R-Ras promotes metastasis of cervical cancer epithelial cells

Cancer Immunol Immunother (2007) 56:535–544
DOI 10.1007/s00262-006-0205-z
ORIGINAL ARTICLE

R-Ras promotes metastasis of cervical cancer epithelial cells

Nancy Mora · Ricardo Rosales · Carlos Rosales

Received: 29 May 2006 / Accepted: 22 June 2006 / Published online: 22 July 2006© Springer-Verlag 2006

Abstract Mutations in the small GTPase R-Ras thatpromote constitutive activation of this signaling mole-cule have been observed in a variety of invasive cancercell types. We previously reported that expression ofan oncogenic form of R-Ras (R-Ras87L) in a cell lineof cervical cancer (C33A cells) augments cell growthin vitro and tumorigenicity in vivo. Because increasedtumorigenicity in vivo often precedes metastasis, wenow examined whether the expression of R-Ras87Lalso increased the metastatic potential of C33A cells.Accelerated tumor growth was observed in athymicmice after subcutaneous injection of R-Ras87L-expressing C33A cells. In addition, increased metas-tasis to the liver, in immunodeWcient SCID mice, wasobserved after intravenous injection of R-Ras87L-expressing C33A cells. Also, R-Ras87L-expressingcells presented decreased membrane expression ofMHC class I molecules, and �1 integrins, but increasedlevels of PI 3-K and Akt activities. C33A cells express-ing R-Ras87L also migrated more over collagen I inwound assays. Inhibition of the PI 3-K/Akt/mTORpathway by pharmacological means blocked R-Ras87L-induced accelerated growth and migration overcollagen I. These results suggest oncogenic R-Ras has a

central role in cancer progression towards a metastaticphenotype, through the activation of the PI 3-K/Akt/mTOR signaling pathway.

Keywords Oncogene · Migration · PI 3-K · Akt

Introduction

Cervical carcinoma is the second most common malig-nant disease among women worldwide, accounting for15% of all deaths from malignant disease [35, 40]. Thehighest incidence rates are observed in parts of Africa,Southeast Asia, and Latin America [29]. Over 95% ofall cervical carcinomas contain DNA of some humanpapillomavirus [20, 21, 35]. Cervical tumors usually areinvasive and cause many deaths each year, close to2,000 in the United Kingdom [41] and 12,000 in Mexico[40]. Thus there is a lot of interest in understanding theadhesion and migration properties of epithelial tumorcells in an eVort to control the most invasive and malig-nant forms of this type of carcinoma.

The oncogene R-Ras, a member of the super familyof small GTPases, has been implicated in several cellfunctions [36]. The R-Ras protein is 55% identical toH-Ras and has an extension of 26 amino acids in itsamino terminal end [17]. R-Ras reportedly inXuencesintegrin activation by both direct [44] and indirectmechanisms [37]. Oncogenic R-Ras induced cell trans-formation in Wbroblasts [3, 25] but not in other celltypes [36]. This diVerence is due to the fact that distinctmolecular activators from those that control H-Ras[13] regulate R-Ras. Activated R-Ras also wasreported to promote migration and invasion in vitro,of breast epithelial cells [16]. Moreover, the eVects of

N. Mora · C. Rosales (&)Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Apdo. Postal 70228, Cd. Universitaria, México City 04510, Mexicoe-mail: [email protected]

R. RosalesMolecular Biology and Biotechnology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico

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536 Cancer Immunol Immunother (2007) 56:535–544

R-Ras on cell migration seem to involve changes in thecytosqueleton through activation of the GTPases Rhoand Rac [10, 14, 42]. We previously reported that cervi-cal epithelial cells expressing a constitutively activeform of R-Ras grew, as tumors in nude mice, fasterthan control cells [32]. These cells also presented amarked increase in cell spreading and migrated moreover collagen I-coated Wlters [32].

Because increased tumorigenicity in vivo often pre-cedes metastasis, we now examined whether theexpression of an active form of R-Ras also increasedthe metastatic potential of cervical epithelial cells. Wefound increased metastasis to the liver in immunodeW-cient SCID mice, injected intravenously with cervicalepithelial cells expressing active R-Ras. Also, thesecells presented decreased membrane expression ofMHC class I molecules, and �1 integrins, and increasedlevels of phosphatidylinositol 3-kinase (PI 3-K) andAkt activities; all markers of a more malignant pheno-type. In addition, pharmacological inhibition of thePI 3-K/Akt/mTOR pathway blocked active R-Ras-induced accelerated cell growth and migration overcollagen I. These data suggest that oncogenic R-Rashas a central role in cancer progression towards ametastatic phenotype, through activation of the PI3-K-Akt-mTOR signaling pathway.

Materials and methods

Cell culture

The C33A cervical carcinoma epithelial cell line (cata-log no. HTB-31; American Type Culture Collection;Manassas, VA, USA) was grown in Dulbecco modiWedEagle medium (DMEM; Gibco BRL, Grand Island,NY, USA) supplemented with 10 % heat-inactivatedfetal bovine serum (FBS; Gibco BRL) and 20 �M glu-tamine.

