Yeming Wu

HIF-1α contributes to proliferation and invasiveness of neuroblastoma cells via SHH signaling.

The aim of this study was to investigate the effects of hypoxia-inducible factor-1α (HIF-1α) on the proliferation, migration and invasion of neuroblastoma (NB) cells and the mechanisms involved. We here initially used the real-time polymerase chain reaction (real-time PCR), Western blotting and immunohistochemistry (IHC) to detect the expression of HIF-1α and components of the sonic hedgehog (SHH) signaling pathway in NB cells and human specimens. Subsequently, cell proliferation, migration and invasion were analyzed using the cell counting assay, wound healing assay and Transwell system in two types of human NB cell lines, SH-SY5Y and IMR32. In addition, the role of HIF-1α in NB cells growth was determined in a xenograft nude mouse model. We found that the level of HIF-1α was significantly upregulated during NB progression and was associated with the expression of two components of SHH signaling, SHH and GLI1. We next indicated that the proliferation, migration and invasiveness of SH-SY5Y and IMR32 cells were significantly inhibited by HIF-1α knockdown, which was mediated by small interfering RNAs (siRNAs) targeting against its mRNA. Furthermore, the growth of NB cells in vivo was also suppressed by HIF-1α inhibition. Finally, the pro-migration and proliferative effects of HIF-1α could be reversed by disrupting SHH signaling. In conclusion, our results demonstrated that upregulation of HIF-1α in NB promotes proliferation, migration and invasiveness via SHH signaling.

Preliminary analysis of stem cell-like cells in human neuroblastoma

BackgroundNeuroblastoma is an embryonic neoplasm originating from the neural crest with cellular heterogeneity as one of its oncobiological characteristics. This study was undertaken to determine whether human neuroblastoma contains stem cell-like cells.MethodsTwenty patients with neuroblastoma who have been treated in our hospital since January 2005 were divided into pre-operative chemotherapy (10 patients) and non-chemotherapy (10) groups. Tumor specimens of the patients were taken and paraffin sections were made. The expressions of stem cell markers CD133, ABCG2, CD117 and nestin were immunohistochemically detected in the specimens. Neuroblastoma cells were stained with Hoechst 33342 and PI. The side population (SP) cells were analyzed by the fluorescence-activated cell sorter. The disparity drug resistance to cisplatin (DDP) of SP and non-SP cells was measured with MTT colorimetric assay. The oncogenicity of SP and non-SP cells was identified in nude mice.ResultsThere was no significant difference in the expression intensity of CD117 and nestin between the two groups of specimens (P>0.05). There was a significant difference between the two groups in terms of the expression intensity of CD133 and ABCG2 (P<0.05). The SP cells accounted for 0.2%–1.3% of the total human neuroblastoma cells and were decreased to 0.1%–0.5% after verapamil treatment. The SP and non-SP cells showed disparity in cell growth experiment and drug resistance to DDP. Oncogenicity experiment revealed that nude mice could erupt tumor by an injection of l×106 SHSY5Y and WIV SP cells. However, the nude mice could not form tumor by an injection of l×106 non-SP cells.ConclusionNeuroblastoma might contain cancer stem cell-like cells.

Role of surgery in the treatment of patients with high-risk neuroblastoma who have a poor response to induction chemotherapy

BACKGROUND In instances of high-risk neuroblastoma that do not show a clinical response to induction therapy, whether it is worth performing surgical resection or not and whether gross total resection (GTR) is more important than subtotal resection (STR) remain controversial. METHODS We retrospectively analyzed the data of patients with stage 4 neuroblastoma aged 18 months or older at diagnosis. Primary tumor volumes were measured both at diagnosis and at the first tumor response evaluation (after 6 cycles of induction chemotherapy). If the tumor volume at the first response evaluation was > 50% of the initial tumor volume, the patient was categorized as a poor responder. Otherwise, the patient was categorized as a good responder. Only poor responders were included. Patients were evaluated for event-free survival (EFS), overall survival (OS), and complications of surgery based on extent of surgical intervention. RESULTS Sixty-five patients were included in this study. The 41 patients who underwent surgical intervention had a higher 3-year OS than the 24 patients who had a biopsy only (55.4% ± 8.1% vs. 31.3% ± 10.2%, P=0.02). However, there was limited improvement in 3-year EFS following surgical intervention. Three-year EFS rates of BX group (biopsy only) and OP group (surgical resection) were 24.2% ± 9.3% and 37.7% ± 7.9%, respectively (P=0.063). The extent of resection had no impact on 3-year OS (P=0.631) and 3-year EFS (P=0.796). Patients in the GTR group trended to have more severe surgical complications than patients in the STR group (P=0.105). CONCLUSIONS For high-risk neuroblastomas that do not show a clinical response to induction therapy, surgical resection is important in predicting outcome, but the extent of resection is not.

