The Underlying Mechanism Of FoxM1-β-catenin Co-regulation Involved In The Self-renewal And Differentiation Of Glioblastoma Stem Cell

Glioblastoma is one of the most common brain tumors in adults, without distant metastasis but the median survival is only 12 to 18 months for patients. Despite the conventional method of glioblstoma treatment is microscopic surgery with adjuvant chemotherapy, the glioblastoma cells are always resistant to radiation therapy and anticancer drugs, Glioblastoma is usually fatal within a year of diagnosis. Advances in the treatment of glioblastoma and find new drug target will require an improvement in the understanding of the biology and molecular mechanisms of glioma development and progression.Currently,the etiology of glioma focused on the cancer stem cells. This hypothesis suggests that, glioma stem cells of self-renewal and differentiation ability exist in the malignant glioma(lower than 1% of tumor cells).after treatment of surgery or radiotherapy and chemotherapy, glioma stem cells will rapidly proliferate for the internal environment disorder. Some of the cells will remain the character of stem cells-self-renewal, the others will differentiate to mature glioma cells, and become the original of glioma recurrence. Generally, the hypothesis of glioma stem cells has important implications for understanding of glioma biology. These cells may be crucial cellular targets for curative tumor therapy generally and particularly in treatment of brain malignant tumors.Wnt/β-catenin signaling plays a critical role in cancer formation, including regulation of transformation, cell proliferation, and invasion. Persistent activation ofβ-catenin has been implicated in a variety of human cancers, such as glioblastoma multiforme (GBM), the most malignant form of glioma. Indeed, (3-catenin is expressed at markedly higher levels in GBM than in normal brain tissue, and other components of the Wnt pathway, including Wnt ligands and receptors, are also significantly upregulated in GBM. It is generally believed that a crucial step in Wnt/β-catenin pathway activation is the translocation ofβ-catenin into the nucleus to its ultimate destinations, the Wnt target genes. However, the molecular mechanisms that regulateβ-catenin nuclear localization GBM are unclear. Unlike colorectal cancer, in which high levels ofβ-catenin are frequently found as a result of mutational loss of the tumor suppressor protein adenomatous polyposis coli (APC) gene or stabilizing mutations in the CTNNB1 (β-catenin) gene itself, in sporadic gliomas the frequency of APC gene loss or CTNNB1 gene mutations appears to be substantially lower than that ofβ-catenin nuclear accumulation, suggesting that genetic mutation may not be the major molecular event that leads to elevated p-catenin nuclear accumulation, and hence activation, in GBM. Moreover, the Wnt/β-catenin pathway has been shown to critically regulate self-renewal and differentiation of neural stem/progenitor cells. These observations prompted us to examine the molecular mechanism of Wnt/β-catenin signaling activation and its potential role in tumorigenicity of GBM-initiating cells (GICs).The forkhead box M1 (FoxM1) transcription factor is an important regulator of the cell cycle. Likeβ-catenin, FoxM1 also plays a critical role in the biogenesis of neural stem cells (NSCs), including regulation of mitotic entry in cerebellar granule neuron precursors. FoxMl is commonly overexpressed in most human tumors, including glioma. For example, a study that analyzed gene expression data of GBM clinical specimens derived from the Cancer Genome Atlas (TCGA) revealed that FoxMl is overexpressed in GBMs compared with nontumor controls.In the current study, we first generate primary glioma stem cells from the tumor lesion of glioblastoma patients, and identify the cell morphology, tumor initiating ability and differentiation ability of the primary cell lines. Then pick three of them to do the experiments. Given that overactivation of FoxM 1 andβ-catenin occurs in many human cancers, including glioma.we examined whether they have function in the glioma stem cells. We found that depletion of FoxM1 