[CANCER RESEARCH 60, 2104–2107, April 15, 2000]

Advances in Brief

U0126 Reverses Ki-ras-mediated Transformation by Blocking Both Mitogen-activated Protein Kinase and p70 S6 Kinase Pathways1

Hidesuke Fukazawa and Yoshimasa Uehara2

Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

Abstract

U0126, a recently introduced mitogen-activated protein kinase kinase(MAPK)/extracellular signal-regulated kinase kinase inhibitor reversedmorphology and inhibited anchorage-independent growth of Ki-ras-trans-formed rat fibroblasts. Immunoblot analyses with phosphospecific anti-bodies indicated that in addition to MAPK, U0126 suppressed activationof p70S6K, but not Akt, at concentrations at which it normalized thetransformed phenotypes. Another MAPK/extracellular signal-regulatedkinase kinase inhibitor, PD98059, showed only marginal effects on p70S6K

phosphorylation and did not effectively block Ki-ras-induced transforma-tion. However, simultaneous inhibition of the MAPK pathway and thep70S6K pathway by PD98059 in conjunction with the p70S6K inhibitorrapamycin essentially restored the normal phenotype. U0126 or the com-bination of PD98059 and rapamycin flattened morphology of v-src-trans-formed cells, but did not reverse anchorage independence, although acti-vation of both MAPK and p70S6K was blocked. The results suggest thatnormalization of Ki-ras-induced transformed phenotypes by U0126 is aconsequence of concurrent inhibition of the MAPK and p70S6K pathways.Intervention of other pathway(s) appears to be required to completelyantagonize transformation by v-src. Simultaneous blockade of more thanone signal transduction pathway by combining selective inhibitors mightbe effective in suppressing uncontrolled tumorigenic growth.

Introduction

Much of the current effort in anticancer drug development focuseson signal transduction pathways. The strategy of precisely targetingabnormalities that propel uncontrolled growth is more rational anddisease oriented than the traditional cell-killing approach. A potentialdifficulty in development of target therapies is assessment of theirefficacy. Signal transduction inhibitors are inherently cytostatic, andsimple cytotoxicity assays may not be relevant. Anchorage-indepen-dent growth is the hallmark of uncontrolled tumorigenic proliferation(1, 2). In theory, specific blockade of aberrant signal transductionwould normalize the transformed phenotypes and thus render tumorcells anchorage dependent. The cells are expected to lose the ability toproliferate without firm attachment but still to be competent forgrowth on solid support. We developed a simple, objective, andquantitative method to measure anchorage-independent growth usingmicrotiter plates coated with the antiadhesive polymer polyHEMA3

(3, 4). We anticipate the polyHEMA-coated plates will provide asystem for evaluating inhibitors of oncogenic signal transduction.

The MAPK (extracellular signal-regulated kinase 1/2) pathway isconsidered to be one of the promising targets for signal transduction-

based cancer chemotherapy. We examined the effects of U0126, arecently introduced MEK inhibitor (5), on growth properties of Ki-ras- and v-src-transformed rat fibroblasts. U0126 selectively re-pressed anchorage-independent growth of Ki-ras transformed cells.Subsequent analyses revealed that U0126 blocked not only the MAPKpathway but also the p70S6K pathway. Experiments with PD98059 (6)and rapamycin (7) suggested that separate intervention of the MAPKpathway or the p70S6K pathway has little effect on Ki-ras-inducedtransformation, but simultaneous blockade of the two pathways re-stores the normal phenotype. The results imply significant benefits ofcombining inhibitors of signal transduction pathways. Synergy arisingfrom combinations of selective pharmacological agents might extendthe range of signal transduction-based cancer chemotherapy.

Materials and Methods

Materials. U0126 was from Promega (Madison, WI); PD98059 and rapa-mycin were from Calbiochem (San Diego, CA); and polyHEMA was fromSigma Chemical Co. (St. Louis, MO). Antibody against phosphorylatedMAPK was from Promega, and other phosphospecific antibodies were fromNew England Biolabs (Beverly, MA). Antibodies against MAPK, p70S6K, andAkt were from Zymed Laboratories (South San Francisco, CA), Santa CruzBiotechnology (Santa Cruz, CA), and New England Biolabs, respectively.

Cell Culture and Measurement of Anchorage-independent Growth.Cell lines used in the experiments have been described (3). Anchorage-independent growth was measured on polyHEMA-coated 96-well plates asdescribed (3, 4). Briefly, 50ml of polyHEMA solution (5 mg/ml in 95%ethanol) were pipetted into wells of 96-well plates and dried for 2 days withlids in place. Cells were inoculated in a volume of 135ml at a density of 1000cells per well for Ki-ras/NRK and 5000 per well for others. Inhibitors dis-solved in 15ml of medium were added, and the cells were cultured for 4 days.Fifteen microliters of 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bro-mide solution (5 mg/ml in PBS) were added, and the mixture was furtherincubated for 4 h. The resulting 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetra-zolium bromide formazan was solubilized by addition of 100ml of SDSsolution (20% in 10 mM HCl), and the absorbance was measured after 24 h at570 nm and a reference wavelength of 690 nm using a microplate reader.