Plasmids and reagents

The following antibodies were used: monoclonal anti-body (mAb) TS2/16, anti-�1 integrin (donated byDr. Martin Hemler; Dana Farber Cancer ResearchInstitute, Boston, MA, USA); mAb IB4, anti-�2 integrin(a gift from Dr. Eric J. Brown; University of California,San Francisco, CA, USA); mAb W6/32, anti-majorhistocompatibility complex (MHC) class I (fromAmerican Type Culture Collection). mAb AP3, anti-�3integrin; mAb R6G9, anti-�6 integrin; mAb P1E6, anti-�2 integrin; mAb P1B5, anti-�3 integrin; mAb P4G9,anti-�4 integrin; mAb P1D6, anti-�5 integrin; and mAb

VNR147, anti-�v integrin, were from Gibco, BRL(Grand Island). Anti-PI 3-K p110� rabbit polyclonalIgG (catalog no. sc-7189) was from Santa Cruz Bio-technology (Santa Cruz, CA, USA). Rabbit polyclonalanti-Akt (catalog no. 9272) and rabbit polyclonal antiphospho-Akt (catalog no. 4058) were from Cell Signal-ing Technology (Beverly, MA, USA). FITC-conju-gated F(ab’)2 goat anti-mouse IgG (catalog no. 55522),and F(ab’)2 goat anti-rabbit IgG (catalog no. 55665)were from ICN-Cappel (Aurora, OH, USA). Plasmidsthat direct the synthesis of normal (wild-type) or mutantforms of R-Ras were a generous gift from Dr. AdrienneD. Cox (University of North Carolina, Chapel Hill,NC, USA) and have been previously described [3, 13,16]. The constitutively active PI 3-K p110 K227E [33]was a generous gift from Dr. Julian Downward (TheImperial Cancer Research Fund, London, UK). Colla-gen I was donated by Dr. Jesus Chimal (Instituto deInvestigaciones Biomédicas, UNAM). Fibronectin wasfrom Roche Molecular Biochemicals (Indianapolis, IN,USA). The speciWc PI 3-K inhibitor LY294002 wasobtained from Calbiochem (San Diego, CA, USA). Allother chemicals were from Sigma Chemical Company(St. Louis, MO, USA).

Transfection

Cells were transfected with the calcium phosphate-DNA coprecipitation method using 10 �g of plasmidfor each 60-mm culture dish and individual clonesselected in the presence of G418 as described [32].

PI 3-K activity assay

PI 3-K activity was determined by in vitro kinase assaysas previously described [31].

Tumor growth in nude mice

R-Ras-, PI 3-K p110-, or empty vector-transfectedC33A cells (5 £ 106 cells) were injected subcutaneouslyinto athymic mice (Harlam, Mexico City, Mexico). Fivemice were used for each cell type. Tumor growth wasevaluated by measuring tumor size in two dimensionswith a calibrated caliper every week.

In vivo metastasis assay

A total of 10 £ 106 C33A cells expressing one of thediVerent forms of R-Ras, or the active PI 3-K p110were injected into the tail vein of 6-week old femaleFox Chase C.B-17-scid mice (Taconic; Hudson, NY,USA). Three mice were injected with each cell type.

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Cancer Immunol Immunother (2007) 56:535–544 537

After 42 days, mice were sacriWced, dissected, and ana-lyzed by gross examination. The livers were excisedand placed in saline solution. Metastatic foci werecounted at the liver surface under a dissecting micro-scope.

Flow cytometry

Staining of cells with various antibodies for Xow cytom-etry analysis was done exactly as previously described[30].

Cell migration/wound assay

Six-well tissue culture plaques (Costar; Corning, NY,USA) were coated with a 10 �g/ml solution of eithercollagen I or Wbronectin, and incubated for 8 h at 4°C.Wells were then washed and one million cells wereseeded into each well. After cells have reached conXu-ency, the cell monolayer was “wounded” by scraping itwith a 200 �l pipette tip, and covered with fresh serum-supplemented DMEM medium. Cell migration overthe monolayer wound was analyzed every 12 h. Inselected experiments, cells were treated with 25 �MLY294002, or with 5 nM rapamycin in serum-freeDMEM for 2 h before wound inXiction.

Western blotting

PI 3-K, Akt, and phospho Akt were detected by immu-noblotting with the corresponding antibody: anti-PI 3-Kat 0.05 �g/ml, anti-Akt at 1/1,000 dilution, and anti-phospho-Akt at 1/1,000 dilution, exactly as previouslydescribed [7, 30].

Statistical analysis

Data were compared with an unpaired Student t andANOVA tests using the computer program Kaleida-Graph version 3.6.2 (Synergy Software; Reading, PA,USA). DiVerences were considered statistically signiW-cant when P values · 0.01.

