The Role Of WT1 In The Tumorigenesis And Differentiation Of Neuroblastoma

ObjectiveThe heterogeneity of biological aspect and clinical behavior results in treatment resistance of neuroblastoma (NB), especially high risk group. Toxicity from current treatment modes is already significant, and there is little room to further intensify existing therapies. Alternative treatment strategies are therefore needed in order to improve survival. Immunotherapy is an attractive therapeutic option as it potentially offers a much more specific and less toxic treatment than conventional therapies. Over the past years, the immunotherapy using peptide vaccines against Wilms tumor gene (WT1) in adults with leukemia and various solid cancers was promising. To determine whether WTl vaccines are applicable for neuroblastoma, firstly, we must understand the exact function of WTl in neuroblastoma. In this study, we aimed to elucidate the roles of WT1 in the development of NB and the expression of WT1 in neuroblastomas.MethodsWTl mRNA expression was investigated in the tumor tissue of NBs, GNs and 4 NB cell lines using real-time RT-PCR. The siRNA against WTl was utilized to suppress WT1 gene expression in NB cell lines (NB 19 and NB69), and the effects on cell proliferation were investigated using WST-1 assay. WTl protein expression was examined in neuroblastomas (NBs) and ganglioneuromas (GNs) using immunohistochemistry. To observe the effects of WT1 gene silence on neuroblastic cell differentiation induced by retinoic acid (RA), the siRNA against WT1 was also used. Statistical analysis:The positive rates of WT1 immunostaining in NBs and GNs were compared by using Fisher’s Exact Probability Test. The comparison of WTl mRNA expression in NBs and GNs was performed with Mann-Whitney Test. The Kruskal-Wallis Test was used to analyze the difference of WT1 mRNA expression among stage groups of NBs. The effect of WT1 mRNA expression on survival was analyzed using Kaplan-Meier survival curve and Log-Rank Test. The t-test was introduced to examine the difference of cell proliferation between control and WTl siRNA group. The data were expressed as mean±SD for normally distributed data and median or rage for skewed data. The level of significance was set at 0.05. All data were processed with SPSS for windows software version 13.0 (SPSS Inc, Chicago, IL, USA).Results1. On the basis of comparison of NBs and GNs, there was no elevation of WTl expression in NBs with high histological grade (p=0.261). In NBs, the WTl mRNA expression levels were not correlated with Evans’s stages (stage I,Ⅱ,Ⅲ, and IV) (P=0.412). The median of relative WTl mRNA expression in NBs was 8.55X10-4, which was defined as threshold value. The NBs were divided into two groups:high (more than 8.55×10-4) and low WT1 mRNA expression group (less than 8.55×10-4). Survival analysis indicated that the expression levels did not affect the prognosis (p=0.193).2. Positive WTl mRNA expression in NB1, NB19, NB69 and SK-N-SH was observed. The highest expression appeared in NB69 without MYCN amplification (relatively low malignancy).3. WTl gene expressions were successfully suppressed by siRNA. The efficacies of WT1 gene knockdown were 74.3%±13.3% and 72.3%±10.5%in NB19 and NB69 respectively.4. For NB19 cells, there was no significant difference of cell growth between WT1 and control siRNA group (p=0.937). For NB69 cells, knockdown of WT1 gene with siRNA significantly prompted the cell growth (p=0.001).5. Immunohistochemistry showed that the differentiating ganglion cells were more intensively stained than the primitive neuroblastic cells. The positive rate was significantly higher in GNs than in NBs with WT1 polyclonal antibody (p=0.000) and monoclonal antibody (p=0.009).6. Compared with corresponding RA and RA+Control siRNA group, the outgrowths of neurite were inhibited in RA+Test siRNA group of both NB19 and NB69. This showed that WT1 gene silence suppressed the RA-induced differentiation.Conclusions 1. The levels of WT1 mRNA expression were not correlated with histological grade, clinical stage and prognosis. Moreover, among the four neuroblastic cells, NB69 with relatively low malignancy exhibited the highest WT1 mRNA expression. WT1 gene knockdown promoted cell growth in NB69 cells which had the highest WT1 mRNA expression. These evidences indicate that WT1 does not participate in the oncogenicity of neuroblastoma, and may be an anti-proliferation factor.2. Immunohistochemistry of NBs and GNs showed that ganglion cells possessed a higher expression of WT1 protein. The RA-induced differentiation was suppressed by WT1 gene silence in neuroblastic cells. WT1 gene knockdown promoted cell growth in NB69 cells. Taken together, WT1 may be a pro-differentiation and pro-regression factor which induces neuroblastic cells to differentiate into ganglionic cells or regress.3. Based on the low level expression of WT1 protein in NBs and its anti-proliferation and pro-differentiation function, we can conclude that WT1 vaccines are not applicable for neuroblastoma. It may be a potential target for biological treatment of neurob lastoma.