Tumor and Stem Cell Biology Cancer Research FoxM1B ... · Tumor and Stem Cell Biology FoxM1B Regulates NEDD4-1 Expression, Leading to Cellular Transformation and Full Malignant Phenotype

Published OnlineFirst March 23, 2010; DOI: 10.1158/0008-5472.CAN-09-3909

Tumor and Stem Cell Biology
Cancer
Research

FoxM1B Regulates NEDD4-1 Expression, Leading to CellularTransformation and Full Malignant Phenotype inImmortalized Human Astrocytes

Bingbing Dai1, Russell O. Pieper6, Dawei Li1, Ping Wei1, Mingguang Liu1, Shiao Y. Woo2,Kenneth D. Aldape3,4, Raymond Sawaya1, Keping Xie4,5, and Suyun Huang1,4

Abstract

Authors'Oncology,University oBiology, TSciencesNeurologicFrancisco,

CorresponUnit 1004,Holcombe713-834-62

doi: 10.115

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Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human gliomatissues and that the level of its expression correlates directly with glioma grade. However, whether FoxM1Bplays a role in the early development of glioma (i.e., in transformation) is unknown. In this study, we foundthat the FoxM1B molecule causes cellular transformation and tumor formation in normal human astrocytes(NHA) immortalized by p53 and pRB inhibition. Moreover, brain tumors that arose from intracranial injectionof FoxM1B-expressing immortalized NHAs displayed glioblastoma multiforme (GBM) phenotypes, suggestingthat FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBMformation. Mechanistically, our results showed that overexpression of FoxM1B upregulated NEDD4-1, an E3ligase that mediates the degradation and downregulation of phosphatase and tensin homologue (PTEN) inmultiple cell lines. Decreased PTEN in turn resulted in the hyperactivation of Akt, which led to phosphoryla-tion and cytoplasmic retention of FoxO3a. Blocking Akt activation with phosphoinositide 3-kinase/Aktinhibitors inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, overexpressionof FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E, which areimportant molecules for tumor growth. Collectively, these results indicate that overexpression of FoxM1B,in cooperation with p53 and pRB inhibition in NHA cells, promotes astrocyte transformation and GBMformation through multiple mechanisms. Cancer Res; 70(7); 2951–61. ©2010 AACR.

Introduction

Malignant glioma, including the most common subtype,astrocytoma, is aggressive, highly invasive, and neurologicallydestructive. The prognosis for patients with malignant glio-ma is poor (1, 2). Several pathways have been correlated withhuman gliomas, such as pRb, p53, epidermal growth factorreceptor, platelet-derived growth factor (PDGF)/PDGF recep-tor, p16INK4a, p19ARF, telomerase, and Akt (3–6). Activation ofthe Akt pathway is strongly implicated in glioma develop-ment and progression. In human studies, Akt is activatedin ∼80% of glioblastoma multiformes (GBM; ref. 3). Activa-tion of the Akt pathway in glioma is often achieved by theunderexpression of its negative regulator phosphatase and

Affiliations: Departments of 1Neurosurgery, 2Radiation3Pathology, and 4Gastrointestinal Medical Oncology, Thef Texas M.D. Anderson Cancer Center; 5Program in Cancerhe University of Texas Graduate School of Biomedicalat Houston, Houston, Texas; and 6Department ofal Surgery, The University of California-San Francisco, SanCalifornia

ding Author: Suyun Huang, Department of Neurosurgery,The University of Texas M.D. Anderson Cancer Center, 1515Boulevard, Houston, TX 77030. Phone: 713-834-6232; Fax:57; E-mail: [email protected].

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tensin homologue (PTEN). Studies have shown that PTENis underexpressed in almost all GBM tumors, compared withlevels in normal brain tissue (7). Several mechanisms may beassociated with the underexpression of PTEN in gliomas,such as genetic alterations or promoter methylation (8–11).However, gene deletion, mutation, loss of heterozygosity, orpromoter methylation in the PTEN gene is present in onlyabout 50% of GBMs (7–11). In addition, although PTEN ge-netic alterations are rare in low-grade gliomas, these tumorsexhibit lower PTEN protein levels than does the normal braintissue (7). Furthermore, underexpression of PTEN in the ab-sence of genetic alterations or promoter methylation is seenfrequently in gliomas as well (8). Thus, mechanisms for PTENcontrol at the transcriptional and posttranslational levelsmay be altered in glioma.Ubiquitin-mediated degradation of PTEN has recently

been reported, and neural precursor cell–expressed, develop-mentally downregulated 4-1 (NEDD4-1) has been identifiedas the E3 ligase for PTEN (12). NEDD4-1 negatively regulatesPTEN stability by catalyzing PTEN polyubiquitination. Infact, overexpression of NEDD4-1 has been shown to increasePTEN polyubiquitination in a dose-dependent manner (12).Consistent with the tumor-suppressive role of PTEN,NEDD4-1 was reported to be an oncogenic protein that reg-ulates tumorigenesis (12). Overexpression of NEDD4-1 in-creased cellular transformation by K-Ras. Elimination of