Results

R-Ras promoted accelerated in vitro growth of cervical epithelial cells via PI 3-K

We have previously shown that the expression of anoncogenic form of R-Ras (R-Ras87L) in a cervical epi-thelial cell line (C33A cells) promotes accelerated cellgrowth in vitro and increased tumorigenicity in vivo [32].

It has also been shown for various cell types that PI 3-Kcan act as a down-stream eVector of R-Ras [18, 36],controlling cellular functions such as cell survival,migration, and adhesion. We thus explored the possi-bility that PI 3-K was acting as an R-Ras eVector forincreased tumorigenicity. C33A cells were transfectedwith a constitutively active form of PI 3-K (p110) [33],and then selected for stable expression of this enzyme.Similarly to expression of R-Ras87L in C33A cells,expression of constitutively active PI 3-K resulted inaccelerated cell growth in vitro (Fig. 1a). Pharmacolog-ical inhibition of PI 3-K with the drug LY294002blocked accelerated growth both in R-Ras87L- and PI3-K p110-expressing cells (Fig. 1a). This suggested thatin these cells PI 3-K is indeed a downstream eVector ofoncogenic R-Ras. To conWrm this idea we directlymeasured the activity of PI 3-K in R-Ras87L-express-ing cells. A nearly tenfold increase in PI 3-K activitywas observed in C33A cells expressing R-Ras87L, overC33A control cells (Fig. 1b). As expected, an increasein PI 3-K activity was observed in C33A cells express-ing PI 3-K p110 (Fig. 1b). Taken together these resultsindicate that accelerated in vitro cell growth, inducedby the expression of oncogenic R-Ras, involves a PI 3-K-dependent mechanism.

Expression of oncogenic R-Ras promoted accelerated tumor growth in vivo

To determine whether PI 3-K was also involved in R-Ras87L-mediated accelerated tumor growth in vivo,we injected subcutaneously R-Ras87L- and PI 3-Kp110-expressing C33A cells into athymic mice. All ani-mals developed subcutaneous solid tumors (Fig. 2).Tumors in mice injected with R-Ras87L- and PI 3-Kp110-expressing C33A cells, grew at a faster rate thanthose tumors formed by untransfected C33A cells orcells expressing the dominant negative form of R-Ras,R-Ras43N (Fig. 2). These results thus indicated thatexpression of oncogenic R-Ras could also promoteincreased tumor growth in vivo through a mechanisminvolving PI 3-K.

Expression of oncogenic R-Ras promoted metastasis of C33A cells

The observation that expression of oncogenic R-Ras inC33A cells promoted increased tumorigenicity in vivoraised the question as to whether oncogenic R-Rascould also promote metastasis. To explore this ideawe directly evaluated the metastasis-promoting poten-tial of R-Ras87L by injecting C33A cells expressingthis form of R-Ras into the tail vein of SCID mice.

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Forty-two days after injection, mice were sacriWcedand dissected to look for tumor formation. Severalorgans, including spleen, kidney, heart, stomach, intes-tines, lungs, muscle, and brain were free of tumors(data not shown). In contrast, liver of SCID miceinjected with R-Ras87L-expressing C33A cells wereenlarged and presented extensive metastatic nodules(Fig. 3a). Similarly, intravenous injection of PI 3-Kp110- expressing C33A cells resulted in altered livermorphology and extensive nodule formation (Fig. 3b).In contrast, intravenous injection of dominant negativeR-Ras43N-expressing C33A cells (Fig. 3c) or empty-vector-expressing C33A cells (Fig. 3d) resulted inalmost normal liver morphology with few metastaticnodules. Liver metastasis was quantiWed by countingthe number of nodules in various liver preparations(Fig. 3e). These results thus indicate that expression ofoncogenic R-Ras in C33A cells promotes a more meta-static phenotype, and also suggest that a PI 3-K-depen-dent mechanism is involved.

Expression of oncogenic R-Ras promoted an altered expression pattern of membrane receptors

Altered integrin expression patterns have been found invarious types of cancer cells [15, 26] and these changesare often related to a more metastatic phenotype [5, 11,19]. We thus explored the possibility that expression ofR-Ras87L could alter the pattern of integrin expressionin C33A cells. Expression of the �1 intregrin subunit, amolecule with a major role in cell attachment to theextracellular matrix, was considerably reduced in cellsexpressing R-Ras87L or PI 3-K p110 (Table 1), but notin R-Ras43N-expressing cells. In addition, R-Ras87L-and PI 3-K p110-expressing cells also had a signiWcantreduction in membrane expression of MHC class I mole-cules (Table 1). Expression of integrin �2, �3, and �6,but not �5 subunits was also reduced in cells expressingR-Ras87L, and PI 3-K p110 (Table 1). Our resultsstrongly suggest that expression of oncogenic R-Rasinduces alterations in the expression pattern of

