BET inhibition therapy counteracts cancer cell survival, clonogenic potential and radioresistance mechanisms in rhabdomyosarcoma cells.

The antitumour effects of OTX015, a first-in-class BET inhibitor (BETi), were investigated as a single agent or in combination with ionizing radiation (IR) in preclinical in vitro models of rhabdomyosarcoma (RMS), the most common childhood soft tissue sarcoma. Herein, we demonstrated the upregulation of BET Bromodomain gene expression in RMS tumour biopsies and cell lines compared to normal skeletal muscle. In vitro experiments showed that OTX015 significantly reduced RMS cell proliferation by altering cell cycle modulators and apoptotic related proteins due to the accumulation of DNA breaks that cells are unable to repair. Interestingly, OTX015 also impaired migration capacity and tumour-sphere architecture by downregulating pro-stemness genes and was able to potentiate ionizing radiation effects by reducing the expression of different drivers of tumour dissemination and resistance mechanisms, including the GNL3 gene, that we correlated for the first time with the RMS phenotype. In conclusion, our research sheds further light on the molecular events of OTX015 action against RMS cells and indicates this novel BETi as an effective option to improve therapeutic strategies and overcome the development of resistant cancer cells in patients with RMS.

MiR-200c sensitizes Olaparib-resistant ovarian cancer cells by targeting Neuropilin 1.

BACKGROUND: Ovarian cancer (OC) is the most lethal gynecological malignancy and the second leading cause of cancer-related death in women. Treatment with PARP inhibitors (PARPi), such as Olaparib, has been recently introduced for OC patients, but resistance may occur and underlying mechanisms are still poorly understood. The aim of this study is to identify target genes within the tumor cells that might cause resistance to Olaparib. We focused on Neuropilin 1 (NRP1), a transmembrane receptor expressed in OC and correlated with poor survival, which has been also proposed as a key molecule in OC multidrug resistance. METHODS: Using three OC cell lines (UWB, UWB-BRCA and SKOV3) as model systems, we evaluated the biological and molecular effects of Olaparib on OC cell growth, cell cycle, DNA damage and apoptosis/autophagy induction, through MTT and colony forming assays, flow cytometry, immunofluorescence and Western blot analyses. We evaluated NRP1 expression in OC specimens and cell lines by Western blot and qRT-PCR, and used RNA interference to selectively inhibit NRP1. To identify miR-200c as a regulator of NRP1, we used miRNA target prediction algorithms and Pearsons' correlation analysis in biopsies from OC patients. Then, we used a stable transfection approach to overexpress miR-200c in Olaparib-resistant cells. RESULTS: We observed that NRP1 is expressed at high levels in resistant cells (SKOV3) and is upmodulated in partially sensitive cells (UWB-BRCA) upon prolonged Olaparib treatment, leading to poor drug response. Our results show that the selective inhibition of NRP1 is able to overcome Olaparib resistance in SKOV3 cells. Moreover, we demonstrated that miR-200c can target NRP1 in OC cells, causing its downmodulation, and that miR-200c overexpression is a valid approach to restore Olaparib sensitivity in OC resistant cells. CONCLUSIONS: These data demonstrate that miR-200c significantly enhanced the anti-cancer efficacy of Olaparib in drug-resistant OC cells. Thus, the combination of Olaparib with miRNA-based therapy may represent a promising treatment for drug resistant OC, and our data may help in designing novel precision medicine trials for optimizing the clinical use of PARPi.

Bevacizumab-containing regimen in relapsed/progressed brain tumors: a single-institution experience.