A novel HLXB9 mutation in a Chinese family with Currarino syndrome.

Introduction Currarino syndrome (CS), first described in 1981,1 is a congenital malformation typically associated with sacral agenesis, anorectal malformations, and a presacral mass. Patients affected by CS display a phenotypic variability, whereby the spectrum of phenotypes ranges from a severe triad to asymptomatic features.2,3 A familial tendency with autosomal dominant inheritance was noted by Yates et al. in 1983.4 Ross et al. further reported that mutations in the homeobox gene HLXB9 are the major cause of CS.5 Mutations in the HLXB9 gene have been identified in almost all reported cases of familial CS, and in approximately 30% of patients with sporadic CS.6 The HLXB9 gene is essential for proper pancreatic development and for the differentiation of motor neurons in the spinal cord.7,8 It has 3 exons and encodes a 403-amino acid transcription factor HB9 protein. The HB9 protein contains a homeodomain preceded by a highly conserved 82-amino acid domain and a poly-alanine region.9 In this article, we report a new HLXB9 gene mutation identified in a Chinese family with members suffering from CS, together with the clinical characteristics of the affected individuals.

Glial fibrillary acidic protein expression is an indicator of teratoma maturation in children

BackgroundThe present diagnosis of teratomas is limited to visual examination of their tissues. For the sake of treatment, teratomas are graded according to degrees of nerve tissue maturation. Mature fetal nerve tissue contains the astrocyte-specific intermediate filament protein, the glial fibrillary acidic protein (GFAP). This study aimed to investigate GFAP expressions in the nerve tissue of immature and mature teratomas, and to evaluate if GFAP is indicative of teratoma maturation in pediatric patients.MethodsNerve tissue specimens were collected from immature (10 children) and mature teratomas (45 children). Nerve tissue specimens as a control group were taken from 33 children with neuroblastoma. GFAP expression of the specimens was studied by immunohistochemical and semi-quantitative analyses.ResultsGFAP expression was low in the nerve tissue of immature teratomas and high in that of mature ones. A semi-quantitative analysis confirmed statistically significant difference between the GFAP expressions of immature and mature teratomas (P=0.0001).ConclusionGFAP is highly expressed in the nerve tissue of mature teratomas and low in that of immature ones, suggesting that the GFAP expression is a meaningful indicator of teratoma maturation. It is helpful for pathologists to diagnose and classify teratomas.

Delivery room surgery: an applicable therapeutic strategy for gastroschisis in developing countries

BackgroundThe survival rate of infants with gastroschisis has improved significantly. It is over 90% in developed countries, but 50% in developing countries. This study aimed to investigate the factors improving the survival rate of infants with gastroschisis in developing countries.MethodsNeonates meeting the inclusion criteria, who presented to our center since the establishment of delivery room surgery, were enrolled into this retrospective study. Data were evaluated specifically to determine the role of delivery room surgery in reducing the mortality and morbidity of infants with gastroschisis and to identify factors optimizing the conditions of outborn infants.ResultsA total of 64 infants were identified. The overall survival rate of the infants was 60.9%. The survival rate of infants in inborns was 76.5%, and the survival rate of infants in outborns was 43.3%. Infants of the outborn group took more time to reach full enteral feeding, and were more likely to require a prolonged stay in hospital when compared with those of the inborn group. Logistic analysis identified that the surgical technique, the presence of sepsis and intestinal necrosis could be expected to influence the outcome of gastroschisis.ConclusionsThe strategy of delivery of patients in a center prepared to perform delivery room closure of gastroschisis appears to improve the survival of patients with gastroschisis. Further reduction in mortality rates will depend on improved conditions of outborn infants.

Successful Sleeve Lobectomy of Inflammatory Myofibroblastic Tumor in a 4-year-old Child

Primary pulmonary tumors in small children have remained a challenge for pediatric surgeons. Pneumonectomy and radical lobectomy are limitedly indicated due to surgical difficulties and sequelae. Here, we present our experience with a 4-year-old patient who suffered from an inflammatory myofibroblastic tumor. A left lower sleeve lobectomy was performed, and the patient recovered significantly after surgery. At the last follow-up, the child was growing well without any sequel, which supports our hypothesis that in small children, sleeve resection is the preferred treatment for tumors on the main stem bronchus and presents an alternative to an otherwise unavoidable pneumonectomy.

