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FoxM1 moderately affects the growth of neuroblastoma cells. Viable cell number was measured by using CellTiter-Glo luminescent cell viability assay kit Promega every two days. The fold change in cell count was presented by luminescence unit and was normalized by the value at day zero. B, Seventy-two hours after FoxM1 or control siRNA transfection, cells were fixed and stained with propidium iodide PI and analyzed by flow cytometry for sub-G1 population. C, Seventtwo hours after FoxM1 or control siRNA transfection, cell lysates were collected and assayed for cleaved caspase-3 by immunoblot. A, Colony formation is induced in cells stably expressing FoxM1.

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FoxM1 moderately affects the growth of neuroblastoma cells. Viable cell number was measured by using CellTiter-Glo luminescent cell viability assay kit Promega every two days.

The fold change in cell count was presented by luminescence unit and was normalized by the value at day zero. B, Seventy-two hours after FoxM1 or control siRNA transfection, cells were fixed and stained with propidium iodide PI and analyzed by flow cytometry for sub-G1 population. C, Seventtwo hours after FoxM1 or control siRNA transfection, cell lysates were collected and assayed for cleaved caspase-3 by immunoblot. A, Colony formation is induced in cells stably expressing FoxM1.

Colonies were stained and quantified after 2 weeks. Cells were plated a density of 1. Colonies were stained and counted after three weeks. FoxM1 and ESC-like gene signature. Cyclophilin was used as a loading control. The intensity of the bands was determined by ImageJ software and the intensity of control samples were set at one. Sox2 target genes were reported from chip array study 49 , 39 , table S1 and core ESC-like module was previously purposed 49 table S6. Among them, genes that were also Sox2 targets were highlighted in red.

FoxM1 mediated anchorage-independent growth requires expression of Sox2. Cell lysates were collected at four days after transfection. The fold change in cell count was presented by luminescence unit and was normalized by day zero. Colonies were stained and quantified after three weeks.

D, Quantification of colonies numbers derived from C. Malignant neuroblastomas contain stem-like cells. These tumors also overexpress the Forkhead box transcription factor FoxM1. In this study, we investigated the roles of FoxM1 in the tumorigenicity of neuroblastoma.

We showed that depletion of FoxM1 inhibits anchorage-independent growth and tumorigenicity in mouse xenografts. Moreover, knockdown of FoxM1 induces differentiation in neuroblastoma cells, suggesting that FoxM1 plays a role in the maintenance of the undifferentiated progenitor population. We showed that inhibition of FoxM1 in malignant neuroblastoma cells leads to the downregulation of the pluripotency genes sex determining region Y box 2 Sox2 and Bmi1. We provided evidence that FoxM1 directly activates expression of Sox2 in neuroblastoma cells.

Together, our observations suggested that FoxM1 plays an important role in the tumorigenicity of the aggressive neuroblastoma cells through maintenance of the undifferentiated state. Neuroblastoma, a malignancy derived from neural crest of the sympathetic nervous systems, is the second most common solid tumor in childhood and the most common tumor of infancy with an incidence of The origin of neuroblastoma is thought to be incompletely committed precursor cells derived from neural crest tissues 2.

Neuroblastoma is unique in terms of its clinical bipolarity. The unfavorable prognosis has been associated with several factors including MYCN and TrkB gene amplification, chromosome 1p losses, and so on 4 - 6.

However, the molecular pathways mediating tumorigenicity of aggressive neuroblastoma remain largely unclear. Consistent with its clinical bipolarity, neuroblastomas are heterogeneous in terms of pathologic features, ranging from tumors containing predominantly undifferentiated neuroblast cells to those that are mainly well-differentiated neurons surrounded by Schwann stroma cells 1 , 7.

This heterogeneous feature is manifested in the cell lines established in vitro. The less malignant S-type cells substrate adherent and non-neuronal are usually flattened and attach strongly to the substrate. The N-type cells neuroblastic , which grow as poorly attached aggregates of small and rounded cells, are tumorigenic and rapidly proliferating.

The I-type cells intermediate , which are less differentiated than the N type, represent malignant, multipotent neural crust stem cells 8 - The I-type neuroblastoma cells possess self-renewal ability and have significantly higher tumor-forming capacity, as determined by soft agar colony formation and tumor growth in immunodeficient mice 7 , 9 , Studies from both patient samples and in vitro cell culture system suggested that neuroblastoma contains pluripotent tumor initiating cells TIC; refs.

The existence of TICs may account for both the heterogeneity nature of neuroblastoma as well as the tumor relapse 11 , 13 , It is also consistent with the observation that the I-type neuroblastoma cells, the most aggressive type of neuroblastoma cells, are malignant neural crest stem cells that possess the ability to self-renewal High frequency of the I-type cells in tumor is associated with increased recurrence 9.

A better understanding of the tumorigenicity mechanism of the neuroblastoma possessing stem cell properties will be critical to improve therapeutic outcomes. Sox2 sex determining region Y box 2 is a transcription factor that is essential for the maintenance of self-renewal and growth of both embryonic and adult stem cells Recent evidences imply that Sox2 is involved in promoting tumorigenicity in malignant tissues.

