FoxM1 Interacts Directly with β-Catenin In Vitro and In Vivo
A Flag-tag affinity procedure was used to purify a FoxM1-containing complex, which was subjected to LC-MS/MS analysis. The peptide sequences of β-catenin found in the complex suggested β-catenin as a potential binding partner of FoxM1 ( and Figures S1A and S1B
). The interaction between β-catenin and FoxM1 was confirmed by immunoprecipitation (IP) analysis of 293T cells transfected with HA-tagged β-catenin and Flag-FoxM1 (Figure S1C
). The physical interactions between β-catenin and FoxM1 were further analyzed in vitro using recombinant GST-β-catenin and His-FoxM1. In a GST pull-down assay, purified His-FoxM1 bound directly to GST-β-catenin (, left). Reciprocally, in an IP assay using an anti-FoxM1 antibody, GST-β-catenin also bound to His-FoxM1 (, right). Thus, β-catenin binds to FoxM1 directly in vitro.
FoxM1 and β-Catenin Directly Interact In Vitro and In Vivo
We detected endogenous FoxM1 expression in 293T cells, human glioma cell lines Hs683 and SW1783 (grade III), and a panel of GIC cell lines (MD 11, 20s, 23, 7-2, and 6-27). These GICs are primary neurosphere-derived cells isolated from fresh surgical specimens of GBM and have an enriched GIC population (Figures S1D-G
). FoxM1B is the predominant FoxM1 isoform in 293T cells and in the human glioma cells including the GICs (Figures S1H-J
). Moreover, GICs expressed substantially higher levels of FoxM1 than Hs683 and SW1783 cells (). Further, Wnt activity was substantially higher in GICs than in Hs683 and SW1783 cells, as determined using the TOP-Flash reporter (), indicating that Wnt activity is deregulated and correlated with the FoxM1 levels in the cells.
We next determined whether β-catenin and FoxM1 interact in glioma cells. In MD11 and MD20s cells, endogenous FoxM1 bound to endogenous β-catenin as determined by co-IP assays (). When Hs683 and SW1783 cells overexpressed T7-tagged FoxM1, endogenous β-catenin bound to FoxM1 (, left) and T7-FoxM1 (, right). Moreover, endogenous TCF4 protein, a well-known binding partner and transcriptional target of β-catenin, could be detected in the FoxM1 and T7-FoxM1 precipitates, and its level was increased upon FoxM1 overexpression (). Thus, β-catenin and FoxM1 interact in tumor cells. We also examined the specificity of FoxM1 isoforms interacting with β-catenin and found that the FoxM1B binding ability is higher than that of FoxM1C (Figure S1K & S1L
Wnt Promotes Nuclear Translocation of both FoxM1 and β-Catenin
Because Wnt3a is known to stabilize β-catenin and increase its nuclear accumulation, we examined the interaction of FoxM1 with β-catenin upon Wnt3a stimulation in 293T cells that had been cotransfected with HA-β-catenin and Flag-FoxM1. The amount of β-catenin that bound to FoxM1 in the nucleus increased substantially with Wnt3a treatment in a time-dependent manner (). Surprisingly, the amount of Flag-FoxM1 that translocated to the nucleus also increased with Wnt3a treatment in a time-dependent manner (). Thus, β-catenin interacts with FoxM1 in both the cytoplasm and the nucleus, and Wnt activation increases the translocation of both proteins to the nucleus.
