Study On The Functioning Of Astrocyte Elevated Gene-1 In Neuroblastoma And Effects Of Gene Silencing On Biological Behavior Of Neuroblastoma Cells

BackgroundNeuroblastoma is a malignant tumour consisting of undifferentiated neuroectodermal cells, derived from the neural crest. As is typical of embryonic tumours, neuroblasts are indistinguishable from normal developing neuroblastic cells in the embryo. It represents 9% of all neoplasias in children and 90% of cases are diagnosed in the first 5 years of life. Neuroblastoma has diverse clinical features related to its variable sites of origin, the frequent presence of distant metastases (around 50% of cases at diagnosis), secretion of catecholamine metabolites and also paraneoplastic syndromes. Generally, most patients with neuroblastoma are treated with conventional therapeutic approaches that include surgery, external beam radiation therapy and cytotoxic chemotherapy.As the genes, proteins and pathways relevant for the pathogenesis of neuroblastoma are identified and characterized, better understanding of the unique biological basis of neuroblastoma is expected to provide insight into the development of novel, more biologically based therapies. Although a large body of basic research into genes and oncogenes has accumulated up till the present, increased/decreased expression of the molecular factors, MYCN, H-ras and trkA is well known in neuroblastoma, the molecular mechanism of its development and progression remains poorly understood, and thus far, no specific signature of neuroblastoma gene expression has been reported to allow for patient-tailored therapy strategies. Furthermore, the clinical hallmark of neuroblastoma is heterogeneity, with the likelihood of cure varying widely according to age at diagnosis, clinical stage, and tumor histology. Hence, it is of great clinical value to further understand the molecular mechanisms underlying the progression of neuroblastoma and to identify effective markers for the diagnosis and prognosis of the disease as well as novel therapeutic targets.The accumulation of mutations during carcinogenesis results in 6 essential alterations in cell physiology that collectively dictate malignant growth: self sufficiency in cell growth, insensitivity to growth-inhibitory signals, evasion of cell death, limitless replicative proliferation, sustained angiogenesis, and tissue invasion and metastasis. Most of these alterations affect cell signaling pathways, and the majority of oncogenes are integral components of cellular signaling circuits, which are up-regulated or constitutively activated in malignant cells. Recent studies highlight a potential role of astrocyte elevated gene (AEG) -1 in promoting tumor progression and metastasis. AEG-1 was cloned as an human immunodeficiency virus (HIV)-1-inducible and tumor necrosis factor-α(TNF-α)-inducible transcript in primary human fetal astrocytes (PHFA) by a rapid subtraction hybridization approach. AEG-1 down-regulates the expression of the glutamate transporter EAAT2; thus, it is implicated in glutamate-induced excitotoxic damage to neurons as evident in HIV-associated neurodegeneration. Interestingly, AEG-1 expression is elevated in subsets of breast cancer, glioblastoma multiforme and melanoma cells, and AEG-1 cooperates with Ha-ras to augment the transformed phenotype of normal immortal cells. Moreover, AEG-1 is overexpressed in 95% of human malignant glioma samples when compared with normal human brain. Overexpression of AEG-1 increases and siRNA inhibition of AEG-1 decreases migration and invasion of human glioma cells, respectively. AEG-1 contains a lung-homing domain facilitating breast tumor metastasis to lungs. These findings indicate that AEG-1 might play a pivotal role in the pathogenesis, progression and metastasis of diverse cancers.AEG-1 is a downstream target molecule of Ha-ras and c-myc mediating their tumor promoting effects. AEG-1 can physically interact with p65 and modulate its function in the nucleus and increased expression of adhesion molecules by activating the NF-κB pathway. Knockdown of AEG-1 inhibited prostate cancer progression through up-regulation of FOXO3a activity, and that such an alteration of FOXO3a activity was dependent on the reduction of protein kinase B (AKT) activity. AEG-1 regulates several hallmarks of metastatic cancers including aberrant proliferation, increased adhesion and invasiveness and resistance to apoptosis under certain stressful condition. Although published studies have suggested that AEG-1 expression is up-regulated in subsets of breast cancer, multiform glioblastoma, melanoma, and prostate cancer, implicating the possibility of using AEG-1 as an indicator of cancer development or progression, the functionality of overexpression in carcinoma progression and pathogenesis remains to be elucidated. Recently, Li et al. documented that high expression of AEG-1 significant correlated with the clinical staging of the patients with breast cancer. Till now, there has been no report on whether AEG-1 is expressed in neuroblastoma and the expression of AEG-1 is correlated