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NEDD4-1 expression inhibited xenotransplanted tumorgrowth. Furthermore, NEDD4-1 regulates tumorigenesis byregulating Akt signaling through PTEN and the oncogenicactivity of NEDD4-1 is PTEN dependent (12). However, littleis known about the regulation of NEDD4-1.FoxM1 is a member of the Forkhead box (Fox) transcription

factor family known for its function as a regulator of the cellcycle (13–17). Results from recent studies have revealed thatFoxM1 is substantially elevated in several aggressive humancarcinomas and can contribute to oncogenesis in many tissuetypes (18–25). For example, using transgenic and knockoutmouse models, studies have shown that FoxM1B plays essen-tial roles in the development of hepatocellular carcinoma (20),prostate carcinoma (21), lung cancer (22, 23), and colorectalcancer in mice (24). Moreover, FoxM1 is an essential moleculein the regulation of oxidative stress, which contributes tomalignant transformation and tumor cell survival (16).Increasing evidence indicates that FoxM1 may also con-

tribute to oncogenesis in glioma. For example, several studiesfound that aberrant expression of FoxM1 was a common mo-lecular alteration in malignant glioma (25–27). Another studythat analyzed expression data from 201 GBM clinical speci-mens derived from TCGA showed the overexpression ofFoxM1 in GBM specimens compared with nontumor con-trols (28). In our own studies of the functional importanceof this factor, we found that FoxM1B was the predominantFoxM1 isoform in human gliomas (25). FoxM1 levels in hu-man glioma tissue directly correlated with glioma grade andinversely correlated with the survival of GBM patients. Inanimal models, increased levels of FoxM1B expression inglioma cells enhanced the tumorigenicity, invasiveness, andangiogenesis of these cells (25, 29, 30).In this study, we sought to determine whether overexpres-

sion of FoxM1B induces the transformation of human astro-cytes into glioma cells because FoxM1 expression is detectedin low-grade glioma. We used immortalized NHA-E6/E7/hTERT cells because human astrocytes seem to bemore genet-ically stable than rodent cells (31) and individual eventsbelieved to be important in gliomagenesis might be more read-ily studied in the human cells (31–33). Moreover, the fact thatnormal human astrocyte (NHA) cells were not transformed byE6/E7/hTERT suggests that inactivation of the p53 and pRbpathways was not sufficient to create malignant astrocytesand that additionalmolecular events are needed (31).We foundfor the first time, to our knowledge, that FoxM1B expression inNHA-E6/E7/hTERT cells resulted in cellular transformationand GBM formation in nudemice due, at least in part, to its rolein the regulation of NEDD4-1, the activation of Akt, and theinduced expression of survivin, cyclin D1, and cyclin E.

Materials and Methods

Cell lines and culture conditions. The NHA-E6/E7/hTERTcell line and the Hs683 and HFU-251 glioma cell lines werepreviously described (25, 31, 33). COS-1 cells were obtainedfrom the American Type Culture Collection. All of the celllines were cultured in DMEM supplemented with 10% fetalbovine serum (FBS).

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Retrovirus production and transduction. Retrovirus wasproduced with pLXSN-FoxM1B or pLXSN plasmids using thePhoenix A retrovirus production system. NHA-E6/E7/hTERTcells were infected with the virus (10 multiplicity of infection)with 4 μg/mL polybrene for 24 h. To generate stable trans-duced cell lines, some infected cells were cultured with neo-mycin for 2 wk.Northern blot analysis. Northern hybridization was done

by using a [32P]dCTP-radiolabeled FoxM1 cDNA probe (25).Equal mRNA loading was monitored by hybridizing the samemembrane with a β-actin cDNA probe.Western blot analysis. Standard Western blotting was

done with antibody against FoxM1, cyclin E, cyclin D1, PTEN(Santa Cruz Biotechnology) or NEDD4-1, Akt, phospho-Akt,FoxO3a, phospho-FoxO3a (Cell Signaling Technology), andsurvivin (Novus Biologicals).Animals. Female athymic BALB/c nude mice were pur-

chased from the National Cancer Institute and were main-tained in institutional facilities approved by the Associationfor Assessment and Accreditation of Laboratory Animal Care.Colony formation in soft-agar assay. Cells (5 × 103) were

mixed with 0.3% agar solution in DMEM containing 10% FBSand 200 μg/mL neomycin, and the solution was poured on topof a 0.60% agar layer in six-well tissue culture plates. Plateswere incubated at 37°C in a humidified atmosphere containing5% CO2 for 21 d. Colonies were then stained with p-iodonitro-tetrazolium violet (1mg/mL) and examined microscopically.Subcutaneous tumor growth. Cells (1 × 107) were injected

s.c. into nude mice (25). The length (L), width (W), and height(H) of the resulting tumors were measured once every 5 d.The volume of each tumor was calculated by using the for-mula 1/2 × L × W × H at every time point.Intracranial human glioma xenograft model. Cells (1 ×

