Hyperactivation of 4E-Binding Protein 1 as a Mediator of ......translation initiation has also been shown to play an important role in the UPR, especially at the late stage during

Therapeutic Discovery

Hyperactivation of 4E-Binding Protein 1 as a Mediator ofBiguanide-Induced Cytotoxicity during Glucose Deprivation

Junichi Matsuo1,2, Yoshinori Tsukumo1, Sakae Saito1, Satomi Tsukahara1, Junko Sakurai1, Shigeo Sato1,Hiromichi Kondo1,3, Masaru Ushijima4, Masaaki Matsuura4, Toshiki Watanabe2, and Akihiro Tomida1

AbstractBiguanides, including metformin, buformin, and phenformin, are potential antitumorigenic agents and

induce cell deathduringglucosedeprivation, a cell condition that occurs in the tumormicroenvironment.Here,

we show that this selective killing of glucose-deprived cells is coupled with hyperactivation of eukaryotic

initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of translation initiation. We found, in fact,

that the 4E-BP1 hyperactivation led to failure of the unfolded protein response (UPR), an endoplasmic

reticulum–originated stress signaling pathway for cell survival. We also found that the 4E-BP1–mediated

UPR inhibition occurred through a strong inhibition of the mTOR signaling pathway, a proven antitumor

target. Importantly, the 4E-BP1 hyperactivation can be also seen in xenografted cancer cells through an in vivo

biguanide treatment. Our findings indicate that antitumor action of biguanides can be mediated by 4E-BP1

hyperactivation, which results in UPR inhibition and selective cell killing when glucose is withdrawn. Mol

Cancer Ther; 11(5); 1082–91. �2012 AACR.

IntroductionTranslation initiation, a rate-limiting process in mRNA

translation, is essential for cell growth and survival but isoftendysregulated in cancer (1). Theprocess of translationinitiation has 2 major regulatory steps. One is assembly ofa ternary complex consisting of methionine-chargedtRNA, eukaryotic initiation factor (eIF) 2, and GTP, andthis assembly is negatively regulated by phosphorylationof eIF2a (a-subunit of eIF2; ref. 1). The other regulatorystep is formation of an eIF4F complex, which binds to the7-methylguanosine triphosphate (7mGTP)-cap structureat the 50 terminus of mRNA and recruits 40S ribosome (1).The eIF4F complex consists of eIF4E, eIF4G, and eIF4A,and this assembly is inhibited by eIF4E-binding protein 1(4E-BP1). 4E-BP1 binds to eIF4E and disturbs the eIF4Eand eIF4G association. The binding activity of 4E-BP1 isregulated and turned off by phosphorylationmediated bya kinase, mTOR (1). Recent clinical studies show that

mTOR inhibitors can be effective as antitumor agents(2) and that 4E-BP1plays an important role in suppressingtumor growth by mTOR inhibition (2, 3).

Glucose deprivation is a cell condition that occurs in themicroenvironment of solid tumors due to insufficientblood supply and the large glucose consumption of gly-colytic tumor cells (4, 5). During glucose deprivation, thetranslation initiation system can be involved in an adap-tive cell survival response. In fact, glucose deprivationattenuates themTOR signaling pathway,which decreasestranslation initiation by activating the inhibitory protein4E-BP1 (6). Glucose deprivation also elicits the unfoldedprotein response (UPR), an endoplasmic reticulum (ER)–originated stress signaling pathway that reduces globalprotein synthesis with decreased translation initiationand produces several transcription factors to induce theUPR target genes, such as the ER-resident molecularchaperones glucose-regulated protein 78 (GRP78; refs.7, 8).