Immunoblotting Analysis. Cells were seeded in a volume of 1 ml in24-well plates at a density of 13 105 per well and cultured for 24 h. Cells werethen treated with inhibitors for 24 h, washed with cold PBS, fixed for 10 minwith 10% cold trichloroacetic acid, and lysed with 80ml of 9 M urea and 2%Triton X-100 and 20ml of 10% lithium dodecyl sulfate. Lysates were neu-tralized with 2M Tris, passed through a 250-ml syringe (Hamilton, Reno, NV)to reduce viscosity and normalized for protein using a Pierce (Rockford, IL)bicinchoninic acid kit. After addition of bromphenol blue and DTT to 0.001%and 50 mM, respectively, proteins were electrophoresed through 10% SDS-PAGE and analyzed by immunoblotting using phosphospecific antibodies.Proteins were visualized with Renaissance Western blot chemiluminescencereagent (DuPont New England Nuclear, Boston, MA).

Results

U0126 Inhibits Anchorage-independent Growth and Normal-izes Morphology of Ki-ras-transformed Cells. Anchorage inde-pendence is the primein vitro parameter of transformation (1, 2). Wedeveloped a microplate assay for efficient quantitation of anchorage-

Received 9/3/99; accepted 3/6/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a grant-in-aid for Cancer Research from the Ministry of Education,Science, Sports and Culture of Japan, by the Social Institute Agency Contract Fund of theJapan Health Science Foundation, and by the Promotion of Fundamental Studies in HealthSciences of the Organization for Pharmaceutical Safety and Research of Japan.

2 To whom requests for reprints should be addressed.3 The abbreviations used are: HEMA, 2-(hydroxyethyl methacrylate); MAPK, mito-

gen-activated protein kinase; MEK, MAPK/extracellular signal-regulated kinase kinase;NRK, normal rat kidney; mTOR, mammalian target of rapamycin.

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independent growth using plates coated with an antiadhesive polymer,polyHEMA (3, 4). This method can substitute for soft agar colonyformation in many experimental situations and is more practical whenmany samples are to be tested.

We assessed the growth-inhibitory effects of MEK inhibitors onKi-ras/NRK and v-src/NRK cells in polyHEMA-coated and normaltissue culture plastic plates. PD98059 inhibited growth of Ki-ras/NRKcells comparably in polyHEMA-coated and plastic plates (Fig. 1A,middle panel). In contrast, U0126 clearly suppressed the growth ofKi-ras/NRK cells on the nonadhesive polyHEMA surface at concen-trations lower than on plastic (Fig. 1A,top panel). IC50 was 2mM onpolyHEMA, compared with 8mM on plastic. Against the v-src/NRKcells, on the other hand, neither PD98059 nor U0126 showed prefer-ence for polyHEMA to tissue culture plastic (Fig. 1B,top andmiddlepanels).

Treatment of Ki-ras/NRK cells with U0126 caused apparent mor-phological reversion to a flat, normal appearance (Fig. 2A; 24-htreatment). Although not as clear, U0126 also flattened morphology ofv-src/NRK cells (Fig. 2B; 48-h treatment). PD98059 induced a slightmorphological change in Ki-ras/NRK cells at high concentrations(Fig. 2A), but the morphology of v-src/NRK cells did not change (Fig.2B).

U0126 Inhibits Both MAPK and p70S6K Pathways. The aboveresults indicate that the two MEK inhibitors U0126 and PD98059differ in ability to block transformation by Ki-ras. Because the selec-tivity of PD98059 has been well tested (8), we speculated that U0126inhibits other pathway(s) in addition to the MAPK pathway.

To dissect the issue, we monitored the effects of the two com-pounds on activation of three serine/threonine kinases that are down-stream targets of Ras (9). Cell lysates from Ki-ras/NRK cells treatedfor 24 h were examined by immunoblotting with phosphospecificantibodies that recognize the activated form of MAPK, p70S6K or Akt.As shown in Fig. 3A, both U0126 and PD98059 inhibited activation ofMAPK, U0126 being more potent as reported (5). Neither compounddisplayed any effect on phosphorylation of Akt. PD98059 at 25mM

only slightly inhibited activation of p70S6K. On the other hand,reduction of p70S6K phosphorylation by U0126 was much moreapparent. Although U0126 did show selectivity toward MAPK overp70S6K, it clearly blocked p70S6K activation at high concentrations.Reduction of p70S6K phosphorylation was further manifested by theincreased electrophoretic mobility of total p70S6K.