106) were injected intracranially into nude mice as previouslydescribed (25). Animals showing general or local symptomswere killed; the remaining animals were killed 90 d after gli-oma cell injection.Histologic and immunohistochemical analysis of

tumors. Tissue sections of brain tissue specimens werestained with H&E according to standard protocols. Tissuesections were also used for immunostaining with antibodiesagainst CD31 (BD Biosciences), glial fibrillary acidic protein(GFAP, DAKO), phospho-Akt (Cell Signaling Technology),PTEN, cyclin E, cyclin D1 (Santa Cruz Biotechnology), andsurvivin (Novus Biologicals).Cycloheximide chase experiments. Cells (5 × 105) were

plated into six-well plates. Twenty-four hours later, 50 mg/mL of cycloheximide were added to the cells and cyclohexi-mide treatment was terminated at the indicated time points.Cell lysates were then prepared and the expression of PTENwas analyzed by Western blotting.Chromatin immunoprecipitation assay. Cells (5 × 106)

were prepared for the chromatin immunoprecipitation(ChIP) assay as according to the manufacturer's instruc-tions with the ChIP assay kit (Cell signaling). ResultingDNA complexes were first immunoprecipitated using theindicated antibodies and then DNA was purified fromthe complexes. The DNA was then subjected to PCR to

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FoxM1B in Astrocytoma Transformation


amplify the putative FoxM1 binding region 1 (−642 to −828bp) or region 2 (−841 to −993 bp) of the NEDD4-1 promoterusing the primers 5′-TCTTCGTCACTGCCTTCTG-3′ and5′-TTGGACT‐CGGGATCTGAAA-3′ or the primers 5′-TATG-GATGCCTTATTTGGTG-3′ and 5′-TGAACTAGGACCTCCTG-TAGAAT-3′, respectively.Statistical analyses. The significance of the in vitro results

was determined by using the Student t test (two tailed),whereas the significance of the in vivo data was determinedby using the Mann-Whitney U test.

Results

FoxM1 induces malignant transformation of immortal-ized NHAs.We infected NHA-E6/E7/hTERT cells with pLXSN-FoxM1B or control pLXSN retrovirus. The infected cellswere plated for growth in soft agar. Both NHA-E6/E7/hTERTcells and NHA-E6/E7/hTERT cells infected with pLXSN wereunable to grow in soft agar (Table 1; Fig. 1A). However, colo-nies were obtained with NHA-E6/E7/hTERT cells infectedwith the virus containing FoxM1B (Table 1; Fig. 1A). Theseresults suggest that although inactivation of the p53 andpRb pathways is not sufficient to convert normal astrocytesinto malignant glioma cells, such inactivation acts coopera-tively with FoxM1B to achieve cellular transformation.Immortalized NHAs that express FoxM1 are tumorige-

nic. Next, we created a series of cell lines immortalized bythe expression of E6/E7/hTERT and expressing FoxM1B. Toavoid clonal selection and variation, we carried out three in-dependent infections of pLXSN-FoxM1B in NHA-E6/E7/

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hTERT cells and pooled G418-resistant colonies to establishstable cell lines, designated as FoxM1B-transduced cell lines(NHA-E6/E7/hTERT/FoxM1B-1, NHA-E6/E7/hTERT/FoxM1B-2, and NHA-E6/E7/hTERT/FoxM1B-3). FoxM1 pro-tein expression was increased in FoxM1B-transduced NHA-E6/E7/hTERT cell lines (Fig. 1B).We then evaluated FoxM1-infected NHA-E6/E7/hTERT

cells for their ability to form subcutaneous tumors in nudemice; tumors formed within 3 wk (Fig. 1C). No tumors devel-oped by 8 wk in mice injected with parental NHA-E6/E7/hTERT or pLXSN-infected cells. Thus, the results show thatNHA-E6/E7/hTERT/FoxM1 cells are tumorigenic.FoxM1 induces GBM formation of immortalized NHAs.