AIM: The aim of the study is to assess tumor response, treatment-related toxicities, progression-free survival (PFS), and overall survival (OS) in patients with relapsed/refractory brain tumors treated with bevacizumab-containing regimen. METHODS: Patients that had received I and II line treatments with or without megatherapy were included. Doses and schedule were as follows: bevacizumab (BVZ) 10 mg/kg i.v. with irinotecan (IRI) 150 mg/m2 i.v. every 2 weeks ± temozolamide (TMZ) 200 mg/m2 p.o. daily for 5 days every 4 weeks. TMZ was omitted in heavily pretreated cases. RESULTS: Between 2013 and 2018, 12 patients (3F/9M), median age 161 months (range 66-348), affected with medulloblastoma (n 7), or low-grade glioma (n 2), or high-grade glioma (n 3), received BVZ/IRI association (median courses 20, range 4-67); 3 of them continued single-agent BVZ (median courses 23, range 8-39). TMZ (median courses 8, range 2-26) was administered in eight patients and then stopped in three of them because of myelotoxicity or lack of compliance. Treatment was well tolerated. After 3 months, two complete responses, two partial responses, seven stable diseases, and one progressive disease were observed. Nine cases experienced an improvement in neurological symptoms. Median time to progression was 11 months (95% confidence interval, 4-18 months). Six-month and 2-year PFS were 75% and 42%, respectively. The OS is 33%; interestingly, two cases (one medulloblastoma and one high-grade glioma) are progression-free off-therapy since 30 and 48 months, respectively. CONCLUSIONS: BVZ/IRI association ± TMZ showed encouraging therapeutic activity and low toxicity in this series of relapsed/refractory brain tumors.

NRF2 orchestrates the redox regulation induced by radiation therapy, sustaining embryonal and alveolar rhabdomyosarcoma cells radioresistance.

PURPOSE: Tumor cells generally exhibit higher levels of reactive oxygen species (ROS), however, when stressed, tumor cells can undergo a process of 'Redox Resetting' to acquire a new redox balance with stronger antioxidant systems that enable cancer cells to become resistant to radiation therapy (RT). Here, we describe how RT affects the oxidant/antioxidant balance in human embryonal (RD) and alveolar (RH30) rhabdomyosarcoma (RMS) cell lines, investigating on the molecular mechanisms involved. METHODS: Radiations were delivered using an x-6 MV photon linear accelerator and their effects were assessed by vitality and clonogenic assays. The expression of specific antioxidant-enzymes, such as Superoxide Dismutases (SODs), Catalase (CAT) and Glutathione Peroxidases 4 (GPx4), miRNAs (miR-22, -126, -210, -375, -146a, -34a) and the transcription factor NRF2 was analyzed by quantitative polymerase chain reaction (q-PCR) and western blotting. RNA interference experiments were performed to evaluate the role of NRF2. RESULTS: Doses of RT higher than 2 Gy significantly affected RMS clonogenic ability by increasing ROS production. RMS rapidly and efficiently brought back ROS levels by up-regulating the gene expression of antioxidant enzymes, miRNAs as well as of NRF2. Silencing of NRF2 restrained the RMS ability to counteract RT-induced ROS accumulation, antioxidant enzyme and miRNA expression and was able to increase the abundance of γ-H2AX, a biomarker of DNA damage, in RT-treated cells. CONCLUSIONS: Taken together, our data suggest the strategic role of oxidant/antioxidant balance in restraining the therapeutic efficiency of RT in RMS treatment and identify NRF2 as a new potential molecular target whose inhibition might represent a novel radiosensitizing therapeutic strategy for RMS clinical management.

Circ-ZNF609 regulates G1-S progression in rhabdomyosarcoma.

Circular RNAs (circRNAs) represent a class of covalently closed RNAs, derived from non-canonical splicing events, which are expressed in all eukaryotes and often conserved among different species. We previously showed that the circRNA originating from the ZNF609 locus (circ-ZNF609) acts as a crucial regulator of human primary myoblast growth: indeed, the downregulation of the circRNA, and not of its linear counterpart, strongly reduced the proliferation rate of in vitro cultured myoblasts. To deepen our knowledge about circ-ZNF609 role in cell cycle regulation, we studied its expression and function in rhabdomyosarcoma (RMS), a pediatric skeletal muscle malignancy. We found that circ-ZNF609 is upregulated in biopsies from the two major RMS subtypes, embryonal (ERMS) and alveolar (ARMS). Moreover, we discovered that in an ERMS-derived cell line circ-ZNF609 knock-down induced a specific block at the G1-S transition, a strong decrease of p-Akt protein level and an alteration of the pRb/Rb ratio. Regarding p-Akt, we were able to show that circ-ZNF609 acts by counteracting p-Akt proteasome-dependent degradation, thus working as a new regulator of cell proliferation-related pathways. As opposed to ERMS-derived cells, the circRNA depletion had no cell cycle effects in ARMS-derived cells. Since in these cells the p53 gene resulted downregulated, with a concomitant upregulation of its cell cycle-related target genes, we suggest that this could account for the lack of circ-ZNF609 effect in ARMS.