orβ-catenin dramatically decreased the colony formation ability of stem cells, and subsequently inhibited the expression of CD 133 and neural precursor markers (Nestin, Sox2, and Musashi-1) and upregulated the differentiation markers of neuronal (Tuj1) and astrocyte (GFAP) cells in GSCs analyzed by IB on cells cultured under sphere-formation conditions or by IF on dissociated cells from spheres. Also the tumor-initiating ability of the glioma stem cells were also reduced in cells with suppressed FoxM1orβ-catenin expression. Then we analyzed the significance of FoxM1 andβ-catenin activation in human GBM using a panel of 40 GBM samples. The expression levels of nuclear FoxM1 directly correlated with those of nuclearβ-catenin. We also performed IF staining on 8 frozen GBM tumor samples that were FoxM1-positive. Sections from the same tumor tissues were used for co-staining for FoxM1 andβ-catenin and for Nestin and GFAP. We found that nuclear FoxM1 colocalized with nuclearβ-catenin in tumor cells. Moreover, FoxM1 expression correlated directly with Nestin expression but inversely with GFAP expression in tumors. Whether there is any functional connection between these two intensively studied proteins. We found that FoxM1 tumor promotion depends onβ-catenin and FoxM1 has effect on theβ-catenin nuclear localization. The endogenous FoxM1 expression level in 293T cells, human glioma Hs683 and SW1783 (gradeⅢ) cells, and a panel of GIC lines (MD 11,20s,23,7-2, and 6-27) is correlated with the Wnt activity. Wnt3a could increase the expression level of FoxM1 andβ-catenin and promote the nulear translocation of both them. ChIP assay experiment indicated that FoxM1 andβ-catenin mutually depend on each other for recruitment to TBEs/WREs occupied by TCF/LEF in Wnt target-gene promoters. Collected all the data, our study uncovers FoxM1 forms a tripartite complex withβ-catenin and TCF/LEF on Wnt target genes, transcriptional regulate the expression of wnt target genes, and establishes the central role of FoxM1 andβ-catenin in maintain the self-renewal ability of glioma stem cells.Part1 Separation, primary culture and identification of primary human glioma stem cellsAccording to the reported method of separating human glioma stem cells, we generated several glioma stem cells from different glioblastoma patients, and cultured in the DMEM F12 with EGF and bFGF. Most of the cells could form a colonsphere and express high level of neural stem cell marker Nestin and low level of astrocyte cell marker GFAP. To detect the tumor initiating ability of the primary stem cells, we use an intracranial injection model, and found 1000 MD11cells and MD20s cells, 5000MD23cells can induce the brain tumor, and perform the similar characteristic GBM features with original glioblastoma patients(highly infiltrative, pseudopalisading necrosis and tumor microvascular proliferation). Given the tumor stem cell has the ability of differentiation; we next identify this ability of our primary stem cells. We cultured the cells from three different GBM patients in the differentiation medium which contains2%FBS for 7-10 days, we found that all the cells began to adherent grow. rtPCR results showed the neuron cell marker Tuj-1 and the astrocyte marker GFAP time-dependent increased. This result was also confirmed by IF staining in dissociated cells and IB analysis in cultured cells. Also we found the expression level of FoxM1 in stem cell medium cultured cells was dramatically higher than differentiation cells. Thus, from the cell morphology, tumor initiating ability and differentiation ability, we think these three cells are glioma stem cells and can be used in the experiments to study the mechanism of glioma stem cell regulation.Part2 The role of FoxM1 andβ-catenin in the glioma stem cellsTo study the function of FoxM1 andβ-catenin in GSCs, we first generated the lentivirus that expressed FoxM1-shRNA andβ-catenin-shRNA, then stable transfected into MD11 and MD20s cells. We tested whether FoxM1 andβ-catenin are required for GSCs self-renewal and differentiation. Knockdown of FoxM1 or p-catenin in MD11 and MD20s cells substantially decreased the size and numbers