with clinical staging as well as survival in neuroblastoma patients. In the present study, we examined the characterization of AEG-1 expression in neuroblastoma of various clinicopathologic grades, correlation of AEG-1 expression to long-term survival of patients, and expression and localization of AEG-1 in fresh neuroblastoma tissues and cell lines.RNA interference (RNAi) is a phenomenon in which double strand RNA (dsRNA) silences the homologous gene expression. It has been a powerful tool in the research of specific gene function. It has also been used in gene therapy research of cancer and viral infections. It is practical that lentiviral vectors are used in mediating RNAi for two features: more efficient transduction of non-dividing cells and stable RNAi by integrating shRNA expression cassette into genome of host cells.In the light of the previous studies showing that AEG-1 might play a pivotal role in the pathogenesis, progression and metastasis of diverse cancers, we hypothesized that the interference of AEG-1 could not only inhibit proliferation, and apoptosis, but also and interfere the metastasis of neuroblastoma cells. To begin with our study, two stable AEG-1 silencing neuroblastoma cell lines, M17/AEG-1(-) and SK-N-SH/AEG-1(-), were established with lentiviral vector mediated RNAi technology. Then we determined whether AEG-1 silencing could inhibit proliferation and apoptosis of neuroblastoma cells and whether AEG-1 silencing interfere the metastasis of neuroblastoma cells.Chapter One: Overexpression of astrocyte elevated gene-1 predicted poorprognosis in neuroblastoma1. Aim(1) To dect the expression of AEG-1 in neuroblastoma tissue samples.(2) To study the relationship between AEG-1 and clinical characteristics. (3) To clarity the independent factor of neuroblastoma to the patients' survival.2. Materials and Methods(1) This study was conducted on a total of 32 paraffin-embedded neuroblastoma samples, which were clinically diagnosed at the Qilu Hospital of Shandong University from January 1993 to October 2004.(2) Immunohistochemistry for AEG-1 protein expression using a rabbit polyclonal anti-AEG-1 antibody(3) Intracellular localization of AEG-1 was examined in neuroblastoma, using a rabbit polyclonal anti-AEG-1 antibody and immunofluorescence microscopy.3. Results(1) AEG-1 was strongly positive (+2/+3) in 24 of 32 neuroblastomas (75%).(2) We analyed the clinical data of 32 neuroblastoma patients. AEG-1 expression was 37.5% (3/8) among patients younger than one year old, and 87.5% (21/24) among patients older than one year old. Compared to the group with positive AEG-1 expression, AEG-1 expression was significantly associated with the age (P=0.012); AEG-1 expression was 77.8% (14/18) among male patients, and 71.4% (10/14) among female patients. Compared to the group with positive AEG-1 expression, AEG-1 expression was not associated with the gender (P=0.703); AEG-1 expression was 78.9% (13/19) among patients whose primary tumor site was adrenal, and 69.2% (11/13) among patients whose primary tumor site was extral-adrenal. Compared to the group with positive AEG-1 expression, AEG-1 expression was not associated with the age (P=0.420); AEG-1 expression was 50% (5/10) among early stage (I+II+IVs) patients, and 90% (18/20) among advanced stage (III+IV) patients. Compared to the group with positive AEG-1 expression, AEG-1 expression was significantly associated with clinical staging (P=0.030); AEG-1 expression was 58.8% (10/17) among patients with favourable histology (FH), and 93.3% (14/15) among patients with unfavourable histology (UFH). Compared to the group with positive AEG-1 expression, AEG-1 expression was significantly associated with histology classification (P=0.041).(3) Positive expression of AEG-1 was associated with age (r =0.404, P=0.022), clinical stage (r =0.447, P=0.010) and histology classification (r =0.389, P=0.024), while not with gender and the primary tumor site by spearman correlation analysis.(4) Survival analysis was done by the Kaplan-Meier method and log-rank test. The expression of AEG-1 (P=0.031), age (P=0.020), clinical stage (P=0.015) and histology classification (P=0.003) were associated with clinical outcome. While gender (P=0.902) and the primary tumor site (P=0.242) was not associated with clinical outcome. The cumulative survival percentage for the patients with and without AEG-1 expression at evaluation year 5 were 17.7% and 62.5%, respectively, significantly different (P=0.031) from each other. The cumulative survival percentage for patients younger and older than one year at evaluation year 5 were 17.7% and 62.5%, respectively, significantly different (P=0.020) from each other. The cumulative survival percentage for patients with early stage and with advanced stage, at evaluation year 5 were 69.4% and13.8%, respectively, significantly different (P=0.015) from each other. The cumulative survival percentage for patients with favorable histology and with unfavorable histology, at evaluation year 5 were 45.7% and 15.6%, respectively, significantly different (P=0.003) from each other.