Fig. 1 R-Ras promotes accelerated in vitro growth of cervicalepithelial cells via PI 3-K. a 2 £ 104 vector-transfected C33A cells(open circles), activated R-Ras87L-transfected C33A cells (solidcircles), dominant negative R-Ras43N-transfected C33A cells(open triangles), or activated PI 3-K p110-transfected C33A cells(closed triangles), were plated in wells of a six-well plate and cul-tured for 1 week. Cells from each well were trypsinized, resus-pended, and counted every day. Some cells were cultured in the

presence of 25 �M LY294002 (dashed lines). Data are from one offour diVerent experiments that yielded similar results. b PI 3-Kwas immunoprecipitated from cell lysates of 1 £ 107 vector-trans-fected C33A cells (vector), activated R-Ras-transfected C33Acells (R-Ras87L), or activated PI 3-K p110-transfected C33A cells(PI 3-K p110). PI 3-K was then measured by an in vitro kinaseassay. Phosphatidylinositol-3-phosphate (PI3P). Data are repre-sentative of two separate experiments

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membrane receptors that contribute to an increasedmalignant phenotype of cervical epithelial cells.

Expression of oncogenic R-Ras promoted increased cell migration over collagen I

Metastatic cells often show increased migratorycapacity over a variety of substrates. Expression ofR-Ras87L resulted in elevated cell migration over col-lagen I in wound assays, compared to untransfectedC33A cells and cells expressing R-Ras43N (Fig. 4).Similarly, expression of PI 3-K p110 in C33A cells alsoresulted in increased cell migration over collagen I(Fig. 4). Consistent, with a PI 3-K-dependent mecha-nism involved in R-Ras87L-induced metastasis, thisincrease in migration was blocked by the PI 3-K inhibi-tor LY294002 (Fig. 5). In contrast, cell migration overWbronectin was not observed in untransfected cells, norin C33A cells transfected with either R-Ras87L, R-Ras43N, or PI 3-K p110 (data not shown).

Expression of oncogenic R-Ras promotes increased cell motility through activation of Akt/mTOR

PI 3-K-dependent activation of Akt and of its down-stream eVector mammalian Target of Rapamycin

(mTOR), has been reported to be of importance in theregulation of cell migration in various systems [4, 6,39]. Western-blotting experiments showed that, com-pared to untransfected C33A cells, cells expressingeither R-Ras87L or PI 3-K p110 had higher levels ofactive (phosphorylated) Akt (data not shown). Todirectly evaluate the participation of mTOR in R-Ras-dependent migration of C33A cells, we repeated thewound assays in the presence of the mTOR inhibitorrapamycin. Migration of R-Ras87L- or PI 3-K p110-expressing cells over collagen I was indeed blocked byrapamycin (Fig. 6). In contrast, rapamicyn had noeVect on the slow migration of untransfected C33Acells, or cells transfected with R-Ras43N (data notshown). These results suggested that induction of amore metastatic phenotype by oncogenic R-Ras mightbe related, at least in part, to increased cell migrationthrough activation of the PI-3K/Akt/mTOR pathway.

Discussion

In this report, we have investigated the ability of theoncogene R-Ras to induce a more metastatic pheno-type in human cervical epithelium cells. We found thatthe expression of an active mutant of R-Ras (R-Ras87L) resulted in faster tumor growth in nude mice,and in augmented metastasis to the liver in SCID mice.In addition, cells that expressed the active oncogene R-Ras87L had an important reduction of integrin andmajor histocompatibility complex (MHC) class I mole-cules on their membrane. R-Ras87L-expressing cellsalso presented increased cell migration over collagen I.The increase in cell migration was dependent on PI 3-Kand mTOR. Moreover, the activity of PI 3-K and Aktwas augmented in cells that expressed R-Ras87L.These results indicate that the oncogene R-Rasdepends on PI 3-K, Akt, and mTOR to induce cellmigration and tumor metastasis of cervical epithelialcells.

R-Ras reportedly induces cell transformation inWbroblasts [3, 34] and induces a more invasive pheno-type in breast epithelial cells [16]. NIH-3T3 cells thatexpress an active R-Ras could form foci, although theydid so much less eYciently than cells expressing thebetter-studied oncogne H-Ras. R-Ras-expressing Wbro-blasts [3, 25] also grew in nude mice and had elevatedlevels of ERK kinases [3]. These data suggested that R-Ras, like H-Ras, plays an important role in cell growthcontrol. We found that active R-Ras87L-expressingC33A cells grew faster than control cells or dominantnegative R-Ras43N-expressing cells, in vitro and innude mice. The mechanism whereby R-Ras induces

Fig. 2 Activated R-Ras increased tumor growth of cervical epi-thelial cells in athymic mice. 5 £ 106 vector-transfected C33Acells (open circles), activated R-Ras87L-transfected C33A cells(solid circles), dominant negative R-Ras43N-transfected C33Acells (open triangles), or activated PI 3-K p110-transfected C33Acells (closed triangles), were injected subcutaneously into athymicmice. Tumor size was measured every week in two dimensionswith a calibrated caliper. Data are the mean § standard error val-ues from Wve animals for each cell type

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cell cycle progression and a more malignant phenotyperemains unknown. However, it does not seem toinvolve ERK kinases, because C33A cells thatexpressed active R-Ras87L did not have increasedERK activity [32], although ERK could be easily acti-vated by phorbol esters in these cells [32]. Othergroups also have not been able to detect R-Ras-induced ERK activation [16, 18, 36].