PARP inhibitors affect growth, survival and radiation susceptibility of human alveolar and embryonal rhabdomyosarcoma cell lines.

PURPOSE: PARP inhibitors (PARPi) are used in a wide range of human solid tumours but a limited evidence is reported in rhabdomyosarcoma (RMS), the most frequent childhood soft-tissue sarcoma. The cellular and molecular effects of Olaparib, a specific PARP1/2 inhibitor, and AZD2461, a newly synthesized PARP1/2/3 inhibitor, were assessed in alveolar and embryonal RMS cells both as single-agent and in combination with ionizing radiation (IR). METHODS: Cell viability was monitored by trypan blue exclusion dye assays. Cell cycle progression and apoptosis were measured by flow cytometry, and alterations of specific molecular markers were investigated by, Real Time PCR, Western blotting and immunofluorescence experiments. Irradiations were carried out at a dose rate of 2 Gy (190 UM/min) or 4 Gy (380 UM/min). Radiosensitivity was assessed by using clonogenic assays. RESULTS: Olaparib and AZD2461 dose-dependently reduced growth of both RH30 and RD cells by arresting growth at G2/M phase and by modulating the expression, activation and subcellular localization of specific cell cycle regulators. Downregulation of phospho-AKT levels and accumulation of γH2AX, a specific marker of DNA damage, were significantly and persistently induced by Olaparib and AZD2461 exposure, this leading to apoptosis-related cell death. Both PARPi significantly enhanced the effects of IR by accumulating DNA damage, increasing G2 arrest and drastically reducing the clonogenic capacity of RMS-cotreated cells. CONCLUSIONS: This study suggests that the combined exposure to PARPi and IR might display a role in the treatment of RMS tumours compared with single-agent exposure, since stronger cytotoxic effects are induced, and compensatory survival mechanisms are prevented.

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype, induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells.

BACKGROUND: EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines. METHODS: EPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts. RESULTS: Our study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts. CONCLUSIONS: Taken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

A sketch of known and novel MYCN-associated miRNA networks in neuroblastoma.

Neuroblastoma (NB) originates from neural crest-derived precursors and represents the most common childhood extracranial solid tumour. MicroRNAs (miRNAs), a class of small non-coding RNAs that participate in a wide variety of biological processes by regulating gene expression, appear to play an essential role within the NB context. High-throughput next generation sequencing (NGS) was applied to study the miRNA transcriptome in a cohort of NB tumours with and without MYCN-amplification (MNA and MNnA, respectively) and in dorsal root ganglia (DRG), as a control. Out of the 128 miRNAs differentially expressed in the NB vs. DRG comparison, 47 were expressed at higher levels, while 81 were expressed at lower levels in the NB tumours. We also found that 23 miRNAs were differentially expressed in NB with or without MYCN-amplification, with 17 miRNAs being upregulated and 6 being downregulated in the MNA subtypes. Functional annotation analysis of the target genes of these differentially expressed miRNAs demonstrated that many mRNAs were involved in cancer-related pathways, such as DNA-repair and apoptosis as well as FGFR and EGFR signalling. In particular, we found that miR-628-3p negatively affects MYCN gene expression. Furthermore, we identified a novel miRNA candidate with variable expression in MNA vs. MNnA tumours, whose putative target genes are implicated in the mTOR pathway. The present study provides further insight into the molecular mechanisms that correlate miRNA dysregulation to NB development and progression.

HDAC4 and HDAC6 sustain DNA double strand break repair and stem-like phenotype by promoting radioresistance in glioblastoma cells.