The UPR signaling pathway is initiated when the ER-localized transmembrane proteins PKR-like ER kinase(PERK), inositol-requiring 1 (IRE1), and activating tran-scription factor 6 (ATF6) are activated (8). Among them,PERK plays a major role in the UPR translational controlby phosphorylating eIF2a, an important regulatorymech-anism of translation initiation (9, 10). Phosphorylation ofeIF2a reduces global translation and, paradoxically,directs preferential translation of ATF4, a UPR transcrip-tion activator (10). The PERK signaling pathway is furtherregulated by an ATF4-directed feedback system for eIF2adephosphorylation, thereby restoring translation to theUPR target transcripts (11). In addition to this eIF2a-mediated regulation, 4E-BP1–mediated regulation of

Authors' Affiliations: 1Cancer Chemotherapy Center, Japanese Founda-tion for Cancer Research; 2Laboratory of Tumor Cell Biology, Departmentof Medical Genome Sciences, Graduate School of Frontier Sciences, TheUniversity of Tokyo; 3Hitachi Aloka Medical, Ltd.; and 4Genome Center,Japanese Foundation for Cancer Research, Tokyo, Japan

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

J. Matsuo and Y. Tsukumo contributed equally to this work.

Corresponding Author: Akihiro Tomida, Cancer Chemotherapy Center,Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo135-8550. Phone: 81-3-3570-0514; Fax: 81-3-3570-0484; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-11-0871

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 11(5) May 20121082

on February 7, 2021. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst March 8, 2012; DOI: 10.1158/1535-7163.MCT-11-0871

http://mct.aacrjournals.org/

translation initiation has also been shown to play animportant role in the UPR, especially at the late stageduring prolonged ER stress (12). Thus, translation initia-tion is an important regulatory mechanism for the UPRthat is involved in cell survival under stress conditions(13–16).Antidiabetic biguanides, including metformin, bufor-

min, and phenformin, are known as small molecule com-pounds that reduce blood glucose level by inhibitingglucose synthesis and enhancing its uptake into cells(17, 18). These behaviors have been associated with acti-vation of AMP-activated protein kinase (AMPK; ref. 17,18). It was recently reported that antidiabetic biguanidescan exert antiproliferative and proapoptotic effects ondifferent cancer types through AMPK-dependent andAMPK-independent mechanisms (19, 20). These antidia-betic biguanides have also been shown to inhibit UPRactivation and to induce selective cancer cell killing dur-ing glucose deprivation (21). Previous studies havefocused on the actions of biguanides under normalgrowth conditions; however, it is largely unknown howbiguanides affect cellular response to glucosedeprivation,a representative tumor microenvironmental stress condi-tion. In this study, we show that, during glucose depri-vation, biguanides induce hyperactivation of 4E-BP1through a strong inhibition of the mTOR signaling path-way, which is closely associated with UPR inhibition andselective cytotoxicity.

Materials and MethodsCell cultures and treatmentCells were cultured in either RPMI1640 (Wako Pure

Chemical Industry; human fibrosarcoma HT1080 andstomach cancer MKN-74 cells) or Dulbecco’s ModifiedEagle’s Medium (Wako Pure Chemical Industry; humancervical cancer HeLa and embryonic kidney 293T cells)supplemented with 10% FBS and 100 mg/mL of kanamy-cin andwere cultured at 37�C in a humidified atmosphereof 5%CO2 (20, 21). Cell authenticationwas not done by theauthorswithin the last 6months. To analyze several stressconditions, cells were cultured for the indicated timeperiods in glucose-containing medium in the presence of10 mmol/L 2-deoxyglucose (2DG; Sigma), 5 or 10 mg/mLof tunicamycin (Nacalai Tesque) or in glucose-free medi-um (Sigma). Cells were also treated with various concen-trations of phenformin (5–500 mmol/L; Sigma), buformin(30–1000 mmol/L; Wako), 10 mmol/L metformin (Sigma)rapamycin (0.3–100 nmol/L; Sigma), and PP242 (0.03–10mmol/L; Sigma). These compoundswere added to culturemedium, with the solvent being less than 0.5% of themedium’s volume.

RNA interferenceControl short interfering RNA (siRNA) and Stealth

siRNAs against human 4E-BP1 were purchased fromInvitrogen. Lipofectamine RNAi MAX (Invitrogen) wasused for transfection.

Immunoblot analysisCell lysateswere prepared as previously described (22).

The following antibodies were used for immunoblotting:anti-eIF2a (Abcam), anti-phospho eIF2a, anti-4EBP1,anti-phospho-4EBP1 (Ser65; 174A9), anti-phospho-4EBP1(Thr70), anti-eIF4E, anti-eIF4G, anti-AMPK, anti-phos-pho-AMPK (Cell Signaling Technology), anti-KDEL (forGRP78 detection; Stressgen), anti-ATF4 (Santa Cruz Bio-technologies), and anti–b-actin (Sigma).