Annexin A2 could enhance multidrug resistance by regulating NF-κB signaling pathway in pediatric neuroblastoma

BackgroundChemotherapy is one of major therapeutic regimens for neuroblastoma (NB) in children. However, recurrence and metastasis associated with poor prognosis caused by acquired multidrug resistance remains a challenge. There is a great need to achieve new insight into the molecular mechanism of drug resistance in NB. The aim of this study is to identify novel drug sensitivity-related biomarkers as well as new therapeutic targets to overcome chemoresistance.MethodsWe proteome-wide quantitatively compared protein expression of two NB cell lines with different drug sensitivities, isolated from the same patient prior to and following chemotherapy. Annexin A2 (ANXA2) emerged as a key factor contributing to drug resistance in NB. Then, we assessed the correlation of ANXA2 expression and clinical characteristics using a tissue microarray. Further, the roles of ANXA2 in chemoresistance for NB and the underlying mechanisms were studied by using short hairpin RNA (shRNA) in vitro and vivo.ResultsFirst in total, over 6000 proteins were identified, and there were about 460 significantly regulated proteins which were up- or down-regulated by greater than two folds. We screened out ANXA2 which was upregulated by more than 12-fold in the chemoresistant NB cell line, and it might be involved in the drug resistance of NB. Then, using a tissue chip containing 42 clinical NB samples, we found that strong expression of ANXA2 was closely associated with advanced stage, greater number of chemotherapy cycles, tumor metastasis and poor prognosis. Following knockdown of ANXA2 in NB cell line SK-N-BE(2) using shRNA, we demonstrate enhanced drug sensitivity for doxorubicin (2.77-fold) and etoposide (7.87-fold) compared with control. Pro-apoptotic genes such as AIF and cleaved-PARP were upregulated. Inhibiting ANXA2 expression attenuated transcriptional activity of NF-κB via down-regulated nuclear translocation of subunit p50. Finally, simulated chemotherapy in a xenograft NB nude mouse model suggests that ANXA2 knockdown could improve clinical results in vivo.ConclusionOur profiling data provided a rich source for further study of the molecular mechanisms of acquired drug resistance in NB. Further study may determine the role of ANXA2 as a prognostic biomarker and a potential therapeutic target for patients with multidrug-resistant NB.

Phosphoproteomics reveals ALK promote cell progress via RAS/ JNK pathway in neuroblastoma

Emerging evidence suggests receptor tyrosine kinase ALK as a promising therapeutic target in neuroblastoma. However, clinical trials reveal that a limited proportion of ALK-positive neuroblastoma patients experience clinical benefits from Crizotinib, a clinically approved specific inhibitor of ALK. The precise molecular mechanisms of aberrant ALK activity in neuroblastoma remain elusive, limiting the clinical application of ALK as a therapeutic target in neuroblastoma. Here, we describe a deep quantitative phosphoproteomic approach in which Crizotinib-treated neuroblastoma cell lines bearing aberrant ALK are used to investigate downstream regulated phosphoproteins. We identified more than 19,500—and quantitatively analyzed approximately 10,000—phosphorylation sites from each cell line, ultimately detecting 450–790 significantly-regulated phosphorylation sites. Multiple layers of bioinformatic analysis of the significantly-regulated phosphoproteins identified RAS/JNK as a downstream signaling pathway of ALK, independent of the ALK variant present. Further experiments demonstrated that ALK/JNK signaling could be inactivated by either ALK- or JNK-specific inhibitors, resulting in cell growth inhibition by induction of cell cycle arrest and cell apoptosis. Our study broadly defines the phosphoproteome in response to ALK inhibition and provides a resource for further clinical investigation of ALK as therapeutic target for the treatment of neuroblastoma.

Proteomic profiling of isogenic primary and metastatic medulloblastoma cell lines reveals differential expression of key metastatic factors

Medulloblastoma is the most common malignant brain tumor in children. Around 30% of medulloblastoma patients are diagnosed with metastasis, which often results in a poor prognosis. Unfortunately, molecular mechanisms of medulloblastoma metastasis remain largely unknown. In this study, we employed the recently developed deep proteome analysis approach to quantitatively profile the expression of >10,000 proteins from CHLA-01-MED and CHLA-01R-MED isogenic cell lines derived from the primary and metastatic tumor of the same patient diagnosed with a group IV medulloblastoma. Using statistical analysis, we identified ~1400 significantly altered proteins between the primary and metastatic cell lines including known factors such as placental growth factor (PLGF), LIM homeobox 1 (LHX1) and prominim 1 (PROM1), as well as the negative regulator secreted protein acidic and cysteine rich (SPARC). Additional transwell experiments and immunohistochemical analysis of clinical medulloblastoma samples implicated yes-associated protein 1 (YAP1) as a potential key factor contributing to metastasis. Taken together, our data broadly defines the metastasis-relevant regulated proteome and provides a precious resource for further investigating potential mechanisms of medulloblastoma metastasis. SIGNIFICANCE This study represented the first deep proteome analysis of metastatic medulloblastomas and provided a valuable candidate list of altered proteins in metastatic medulloblastomas. The primary data suggested YAP1 as a potential driver for the metastasis of medulloblastoma. These results open up numerous avenues for further investigating the underlying mechanisms of medulloblastoma metastasis and improving the prognosis of medulloblastoma patients.