We used an orthotopic xenograft model of human gliomato study the growth of these cells in the brains of nudemice. Intracranially implanted NHA-E6/E7/hTERT orpLXSN-infected cells did not form tumors by 90 d afterinjection (Table 1). In contrast, intracranially injectedFoxM1B-infected NHA-E6/E7/hTERT cells formed brain tu-mors in nude mice and significantly shortened their survivalduration (Table 1). We also examined the histologic fea-tures of brain tumors that arose from NHA-E6/E7/hTERT/FoxM1 cells in the brains of nude mice. The lesions werecharacterized by high cellularity and invasiveness, werepleomorphic, and exhibited microvessel proliferation andnecrosis (Fig. 1D), all distinct histologic features of humanGBM. In addition, the brain tumor cells were positive forGFAP, but the level of expression was less than that inastrocytes in the surrounding tissue, indicating a humanastrocytic origin of the tumors (Fig. 1D). These findings

Table 1. Effect of FoxM1B on transformation of immortalized NHAs and tumor growth in the brain ofnude mice

Transformation

Cell type
Colony number*
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NHA-E6/E7/hTERT
0 NHA-E6/E7/hTERT/pLXSN 0 NHA-E6/E7/hTERT/FoxM1B 1,512 ± 91
Tumor growth in the brains of nude mice

Cell line
Incidence of tumor formation† Median days of survival (range)
NHA-E6/E7/hTERT
0/5 90 (all >90) NHA-E6/E7/hTERT/pLXSN 0/5 90 (all >90) NHA-E6/E7/hTERT/FoxM1B-1 5/5 51 (47–58)‡
NHA-E6/E7/hTERT/FoxM1B-2
5/5 54 (52–64)‡
NHA-E6/E7/hTERT/FoxM1B-3
4/5 63 (52 to >90)‡
*NHA-E6/E7/hTERT cells were infected with retroviral constructs containing the FoxM1 expression unit (pLXSN-FoxM1B) or controlpLXSN. Parental and infected NHA-E6/E7/hTERT cells (1 × 104) were plated in soft agar and colonies were scored after 3 wk.Results represent mean ± SD from three separate experiments.†Cells (1 × 106) were implanted intracranially into nude mice. Mice were killed when they were moribund or on day 90. Brains wereharvested and tumor formation was histologically analyzed. Results were shown for one representative experiment of two.‡P < 0.001.

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suggest that the overexpression of FoxM1B in immortalizedNHA cells leads to the formation of GBM in nude mice.Increased FoxM1 expression leads to Akt activation. In

animal studies, activation of Akt seems to be essential for theformation of GBMs from genetically modified neural progeni-tors and NHAs (33, 34). Thus, we examined whether overex-pression of FoxM1 affects the activation status of Akt inNHA-E6/E7/hTERT cells. We found that pLXSN-FoxM1B in-fection induced more phosphorylation and activation of Aktthan did infection with retroviral pLXSN or Ras (Fig. 2A). Incontrast, overexpression of FoxM1 did not affect extracellularsignal-regulated kinase (ERK)-1/2 activation, a different path-way from Akt (Fig. 2A). NHA-E6/E7/hTERT/FoxM1B brain



tumors also expressed phosphorylated Akt (p-Akt; Fig. 2B).Furthermore, overexpression of FoxM1 in NHA-E6/E7/hTERT by transient pLXSN-FoxM1B infection induced thenuclear translocation of Akt and the cytoplasmic retentionof FoxO3a protein (Fig. 2C), which have been reported assignificant predictors of p-Akt status in gliomas (35). Theseresults indicate that increased FoxM1 expression leads to Aktactivation and subsequently inhibits FoxO3a function.Blocking Akt activation inhibits FoxM1-induced trans-

formation of immortalized NHAs. To determine whetherFoxM1 induces transformation of immortalized NHAsthrough the Akt pathway, we treated NHA-E6/E7/hTERT/FoxM1 cells with phosphoinositide 3-kinase (PI3K) inhibitors

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Figure 1. Effects of FoxM1Bexpression on transformationand glioma growth in vivo.A, colony formation assays weredone with NHA-E6/E7/hTERTcells infected with pLXSN-FoxM1Bor pLXSN retrovirus and withoutretroviruses (mock) as indicated.A, typical colony formation assayresult. B, determination ofoverexpression of FoxM1B in threeindependent pLXSN-FoxM1retrovirus–transduced E6/E7/hTERT cell lines by Northern (top)and Western (bottom) blotanalyses. C, cells (1 × 107 permouse) from NHA-E6/E7/hTERT,pLXSN, and three independentpLXSN-FoxM1 B-cell lines wereinjected s.c. into nude mice (n = 5)and tumor formation wasdetermined. Representative resultsof two experiments. D, histologicfeatures of NHA-E6/E7/hTERT-FoxM1B brain tumors. a, a highlyinvasive phenotype of the tumors(arrows, invasive cells; H&E stains,magnification ×100). b, increasedmicrovessel proliferation in thetumors (CD34 stains, magnification×100). c, microscopic necrosiswith pseudopalisading (H&Estaining, magnification ×100). d,brain tumor cells are GFAPpositive (arrows, astrocytes in thesurrounding tissue; GFAP stains,magnification ×200).