As shown above, anchorage-independent growth of v-src/NRKcells was not preferentially inhibited by U0126. However, U0126 alsoinhibited activation of MAPK and p70S6K of the v-srctransformant,although p70S6K was less attenuated than in Ki-ras/NRK (Fig. 3B).Phosphotyrosine content of v-src/NRK cells did not change (data notshown).

PD98059 plus Rapamycin Blocks Transformation by Ki-ras.The above results raised a possibility that U0126 normalizes Ki-ras-mediated transformation by blocking both MAPK and p70S6K path-ways. The p70S6K pathway is selectively blocked by the immunosup-pressant rapamycin (7, 10). Activation of p70S6K is mediated by thephosphatidylinositol kinase-related kinase mTOR/FK506-bindingprotein rapamycin-associated protein (11). Rapamycin inhibits mTORand hence p70S6K by forming a stable complex with FK506-bindingprotein 12, which binds to mTOR (12). We tested whether simulta-neous blockade of MAPK and p70S6K pathways by combination ofPD98059 and rapamycin would result in similar consequences.

Rapamycin alone did not selectively inhibit anchorage-independentgrowth (data not shown) or alter morphology (Fig. 2A) of Ki-ras/NRKcells. As expected, rapamycin completely blocked activation ofp70S6K in Ki-ras/NRK cells without any effect on MAPK or Akt, and

the combination of PD98059 and rapamycin reduced phosphorylationof both MAPK and p70S6K (Fig. 3A).

We examined the effect of PD98059 plus rapamycin on anchorage-independent growth and morphology of Ki-ras/NRK cells. At 25 nM,rapamycin by itself reduced the growth of Ki-ras/NRK cells to57.8 6 3.1 and 64.86 4.8% of control in plastic plates and poly-HEMA plates, respectively. As shown above, PD98059 did not showa marked effect on anchorage independence. However, whenPD98059 was combined with 25 nM rapamycin, anchorage-indepen-dent growth on polyHEMA was noticeably inhibited over anchorage-dependent growth on plastic (Fig. 1A,bottom panel). Ki-ras/NRKcells treated with PD98059 plus rapamycin appeared morphologicallynormal, similar to those treated with U0126 (Fig. 2A).

PD98059 plus rapamycin also inhibited MAPK and p70S6K inv-src/NRK (Fig. 3B). Cells treated with the combination were flatterthan cells treated with U0126, which probably reflects the more potentinhibition of p70S6K phosphorylation. Despite the clear morphologicalalterations, anchorage-independent and -dependent growth was stillequally inhibited (Fig. 1B,bottom panel).

U0126 Is Effective against Otherras-transformed Cells. Wenext examined whether U0126 could inhibit anchorage-independentgrowth of otherras-transformed fibroblasts. Results of two represent-ative cell lines, Ki-ras/3Y1 and Ha-ras/3Y1, are shown in Fig. 4.U0126 suppressed anchorage-independent growth (Fig. 4) and flat-tened morphology (data not shown) of these two cell lines as well asof pMAM-Ki-ras, another Ki-rastransformed fibroblast tested (datanot shown). On the contrary, PD98059 was ineffective in normalizingtransformed phenotypes of Ki-ras/3Y1 or pMAM-Ki-ras(Fig. 4; datanot shown). The results further support the hypothesis that normal-ization of Ki-ras-induced transformation requires inhibition of both

Fig. 1. Effects of U0126, PD98059, and PD98059 plus rapamycin (RPM, 25 nM) onanchorage-independent growth in polyHEMA-coated (F) or anchorage-dependent growthin uncoated (E) 96-well plates.A, Ki-ras/NRK cells. B, src/NRK cells. Each plotrepresents mean6 SD of quadruplicate wells.

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MAPK and p70S6K pathways. PD98059, however, inhibited anchor-age-independent growth (Fig. 4) and induced morphological reversion(data not shown) of Ha-ras/3Y1 cells.

Discussion

We have shown here that the MEK inhibitor U0126 normalizesmorphology and inhibits anchorage-independent growth of Ki-ras-transformed rat fibroblasts. U0126 has been introduced as a potent andselective inhibitor of the MAPK pathway (5). However, our immu-noblot analyses with phosphospecific antibodies suggested thatU0126 also blocks the p70S6K pathway, and the reversion of trans-formation appeared to coincide with concurrent inhibition of MAPKand p70S6K activation. The MEK inhibitor PD98059 did not reverse

Ki-ras-induced transformation by itself but essentially restored thenormal phenotype in conjunction with the p70S6K pathway inhibitorrapamycin. We conclude that simultaneous inhibition of the MAPKand p70S6K pathways is necessary and sufficient to block transforma-tion induced by Ki-ras.