7mGTP affinity purificationHeLa cells were treated in the lysis buffer [50 mmol/L

Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA,1% Triton X-100, Phosphatase Inhibitor Cocktail 1 and 2(Sigma), and Protease Inhibitor (Sigma)] at 4�C for 30minutes. Cell extracts (500 mg) were incubated with7mGTP-conjugated Sepharose beads (GE Healthcare UKLtd.) in the lysis buffer at 4�C for 2 hours (22). After 3washings in the lysis buffer, the beads were boiled with2�SDS sample loading buffer at 100�C. Each sample wasanalyzed by immunoblot.

Measurement of cells viabilityHeLa cells were treated with phenformin, rapamycin,

or PP242 in the presence or absence of ER stress inducers(2DG or tunicamycin) for 24 hours. Themediumwas thenreplaced with fresh growth medium, and cells were cul-tured for a further 24 hours.MTT (Sigma)was then addedto the culture medium. After 3 hours, the absorbance ofeach well was determined, as described previously (22).Relative cell survival was calculated by setting each con-trol absorbance from untreated cells as 100%.

In the case of 4E-BP1 knockdown, HeLa cells weretreated with phenformin or buformin in the presence orabsence of 2DG for 14 hours. Then, the cellswere reseededto 12-well plates and incubated for 5 days. Afterward, thecell viability was determined by MTT assay.

Reporter assayHT1080 cells were transfected with a firefly luciferase-

containing reporter plasmid (pGRP78pro160-Luc) thatcontained the promoter region of grp78 (23) and Renillaluciferase–containing plasmid phRL-CMV (Promega) asan internal control. Relative activity of firefly luciferase toRenilla luciferase (mean� SD of triplicate determinations)was determined using the Dual-Glo Luciferase AssaySystem (Promega).

Nuclear and cytoplasmic extract preparationCells were fractionated using NE-PER nuclear and

cytoplasmic extraction reagents (Pierce), following man-ufacturer’s instruction.

MKN74 xenograft tumorsMKN-74 cells were implanted subcutaneously in the

right flank region of 9-week-old BALB/cAJcl-nu/numice(n ¼ 6 for evaluation of tumor growth; Charles RiverJapan, Inc.). The experiments were started approximately

Hyperactivation of 4E-BP1 by Biguanide

www.aacrjournals.org Mol Cancer Ther; 11(5) May 2012 1083




10 days after cell implant when tumors, measured withcalipers, reached 150 mm3 (day 0). Buformin was injectedinto the tumor for immunohistochemical analysis or wasadministered daily by oral gavage to evaluate the thera-peutic effects of the drug. The length (L) and width (W) ofthe tumor were measured, and the tumor volume (TV)was calculated as TV ¼ (L � W � W)/2. The data areexpressed as mean � SE. After checking the linearity ofgrowth curves of TVs, we conducted a Welch t testbetween the treatment and control groups using theslopes of the growth curves based on the linear regression.

ResultsHypophosphorylation of 4E-BP1 by biguanides

We carried out immunoblot analysis to determine 4E-BP1 phosphorylation status in HT1080 cells that had beentreated for 4 or 8 hourswith phenformin in the presence orabsence of the hypoglycemia-mimicking agent 2DG (Fig.1A). The phosphorylation status was monitored both byband shifts from higher (hyperphosphorylated) to lowerapparent molecular weight (hypophosphorylated) withanti-4E-BP1 antibody and by signal intensity with eachphosphospecific anti–4E-BP1 antibody at Thr37/46,Ser65, and Thr70. We found that phenformin in combi-nation with 2DG strongly induced hypophosphorylationof 4E-BP1, whereas each single phenformin and 2DG

treatment had a weak or marginal effect (Fig. 1A). Den-sitometric analysis of band intensities revealed that Ser65phosphorylation levels of 4E-BP1 reduced to the levels ofless than 5% by the combination, although remained 50%or more in the case of each single treatment (Supplemen-tary Table S1). Under 2DG stress conditions, induction ofATF4 and GRP78, typical UPRmarker proteins, was seenwithin the time periods, but induction was suppressed inthe presence of phenformin (Fig. 1A, SupplementaryTable S1). We noted that both hypophosphorylation of4E-BP1 and suppression of GRP78 induction in 2DG-stressed HT1080 cells occurred at the same phenformindose (Fig. 1B, Supplementary Table S1).