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LY294002 and wortmannin. Both treatments inhibited the ex-pression of p-Akt in the cells (Fig. 2D, left). Furthermore,blockade of Akt activation by LY294002 and wortmanninsuppressed the transformation of immortalized NHAs, as de-termined by colony formation assay (Fig. 2D, right). There-fore, these data suggest that Akt activation is an importantstep in the FoxM1-mediated transformation of immortalizedNHAs into glioma cells.



Increased FoxM1 expression leads to PTEN reductionand Akt activation. It is known that PTEN is an importantmolecule that regulates Akt activity by inhibiting PI3K (36,37). To further define how FoxM1 activates the Akt pathwayin NHA cells, we examined PTEN expression in these cells.Levels of PTEN expression were substantially lower inNHA-E6/E7/hTERT/FoxM1B cells than in NHA-E6/E7/hTERT or NHA-E6/E7/hTERT/pLXSN control cells (Fig. 3A,

Figure 2. Overexpression ofFoxM1 increases p-Akt but notp-ERK-1/2. A, the expression ofFoxM1, p-Akt, total Akt, FoxO3a,p-FoxO3a, ERK-1/2, andp-ERK-1/2 on NHA-E6/E7/hTERT/FoxM1B cells or parental andNHA-E6/E7/hTERT/pLXSN cellswas analyzed by Western blotting.B, paraffin sections from braininjected with pLXSN- andpLXSN-FoxM1B–transducedNHA-E6/E7/hTERT cells (tumors)were stained with antibody againstp-Akt. C, immunofluorescentmicroscopic analyses of Aktactivation and FoxO3a localization.NHA-E6/E7/hTERT cells weretransiently infected withpLXSN-FoxM1B or pLXSN. Then,the cells were immunostainedfor Akt (green) and their nucleiwere stained with4′,6-diamidino-2-phenylindole(DAPI; blue). The cells were alsoimmunostained for FoxO3a (green)and their nuclei were stained withDAPI (blue). Representative ofthree experiments. D, blocking Aktactivation decreases the colonyformation induced by FoxM1.NHA-E6/E7/hTERT cells wereinfected with retroviral-pLXSN orpLXSN-FoxM1B for 24 h and thentreated with LY294002 (25 μmol/L)or wortmannin (0.1 μmol/L) for 2 h.Left, the expression of FoxM1,p-Akt, total Akt, FoxO3a, andp-FoxO3a was analyzed byWestern blotting. Right, thecolony-forming ability of the cellswas determined by colonyformation assay. Columns, meancolony numbers from arepresentative experiment done intriplicate; bars, SD. *, P < 0.01,compared with the no-treatmentgroup.

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left). The observed decrease in PTEN expression in these cellswas accompanied by enhanced Akt pathway signaling, as wasevident by a concomitant increase in the level of p-Akt, butnot in the level of total Akt (Fig. 3A, left). NHA-E6/E7/



hTERT/FoxM1B brain tumors also had lower levels of PTENexpression than did brain tissues from the mice injected withNHA-E6/E7/hTERT/pLXSN cells (Fig. 3A, right). Moreover,knockdown of FoxM1 in NHA-E6/E7/hTERT/FoxM1B cells

Figure 3. FoxM1 downregulates PTEN expression in NHA cells. A, the expression of FoxM1, PTEN, p-Akt, and Akt in NHA-E6/E7/hTERT/FoxM1B orparental and NHA-E6/E7/hTERT/pLXSN cells was analyzed by Western blotting (left). Paraffin sections from brain injected with pLXSN- andpLXSN-FoxM1B–transduced NHA-E6/E7/hTERT cells were stained with antibodies against PTEN (right). B, NHA-E6/E7/hTERT-FoxM1B cells weretransfected with FoxM1 siRNA or control siRNA (50 nmol/L) for 48 h; the expression of FoxM1, PTEN, p-Akt, and Akt was analyzed by Western blotting (left).Hs683 cells were transfected with pcDNA3.1-FoxM1 and pcDNA3.1 control vector and the expression of FoxM1, PTEN, p-Akt, and Akt was analyzedby Western blotting (right). C, real-time PCR analysis of relative mRNA levels of FoxM1 and PTEN in pLXSN- and FoxM1B-transduced NHA-E6/E7/hTERTcells. Columns, mean of experiments done in triplicate; bars, SD. *, P < 0.01. D, cycloheximide chase experiment (left). FoxM1B-transducedNHA-E6/E7/hTERT cells and pLXSN-transduced NHA-E6/E7/hTERT cells were treated with cycloheximide (CHX; 50 mg/mL) and cells were collected at theindicated time point for Western blot analysis of PTEN expression. Relative PTEN protein stability (middle). Intensities of the bands in the Western blotwere analyzed by using the NIH Image software and the integrated absorbances of the bands at time zero were set as 100%. Blocking of PTENdecrease by MG132 (right). pLNCX-PTEN plasmid was cotransfected with pcDNA3.1-FoxM1 or pcDNA3.1 plasmids into COS-1 cells for 24 h. MG132(0–5 μmol/L) was added to the medium as indicated. The cells were cultured for another 24 h and Western blotting was done.