The role of histone deacetylase (HDAC) 4 and 6 in glioblastoma (GBM) radioresistance was investigated. We found that tumor samples from 31 GBM patients, who underwent temozolomide and radiotherapy combined treatment, showed HDAC4 and HDAC6 expression in 93.5% and 96.7% of cases, respectively. Retrospective clinical data analysis demonstrated that high-intensity HDAC4 and/or HDAC6 immunostaining was predictive of poor clinical outcome. In vitro experiments revealed that short hairpin RNA-mediated silencing of HDAC4 or HDAC6 radiosensitized U87MG and U251MG GBM cell lines by promoting DNA double-strand break (DSBs) accumulation and by affecting DSBs repair molecular machinery. We found that HDAC6 knock-down predisposes to radiation therapy-induced U251MG apoptosis- and U87MG autophagy-mediated cell death. HDAC4 silencing promoted radiation therapy-induced senescence, independently by the cellular context. Finally, we showed that p53WT expression contributed to the radiotherapy lethal effects and that HDAC4 or HDAC6 sustained GBM stem-like radioresistant phenotype. Altogether, these observations suggest that HDAC4 and HDAC6 are guardians of irradiation-induced DNA damages and stemness, thus promoting radioresistance, and may represent potential prognostic markers and therapeutic targets in GBM.

DNMT3B in vitro knocking-down is able to reverse embryonal rhabdomyosarcoma cell phenotype through inhibition of proliferation and induction of myogenic differentiation.

Aberrant DNA methylation has been frequently observed in many human cancers, including rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. To date, the expression and function of the de novo DNA methyltransferase (DNMT) 3B in RMS have not yet been investigated. Our study show for the first time a significant up-regulation of DNMT3B levels in 14 RMS tumour samples and 4 RMS cell lines in comparison to normal skeletal muscle. Transfection of RD and TE671 cells, two in vitro models of embryonal RMS (ERMS), with a synthetic DNMT3B siRNA decreased cell proliferation by arresting cell cycle at G1 phase, as demonstrated by the reduced expression of Cyclin B1, Cyclin D1 and Cyclin E2, and by the concomitant up-regulation of the checkpoint regulators p21 and p27. DNMT3B depletion also impaired RB phosphorylation status and decreased migratory capacity and clonogenic potential. Interestingly, DNMT3B knock-down was able to commit ERMS cells towards myogenic terminal differentiation, as confirmed by the acquisition of a myogenic-like phenotype and by the increased expression of the myogenic markers MYOD1, Myogenin and MyHC. Finally, inhibition of MEK/ERK signalling by U0126 resulted in a reduction of DNMT3B protein, giving evidence that DNMT3B is a down-stream molecule of this oncogenic pathway.Taken together, our data indicate that altered expression of DNMT3B plays a key role in ERMS development since its silencing is able to reverse cell cancer phenotype by rescuing myogenic program. Epigenetic therapy, by targeting the DNA methylation machinery, may represent a novel therapeutic strategy against RMS.

Down-regulation of the Lamin A/C in neuroblastoma triggers the expansion of tumor initiating cells.

Tumor-initiating cells constitute a population within a tumor mass that shares properties with normal stem cells and is considered responsible for therapy failure in many cancers. We have previously demonstrated that knockdown of the nuclear envelope component Lamin A/C in human neuroblastoma cells inhibits retinoic acid-mediated differentiation and results in a more aggressive phenotype. In addition, Lamin A/C is often lost in advanced tumors and changes in the nuclear envelope composition occur during tumor progression. Based on our previous data and considering that Lamin A/C is expressed in differentiated tissues, we hypothesize that the lack of Lamin A/C could predispose cells toward a stem-like phenotype, thus influencing the development of tumor-initiating cells in neuroblastoma. This paper demonstrates that knockdown of Lamin A/C triggers the development of a tumor-initiating cell population with self-renewing features in human neuroblastoma cells. We also demonstrates that the development of TICs is due to an increased expression of MYCN gene and that in neuroblastoma exists an inverse relationship between LMNA and MYCN expression.