To examine whether phenformin induced 4E-BP1hypophosphorylation under different ER stressors, wetreated HT1080 cells for 4 hours with phenformin,together with 2DG or an N-glycosylation inhibitor tuni-camycin (Fig. 1C, Supplementary Table S1). In contrastto what we saw in 2DG-stressed cells, phenformin hadlittle effect on either 4E-BP1 phosphorylation status orGRP78 induction in tunicamycin-stressed cells (Fig. 1C,Supplementary Table S1). On the other hand, effectssimilar to those seen for phenformin on 4E-BP1 andGRP78 were also observed in HT1080 cells subjected toglucose withdrawal for 20 hours and in HeLa cellsstressed for 4 hours with 2DG (Fig. 1D and E, Supple-mentary Table S1). In LKB1-defective HeLa cells, LKB1

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Figure 1. Hyperactivation of4E-BP1 by phenformin. A, HT1080cells were treated with phenformin(100 mmol/L) in the presence (þ) orabsence (–) of 2DG (10 mmol/L) forthe indicated times. B, the cellswere treated with indicatedconcentration of phenformin aloneor together with 2DG (10 mmol/L)for 4 hours. C, the cells weretreated with phenformin(100 mmol/L) alone or together with2DG (10 mmol/L) or tunicamycin(10 mg/mL) for 6 hours. D, the cellswere treated with phenformin(100 mmol/L) under glucosefree (GFþ) or normal (GF�)conditions for 20 hours. E and F,HeLa or HT1080 cells were treatedwith phenformin (100 mmol/L) aloneor together with 2DG (10 mmol/L)for 4 hours.

Matsuo et al.

Mol Cancer Ther; 11(5) May 2012 Molecular Cancer Therapeutics1084




being an upstream kinase of AMPK (24), phenforminhad marginal effect on AMPK phosphorylation (Fig. 1E,Supplementary Table S1), suggesting that AMPKactivation was unnecessary for phenformin-induced4E-BP1 hypophosphorylation. Phenformin had only amarginal effect on the phosphorylation states of eIF2a,another translation initiation regulator important forthe UPR, in HT1080 cells (Fig. 1F, Supplementary TableS1).We also examined the effects of other antidiabetic

biguanides buformin and metformin on 4E-BP1 phos-phorylation status in HT1080 cells (Fig. 2). Buforminshowed essentially the same effects on 4E-BP1 and onATF4 and GRP78 in 2DG-stressed HT1080 cells, althoughsomewhat higher concentrations than phenformin wererequired (Fig. 2A). Metformin also induced 4E-BP1 hypo-phosphorylation and suppressed GRP78 induction at ashigh as 10 mmol/L (Fig. 2B). Thus, the biguanides, whichhave an inhibitory activity on UPR activation, can inducestrong hypophosphorylation of 4E-BP1 under conditionsof 2DG stress and glucose withdrawal.

Involvement of 4E-BP1 in cell death under stressconditionsTo examine whether 4E-BP1 has any impact on cyto-

toxicity induced by 2DG stress and biguanides, wesilenced 4E-BP1 expression in HeLa cells using siRNA(Fig. 3A). The 4E-BP1 knockdown enhanced GRP78induction under 2DG stress conditions (Fig. 3B), as wesaw in our previous study (22). Consistent with the pro-tective role of GRP78, the 4E-BP1–silenced cells showedan enhanced cell survival during 2DG stress, as comparedwith nonsilenced control cells (Fig. 3C and D). Interest-ingly, the 4E-BP1–silenced cells also showed enhancedcell survival even after treatment for 18 hours with phen-

formin (Fig. 3C) or buformin (Fig. 3D) in the presence of2DG. In the absence of 2DG, biguanide cytotoxicity wasscarcely observed in both control and 4E-BP1–silencedcells. These results suggested that 4E-BP1 could beinvolved in cell death regulation under the stressconditions.