(5) Multivariate analysis revealed that histology classification (INPC) was a significant prognostic factor (P=0.022), independent of the other conventional prognostic factors to neuroblastoma patients. While the expression of AEG-1 (P=0.176), age (P=0.247), and clinical stage (P=0.694) was not independent prognostic factor.(6) AEG-1 protein had a diffuse distribution in the cytoplasm and the staining was very intense in the perinuclear region, but not at the nucleus in neuroblastoma cells.4. Conclusions(1) AEG-1 was strongly positive (+2/+3) in 24 of 32 neuroblastomas (75%).(2) AEG-1 expression was significantly associated with the age, clinical stage and histology classification of the disease by x² test. In contrast, no correlation was found between gender or primary tumor site and the expression level of AEG-1 protein of neuroblastoma patient.(3) Positive expression of AEG-1 was associated with age, clinical stage and histology classification, while not with gender and the primary tumor site by spearman correlation analysis. (4) Survival analysis was done by the Kaplan-Meier method and log-rank test. The expression of AEG-1, age, clinical stage and histology classification were associated with clinical outcome. While gender and the primary tumor site was not associated with clinical outcome.(5) Multivariate analysis revealed that histology classification (INPC) was a significant prognostic factor, independent of the other conventional prognostic factors to neuroblastoma patients.(6) AEG-1 protein had a diffuse distribution in the cytoplasm and the staining was very intense in the perinuclear region, but not at the nucleus in neuroblastoma cells.Chapter Two: Silencing of AEG-1 in Neuroblastoma Cells Mediated byLentiviral Vector1. Aim(1) To constructed AEG-1 RNAi lentiviral vectors.(2) To determine the functional titre and molecular copies of packaged AEG-1 RNAi lentiviral vectors.(3) To constructe AEG-1 gene specific silenced M17 and SK-N-SH cells and the non-specific controls.(4) To determine AEG-1 mRNA level.(5) To analyze AEG-1 protein.2. Materials and Methods(1) AEG-1 RNAi lentiviral vectors were constructed with BLOCK-iT Lentiviral RNAi Expression System.(2) AEG-1 RNAi lentiviral vectors were packaged by co-transfecting 293FT with 4 plasmids. The functional titre and molecular copies were determined with transduction assay and real time PCR respectively.(3) AEG-1 gene specific silenced M17 and SK-N-SH cells and the non-specific controls were constructed by virus transduction and blasticidin screening.(4) AEG-1 mRNA level was determined by real time RT-PCR. (5) AEG-1 protein was analyzed by Western Blot3. Results(1) Two entry plasmids targeting 2 AEG-1 mRNA sequences were constructed successfully. Both of them can mediate AEG-1RNAi effectively. (2) The functional titre of 2×10¹⁰copies/ml Lenti6-AEG-1 and Lenti6-NS in M17 were 1.8×10⁶TU/ml and 1.2×10⁶TU/ml, and 1.7×10⁶TU/ml 1.2×10⁶TU/ml in SK-N-SH cells, respectively.(3) AEG-1 gene specific silenced M17 and SK-N-SH cells were established constructed and named M17/AEG-1(-) and SK-N-SH/AEG-1(-), the corresponding non-specific controls were named M17/NS and SK-N-SH/NS respectively.(4) Transduction of cells with AEG-1 RNAi vector resulted in sequence specific silencing with 80% decreases of AEG-1 mRNA transcription and 90% of protein expressions respectively.4. Conclusions(1) The construction and production of lentiviral vectors was successful.(2) The selected cells demonstrated significant disruption of AEG-1 expression at both mRNA and protein levelsChapter Three: Effects of AEG-1 gene silence on biological behavior ofneuroblastoma cells1. Aim(1) To test the function of AEG-1 in proliferation of neuroblastoma cells. (2) To determine the function of AEG-1 in apoptosis.(3) To illustrate the function of AEG-1 in cell cycle.(4) To elucidate the function of AEG-1 in invasion.(5) To analyse the function of AEG-1 in drug sensitivity.2. Materials and Methods(1) Proliferation of neuroblastoma cells were tested by MTT and clonogenic forming assay(2) Apoptosis of cells were tested by flow cytometry with Annexin-V and PI staining.(3) Cell cycle analysis is performed by flow cytometry. (4) Invasion of cells were tested by transwell method.(5) Drug sensitivity of neuroblastoma cells were tested by MTT assay.3. Results(1) Knockdown AEG-1 reduces proliferation to 45.1%, 57.4% and inhibits the colony forming to 43.5%, 56.7% compare with parental cells. (2) AEG-1 silencing could increase apoptosis level to 7.5% and 6.4%. (3 ) Cell cycle distribution of M17 and SK-N-SH was similar. More silencing cells were at G1/G0 phase and less were at S phase, P<0.05.(4) Knock down of AEG-1 decreased the invasion of cells to 56.8% and 50.0%.(5) The sensitivity of AEG-1 silenced cells to doxorubicin or cisplatin were increased compared parents cells.4. Conclusions(1) AEG-1 serves in regulating cell proliferation and apoptosis.(2) Silencing of AEG-1 interfered with cell cycle control.(3) AEG-1 serves in regulating cell invasion.(4) Cisplatin chemotherapy could be more effective in combination with RNAi mediated knockdown of AEG-1.