To further investigate the nuclear translocation of FoxM1 and β-catenin during Wnt activation, we cotransfected 293T cells with DsRed2-N1-FoxM1 and CFP-β-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 primarily β-catenin translocation rather than stabilization of β-catenin that was already present as a result of overexpression. FoxM1 and β-catenin were colocalized in both the cytoplasm and nucleus, and Wnt3a increased the levels and nuclear translocation of both proteins ( and S2A
). 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 probably underestimated, considering the effect of photo-bleaching during live imaging (Figure S2B
). Indeed, Wnt3a promoted the nuclear translocation of endogenous FoxM1 and β-catenin () and increased FoxM1 levels () in 293T cells as determined by immunoblotting (IB) analysis. Wnt3a also promoted the nuclear translocation of endogenous FoxM1 and β-catenin and increased FoxM1 levels in MD11 cells as determined by immunofluorescence (IF) analysis (). Thus, Wnt activation increases the level and nuclear translocation of FoxM1, indicating that FoxM1 is a downstream component of Wnt signaling. However, FoxM1 mRNA levels did not increase upon Wnt3a treatments in MD11 cells (Figure S2C
). Next, we inhibited new protein synthesis in the cells by cycloheximide, and found that Wnt3a treatment resulted in a decrease in the endogenous FoxM1 degradation compared with control treatment (). These results indicated that Wnt3a increased FoxM1 levels through, at least in part, inhibition of FoxM1 protein degradation.
FoxM1 Promotes β-Catenin Nuclear Accumulation
FoxM1 Is Required for β-Catenin Nuclear Localization in Tumor Cells
FoxM1 contains a functional nuclear localization signal (NLS) domain and shuttles between the cytoplasm and the nucleus (Ma et al., 2005
). Because the amount of β-catenin that bound to FoxM1 was directly proportional to the amount of FoxM1 in the nucleus (), we examined whether FoxM1 plays a role in β-catenin nuclear localization. Expression levels of the endogenous FoxM1 and β-catenin increased in the cytoplasm and nucleus of Wnt3a-treated cells (), consistent with the findings of the IP assay (), of live imaging of the Wnt3a-treated 293T cells (), and of the IF assay of the Wnt3a-treated MD11 cells (). Overexpression of Flag-FoxM1 was sufficient to increase nuclear β-catenin while concomitantly decreased cytoplasmic β-catenin (). These data suggest that the FoxM1 expression level is increased upon Wnt signaling and that increased expression of FoxM1 directly increases β-catenin nuclear accumulation.
FoxM1 Is Required for Constitutive and Wnt3a-induced β-Catenin Nuclear Accumulation
To ascertain the role of FoxM1 in β-catenin nuclear accumulation, we examined β-catenin nuclear localization in FoxM1-null cells. Deletion of FoxM1 in FoxM1fl/fl immortalized neural stem cells (NSCs) did not change the total level of β-catenin expression but abolished β-catenin nuclear localization (). Moreover, β-catenin nuclear localization induced by Wnt3a was abolished in FoxM1-null immortalized MEFs derived from FoxM1–/– mice (). These results suggest that FoxM1 is critical for β-catenin nuclear localization.
Consistently, knockdown of FoxM1 by a FoxM1-siRNA, or via two independent FoxM1-shRNAs (Liu et al., 2006
) in MD11 and MD20s cells substantially decreased the levels of nuclear β-catenin as determined by IB and IF analyses (). In contrast, when FoxM1 expression was restored in sh-FoxM1 MD11 cells, β-catenin nuclear localization was restored ().
We also analyzed the effect of reducing FoxM1 on Wnt3a-induced β-catenin nuclear localization in glioma cells. Wnt3a increased β-catenin nuclear accumulation in MD11 and MD20s cells expressing the control shRNA (). In contrast, expression of either of the two shRNAs against FoxM1 abolished Wnt3a-induced β-catenin nuclear but not cytoplasmic accumulation (). Conversely, overexpression of FoxM1 counteracted the inhibitory effect of Wnt antagonist DKK1 on β-catenin nuclear accumulation (Figure S2D
). However, total β-catenin protein levels remained unchanged in MD11 and MD20s treated with a FoxM1-siRNA (). Thus, these data indicate that FoxM1 does not affect β-catenin level but is required for β-catenin nuclear accumulation in tumor cells.