Although, most cervical carcinomas contain DNA ofsome human papillomavirus (HPV) [20, 21, 35], we

used the C33A cervical carcinoma epithelial cell lineprecisely because it does not have HPV. This choicewas made to look at the eVects of R-Ras on a cervicalepithelial cell without interference from the eVects ofviral oncoproteins. We found that oncogenic R-Ras87L indeed induced cell cycle progression and amore malignant phenotype in cervical epithelialcells. However, there is not information on the level ofR-Ras activity in HPV-infected cells. R-Ras has beenfound altered in other types of tumors. For example,

Fig. 3 Activated R-Ras in-creased metastatic potential of cervical epithelial cells. Liv-ers of female Fox Chase C.B-17-scid mice 42 days after i.v. injection of 10 £ 106 C33A cells transfected with a R-Ras87L, b PI 3-K p110, c R-Ras43N, or d empty vec-tor. e Number of metastatic foci at the liver surface. Data shown are the mean § stan-dard error values from three animals for each cell type. DiVerences were statistically signiWcant (*) at P · 0.003 in ANOVA test

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elevated levels of R-Ras are reported to be suYcientfor inducing estrogen-independent proliferation ofbreast cells [43] and the progression of breast cancercells to tamoxifen resistance [9]. Also, upregulation ofR-Ras was found in transformed colorectal crypt cells[45] and in gastric cancers [23]. In addition, functionalblocking of R-Ras in these cells resulted in the disap-pearance of adhered cells, conWrming the role of R-Ras

in clinal gastric tumors [23]. Moreover, R-Ras expres-sion and phosphorylation correlated with increasinggrade of gliomas in human brain tumor specimens [22].These reports show that R-Ras has a relevant role intumor progression of varios types of cancer. Our datashow that activated (oncogenic) R-Ras can also inducea malignant phenotype in human cervical cells. Itwould be now very interesting to determine the statusof R-Ras, either overexpression or mutants, in clinicalHPV-positive cervical tumors to determine a possibleconnection between R-Ras and HPV oncoproteins E6and E7.

Early studies suggested that R-Ras could increasethe migration and invasion potential of breast epithe-lial cells [16]. Later, we found that R-Ras could alsoincrease migration in vitro of cervical epithelial cells[32]. More recently several reports have indicated thatR-Ras is an important regulator of cell migration inother cell types [10, 12, 24, 42]. These reports underlinethe fact that R-Ras is an important regulator of themigratory capacity of many cell types. Now, we alsoreport here that active R-Ras87L indeed promotes amore migratory and metastatic phenotype in cervicalepithelial cells. The mechanism by which R-Rasinduces augmented cell migration is complex andremains obscure. The eVects of R-Ras are clearly

Table 1 Integrin and MHC class I expression on C33A cellstransfected with R-Ras or PI 3-K

Mean Xuorescence intensity. Average of two independent mea-surements

– Molecule not expressed on cell membrane

Molecule Empty-vector R-Ras43N R-Ras87L PI 3-Kp110

�2 61.3 60.6 40.9 28.3�3 62.2 40.4 19.9 15.8�4 – – – –�5 60.3 50.1 58.7 57.5�6 58.3 71.2 25.9 21.8�v – – – –�1 74.8 56.7 25.5 19.3�2 – – – –�3 – – – –�4 – – – –�6 – – – –MHC-I 280.6 223.7 96.1 55.8

Fig. 4 Activated R-Ras and activated PI 3-K increased migrationof cervical epithelial cells on collagen I. Monolayers of C33Acells transfected with empty plasmid (vector), dominant negativeR-Ras (R-Ras43N), activated R-Ras (R-Ras87L), or activatedPI 3-K (PI 3-K p110), were grown on collagen I-covered six-

well plates, and scratched (wounded) with a pipette tip (0 h).Cells were then covered with fresh medium and monolayerswere allowed to recover. Wound area was photographed 24 and48 h later. Data are from one of three diVerent experiments thatyielded similar results

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multifactorial involving adhesion molecules such asintegrins [11], signaling molecules such as PI 3-K [36],and GTPases to regulate the cytoskeleton [10, 42].