of spheres formed in primary sphere formation assays, which measure self-renewal. Next, the established spheres were dissociated into single cells, which were assayed for secondary sphere formation, a more stringent test for the presence of self-renewing cells. Knockdown of FoxM1 orβ-catenin substantially reduced the size and efficiency of secondary sphere formation in GSCs. These results suggest that both FoxM1 andβ-catenin are required for GSC self-renewal. Next, we investigated whether FoxM1 andβ-catenin also affect GSC differentiation by measuring the expression of neural precursor or differentiation markers. FoxM1 knockdown substantially inhibited the expression of CD133 and neural precursor markers (Nestin, Sox2, and Musashi-1) and up regulated the differentiation markers of neuronal (Tuj1) and astrocyte (GFAP) cells in GICs analyzed by IB on cells cultured under sphere-formation conditions or by IF on dissociated cells from spheres. Depletion ofβ-catenin in GSCs produced almost identical results, inhibiting the expression of CD133, Nestin, and Musashi-1 but up regulating the differentiation marker GFAP.Part3 FoxM1 andβ-Catenin controls tumor formation by glioma cells and nuclearβ-Catenin expression in human GBM correlates with levels of nuclear FoxM1.We further examined the effect of FoxM1 or P-catenin depletion on the tumor-initiating ability of MD11 and MD20s cells by using an intracranial injection model. All mice injected with sh-control MD11 and MD20s cells displayed brain tumors that showed characteristic GBM features. In contrast, mice bearing sh-FoxM1 cells did not develop brain tumors during the studied time frame (120 days). Therefore, the tumor-initiating ability was substantially reduced in cells with suppressed FoxM1 expression (100% decreases) compared with their control counterparts. The tumor-initiating ability was also substantially reduced in cells with suppressedβ-catenin expression (82% and 91% decrease in groups injected with MD11 and MD20s cells, respectively) compared with their control counterparts. In addition, the mice bearing sh-FoxM1 or sh-β-catenin cells had significantly longer survival times (P< 0.001). Moreover, FoxM1 andβ-catenin levels were highly elevated in the brain tumors that arose from control cells, and the proteins colocalized in tumor cell nuclei. In contrast, there was little detectable nuclear FoxM1 orβ-catenin in brain tissues from the mice injected with sh-FoxMl1 cells. These results support a role for the FoxM1-β-catenin complex in the induction of GBM in vivo. To further ascertain whetherβ-catenin mediates the tumorigenic effect of FoxM1, we examined the tumorigenicity of glioma cells that overexpress FoxM1 but are deficient inβ-catenin.SW1783 and Hs683 cells did not form brain tumors in nude mice, but both SW1783-FoxM1 and Hs683-FoxM1 cells did, indicating that FoxM1 overexpression is responsible for tumor formation. However,β-catenin knockdown in SW1783-FoxM1 or Hs683-FoxM1 cells diminished their tumorigenicity, indicating that FoxM1 tumor promotion depends onβ-catenin. Moreover, in the SW1783-FoxM1 brain tumors,β-catenin mostly localized in nuclei, and it colocalized with FoxM1. These results together show that the FoxM1-β-catenin controls tumorigenesis of glioma cells.We analyzed the significance of FoxM1-β-catenin in human GBM using a panel of 40 GBM samples. FoxM1 was moderately expressed in 14 samples and highly expressed in 18 samples. The expression levels of nuclear FoxMl directly correlated with those of nuclearβ-catenin, further supporting the critical role of FoxM1 inβ-catenin nuclear accumulation in human GBM. We also performed IF staining on 8 frozen GBM tumor samples that were FoxM1-positive. Sections from the same tumor tissues were used for co-staining for FoxM1 andβ-catenin and for Nestin and GFAP. We found that nuclear FoxMl colocalized with nuclearβ-catenin in tumor cells. Moreover, FoxMl expression correlated directly with Nestin expression but inversely with GFAP expression in tumors. Together, our data suggest that in GBM tumors that express high levels of FoxMl,β-catenin and FoxMl likely promote GIC self-renewal, as evidenced by the