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Figure 4. FoxM1 downregulates PTEN expression by regulating NEDD4-1. A, NHA-E6/E7/hTERT-FoxM1B cells were transfected with NEDD4-1-siRNA(50 nmol/L) for 24 h and then treated with cycloheximide (50 mg/mL) for 6 h; cells were collected for Western blotting of NEDD4-1 and PTEN expressions.B, the expression of FoxM1, NEDD4-1, and PTEN in parental and pLXSN- or FoxM1B-transducedNHA-E6/E7/hTERTcells was analyzedbyWestern blotting (left).Real-time PCR analysis of relative mRNA levels of NEDD4-1 in NHA-E6/E7/hTERT/pLXSN cells and FoxM1B cells (middle). NHA-E6/E7/hTERT-FoxM1Bcells were transfected with FoxM1 siRNA or control siRNA (50 nmol/L) for 48 h and Western blot analyses were done using the indicated antibodies (right).C, top, the schematic structure of NEDD4-1 promoter shows the sequences and positions of putative FoxM1 binding sites on the promoter. Gray boxes,putative FoxM1binding regions 1 and 2 inChIP assay. Bottom,ChIP assayswere donewithNHA-E6/E7/hTERT/pLXSN andFoxM1Bcells using an anti-FoxM1antibody or anti-FoxO3a antibody (as a control). One percent of the total cell lysates was subjected to PCR before immunoprecipitation (IP) as inputs. D, Hs683cells were transfected with pcDNA3.1-FoxM1 and control vector (left). COS-1 cells were cotransfected with 2 μg of pLNCX-PTEN plasmid with differentdosages of pcDNA3.1-FoxM1 plasmids (right). Cell lysates were collected 48 h after the transfections for Western blotting using the indicated antibodies.

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restored PTEN expression and reversed the upregulation ofp-Akt in the cells (Fig. 3B, left). Furthermore, overexpressionof FoxM1 in Hs683 glioma cells downregulated PTEN andupregulated p-Akt expression (Fig. 3B, right).FoxM1 regulates PTEN expression at the posttranscrip-

tional level. To study the mechanism that may be responsi-ble for the downregulation of PTEN by FoxM1, we firstexamined the RNA levels of PTEN and found that the tran-scriptional levels of PTEN were similar in both the NHA-E6/E7/hTERT/FoxM1 and NHA-E6/E7/hTERT/pLXSN controlcells (Fig. 3C). Next, we inhibited protein translation in cellswith the use of cycloheximide treatment and analyzed PTENprotein levels at the indicated times. As shown in Fig. 3D (leftand middle), overexpression of FoxM1 in NHA-E6/E7/hTERT/FoxM1 cells resulted in an increase in the endoge-nous PTEN degradation rate compared with NHA-E6/E7/hTERT/pLXSN control cells. Overexpression of FoxM1 inCOS-1 cells also resulted in a decreased level of exogenousPTEN protein (Fig. 3D, right). Such a decrease in PTEN pro-tein level can be blocked by the proteasomal inhibitor MG132(Fig. 3D, right). These results indicated that the decreasedPTEN expression in FoxM1 overexpression cells was due, atleast in part, to the increased stability of PTEN protein.FoxM1 downregulates PTEN expression by regulating

NEDD4-1. To study how FoxM1 regulates PTEN degradation,we first performed protein-protein binding experiments onFoxM1 and PTEN. We did not find direct binding of FoxM1with PTEN (data not shown), indicating that the regulationof PTEN by FoxM1 is probably an indirect mechanism. Onthe other hand, NEDD4-1 has been identified as an E3 ligasethat can promote PTEN ubiquitination and degradation. Wedetermined that endogenous NEDD4-1 can indeed regulatePTEN in NHA-E6/E7/hTERT/FoxM1 by showing that theknockdown of NEDD4-1 by small interfering RNA (siRNA) inthe cells caused an increase in endogenous PTEN (Fig. 4A).Therefore, this result confirmed the role of NEDD4-1 as aPTEN E3 ligase in the cells.We next examined the level of NEDD4-1 in NHA cells. As

shown in Fig. 4B, the protein level of NEDD4-1 was upregu-lated in NHA-E6/E7/hTERT/FoxM1B cells compared withthe levels in NHA-E6/E7/hTERT and NHA-E6/E7/hTERT/pLXSN cells, whereas the protein level of PTEN was downre-gulated in NHA-E6/E7/hTERT/FoxM1B cells. Moreover, themRNA level of NEDD4-1 was upregulated in NHA-E6/E7/hTERT/FoxM1B cells (Fig. 4B, middle). In contrast, knockingdown FoxM1 in NHA-E6/E7/hTERT/FoxM1B cells inhibitedthe expression of NEDD4-1, restored PTEN expression, andinhibited p-Akt in the cells (Fig. 4B, right).To determine whether NEDD4-1 could be a direct tran-