Hyperactivation of 4E-BP1 by biguanidesTo examine whether the hypophosphorylation of 4E-

BP1 by biguanides led to increased associationwith eIF4E,we carried out an mRNA cap structure 7mGTP-bindingassay. The 7mGTP-Sepharose was able to pull downessentially the same levels of eIF4E from any lysates ofHeLa cells cultured under various conditions (Fig. 4A).Consistent with the 4E-BP1 hypophosphorylation levels,phenformin caused 4E-BP1 binding to eIF4E in 2DG-stressed cells, but not in tunicamycin-stressed cells (Fig.4A). Buformin also increased 4E-BP1 binding to eIF4E in2DG-stressed cells (Fig. 4B). Conversely, fewer eIF4Gproteins were detected in the eIF4E-containing initiationcomplex of the phenformin-treated, 2DG-stressed cells(Fig. 4A). Thus, the hypophosphorylated 4E-BP1 becamestrongly activated, resulting in disruption of the eIF4E-containing translation initiation complex.

Hyperactivation of 4E-BP1 with mTOR inhibitionRapamycin and PP242 are different classes of mTOR

inhibitors; an allosteric and an active site inhibitors,respectively, that can activate 4E-BP1 by inhibitingmTORkinase activity (25). To compare the actions of mTORinhibitors with those of the biguanides, we conducted a7mGTP-binding assay using HeLa cells that had beentreated for 4 hours with rapamaycin, PP242, or buforminin the presence or absence of 2DG (Fig. 4B). Consistentwith previous observations (25, 26), bothmTOR inhibitors

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Figure 2. Buformin and metformin exhibit similar effect. A, HT1080 cells were treated with 300 mmol/L of buformin for the indicated time (left) or with theindicated concentration (mmol/L) of buformin (right) or B, 10 mmol/L metformin, in the presence (þ) or absence (–) of 2DG (10 mmol/L) for 4 hours.






were able to activate 4E-BP1 regardless of 2DG addition,but PP242 caused much more 4E-BP1 binding to eIF4Ethan rapamycin did. The levels of 4E-BP1 activationinduced by PP242 were comparable with those inducedby buformin under 2DG stress conditions. The hyperac-tivation of 4E-BP1was associatedwith strong cytotoxicityin HeLa cells; indeed, PP242, but not rapamycin, underany conditions tested and buformin under 2DG stressconditions were highly toxic (Fig. 5A).

We further examined the effects of PP242 on 4E-BP1hypophosphorylation and UPR activation in HT1080cells, using 3 different ER stress conditions, 2DG, TM,and glucose withdrawal (Fig. 5B). PP242 induced 4E-BP1hypophosphorylation and substantially prevented induc-

tion of GRP78 and GRP94, regardless of culture condi-tions. This observation was further confirmed by ourreporter assay, using pGRP78-Luc, which contained aGRP78 promoter region (–160 to þ7) immediatelyupstream of the firefly luciferase (ref; Fig. 5C). Indeed,PP242 effectively suppressed 2DG- and tunicamycin-induced GRP78 promoter activities in a dose-dependentmanner.

Hyperactivation and altered localization of 4E-BP1To explore the similarity in the actions between bigua-

nides and PP242 further, we examined subcellular local-ization of 4E-BP1 and eIF4E, using a cell fractionationtechnique followed by immunoblot analysis (Fig. 5D).We

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Figure 3. Involvement of 4E-BP1 incell death under stress conditions.A and B, after 30-hour siRNAtransfection HeLa cells weretreated with the indicatedconcentrations of phenformin inthe presence or absence of 2DG(10 mmol/L) for 18 hours. C and D,after 48-hour siRNA transfectionHeLa cells were treated with theindicated concentrations ofbiguanide with or without 2DG(10 mmol/L) for 14 hours and thenreseeded. Five days later, cellviability was measured by MTTassay.