Crizotinib-induced antitumour activity in human alveolar rhabdomyosarcoma cells is not solely dependent on ALK and MET inhibition.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most commonly diagnosed malignant soft tissue tumour in children and adolescents. Aberrant expression of Anaplastic Lymphoma Kinase (ALK) and MET gene has been implicated in the malignant progression of RMS, especially in the alveolar subtype. This observation suggests that crizotinib (PF-02341066), a kinase inhibitor against ALK and MET, may have a therapeutic role in RMS, although its antitumour activity in this malignancy has not yet been studied. METHODS: RH4 and RH30 alveolar RMS (ARMS) cell lines were treated with crizotinib and then assessed by using proliferation, viability, migration and colony formation assays. Multiple approaches, including flow cytometry, immunofluorescence, western blotting and siRNA-based knock-down, were used in order to investigate possible molecular mechanisms linked to crizotinib activity. RESULTS: In vitro treatment with crizotinib inhibited ALK and MET proteins, as well as Insulin-like Growth Factor 1 Receptor (IGF1R), with a concomitant robust dephosphorylation of AKT and ERK, two downstream kinases involved in RMS cell proliferation and survival. Exposure to crizotinib impaired cell growth, and accumulation at G2/M phase was attributed to an altered expression and activation of checkpoint regulators, such as Cyclin B1 and Cdc2. Crizotinib was able to induce apoptosis and autophagy in a dose-dependent manner, as shown by caspase-3 activation/PARP proteolytic cleavage down-regulation and by LC3 activation/p62 down-regulation, respectively. The accumulation of reactive oxygen species (ROS) seemed to contribute to crizotinib effects in RH4 and RH30 cells. Moreover, crizotinib-treated RH4 and RH30 cells exhibited a decreased migratory/invasive capacity and clonogenic potential. CONCLUSIONS: These results provide a further insight into the molecular mechanisms affected by crizotinib in ARMS cells inferring that it could be a useful therapeutic tool in ARMS cancer treatment.

Noonan syndrome-like disorder with loose anagen hair: a second case with neuroblastoma.

Noonan-like syndrome with loose anagen hair (NSLH), also known as Mazzanti syndrome, is a RASopathy characterized by craniofacial features resembling Noonan syndrome, cardiac defects, cognitive deficits and behavioral issues, reduced growth generally associated with GH deficit, darkly pigmented skin, and an unique combination of ectodermal anomalies. Virtually all cases of NSLH are caused by an invariant and functionally unique mutation in SHOC2 (c.4A>G, p.Ser2Gly). Here, we report on a child with molecularly confirmed NSLH who developed a neuroblastoma, first suspected at the age 3 months by abdominal ultrasound examination. Based on this finding, scanning of the SHOC2 coding sequence encompassing the c.4A>G change was performed on selected pediatric cohorts of malignancies documented to occur in RASopathies (i.e., neuroblastoma, brain tumors, rhabdomyosarcoma, acute lymphoblastic, and myeloid leukemia), but failed to identify a functionally relevant cancer-associated variant. While these results do not support a major role of somatic SHOC2 mutations in these pediatric cancers, this second instance of neuroblastoma in NSLAH suggests a possible predisposition to this malignancy in subjects heterozygous for the c.4A>G SHOC2 mutation.

Deep Sequencing the microRNA profile in rhabdomyosarcoma reveals down-regulation of miR-378 family members.

BACKGROUND: Rhabdomyosarcoma (RMS) is a highly malignant tumour accounting for nearly half of soft tissue sarcomas in children. MicroRNAs (miRNAs) represent a class of short, non-coding, regulatory RNAs which play a critical role in different cellular processes. Altered miRNA levels have been reported in human cancers, including RMS. METHODS: Using deep sequencing technology, a total of 685 miRNAs were investigated in a group of alveolar RMSs (ARMSs), embryonal RMSs (ERMSs) as well as in normal skeletal muscle (NSM). Q-PCR, MTT, cytofluorimetry, migration assay, western blot and immunofluorescence experiments were carried out to determine the role of miR-378a-3p in cancer cell growth, apoptosis, migration and differentiation. Bioinformatics pipelines were used for miRNA target prediction and clustering analysis. RESULTS: Ninety-seven miRNAs were significantly deregulated in ARMS and ERMS when compared to NSM. MiR-378 family members were dramatically decreased in RMS tumour tissue and cell lines. Interestingly, members of the miR-378 family presented as a possible target the insulin-like growth factor receptor 1 (IGF1R), a key signalling molecule in RMS. MiR-378a-3p over-expression in an RMS-derived cell line suppressed IGF1R expression and affected phosphorylated-Akt protein levels. Ectopic expression of miR-378a-3p caused significant changes in apoptosis, cell migration, cytoskeleton organization as well as a modulation of the muscular markers MyoD1, MyoR, desmin and MyHC. In addition, DNA demethylation by 5-aza-2'-deoxycytidine (5-aza-dC) was able to up-regulate miR-378a-3p levels with a concomitant induction of apoptosis, decrease in cell viability and cell cycle arrest in G2-phase. Cells treated with 5-aza-dC clearly changed their morphology and expressed moderate levels of MyHC. CONCLUSIONS: MiR-378a-3p may function as a tumour suppressor in RMS and the restoration of its expression would be of therapeutic benefit in RMS. Furthermore, the role of epigenetic modifications in RMS deserves further investigations.