Expression of the active form R-Ras87L resulted inprofound changes in integrin surface expression. �1 inte-grins were reduced on the cell membrane in about 2/3.This reduction may correspond to the integrins formedwith the �2, �3, and �6 chains, as their surface expres-sion was also reduced (Table 1). Interestingly, theexpression level of �5 integrins did not change. Becausethe �5�1 integrins bind mainly Wbronectin, and �2�1 and�3�1 integrins bind mainly collagen, reduction of the lat-ter integrins may explain in part the increased migrationR-Ras87L-expressing cells had over collagen I, and thetotal absence of migration over Wbronectin. It is likelythat less collagen receptors results in weaker cell attach-ment over this matrix protein, and leads to increased cellmigration. In addition, R-Ras87L-expressing cells pre-sented a signiWcant reduction in major histocompatibilitycomplex (MHC) class I molecules on their membranes(Table 1). Reduction of integrin expression is associatedwith a more migratory phenotype [11, 15], and is also aprerequisite for metastasis [15, 26, 28]. Reduction ofMHC class I molecules often correlates with impaired

recognition and killing by natural killer cells [8, 38].Thus, R-Ras contributes to cancer development byaltering expression of MHC class I molecules and inte-grins on cervical epithelial cells.

Elucidating the signaling molecules downstreamfrom R-Ras is an active line of research. Several groupshave indicated that R-Ras functions through PI 3-K.[1, 2, 16, 18, 25, 32]. Expression of R-Ras87L caused astrong constitutive activation of PI 3-K in C33A cells(Fig. 1 and [32]). This enzymatic activity seems to beresponsible for the eVect of R-Ras87L on cell prolifera-tion and migration, because treatment with the PI 3-Kinhibitor LY294002 prevented cell growth in cultureand inhibited cell migration over collagen I. Moreover,C33A cells expressing a constitutively activated formof PI 3-K (p110) mimicked completely the phenotypeobserved in R-Ras87L-expressing C33A cells. In addi-tion, the kinase Akt was also activated in R-Ras87L-and PI 3-K p110-expressing C33A cells. Thus, R-Ras,via PI 3-K induces Akt, but not ERK activation.Because a major downstream target of Akt is mTOR,we reasoned that inhibition of mTOR would have asimilar eVect on cell migration as the inhibition of PI 3-K.Treatment with rapamycin, a speciWc inhibitor of

Fig. 5 Activated R-Ras increased migration of cervical epithelialcells via PI 3-K. Monolayers of C33A cells transfected with acti-vated R-Ras (R-Ras87L), or activated PI 3-K (PI 3-K p110), weregrown on collagen I-covered six-well plates, and scratched(wounded) with a pipette tip (0 h). Cells were then covered with

fresh medium and monolayers were allowed to recover. Woundarea was photographed 24 and 48 h later. Some cells were cul-tured in the presence of 25 �M LY294002. Data are from one ofthree diVerent experiments that yielded similar results

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mTOR, indeed blocked increased cell migration ofPI 3-K p110- and R-Ras87L-expressing C33A cells.Because mTOR regulates p70 ribosomal S6 kinaseleading to cell cycle progression [27], it is likely thatR-Ras87L-mediated increased tumorigenicity is alsorelated to mTOR activation. Taken together, theseresults suggest that induction of a more metastatic phe-notype by oncogenic R-Ras may be related, at least inpart, to increased cell migration through activation ofthe PI-3K/Akt/mTOR pathway. The mTOR pathwayhas also been implicated in increased proliferation ofmammary and [4] and prostate tumors [6].

In summary, the present report shows that onco-genic R-Ras has a central role in cancer progressionof cervical epithelial cells, towards a metastatic pheno-type, through activation of the PI 3-K-Akt-mTORsignaling pathway.

Acknowledgments We thank Dr. Adrienne D. Cox for R-Rasconstructs, Dr. Julian Downward for the active PI 3-K construct,and Dr. Martin Hemler, and Dr. Eric J. Brown for anti-integrinantibodies. We also thank Dr. Jesus Chimal for helping takingpictures of mouse livers, and Jose Alejandro Marmolejo Valenciafor technical assistance. This work was supported by grant 36407-Mfrom Consejo Nacional de Ciencia y Tecnología, Mexico, and bygrant IN220703 from DGAPA, Universidad Nacional Autónomade México, Mexico.

References

1. Berrier AL, Mastrangelo AM, Downward J, Ginsberg M,LaFlamme SE (2000) Activated R-Ras, Rac-1, PI 3-kinaseand PKCe can each restore cell spreading inhibited by iso-lated integrin b1 cytoplasmic domains. J Cell Biol 151:1549–1560

2. Cole AL, Subbanagounder G, Mukhopadhyay S, BerlinerJA, Vora DK (2003) Oxidized phospholipid-induced endo-thelial cell/monocyte interaction is mediated by a cAMP-dependent R-Ras/PI3-kinase pathway. Arterioscler ThrombVasc Biol 23:1384–1390

3. Cox AD, Brtva TR, Lowe DG, Der CJ (1994) R-Ras pro-motes malignant, but not morphologic, transformation ofNIH3T3 cells. Oncogene 9:3281–3288

4. Debnath J, Walker SJ, Brugge JS (2003) Akt activationdisrupts mammary acinar architecture and enhances prolif-eration in an mTOR-dependent manner. J Cell Biol163:315–326