Matsuo et al.





found that when HT1080 cells were treated with a com-bination of buformin and 2DGorwith PP242 regardless of2DG addition, 4E-BP1 proteins accumulated in the nucle-us. Interestingly, the 4E-BP1 accumulation coincidedwithdecreased accumulation of eIF4E in the nucleus. Phenfor-min, in the presence of 2DG, also induced nuclear accu-mulation of 4E-BP1 in HT1080 cells (Supplementary Fig.S1A). Furthermore, immunofluorescent staining showedthat nuclear accumulation of 4E-BP1 also occurred inHeLa cells treated with phenformin in the presence of2DG (Supplementary Fig. S1B). It is important that, unlikebiguanides and PP242, rapamycin did not cause suchchanges in nuclear localization of 4E-BP1 and eIF4E (Fig.5D and Supplementary Fig. S1).

Activation of 4E-BP1 in xenograftWe further characterized the antitumor activity of

buformin using stomach cancer cells, MKN74. As shownin Fig. 6A, buformin showed selective cytotoxicity under2DG, but not tunicamycin, stress conditions. Buformin

induced hypophosphorylation of 4E-BP1 within 4 hoursand in a dose-dependent manner in 2DG-stressed cells(Fig. 6B and C). This 4E-BP1 hypophosphorylation wasalso seen in cells under glucose withdrawal stress (Fig.6D). Furthermore, buformin suppressed 2DG-inducedGRP78 and GRP94 (Fig. 6C). Thus, the typical effects ofbuformin were seen in MKN74 cells. We also found thatgiving buformin orally to mice caused weak but statisti-cally significant growth retardation ofMKN74 xenografts(Fig. 6E). The weights of buformin-treated animalsdecreased by less than 10% by day 7 but were regainedafterward (data not shown). Althoughwe had carried outimmunohistochemical analysis using MKN74 xenograftsafter giving buformin orally to mice, clear changes in 4E-BP1 phosphorylation statuswere not seenwith orwithoutthedrug treatment. To see thepotential of in vivobuformineffect, we then tried intratumor drug injection. Immuno-histochemical analysis showed that intratumor injectionof buformindecreaseddetection of phosphospecific (T37/46) 4E-BP1, as compared with nonphosphospecific 4E-BP1 (Fig. 6F andSupplementary Fig. S2). In addition, closeexamination of the data revealed that some heterogeneityin the staining for phosphorylated 4E-BP1 was seen evenin the control specimen, suggesting that 4E-BP1 phos-phorylation status could be fluctuated with cellular states(e.g., necrosis) in intratumor conditions.

DiscussionRecent studies have shown that antidiabetic bigua-

nides, particularly metformin, can exert in vitro and invivo antitumor activity through multiple modes of action(19–21). Of interest is their ability to disrupt the UPRduring glucose deprivation because it can lead to selectivecell killing under the particular tumor microenvironment(21, 27). In this study, we have shown that the UPRinhibitory action, as well as cytotoxicity of biguanides,can be mediated by 4E-BP1 hyperactivation during glu-cose deprivation. Such 4E-BP1hyperactivation can also beinduced in xenoplanted cancer cells by intratumor admin-istration of buformin (Fig. 6F). Furthermore, mechanisticanalysis with the different mTOR inhibitors types PP242and rapamaycin suggested that strongly inhibiting themTOR signaling pathway contributes to the 4E-BP1hyperactivation induced by biguanides.

Our present findings showed that hyperactivation of4E-BP1 can be involved in preventing UPR activationunder stress conditions. This UPR inhibitory action maybe associated with the translation repressor activity of 4E-BP1. In fact, we observed strong inhibition of globalprotein synthesis with 4E-BP1 hyperactivation in cellstreated with phenformin and 2DG (Supplementary DataS3). In addition, overexpression of 4E-BP1 by transfectioncan attenuate the UPR activation, depending on the func-tional domain of the eIF4Ebinding site,which is necessaryfor translation repressor activity (22). In this context, it isimportant to note that proper activation of 4E-BP1 isnecessary for translation control at the late stage of the

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Figure 4. Binding of 4E-BP1 to cap structure by phenformin. A, HeLa cellswere treatedwith phenformin (100 mmol/L) alone or together with 2DG (10mmol/L) or tunicamycin (10 mg/mL) or B, buformin (300 mmol/L),rapamycin (100 nmol/L), or PP242 (10 mmol/L) for 4 hours.