FoxM1 Activates Wnt/β-Catenin Signaling
FoxM1 Nuclear Translocation and Binding to β-Catenin Are Required for FoxM1-Mediated β-Catenin Nuclear Accumulation in Tumor Cells
β-Catenin consists of an NH2-terminal domain, a central armadillo (Arm) repeat domain (residues 141–664) composed of 12 Arm repeats, and a COOH-terminal domain (residues 664–782) (, left). Using a series of bacterially expressed GST-β-catenin deletion mutant proteins, we found that Arm repeats 11–12 of β-catenin interacted with FoxM1 (, right). FoxM1 consists of an NH2-terminal domain, a conserved forkhead box domain (residues 232–332), an NLS domain (residues 350–366), and a COOH-terminal domain (, left). We found that the forkhead box domain of FoxM1 interacted with β-catenin (, right). Also, co-IP experiments in 293T cells revealed that β-catenin bound to FoxM1 mutants harboring the forkhead box domain but not to mutants without it (). An artificial fusion protein between the forkhead box domain and the SV40 NLS (235-347+NLS) was sufficient for binding to β-catenin ().
FoxM1 Nuclear Translocation and Binding to β-Catenin Are Required for FoxM1-Mediated β-Catenin Nuclear Accumulation
Next, we determine the effect of FoxM1 mutants on β-catenin nuclear accumulation. β-Catenin accumulated in the nuclei of cells expressing full-length FoxM1 or the mutant with both the forkhead box and NLS domains (). In contrast, β-catenin failed to accumulate in the nuclei of cells expressing FoxM1 mutants lacking the forkhead box domain. The FoxM1 mutant without the NLS domain impaired the nuclear accumulation of both FoxM1 and β-catenin. Importantly, the fusion fragment 235-347+NLS promoted the nuclear accumulation of both the FoxM1 mutant and β-catenin (). Thus, the nuclear localization of β-catenin depends on the interaction of β-catenin with FoxM1 and on FoxM1 nuclear translocation.
FoxM1–β-Catenin Interaction Is Required for β-Catenin–TCF-Mediated Transcription and for the Expression of β-Catenin Target Genes in Tumor Cells
It is well established that nuclear β-catenin associates with members of the TCF/LEF family of DNA-bound transcription factors on TCF-binding elements (TBEs), also called Wnt-responsive elements (WREs), to mediate Wnt target gene expression (Clevers, 2006
; Moon et al., 2004
). We used the TOP-Flash reporter to ascertain whether FoxM1 and its mutants affect β-catenin– TCF/LEF transcriptional activity. Interestingly, overexpression of full-length FoxM1 alone strongly activated the TOP-Flash reporter, and thus the transcriptional activity of β-catenin–TCF (). This TOP-Flash activation was probably mediated by engaging endogenous TCF and β-catenin, as FoxM1 overexpression increased levels of TCF4 () and nuclear β-catenin (). A FoxM1 mutant containing the forkhead box and NLS domains stimulated TOP-Flash, whereas FoxM1 mutants lacking the forkhead box domain or the NLS domain did not (). Importantly, the FoxM1 fragment 235-347+SV40NLS also stimulated TOP-Flash activity (). Thus, the ability of FoxM1 to activate the β-catenin–TCF transcriptional function correlates fully with FoxM1-mediated β-catenin nuclear translocation.
To distinguish the action of FoxM1 as a β-catenin partner from the possibility that FoxM1 acts as a DNA-binding transcription factor, we generated the FoxM1 R286A/H287A mutant, which is incapable of DNA binding, as predicted from crystal structure studies (Littler et al., 2010
). Indeed, this mutant dramatically decreased the ability of FoxM1 to activate a reporter from multimerized FoxM1-binding elements (Figure S3A
) as well as decreased the ability to bind to FoxM1 response elements in the c-Myc promoter (Figure S3C
). However, this mutant maintained the same ability to promote β-catenin nuclear localization (), bind to the β-catenin–TCF binding elements in the c-Myc promoter (Figure S3D
), and activate the β-catenin–TCF reporter (Figure S3B
), indicating that the effect of FoxM1 on β-catenin–TCF transcriptional activity depends on interaction with β-catenin but not on FoxM1's own DNA-binding property.