increase in the number of cells expressing neuroprogenitor/stem cell markers.Part4 The mechanism of FoxMl andβ-Catenin coregulation the glioma stem cellsTo further study the underlysing mechanism of FoxM1 andβ-catenin regulation in the GSCs, we use the FoxM1-null cells to ascertain the role of FoxMl inβ-catenin. FoxM1-null primary NSCs were generated from FoxMlfl/fl mice with Ad-Cre to delete the floxed alleles of FoxM1. Deletion of FoxM1 did not change the total level ofβ-catenin expression but abolishedβ-catenin nuclear localization. Moreover,β-catenin nuclear localization induced by Wnt3a was abolished in FoxM1-null MEFs derived from FoxMl-/-mice. These results suggest that FoxMl is critical forβ-catenin nuclear localization. Consistently, knockdown of FoxM1 by FoxM1-siRNA in MD11 and MD20s cells substantially decreased the levels of nuclearβ-catenin. Also, in MD11 and MD20s cells with FoxM1 knockdown via lentiviruses that carry one of the two independent FoxM1-shRNAs,β-catenin nuclear localization was less than in control cells, as determined by IB and IF analyses. In contrast, when FoxM1 expression was restored in sh-FoxM1 MD11 cells by transient transfection of a FoxM1 expression plasmid,β-catenin nuclear localization was restored.To further investigate the Wnt effect on FoxMl andβ-catenin, we co-transfected 293T cells with DsRed2-Nl-tagged FoxMl and CFP-taggedβ-catenin and monitored the fluorescence intensities by time-lapse live imaging of the cells treated with Wnt3a every 10 sec for 10 min. We reasoned that during this early time window of Wnt treatment, we would observe primarilyβ-catenin translocation rather than stabilization ofβ-catenin that was already present as a result of overexpression. FoxM1 andβ-catenin were colocalized in both the cytoplasm and nucleus, and Wnt3a increased the levels and nuclear translocation of both proteins in the cell. The fluorescence intensities of both proteins increased with time in the nuclei of Wnt3a-treated cells. Note that the nuclear translocation of both proteins was likely underestimated considering the effect of photo-bleaching during live imaging.As FoxMl is required for (3-catenin nuclear translocation and wnt3a can promote FoxMl nuclear translocation, FoxMl might be recruited to a TBE/WRE. We first determined whether FoxMl regulates the activity of the LEF-1 promoter, which is regulated by Wnt/β-catenin signaling and contains three WREs. Overexpression of FoxMl in 293T cells increased the activity of the wild-type LEF-1 promoter but not the mutant LEF-1 promoter, which harbors three mutated WREs, suggesting that FoxM1 activates the promoter via the WREs. Next, we used the chromatin IP (ChIP) assay to determine whether FoxMl bound to the WREs of the LEF-1 promoter. As a negative control, we also examined the LEF-1 ORF region. In MD1 1 cells, FoxMl,β-catenin, and TCF4 bound to the WRE region but not to the ORF region. In 293T cells, in which TCF4 occupied the WRE region regardless of Wnt stimulation, FoxM1 andβ-catenin bound to the WRE region at a background level in the absence of Wnt3a, but addition of Wnt3a caused a dramatic increase in FoxM1 andβ-catenin binding to the LEF-1 promoter. Moreover, by using a re-ChIP assay, we found that FoxM1 associated with the WREs in the LEF-1 promoter that had been pre-immunoprecipitated by the anti-β-catenin or anti-TCF4 antibody.Together, these results indicate that FoxMl,β-catenin, and TCF4 are recruited to WREs in Wnt target-gene promoters as a DNA-binding complex. To understand the role of nuclear FoxM 1 in theβ-catenin-TCF transcriptional complex, we performed ChIP assays in MD11 cells with depletion or reduction of endogenous FoxMl using siRNA. We observed that association ofβ-catenin with the LEF-1 promoter was diminished on FoxM1 depletion. We also examined whetherβ-catenin is required for FoxM1 binding to the Wnt target-gene promoter. Depletion ofβ-catenin diminished the binding of not onlyβ-catenin but also FoxM1 to the LEF-1 promoter. Collectively, the above results suggested that FoxMl and P-catenin mutually depend on each other for recruitment to TBEs/WREs occupied by TCF/LEF in Wnt target-gene promoters.