scriptional target of FoxM1, we scanned ∼2-kb promoter re-gions of the NEDD4-1 gene with the FoxM1 DNA bindingconsensus sequence (13) and two FoxM1 putative bindingsites (−740 to −750 bp and −903 to −913 bp) were found inthe promoter (Fig. 4C). To determine whether FoxM1 bindsdirectly to the human NEDD4-1 promoter, we performedChIP assays using two pairs of PCR primers to DNA se-quences (−642/−828 bp or −841/−993 bp) in the vicinity ofthese potential FoxM1 binding sites. Indeed, ChIP assays in



NHA-E6/E7/hTERT/pLXSN or FoxM1B cells with antibodiesspecific to either FoxM1 or FoxO3a (control) showed thatFoxM1 protein is directly bound to the endogenousNEDD4-1 promoter compared with the FoxO3a control(Fig. 4C). Moreover, in the NHA-E6/E7/hTERT/FoxM1B cellsthat expressed high FoxM1 levels, there is an increased bind-ing of FoxM1 protein to the promoter compared with that inNHA-E6/E7/hTERT/pLXSN (Fig. 4C). These findings showthat FoxM1 binds specifically to FoxM1 binding sites in theNEDD4-1 promoter in vivo, suggesting that NEDD4-1 couldbe a direct transcriptional target of FoxM1.To examine whether FoxM1 regulates NEDD4-1 in other

cells, Hs683 glioma cells were transfected with FoxM1. Overex-pression of FoxM1 in Hs683 glioma cells upregulated NEDD-4,decreased PTEN, and increased p-Akt expressions (Fig. 4D, left).Furthermore, COS-1 cells were cotransfected with PTEN in thepresence or absence of FoxM1 overexpression. Overexpressionof FoxM1 in COS-1 also caused an increase in NEDD4-1 and adecrease in exogenous PTEN protein (Fig. 4D, right). Taken to-gether, the above data indicate that FoxM1 regulates NEDD4-1expression, and hence PTEN expression, in the cells.FoxM1 induces multiple occurrences of cancer-related

gene expression. To further explore the molecular mecha-nisms involved in FoxM1-mediated GBM formation, we mea-sured the expression of cyclin D1, cyclin E, and survivin inFoxM1B-overexpressing cell lines and in the brain tumor tis-sues of mice. Some of these molecules have been previouslyreported to be downstream targets of Akt and/or FoxM1 andto be important factors for glioma proliferation (20, 24, 25, 38,39). Our results showed that cyclin D1, cyclin E, and survivinwere also highly expressed in the FoxM1B-overexpressingcells and in the brain tumors that arose from injection ofNHA-E6/E7/hTERT/FoxM1B cells (Fig. 5A and B). In addi-tion, knockdown of FoxM1 expression downregulated thesegenes significantly in HFU-251 GBM cells (Fig. 5C). These re-sults indicate that overexpression of FoxM1 in NHA cells notonly inhibits PTEN and activates the Akt pathway but alsoinduces the expression of multiple cell cycle–related genes,which are important molecules for tumor growth.

Discussion

Although our previous studies had indicated that aberrantFoxM1B expression is critical to the regulation of tumorigenic-ity, invasion, and angiogenesis of human glioma cells, it wasnot known whether FoxM1B plays an important role in thetransformation of human astrocytes into glioma cells. In thisstudy, we found that overexpression of FoxM1 in immortal-ized human astrocytes not only transforms the cells but alsocauses them to progress into GBM cells in the brain. We alsoprovided evidence that FoxM1 upregulated the expression ofNEDD-4, which resulted in increased PTEN protein degrada-tion. Furthermore, the increased PTEN protein degradation inturn led to the upregulation of activation of the Akt pathway, apotential mechanism for the development of GBM.Glioma formation and progression are widely regarded as

multistep processes resulting from a complex interplaybetween multiple genetic and epigenetic events (3–6, 31–33,