We next determined whether FoxM1 is required for β-catenin–TCF-mediated transcription. As with FoxM1 overexpression, a constitutively stabilized mutant β-catenin (S33Y) activated TOP-Flash, which was inhibited by the FoxM1 siRNA in 293T cells (), suggesting that FoxM1 is required for β-catenin signaling. Conversely, dominant-negative TCF4 (DN-TCF4), which cannot bind to β-catenin, abolished the activation of TOP-Flash by FoxM1 (), suggesting that the effect of FoxM1 is mediated by binding of β-catenin to TCF4. Knocking down FoxM1 also suppressed the activation of the TOP-Flash reporter in MD11 and MD20s cells, likely as a result of endogenous β-catenin–TCF signaling ().
We further examined several endogenous and prototypic Wnt/TCF/β-catenin target genes. Expression levels of Axin2, LEF-1, c-Myc, and cyclin D1 were increased in MD20s cells that overexpressed FoxM1 (), and were decreased in MD11 and MD20s cells with knocking-down of FoxM1 () and in NSCs with genetic deletion of FoxM1 (). Furthermore, overexpression of the FoxM1 R286A/H287A mutant increased the expression of Axin2 in 293T cells (), indicating that the effect of FoxM1 on Wnt target gene expression depends on interaction with β-catenin.
Mutual Recruitment of FoxM1 and β-Catenin to Wnt Target-Gene Promoters
As FoxM1 is required for activation of Wnt target genes and can form a complex with nuclear β-catenin and TCF4, FoxM1 might be recruited to TBEs/WREs in chromatin. We first determined whether FoxM1 regulates the LEF-1 promoter, which contains three WREs (). Overexpression of FoxM1 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. Moreover, FoxM1, β-catenin, and TCF4 bound to the WRE region of LEF-1 promoter in MD11 cells (). Further, Wnt3a stimulation caused a dramatic increase in FoxM1 and β-catenin binding to the LEF-1 promoter, which was occupied by TCF4 constitutively in 293T cells ().
Mutual Recruitment of FoxM1 and β-Catenin to Wnt Target-Gene Promoters
are also well-known Wnt target genes (through characterized TBEs/WREs in their promoters) (Clevers, 2006
; Moon et al., 2004
). We found that in MD11 cells, FoxM1, β-catenin, and TCF4 were recruited to the endogenous Axin2
promoter () and also to a transfected c-Myc
promoter harboring wild-type TBEs (TBE1/2) but not to a c-Myc
promoter harboring mutant TBEs (TBE1m/2m; ). Moreover, FoxM1 associated with the WREs in the LEF-1
promoter that had been immunoprecipitated by the anti-β-catenin or anti-TCF4 antibody (). Together, these results indicate that FoxM1, β-catenin, and TCF4 are recruited to WREs in Wnt target-gene promoters as a DNA-binding complex.
Next, we determined whether nuclear FoxM1 plays a role in assembly of the β-catenin– TCF transcription activation complex. We observed that association of β-catenin with the LEF-1 promoter was diminished upon FoxM1 depletion (). Next, we examined the effect of FoxM1 depletion on the binding between β-catenin and TCF4 using exogenous Myc-TCF4 (since TCF4 is a target gene of FoxM1). We found that the binding between β-catenin and TCF4 was decreased upon FoxM1 depletion (). Thus, the decreased association of β-catenin with the LEF-1 promoter upon FoxM1 depletion was probably because both the nuclear β-catenin level () and the binding between β-catenin and TCF4 were decreased (). These results indicate that FoxM1 is important for assembly of the β-catenin–TCF transcription activation complex.