5. Felding-Habermann B, O’Toole TE, Smith JW, Fransvea E,Ruggeri ZM, Ginsberg MH, Hughes PE, Pampori N, ShattilSJ, Saven A, Mueller BM (2001) Integrin activation controlsmetastasis in human breast cancer. Proc Natl Acad Sci USA98:1853–1888

6. Gao N, Zhang Z, Jiang B-H, Shi X (2003) Role of PI3K/AKT/mTOR signaling in the cell cycle progression of human pros-tate cancer. Biochem Biophys Res Commun 310:1124–1132

7. Garcia-Garcia E, Sanchez-Mejorada G, Rosales C (2001)Phosphatidylinositol 3-kinase and ERK are required forNF-kB activation, but not for phagocytosis. J Leukoc Biol70:649–658

Fig. 6 Activated R-Ras increased migration of cervical epithelialcells via mTOR. Monolayers of C33A cells transfected with acti-vated R-Ras (R-Ras87L), or activated PI 3-K (PI 3-K p110), weregrown on collagen I-covered six-well plates, and scratched(wounded) with a pipette tip (0 h). Cells were then covered with

fresh medium and monolayers were allowed to recover. Woundarea was photographed 24 and 48 h later. Some cells were cul-tured in the presence of 5 nM Rapamycin. Data are from one ofthree diVerent experiments that yielded similar results

123


8. Ghim SJ, Sundberg J, Delgado G, Jenson AB (2001) The path-ogenesis of advanced cervical cancer provides the basis for anempirical therapeutic vaccine. Exp Mol Pathol 71:181–185

9. Gotoh T, Cai D, Tian X, Feig LA, Lerner A (2000) p130Casregulates the activity of AND-34, a novel Ral, Rap1, andR-Ras guanine nucleotide exchange factor. J Biol Chem275:30118–30123

10. Holly SP, Larson MK, Parise LV (2005) The unique N-termi-nus of R-Ras is required for Rac activation and precise regu-lation of cell migration. Mol Biol Cell 16:2458–2469

11. Hood JD, Cheresh DA (2002) Role of integrins in cell inva-tion and migration. Nat Rev Cancer 2:91–100

12. Huang Y, Rangwala F, Fulkerson PC, Ling B, Reed E,Cox AD, Kamholz J, Ratner N (2004) Role of TC21/R-Ras2in enhanced migration of neuroWbromin-deWcient Schwanncells. Oncogene 23:368–378

13. HuV SY, Quilliam TR, Cox AD, Der CJ (1997) R-Ras isregulated by activators and eVectors distinct from those thatcontrol Ras function. Oncogene 14:133–143

14. Jeong H-W, Nam J-O, Kim I-S (2005) The COOH-terminalend of R-Ras alters the motility and morphology of breastepithelial cells through Rho/Rho-kinase. Cancer Res 65:507–515

15. Juliano RL, Varner JA (1993) Adhesion molecules in cancer:the role of integrins. Curr Opin Cell Biol 5:812–818

16. Keely PJ, Rusyn EV, Cox AD, Parise LV (1999) R-Rassignals through speciWc integrin alpha cytoplasmic domains topromote migration and invasion of breast epithelial cells.J Cell Biol 145:1077–1088

17. Lowe DG, Capon DJ, Delwart E, Sakaguchi AY, Naylor SL,Goeddel DV (1987) Structure of the human and murine R-rasgenes, novel genes closely related to ras proto-oncogenes.Cell 48:137–146

18. Marte BM, Rodriguez-Viciana P, Wennstrom S, Warne PH,Downward J (1996) R-Ras can activate the phosphoinositide3-kinase but not the MAP kinase arm of the Ras eVectorpathways. Curr Biol 7:63–70

19. Maschler S, Wirl G, Spring H, Bredow DV, Sordat I, Beug H,Reichmann E (2005) Tumor cell invasiveness correlates withchanges in integrin expression and localization. Oncogene24:2032–2041

20. Motoyama S, Ladines-Llave CA, Luis-Villanueva S, Maruo T(2004) The role of human papilloma virus in the molecularbiology of cervical carcinogenesis. Kobe J Med Sci 50:9–19

21. Münger K, Baldwin A, Edwards KM, Hayakawa H, NguyenCL, Owens M, Grace M, Huh K (2004) Mechanisms of humanpapillomavirus-induced oncogenesis. J Virol 78:11451–11460

22. Nakada M, Niska JA, Tran NL, McDonough WS, Berens ME(2005) EphB2/R-Ras signaling regulates glioma cell adhe-sion, growth, and invasion. Am J Pathol 167(2):565–576

23. Nishigaki M, Aoyagi K, Danjoh I, Fukaya M, Yanagihara K,Sakamoto H, Yoshida T, Sasaki H (2005) Discovery ofaberrant expression of R-RAS by cancer-linked DNA hy-pomethylation in gastric cancer using microarrays. CancerRes 65:2115–2124