Cancer Research





40, 41). Studies have shown that the loss of p53 and pRb is notsufficient to induce glioma and that additional molecularevents are needed such as Ras activation and PTEN loss (40,41). Our results support this finding; specifically, we found thata combination of FoxM1 overexpression, loss of p53 and pRb,and human telomerase reverse transcriptase overexpression issufficient to induce NHA cells to transform into glioma.Moreover, our results support the involvement of PTEN un-

derexpression in transformation and in GBM formation.Brain-specific inactivation of PTEN in a mouse model causedincreased astrocyte proliferation that may render these cellssusceptible to neoplastic transformation (42, 43). PTEN het-erozygous knockout accelerated astrocytoma developmentin mice in which the Rb family proteins were inactivated(44). Moreover, it has been shown that successive loss of eachPTEN allele may contribute to de novo formation of high-grade astrocytoma and progression into glioblastoma (45).Numerous studies have suggested that PTEN can be inac-

tivated not only by gene deletion and mutation but also bydefects in posttranslational regulation during tumorigenesis(12, 46, 47). Recent studies postulated that PTEN is subject toubiquitination mediated by NEDD4-1, an E3 ligase, leadingto PTEN degradation (12, 47). Moreover, overexpression ofNEDD4-1 was shown to induce PTEN degradation and pro-mote K-Ras–mediated transformation (12). In bladder can-cers, the PTEN level was inversely correlated with the levelof NEDD4-1 (12). In breast cancer, NEDD4-1 was shown tobe critical for targeting PTEN for degradation (47). Therefore,these studies suggest that NEDD4-1 shows oncogenic activitythat is PTEN dependent.In the current study, we found that knockdown of NEDD4-1

in NHA-E6/E7/hTERT/FoxM1B cells caused an increase in



endogenous PTEN, suggesting a role of NEDD4-1 in PTENregulation in glioma cells. Furthermore, we showed thatoverexpression of FoxM1 downregulated PTEN expressionin NHA-E6/E7/hTERT, Hs683, and COS-1 cells (all withwild-type PTEN). The mechanism for the overexpression ofFoxM1 that accelerates PTEN degradation is as follows:FoxM1 regulates the expression of NEDD4-1, which promotesPTEN ubiquitination and degradation in these cells.PTEN inactivation could result in uncontrolled PI3K/Akt

signaling activation, a mechanism of tumor formation. Wehave observed that increased expression of FoxM1 in NHA-E6/E7/hTERT caused a significant increase in the level of p-Akt and induced tumor colony formation in a soft-agar assayand tumor formation in a mouse brain tumor model. More-over, the PI3K-specific inhibitors LY294002 and wortmannincan inhibit FoxM1-induced colony formation in vitro. There-fore, the results indicate that PI3K/Akt activation plays animportant role in glioma formation.Several studies have reported that cyclin D1 and cyclin E

expressions correlate significantly with the degree of malig-nancy in astrocytomas (48). Survivin expression is also in-creased in GBMs and correlates with patient survival (49).In this study, we found that FoxM1B overexpression in glio-ma cells significantly increased survivin, cyclin D1, and cyclinE expression, consistent with findings in other types of cells(5, 11). Thus, the molecular mechanisms by which FoxM1regulates the growth of glioma cells are associated with in-creased expression of these molecules that function as cellcycle regulators and/or inhibitors of tumor cell apoptosisand are critical for tumor cell proliferation.In summary, our findings show that FoxM1 plays an im-

portant role in promoting astrocyte transformation and

Figure 5. FoxM1 induces multipleoccurrences of cancer-relatedgene expression. A, the expressionof FoxM1, survivin, cyclin D1, andcyclin E in parental, NHA-E6/E7/hTERT/pLXSN, or FoxM1B cellswas analyzed by Western blotting.B, paraffin sections from brainsinjected with pLXSN- or pLXSN-FoxM1B–transduced NHA-E6/E7/hTERT cells were stained withantibodies against survivin, cyclinD1, and cyclin E (magnification,×200). C, HFU-251 cells weretransfected with FoxM1 siRNA(50 μmol/L) and the expression ofFoxM1, survivin, cyclin D1, andcyclin E was analyzed by Westernblotting.

Cancer Res; 70(7) April 1, 2010 2959



Dai et al.

2960


GBM formation through regulation of multiple factors. Ofmore importance, we have provided a new mechanism ofFoxM1-induced transformation and GBM formation involv-ing the overexpression of FoxM1, which upregulates the ex-pression of NEDD4-1 and hence destabilizes PTEN andactivates the Akt pathway. Our findings strongly suggest thatFoxM1 plays an important role in glioma transformation andmay be a therapeutic target for glioma.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.



Acknowledgments

We thank Tamara Locke for editorial comments.

Grant Support

National Cancer Institute grants R01-CA-116528 and P50-CA-127001, theBrain Tumor Society, and Center for Targeted Therapy at The University ofTexas M.D. Anderson Cancer Center.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10/23/2009; revised 01/12/2010; accepted 01/19/2010; publishedOnlineFirst 03/23/2010.

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2010;70:2951-2961. Published OnlineFirst March 23, 2010.Cancer Res Bingbing Dai, Russell O. Pieper, Dawei Li, et al. Human AstrocytesTransformation and Full Malignant Phenotype in Immortalized FoxM1B Regulates NEDD4-1 Expression, Leading to Cellular

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