Given the binding between β-catenin and FoxM1, we also examined whether β-catenin is required for FoxM1 binding to the Wnt target-gene promoter. Depletion of β-catenin diminished the binding of FoxM1 to the LEF-1 promoter (). Moreover, depletion of β-catenin by a siRNA or genetic deletion reduced or abolished the association between FoxM1 and TCF4 (), indicating that the association of FoxM1 with TCF4 is mediated by β-catenin. Collectively, the above results suggested that FoxM1 and β-catenin mutually depend on each other for recruitment to WREs occupied by TCF4 in Wnt target-gene promoters.
Interaction of FoxM1 and β-Catenin Plays a Critical Role in GIC Self-Renewal and Differentiation
To study the effect of FoxM1 and β-catenin on the self-renewal of GICs, we generated primary cultures of tumor cells (PCTCs) in the presence of serum, and in parallel, neurospheres from human GBM samples. The neurospheres showed enrichment for GICs, because, first, they maintained neurosphere formation ability and thus exhibited self-renewal ability (Lee et al., 2006
; Singh et al., 2003
) (Figure S4A
); secondly, they expressed high levels of the neuroprogenitor markers Nestin, CD133, and SSEA-1 (Figure S4D
); and thirdly, they could undergo multilineage differentiation, acquiring the expression of GFAP (astrocytic marker) and Tuj-1 (neuronal marker) (Figure S4B & 4C
). Importantly, high levels of FoxM1 (Figure S4D
) and Wnt reporter activity (Figure S4E
) were present in these GICs compared with the PCTCs. Thus, we investigated whether FoxM1 and β-catenin were required for GIC self-renewal. Knockdown of FoxM1 or β-catenin in MD11 and MD20s cells substantially decreased the size and number of spheres formed in primary and secondary sphere formation assays (). Next, the cells were assayed for neural colony-forming ability, a more stringent test for the presence of self-renewing cells (Louis et al., 2008
). Knockdown of FoxM1 or β-catenin substantially reduced the efficiency of neural colony formation in GICs (Figures S4F
). Moreover, the inhibitory effect of sh-FoxM1 on self-renewal of GICs was rescued by shRNA-resistant R286A/H287A, which binds to β-catenin but not to FoxM1-response elements or β-catenin-NLS (which translocates into the nucleus constitutively) ( & S4F
FoxM1 and β-Catenin Maintain GIC Self-Renewal and Glioma Formation
We investigated whether FoxM1 and β-catenin also affect GIC differentiation. FoxM1 knockdown substantially inhibited the expression of CD133, Nestin, Sox2, and Musashi-1 and upregulated the differentiation markers of Tuj1 and GFAP in GICs ( & S4G
). Depletion of β-catenin in GICs produced almost identical results (Figure S4H
). Therefore, FoxM1 perturbed the balance between progenitor cell renewal and the commitment to differentiation. Moreover, these effects of sh-FoxM1 were rescued by shRNA-resistant R286A/H287A or β-catenin-NLS (Figures S4I
), supporting that FoxM1–β-catenin interaction controls the self-renewal of GICs.
Next, we examined the cell cycle progression of the above cell lines. An endogenous FoxM1-β-catenin complex and Wnt signal activity were observed in asynchronous GICs and in GICs in cell cycle phases G0/G1, S, and G2/M, with a relative higher level seen in G2/M GICs (Figure S4M-O
). Comparison of cell cycle profiles of the above cell lines revealed a similar cell cycle distribution under asynchronous conditions (Figure S4P
). However, when synchronized and released from G1/S transition, sh-FoxM1 GICs exhibited a delay in mitotic entry (Figure S4P
). Depletion of β-catenin in GICs also resulted in G2/M arrest (Figure S4P
). Expression of shRNA-resistant R286A/H287A or β-catenin-NLS in sh-FoxM1 cells partially restored normal cell-cycle progression. Therefore, these data indicated that the FoxM1/β-catenin interaction is required for G2/M transition and proper mitotic progression.
