the antitumor activity of imatinib may involve the inhibition of other tyrosine kinases and/or pathways.

The cellular response to PPARgamma ligands is related to the phenotype of neuroblastoma cell lines.

Neuroblastoma (NB) is a phenotypically heterogeneous tumor, displaying cells of neuronal, melanocytic, or glial/schwannian lineage. This cellular heterogeneity is also present in vitro, where cells of neuroblastic (N)- or stromal (S)-type may be identified. Ligands of peroxisome proliferator-activated receptor gamma (PPARgamma) have been shown to inhibit growth in different tumor cell lines. The purpose of this study was to determine PPARgamma expression and the response to its ligands in NB cell lines with different phenotypes. We used eight NB cell lines with N-, mixed, and S-phenotype. PPARgamma expression was found in all NB cell lines, regardless of their phenotype. Mutational analysis and transactivation assays showed that PPARgamma is not mutated and remains functional in NB cells. Two PPARgamma ligands, 15-deoxy-delta12,14-prostaglandin J2 (PGJ2) and rosiglitazone, inhibited growth of all cell lines, with PGJ, being the most potent agent. PGJ2, but not rosiglitazone, induced arrest of the cells in the G2/M phase as well as apoptosis. The sensitivity to the two ligands appeared to be more related to the phenotype than PPARgamma expression, with the S-type cells being less sensitive than the N-type, partly because of their lower capability of undergoing apoptosis. No synergistic effect on growth inhibition was observed when all cell lines were co-treated with 9-cis retinoic acid (9-cis RA) and rosiglitazone. Our data indicate that PPARgamma expression and function are maintained in phenotipically different NB cell lines. Activation of PPARgamma by its synthetic ligands might have a therapeutic role in advanced NB.

The PPARgamma ligands PGJ2 and rosiglitazone show a differential ability to inhibit proliferation and to induce apoptosis and differentiation of human glioblastoma cell lines.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is involved in the control of cell proliferation, apoptosis and differentiation in various tumor cells. Among PPARgamma ligands, 15-deoxy-Delta12,14-prostaglandin J2 (PGJ2), the ultimate metabolite of PGD2, plays a role in the biology of brain tumors. It is still unclear to which extent the anti-proliferative and differentiation-promoting activity of PGJ2 is mediated through PPARgamma. We compared the effects of PGJ2 with those of rosiglitazone - the synthetic agonist with the highest affinity for PPARgamma - in 4 human glioblastoma cell lines (A172, U87-MG, M059K, M059J). All cell lines expressed high levels of PPARgamma, consistent with the high levels of PPARgamma protein in 5 tumor samples. Both PGJ2 and rosiglitazone inhibited proliferation of all cell lines with a G2/M arrest and apoptosis, but only PGJ2 up-regulated p21Cip/WAF1. The growth inhibitory effect was partially reversed by the PPARgamma antagonist GW9662. We studied the time sequence of selected molecular events, that lead glioblastoma cells to apoptosis and/or differentiation, after treatment with both agonists. M059K cells committed to undergo apoptosis by PGJ2, initially up-regulated PPARgamma, and then down-regulated PPARgamma as they began apoptosis. Apoptotic cells also increased their expression of retinoic acid receptor beta (RARbeta) and retinoid X receptor alpha (RXRalpha). PGJ2 increased expression of glial fibrillary acidic protein (GFAP) and decreased levels of vimentin, structural proteins modulated during astrocytic differentiation. Unexpectedly, PGJ2 up-regulated the expression of cyclooxygenase-2 (COX-2). Rosiglitazone caused the same pattern of PPARgamma, RARbeta and RXRalpha expression as PGJ2, but no significant modulation of p21Cip/WAF1, cytoskeletal proteins or COX-2 occurred. Our data indicate that PGJ2, and rosiglitazone suppress cell proliferation and cause apoptosis in glioblastoma cell lines, most likely through a PPARgamma-dependent pathway. By contrast, the modulation of differentiation-associated proteins by PGJ2, but not rosiglitazone, suggests that PGJ2 promotes differentiation of glioblastoma cells independently of PPARgamma activation.