Wilms' tumor--lessons and outcomes--a 25-year single center UK experience.

Wilms' tumor (WT) is a common childhood renal cancer. A 25-year single center UK experience is reported. During 1985-2010, 97 children underwent immediate nephrectomy or delayed resection of tumor after chemotherapy. Survival, morbidity, and late effects following treatment are described. Tumor distribution was: Stage I, 25.7% (n = 25); Stage II, 24.7% (n = 24); Stage III, 26.8% (n = 26); Stage IV, 17.5% (n = 17); and Stage V, 5.2% (n = 5). Immediate nephrectomy was performed in 39% (n = 38) patients with elective delayed resection in 61% (n = 59) cases. Ten patients had cavotomy to excise tumor involving vena cava territory. Two cases required cardiopulmonary bypass. Tumor rupture was recorded in eight (8.5%) total operated cases-after immediate (n = 5/37), 13.5% vs delayed nephrectomy-(n = 3/57), 5.2%; X(2) P = .154. From 2001 onwards, one case of tumor rupture was recorded at this center after the universal adoption of UKW3 and SIOP guidelines advocating preoperative chemotherapy and delayed nephrectomy for all WT. Three treatment-related deaths occurred-hepatic veno-occlusive disease (n = 2) with actinomycin D and a single WT fatality due to vascular injury. Overall survival was 84.5% (82/97 cases). Two patients developed "late malignancies" -thyroid cancer and a basal cell carcinoma. This study demonstrates excellent survival for WT comparable with national outcomes and international cooperative studies. Adverse events with chemotherapy and surgery, including "late onset," second malignancies deserve special consideration.

Characterization of Wnt/ß-catenin signaling in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and accounts for about 5% of all malignant paediatric tumours. ß-Catenin, a multifunctional nuclear transcription factor in the canonical Wnt signaling pathway, is active in myogenesis and embryonal somite patterning. Dysregulation of Wnt signaling facilitates tumour invasion and metastasis. This study characterizes Wnt/ß-catenin signaling and functional activity in paediatric embryonal and alveolar RMS. Immunohistochemical assessment of paraffin-embedded tissues from 44 RMS showed ß-catenin expression in 26 cases with cytoplasmic/membranous expression in 9/14 cases of alveolar RMS, and 15/30 cases of embryonal RMS, whereas nuclear expression was only seen in 2 cases of embryonal RMS. The potential functional significance of ß-catenin expression was tested in four RMS cell lines, two derived from embryonal (RD and RD18) RMS and two from alveolar (Rh4 and Rh30) RMS. Western blot analysis demonstrated the expression of Wnt-associated proteins including ß-catenin, glycogen synthase kinase-3ß, disheveled, axin-1, naked, LRP-6 and cadherins in all cell lines. Cell fractionation and immunofluorescence studies of the cell lines (after stimulation by human recombinant Wnt3a) showed reduced phosphorylation of ß-catenin, stabilization of the active cytosolic form and nuclear translocation of ß-catenin. Reporter gene assay demonstrated a T-cell factor/lymphoid-enhancing factor-mediated transactivation in these cells. In response to human recombinant Wnt3a, the alveolar RMS cells showed a significant decrease in proliferation rate and induction of myogenic differentiation (myogenin, MyoD1 and myf5). These data indicate that the central regulatory components of canonical Wnt/ß-catenin signaling are expressed and that this pathway is functionally active in a significant subset of RMS tumours and might represent a novel therapeutic target.

Elevated levels of miR-145 correlate with SMAD3 down-regulation in cystic fibrosis patients.