Interaction of FoxM1 and β-Catenin Controls Tumor Formation by Glioma Cells
Next, we examine the effect of FoxM1 or β-catenin depletion and their interaction on the tumor-initiating ability of GICs in an animal model. All mice injected with sh-control MD11 and MD20s cells displayed brain tumors that showed characteristic GBM features (Figure S1G
). In contrast, mice bearing sh-FoxM1 cells did not develop brain tumors. Therefore, the tumor-initiating ability was substantially reduced in cells with suppressed FoxM1 expression (100% decrease, ). The tumor-initiating ability was also substantially reduced by suppressed β-catenin expression (). In addition, the mice bearing sh-FoxM1 or sh-β-catenin cells survived significantly longer than controls ( and Figure S4J
). Moreover, FoxM1, β-catenin, and Wnt signal activity levels were highly elevated in the brain tumor cells (Figure S4K , S4L & S4Q
), and the proteins colocalized in tumor cell nuclei (Figure S4K
). Furthermore, the inhibitory effect of sh-FoxM1 on tumorigenicity of GICs was rescued by shRNA-resistant R286A/H287A or β-catenin-NLS (). These results support a role for the FoxM1–β-catenin complex in the induction of GBM in vivo.
To further ascertain that β-catenin mediates the tumorigenic effect of FoxM1, we examined the tumorigenicity of glioma cells that overexpress FoxM1 but are deficient in β-catenin. First, overexpression of FoxM1 promoted SW1783 cells to exhibit GIC characteristics, as SW1783-FoxM1 cells (but not control SW1783) were able to form neurospheres and expressed SSEA-1 and Nestin (Figure S5A
). Secondly, SW1783 and Hs683 cells did not form brain tumors in nude mice, but 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 on β-catenin. Moreover, in the SW1783-FoxM1 brain tumors, FoxM1 levels were highly elevated in tumor cell (Figure S5B
), β-catenin mostly localized in the cell nuclei, and it colocalized with FoxM1 (). Further, overexpression of FoxM1 R286A/H287A was able to induce tumor formation (). Consistently, SW1783-FoxM1 and SW1783-R286A/H287A cells exhibited higher levels of TOP-Flash reporter and Axin2, LEF-1, c-Myc, and cyclin D1 expression than SW1783 control cells did (). In contrast, knockdown of β-catenin in SW1783-FoxM1 cells abolished elevated expression of the reporter and the Wnt target genes (). Finally, FoxM1 regulates the nuclear β-catenin level and thus tumor cell growth in mouse high-grade glioma models (Figure S5G-I
). Together, these results show that the FoxM1–β-catenin interaction controls the expression of Wnt/β-catenin target genes and tumorigenesis of glioma cells.
Interaction of FoxM1 and β-Catenin Controls Glioma Formation
Nuclear β-Catenin Expression in Human GBM Correlates with Levels of Nuclear FoxM1
We analyzed the significance of FoxM1-mediated β-catenin activation 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 FoxM1 directly correlated with those of nuclear β-catenin (), further supporting the critical role of FoxM1 in β-catenin nuclear accumulation in human GBM. Also, the expression levels of Wnt targets Axin2 and LEF-1 directly correlated with the levels of FoxM1 (Figures S5C & S5D
Next, we performed IF staining on 8 frozen GBM tumor samples that were FoxM1-positive. Sections from the same tumor tissues were used for costaining for FoxM1 and β-catenin and for Nestin and GFAP. Nuclear FoxM1 colocalized with nuclear β-catenin in tumor cells (Figure S5E & S5F
). Moreover, FoxM1 expression correlated directly with Nestin expression but inversely with GFAP expression in tumors (Figure S5F
). Together, our data suggest that in GBM tumors that express high levels of FoxM1, β-catenin and FoxM1 probably promote GIC self-renewal, as evidenced by the increase in the number of cells expressing neuroprogenitor cell markers.