24. Oinuma I, Ishikawa Y, Katoh H, Negishi M (2004) The Sem-aphorin 4D receptor Plexin-B1 is a GTPase activating proteinfor R-Ras. Science 305:862–965

25. Osada M, Tolkachera T, Li W, Chan TO, Tsichlis PN, Saez R,Kimmelman AC, Chan AM (1999) DiVerential roles of Akt,Rac, and Raf in R-Ras-mediated cellular transformationadhesion and survival. Mol Cell Biol 9:6333–6344

26. Parise LV, Weon J, Juliano RL (2000) New aspects ofintegrin signaling in cancer. Sem Cancer Biol 10:407–414

27. Pene F, Claessens YE, Muller O, Viguie F, Mayeux P,Dreyfus F, Lacombe C, Bouscary D (2002) Role of the

phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinasepathways in the proliferation and apoptosis in multiplemyeloma. Oncogene 21:6587–6597

28. Ponta H, Sleeman J, Herrlich P (1994) Tumor metastasisformation: cell-surface proteins confer metastasis-promotingor -suppressing properties. Biochim Biophys Acta 1198:1–10

29. Potter JD (1997) Food, nutrition and the prevention of can-cer: A global perspective. The American Institute for CancerResearch, Washington DC, p 670

30. Reyes-Reyes M, Mora N, Gonzalez G, Rosales C (2002) b1and b2 integrins activate diVerent signalling pathways inmonocytes. Biochem J 363:273–280

31. Reyes-Reyes M, Mora N, Zentella A, Rosales C (2001)Phosphatidylinositol 3-kinase mediates integrin-dependentNF-kB and MAPK activation through separate signalingpathways. J Cell Sci 114:1579–1589

32. Rincón-Arano H, Rosales R, Mora N, Rodríguez-CastañedaA, Rosales C (2003) R-Ras promotes tumor growth of cervi-cal epithelial cells. Cancer 97:575–585

33. Rodríguez Viciana P, Warne PH, Vanhaesebroeck B, Water-Weld MD, Downward J (1996) Activation of phosphoinositide3-kinase by interaction with Ras and by point mutation.EMBO J 15:2442–2451

34. Saez R, Chan AM-L, Miki T, Aaronson SA (1994) Oncogenicactivation of human R-Ras by point mutations analogous tothose of prototype H-Ras. Oncogene 9:2977–2982

35. Schorge JO, Knowles LM, Lea JS (2004) Adenocarcinoma ofthe cervix. Curr Treat Options Oncol 5:119–127

36. Self AJ, Caron E, Peterson HF, Hall A (2001) Analysis ofR-Ras signalling pathways. J Cell Sci 114:1357–1366

37. Sethi T, Ginsberg MH, Downward J, Hughes PE (1999) Thesmall GTP-binding protein R-Ras can inXuence integrinactivation by antagonizing a Ras/Raf-initiated intetgrin su-pression pathway. Mol Biol Cell 10:1799–1809

38. Sirianni MC, Libi F, Campagna M, Rossi D, Capello D, Scia-ranghella G, Carbone A, Simonelli C, Monini P, Gaidano G,Ensoli B (2005) Downregulation of the major histocompati-bility complex class I molecules by human herpesvirus type 8and impaired natural killer cell activity in primary eVusionlymphoma development. Br J Haematol 130:92–95

39. Sugatani T, Hruska KA (2005) Akt1/Akt2 and mammaliantarget of rapamycin/Bim play critical roles in osteoclast diVer-entiation and survival, respectively, whereas Akt is dispens-able for cell survival in isolated osteoclast precursors. J BiolChem 280:3583–3589

40. Torres Lobaton A, Rojo Herrera G, Torres Rojo A, HurtadoEstrada G, Roman Bassaure E (2004) Cervical cancer. Cur-rent view of its epidemiology and risk factors. Ginecol ObstetMex 72:466–474

41. Woodman CBJ, Collins S, Winter H, Bailey A, Ellis J, PriorP, Yates M, Rollason TP, Young LS (2001) Natural history ofcervical human papillomavirus infection in young women: alongitudinal cohort study. Lancet 357:1831–1836

42. Wozniak MA, Kwong L, Chodniewicz D, Klemke RL, KeelyPJ (2005) R-Ras controls membrane protrusion and cellmigration through the spatial regulation of Rac and Rho. MolBiol Cell 16:84–96

43. Yu Y, Feig LA (2002) Involvement of R-Ras and Ral GTPasesin estrogen-independent proliferation of breast cancer cells.Oncogene 21:7557–7568

44. Zhang Z, Vouri K, Wang HG, Reed JC, Rouslahti E (1996)Integrin activation by R-ras. Cell 85:61–69

45. Zhu Y, Zhong X, Zheng S, Ge Z, Du Q, Zhang S (2005)Transformation of immortalized colorectal crypt cells bymicrocystin involving constitutive activation of Akt andMAPK cascade. Carcinogenesis 26:1207–1214

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R-Ras promotes metastasis of cervical cancer epithelial cells