Although our results demonstrate that U0126 is more effective inblocking the MAPK pathway than the p70S6K pathway, it is obviousthat data obtained from experiments that rely on U0126 as a MEKinhibitor must be cautiously interpreted. The mechanism by whichU0126 inhibits p70S6K activation is not clear at present. PD98059 wasless potent than U0126 in blocking the MAPK pathway but appearedto be more selective, as far as p70S6K is concerned.

Penuel and Martin (13) recently reported that simultaneous inhibi-tion of MAPK and p70S6K pathways blocked transformation by v-src.PD98059 plus rapamycin reduced various parameters of transforma-tion, including morphology and anchorage-independent growth in softagar. However, their soft agar colony formation assay was not done inparallel with colony formation on a solid support. In our experiments,reversal of v-srcmediated transformation was partial;i.e., inhibitionof MAPK and p70S6K pathways flattened morphology to some extentbut equally suppressed anchorage-dependent and -independentgrowth. The possibility that this is due to incomplete blockade of thetwo pathways remains, but we have thus far been unable to selectivelyreduce anchorage-independent growth with increased concentrationsof the inhibitors and have obtained similar results with another v-srctransformant. Intervention of other pathway(s), such as Stat3 signaling(14, 15), might be required to antagonize transformation by v-srccompletely.

In contrast, U0126 or PD98059 plus rapamycin was effectiveagainst all fourras transformants we tested (Fig. 4; data not shown).In the case of a Ha-rastransformant, inhibition of the MAPK pathwayalone appeared to normalize transformation. U0126 also blockedanchorage-independent growth of several human cancer cell lines.Effects of U0126 on human cancer cell lines and cells transformed byother oncogenes will be reported elsewhere.

Activating mutations of theras genes are among the frequentlyfound abnormalities in human cancers (16). MAPK is thought to be

Fig. 2. Morphologies of Ki-ras/NRK (A) and v-src/NRK (B) cells treated with rapamycin (RPM, 25 nM), U0126, PD98059, or PD98059 plus rapamycin (25 nM). Concentrationsof U0126 and PD98059 are as indicated.

Fig. 3. Phosphorylation level of MAPK, p70S6K, and Akt of Ki-ras/NRK and v-src/NRK cells treated with U0126, PD98059, rapamycin (RPM), or PD98059 plus rapamycin.Cells were treated as described in “Materials and Methods” and analyzed by immuno-blotting. Rapamycin was used at 25 nM. pMAPK, phosphorylated MAPK;pp70S6K,phosphorylated p70S6K; pAkt, phosphorylated Akt.

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the key component inras-mediated transformation (9) and is elevatedin various human tumors (17, 18). Consequently, the MAPK cascadehas been the focus of special attention as a target for a new generationof noncytotoxic antitumor drugs. Recently, a highly potent and selec-tive MEK inhibitor was shown to suppress colon tumor growthin vivowithout noticeable side effects, substantiating the concept of theMAPK cascade targeting as a mechanism-based cancer therapy (19).

In Ki-ras transformed cells, however, inhibition of the MAPKpathway was not sufficient to repress disordered growth but requiredadditional inhibition of the p70S6K pathway. Given the fact thatuncontrolled growth of cancer cells involves aberrations of severalmechanisms, the requirement of intervention of multiple downstreampathways in many other tumors can be envisaged. Because completeblockade of all elevated growth-promoting machineries may be harm-ful to normal cell activity, it would be important to identify theminimum subset of signaling pathways required to control oncogenicgrowth. Our results suggest that absolute inhibition of Ras functionmay not be necessary to remedy abnormal growth properties. Block-ing of all Ras function by Ras antagonists such as farnesyltransferaseinhibitors (20) might be overcorrection. Knowledge of necessary andsufficient pathways for reestablishment of regulated growth wouldopen a way to more selective, noncytotoxic signal transduction-basedchemotherapy. In cases in which blockade of a single pathway by a

single agent is ineffectual, targeting of multiple, but a minimal num-ber, of pathways by combinations of selective pharmacological agentscould be a competent strategy for regulation of cancers.

Acknowledgments

We thank Drs. S. Mizuno and Y. Murakami for helpful discussions and Y.Zakabi for technical assistance.

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Fig. 4. Effects of U0126 and PD98059 on anchorage-independent growth in poly-HEMA-coated (Fandf) or anchorage-dependent growth in uncoated (EandM) 96-wellplates.F andE, Ki-ras/3Y1 cells;f andM, Ha-ras/NRK cells.

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2000;60:2104-2107. Cancer Res   Hidesuke Fukazawa and Yoshimasa Uehara  Pathways Both Mitogen-activated Protein Kinase and p70 S6 Kinase

-mediated Transformation by BlockingrasU0126 Reverses Ki-

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