UPR (12, 22, 28, 29). Indeed, prolongedERstress elicits thisnecessary 4E-BP1 activation (22, 28, 29). Thus, dysregu-lated, drug-induced hyperactivation of 4E-BP1 may dis-rupt the translation regulation mechanisms that governthe UPR.

More generally, hyperactivated 4E-BP1 can suppresstranslation initiation through binding to eIF4E, an onco-genic protein important for tumor cell survival. It has beenreported that various cancer cell lines show increasedeIF4E expression and that overexpression of eIF4E causesmalignant transformation of cultured cells (30). It is alsoknown that eIF4Epreferentially stimulates translation of asubset of mRNAs that play important roles in cell growth(30). Consistently, small compounds that inhibit eIF4Efunction have been reported to suppress cancer cellgrowth (30, 31). Therefore, in addition to preventing theUPR, inhibiting eIF4E function can explain the antitumoractivity of biguanides through strong activation of 4E-BP1. In this regard, eIF4E can localize in the cytoplasmandthe nucleus (32), and nuclear eIF4E is known to play a role

in mRNA export from the nucleus (33). Such nucleus-specific function of eIF4E might also be an antitumortarget, because biganides as well as PP242 can decreaseeIF4E protein levels in the nucleus, simultaneously withincreasing activated 4E-BP1 in the nucleus (Fig. 5D andSupplementary Fig. S1).

The biguanide-induced 4E-BP1 hyperactivation duringglucose deprivation is considered to occur throughmTORinhibition. Consistently, biguanides havebeen reported toinhibit mTOR signaling through AMPK-dependent andAMPK-independent mechanisms (19, 20). Glucose with-drawal can also suppress the mTOR signaling pathway(6). Of importance, we found that the inhibitory effects onthe mTOR signaling pathway are strongly enhanced bythe combination of biguanides and glucose withdrawal.The precise mechanisms behind the enhanced mTORinhibition remain to be clarified; however, there are someclues to understanding them. As shown in Fig. 1E, thecombined effects of biguanides and glucose withdrawalseem to be independent of AMPK activation. We recently

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Figure 5. Hyperactivation of 4E-BP1 with mTOR inhibition. A, HeLacells were treated with theindicated concentrations of PP242(left), rapamycin (middle), orbuformin (right) in the presence of2DG (10 mmol/L) or tunicamycin(10 mg/mL) for 24 hours. Cellviability was determined by MTTassay. B, HeLa cells were treatedwith PP242 (10 mmol/L) in thepresence (þ) or absence (–) of 2DG(10 mmol/L) for 4 hours. C, HT1080cells were transfected withpGRP78pro160-Luc and treatedwith the indicated concentrationsof PP242 with 2DG (10 mmol/L) ortunicamycin (10 mg/mL) for 18hours. D, altered localization of 4E-BP1. HeLa cells were treated withbuformin (300 mmol/L), rapamycin(100 nmol/L), or PP242 (10 mmol/L)in the presence (þ) or absence (–) of2DG (10 mmol/L) for 4 hours.

Matsuo et al.





found that mitochondrial dysfunction not only leads tofailure ofUPRactivationwith increased cell death but alsoto 4E-BP1 hyperactivation during glucose deprivation(ref. 34; data not shown). Consistently, it has beenreported that biguanides can induce mitochondrial dys-function (35, 36). Thus, mitochondrial dysfunction mayplay an important role in enhanced mTOR inhibitionleading to 4E-BP1 hyperactivation when cells are treatedwith biguanides during glucose deprivation.In summary, we have shown that hyperactivation of

4E-BP1 is a mediator for biguanides that prevent theUPR and exert antitumor cytotoxicity during glucosedeprivation. Importantly, such biguanide-induced 4E-BP1 hyperactivation can also be induced in xenoplantedcancer cells, suggesting that glucose levels in the xeno-grafts could be low enough for the drug action. Furtherstudies will be needed to show whether the drug-induced 4E-BP1 hyperactivation can be widely seen in

the other tumor models, including orthotopic tumormodels. In the meantime, it is widely recognized thatglucose deprivation generates resistance to many clin-ically important antitumor drugs in a variety of humancancer cells (7, 37, 38). The development of drug resis-tance is closely associated with UPR activation (7). Thus,the action of biguanides via 4E-BP1 may be useful toeliminate otherwise drug-resistant, glucose-deprivedtumor cells. Our findings have also shown that thebiguanide-induced 4E-BP1 hyperactivation can bemediated by a strong inhibition of the mTOR signalingpathway. Recently, the success of rapamycin and itsanalogs as anticancer agents has motivated the devel-opments of other types of mTOR inhibitors (2). In thiscontext, biguanides may be interesting as a unique typeof antitumor agent that suppresses the mTOR signalingpathway during glucose deprivation. Consideringtheir activities on 4E-BP1 activation, buformin and