MicroRNAs (miRNAs) have recently emerged as important gene regulators in Cystic Fibrosis (CF), a common monogenic disease characterized by severe infection and inflammation, especially in the airway compartments. In the current study, we show that both miR-145 and miR-494 are significantly up-regulated in nasal epithelial tissues from CF patients compared with healthy controls (p<0.001 and p<0.01, respectively) by Quantitative Real-Time PCR. Only miR-494 levels showed a trend of correlation with reduced CFTR mRNA expression and positive sweat test values, supporting the negative regulatory role of this miRNA on CFTR synthesis. Using computational prediction algorithms and luciferase reporter assays, SMAD family member 3 (SMAD3), a key element of the TGF-ß1 inflammatory pathway, was identified as a target of miR-145. Indeed, miR-145 synthetic mimics suppressed by approximately 40% the expression of a reporter construct containing the SMAD3 3'-UTR. Moreover, we observed an inverse correlation between SMAD3 mRNA expression and miR-145 in CF nasal tissues (r=-0.68, p=0.0018, Pearson's correlation). Taken together, these results confirm the pivotal role of miRNAs in the CF physio-pathogenesis and suggest that miRNA deregulation play a role in the airway disease severity by modulating CFTR levels as well as the expression of important molecules involved in the inflammatory response. miR-494 and miR-145 may, therefore, be potential biomarker and therapeutic target to specific CF clinical manifestations.

Long-term follow-up of the "wait and see" approach to localized perinatal adrenal neuroblastoma.

BACKGROUND: Evidence-based guidelines for the management of localized perinatal adrenal neuroblastoma are not yet available. We describe our preliminary experience managing this tumor with a "wait and see" policy. METHODS: A single-center prospective study (February 2002 to December 2009) was conducted with 12 consecutive patients in whom an adrenal mass was detected antenatally or within the first 3 months of life. Diagnostic workup included the following investigations: measurement of urine catecholamine metabolites, imaging studies (ultrasonography, magnetic resonance imaging, or computed tomography), metaiodobenzylguanidine scintigraphy, and/or core needle biopsy. RESULTS: The male/female ratio was 1.4:1.0. Median tumor size at presentation was 29 mm (range 10-50 mm). Eight lesions were detected antenatally. Ten lesions were diagnosed as localized neuroblastoma. Of these ten lesions, four were excised because of parental preference (n = 2), tumor enlargement (n = 1) or tumor persistence (n = 1). The remaining six patients underwent watchful clinical observation, which showed progressive tumor shrinkage and complete regression within 10-39 months (median 12.5 months). The final two lesions were small predominantly cystic lesions without a clear-cut diagnosis. They were managed noninvasively. At an overall median follow-up of 109 months (range 30-122 months), all patients are alive and disease-free, although one patient progressed to stage 4 disease despite early excision of the primary tumor. CONCLUSIONS: Spontaneous regression of localized perinatal adrenal neuroblastoma occurs often, and a "wait and see" strategy seems justified in these small infants. Patients with enlarging or stable lesions that have persisted for several months may benefit from surgery, although prompt excision may not prevent tumor progression.

Galectin-3 impairment of MYCN-dependent apoptosis-sensitive phenotype is antagonized by nutlin-3 in neuroblastoma cells.

MYCN amplification occurs in about 20-25% of human neuroblastomas and characterizes the majority of the high-risk cases, which display less than 50% prolonged survival rate despite intense multimodal treatment. Somehow paradoxically, MYCN also sensitizes neuroblastoma cells to apoptosis, understanding the molecular mechanisms of which might be relevant for the therapy of MYCN amplified neuroblastoma. We recently reported that the apoptosis-sensitive phenotype induced by MYCN is linked to stabilization of p53 and its proapoptotic kinase HIPK2. In MYCN primed neuroblastoma cells, further activation of both HIPK2 and p53 by Nutlin-3 leads to massive apoptosis in vitro and to tumor shrinkage and impairment of metastasis in xenograft models. Here we report that Galectin-3 impairs MYCN-primed and HIPK2-p53-dependent apoptosis in neuroblastoma cells. Galectin-3 is broadly expressed in human neuroblastoma cell lines and tumors and is repressed by MYCN to induce the apoptosis-sensitive phenotype. Despite its reduced levels, Galectin-3 can still exert residual antiapoptotic effects in MYCN amplified neuroblastoma cells, possibly due to its specific subcellular localization. Importantly, Nutlin-3 represses Galectin-3 expression, and this is required for its potent cell killing effect on MYCN amplified cell lines. Our data further characterize the apoptosis-sensitive phenotype induced by MYCN, expand our understanding of the activity of MDM2-p53 antagonists and highlight Galectin-3 as a potential biomarker for the tailored p53 reactivation therapy in patients with high-risk neuroblastomas.