Sox2 functions as a lineage survival oncogene in lung and esophageal squamous cell carcinoma, where it promotes oncogenic function of tumor cells Consistently, Sox2 silencing in glioma leads to inhibition of proliferation and loss of tumorigenicity Its expression is also detectable in several other types of malignant tumors including neuroblastoma 18 - FoxM1 is a proliferation-specific transcriptional factor, given the fact that its expression is strongly correlated with the proliferation capacity of the cells.

It is expressed ubiquitously in all proliferating cells, including many tumor-derived cell lines. In normal tissues, FoxM1 is detectable in progenitors with extensive proliferating capacity, whereas its expression is depleted in differentiated or resting cells 24 , Numerous transgenic studies in mouse systems showed that FoxM1 is crucial for the development and progression of tumors of different origins, including liver, prostate, colon, breast, lung, brain, and so on 26 - However, the involvement of FoxM1 in neuroblastoma has not been characterized.

Here, we showed that depletion of FoxM1 inhibits tumorigenicity of neuroblastoma, which is associated with the induction of differentiation. Furthermore, we found FoxM1 is able to directly activate the expression of pluripotency gene Sox2 in neuroblastoma. Oligonucleotides were synthesized by Dharmacon Research. The plasmids and siRNA duplexes were transfected into cells by using Lipofectamine reagent Invitrogen in serum-free tissue culture medium following the manufacturer's protocol.

Neurospheres were dissociated by using chemical dissociation kit following the manufacture's protocol Stemcell Technologies. Dissociated cells were seeded in culture dish with grid Nunc at a clonal density. After 6 to 8 days, the newly generated neurospheres were counted under microscope.

Rabbit polyclonal antibody against FoxM1 was described Horseradish peroxidase-conjugated secondary antibodies were used to amplify the signal from primary antibody Bio-rad. The growth of the cell was monitored by measuring the luminescent signal by using the CellTiter-Glo Kit Promega every other day following manufacture's protocol.

For soft agar assay, cells were counted and plated in 6-well plates in 0. Colonies were stained with crystal violet and counted after 3 weeks. Pictures were taken under dissecting microscope. Picture of the mice were taken 4 weeks after injection. Chromatin immunoprecipitation ChIP assays were conducted as previously described FoxM1 antibody was used for immunoprecipitation.

In all treatments, 3 ng of CMV- Renilla luciferase was cotransfected as an internal control. Cells were harvested 24 hours after transfection, and protein extracts were subjected to dual luciferase assays Promega with firefly luciferase activity normalized to Renilla luciferase activity. Promoter activity was expressed as fold induction of transcription by the FoxM1b expression vector, where the promoter activity resulting from transfection with empty vector was set at 1.

Statistical significance was calculated by the Student's t test 2 tailed. Expression studies with patient samples by several groups had revealed that FoxM1 mRNA is significantly upregulated in neuroblastoma tissue samples compared with noncancerous ganglioneuroma or less aggressive ganglioneuroblastoma 35 - A box plot representing 2 different data sets from publicly available database is shown Fig.

However, the biological function of FoxM1 in neuroblastoma had not been elucidated. To evaluate the role of FoxM1 in neuroblastoma, we first investigated the effects of FoxM1 on anchorage-independent growth, which is a hallmark of tumorigenicity.

After 72 hours siRNA transfection, the protein level of FoxM1 was reduced significantly in these 2 cell lines, as evidenced by the Western blot Fig. The anchorage-independent growth capacity of neuroblastoma cells was checked by carrying out soft agar colony formation assay. For the BE 2 -C line, the reduction was even more severe. FoxM1 is critical for the tumorigenicity of neuroblastoma. White box, benign ganglioneuroma or ganglioneuroblastoma. Grey box, neuroblastoma. P values for the 2 data sets are 2.

Cell lysates were collected at 72 hours after transfection. C, representative pictures and quantification of anchorage-independent growth on soft agar plates. Colonies were stained and counted after 3 weeks. The strong inhibition of tumor growth showed that FoxM1 is critical for the tumorigenicity of the neuroblastoma cells. The loss of tumorigenicity could not be explained sufficiently by the inhibition of cell growth.

A growth curve analysis following depletion of FoxM1 indicated only a partial retardation of growth at the initial time points Supplementary Fig. At later time points, cell counts on FoxM1 silencing increased, most likely because of reexpression of FoxM1. Moreover, we did not see any significant increase in apoptosis based on changes in the sub-G 1 population and caspase-3 activation following depletion of FoxM1 in the BE 2 -C cells Supplementary Fig. S1B and C. The differential effect of transient FoxM1 knockdown on growth curve versus anchorage-independent growth or growth in xenografts suggests that a continuous presence of FoxM1 is critical for the tumorigenicity of the neuroblastoma cells.

Several recent studies, in other tumor models, indicated a link between the state of differentiation of the tumor cells and their tumorigenicity. For example, in human liver cancer, it was shown that the tumorigenicity correlated with the presence of stem cell-like cancer cells Moreover, there is a strong association between poor differentiation and aggressiveness of breast cancers

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