Figure 6. Activation of 4E-BP1 inxenograft. MKN74 cells were used inthese experiments. A, the cells weretreated with the indicatedconcentrations of buformin in thepresence of 2DG (10 mmol/L) ortunicamycin (10 mg/mL) for 24 hours.Cell viability was determined by MTTassay. B, the cells were treated withthe indicated concentration ofbuformin alone or together with 2DG(10 mmol/L) for 4 hours, or C, withbuformin (100 mmol/L) for theindicated time or D, under glucosewithdrawal conditions for 20 hours.E, buformin (100mg/kg) or salinewasadministered orally daily to MKN74tumor-bearing mice (n ¼ 6). Theexperiment was startedwhen tumorshad grown to approximately 150mm3 (day 0). The difference in tumorvolumes between the groups wasstatistically significant (P¼ 0.046). F,intratumor injection of 50 mL ofbuformin solution or saline (as acontrol) was administered. After 4hours tumors were formalin fixed.Each section was stained with HE orspecific antibodies, as indicated. Bar¼ 500 mm.

BA

C

E

D

F

– – –

– + – +

+ + + +– + +– + – +

β-Actin

GRP94GRP78

4E-BP1

P-4E-BP1(Ser65)

P-4E-BP1(Thr37/46)

2440

2DGBu

(h)

Norm

2DG

TM

% o

f su

rviv

alBuformin (mmol/L)

0

25

50

75

100

1010.10.01

4E-BP1P-4E-BP1(Thr37/46)

HE

Saline

Buformin

GF

Bu

Norm

4E-BP1

P-4E-BP1(Ser65)

P-4E-BP1(Thr37/46)β-Actin

Day

Rel

ativ

e tu

mo

r vo

lum

e

0

1

2

3

9630

Saline

Buformin

1 (mmol/L)0.30.10.030

– + – + – + – + – +2DG

4E-BP1

P-4E-BP1(Ser65)

P-4E-BP1(Thr37/46)

β-Actin






phenformin may be more effective as antitumor agentsthan metformin.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Matsuo, Y. Tsukumo, A. Tomida, and T.WatanabeDevelopment of methodology: J. Matsuo, Y. Tsukumo, and A. TomidaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Matsuo, Y. Tsukumo, S. Tsukahara, J. Sakurai,S. Sato, and H. KondoAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): J.Matsuo, Y. Tsukumo,M.Ushijima, andM.MatsuuraWriting, review, and/or revision of the manuscript: J. Matsuo, Y. Tsu-kumo, M. Ushijima, A. Tomida, and T. Watanabe

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y. Tsukumo, and S. SaitoStudy supervision: Y. Tsukumo, A. Tomida, and T. Watanabe

Grant SupportThisworkwas supported in part by a grant-in-aid for scientific research

(B), from the Ministry of Education, Culture, Sports, Science and Tech-nology of Japan,National Cancer Center Research andDevelopment Fund(21-3-1) from the Ministry of Health, Labour and Welfare, a grant fromKobayashi Foundation for Cancer Research, and from the Vehicle RacingCommemorative Foundation.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 29, 2011; revised January 24, 2012; accepted February28, 2012; published OnlineFirst March 8, 2012.

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2012;11:1082-1091. Published OnlineFirst March 8, 2012.Mol Cancer Ther Junichi Matsuo, Yoshinori Tsukumo, Sakae Saito, et al. Biguanide-Induced Cytotoxicity during Glucose DeprivationHyperactivation of 4E-Binding Protein 1 as a Mediator of

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Hyperactivation of 4E-Binding Protein 1 as a Mediator of ......translation initiation has also been shown to play an important role in the UPR, especially at the late stage during