Elevated levels of ornithine decarboxylase cooperate with Raf/ERK activation to convert normal keratinocytes into invasive malignant cells

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1 ORIGINAL ARTICLE Elevated levels of onithine decaoxylase coopeate with Raf/ERK activation to convet nomal keatinocytes into invasive malignant cells CS Hayes, K DeFeo, L Lan, B Paul, C Sell and SK Gilmou Lankenau Institute fo Medical Reseach, Wynnewood, PA, USA (2006) 25, & 2006 Natue Pulishing Goup All ights eseved /06 $ Onithine decaoxylase (ODC) oveexpession coupled with activated Ras is fully sufficient to oncogenically tansfompimay keatinocytes. To detemine the Ras effecto pathways that epesent the minimal essential contiution to full oncogenic tansfomation in this context, we evaluated the coopeativity of diffeent Ras effecto mutants with oveexpessed ODC in an in vivo tacheal xenotansplantation assay fo epithelial cell invasiveness. Pimay keatinocytes, isolated fom eithe K6/ODC tansgenic mouse skin (expessing inceased ODC) o fomnomal littemate skin wee infected with etovius poducing an activated RasV12 o patial lossof-function effecto mutants of RasV12 that selectively induce only the Raf/ERK, RalGDS, o the PI3-kinase signaling pathway. Wheeas keatinocytes expessing a fully activated RasV12 ae not invasive in tacheal xenotansplants, ODC-oveexpessing keatinocytes acquie an invasive phenotype with additional expession of eithe RasV12 o activation of the Raf/ERK pathway. Independent of a mutated as, elevated levels of ODC activate the Akt/mTOR signaling pathway as well as the Rho/Rac pathway in pimay keatinocytes. Thus, Raf/ ERK signaling is sufficient to coopeate with inceased ODC activity in the convesion of nomal keatinocytes to invasive cells. In ode to pomote invasiveness in keatinocytes, elevated levels of ODC may coopeate with Raf/ERK via activation of the Akt and Rho/Rac signaling pathway. (2006) 25, doi: /sj.onc ; pulished online 7 Noveme 2005 Keywods: polyamines; onithine decaoxylase; invasion; Raf/ERK; Akt Intoduction Onithine decaoxylase (ODC) activity and polyamine levels ae elevated in most epithelial tumos compaed to nomal adjacent tissue. ODC is the key enzyme Coespondence: Pofesso SK Gilmou, Lankenau Institute fo Medical Reseach, 100 Lancaste Avenue, Wynnewood, PA 19096, USA. gilmous@mlhs.og Received 17 Mach 2005; evised 15 Septeme 2005; accepted 15 Septeme 2005; pulished online 7 Noveme 2005 esponsile fo the iosynthesis of the diamine putescine that is susequently conveted to the polyamines spemidine and spemine. Polyamines ae essential fo nomal gowth and development of most cell types (Tao and Tao, 1984; Pegg, 1988). We have peviously demonstated that the expession of elevated levels of ODC and an activated Ras is sufficient to tansfom a nomal epithelial cell into a malignant, invasive tumo cell (Smith et al., 1997; Gilmou et al., 2001). Doule tansgenic mice, possessing oth the ODC and v-ha-as tansgenes diected to the skin, develop spontaneous squamous cell cacinomas at an ealy age (Smith et al., 1998). Moeove, tumos fom vaying tissue types possess elevated levels of ODC activity, and a common chaacteistic of all known tumo pomotes is thei aility to induce ODC activity (O Bien et al., 1975). Numeous studies using specific ODC inhiitos suppot the essential ole of ODC/polyamines in pomoting tumo development following initiation with cacinogens such as dimethylenz(a)anthacene, that is known to cause as mutations (Weeks et al., 1982; Takigawa et al., 1983; O Bien et al., 1997; Feith et al., 2001), o with UV iadiation whee Ras mutations ae aely found (Ahmad et al., 2001). Howeve, the key polyamineactivated pathways that dive the malignant phenotype ae not known. Many candidate effectos of Ras function have een identified, including Raf, p120 GAP, guanine nucleotide exchange factos (GEFs) fo the small GTPase Ral (i.e. RalGDS, RGL, and RLF/RGL2), AF6/Canoe, RIN1, and phosphatidylinositol 3-kinase (PI3K)). Stimulation of the Raf/ERK pathway, leading to activation of the cytoplasmic kinases Rsk and Mnk as well as nuclea tansciption factos such as Elk-1, is essential fo cell polifeation (Stugill et al., 1988; Waskiewicz et al., 1997). Activation of memes of the Rho family of GTPases, possily mediated y the Ras GTPaseactivating potein, p120 GAP, is also likely to make a significant contiution to Ras-mediated tansfomation (Settleman et al., 1992; Zohn et al., 1998). Futhemoe, inteaction etween activated Ras and PI3K is equied fo the eaangement of the actin cytoskeleton, which is associated with the tansfomed phenotype (Rodiguez- Viciana et al., 1997). RalGDS egulation of phospholipase D and signaling y AF6/Canoe may additionally contiute to tansfomation-associated cytoskeletal emodeling (Kuiyama et al., 1996).

2 1544 ODC and Raf/ERK tigge keatinocyte invasion One poweful appoach fo elucidating the espective contiution of each of the vaious effecto pathways to Ras-mediated tansfomation has een the use of mutants that selectively activate some ut not all of Ras function. It has een demonstated that single point mutations in the effecto domain of Ras (esidues 32 40) lead to patial loss of function in which Ras inteaction is maintained with some effectos ut lost with othes (White et al., 1995; Magee and Mashall, 1999). Fo example, it has een shown that a mutant of activated Ras (RasV12-C40), which is unale to ind Raf, fails to activate the ERK signaling cascade yet emains fully capale of inducing memane uffling though activation of PI3K and Rac pathways (Rodiguez-Viciana et al., 1997). This demonstates that these two Rasmediated events must e contolled y distinct pathways. Use of the RasV12-C40 mutant has futhe estalished that Raf-independent signaling is sufficient to pomote Ras tansfomation. We have investigated the possile coopeation etween ODC and vaious patial loss-of-function effecto mutants of an activated Ha-RasV12 that selectively induce eithe the Raf/MEK/ERK1/2, RalGDS, o PI3K/Akt pathways. In ode to test fo invasiveness, we have used a tacheal xenotansplant assay whose utility in evaluating in vivo invasive potential has een peviously demonstated (Gilmou et al., 2001). We epot hee that elevated levels of ODC and the Raf/ MEK/ERK pathway confe an invasive phenotype on pimay cultues of nomal keatinocytes. In addition, we show that inceased ODC activity may coopeate with Raf/ERK y activating additional signals associated with the PI3K/Akt and Rho/Rac pathways. expession significantly inceased invasiveness (Figue 1). To assess the involvement of the PI3K/Akt and Raf/ MEK/ERK pathways in the invasiveness of these keatinocytes, cells wee teated with PD98059, an MEK inhiito, o LY294002, a PI3K inhiito. Invasiveness was inhiited y nontoxic concentations of oth inhiitos; howeve, inhiition of PI3K/Akt signaling with LY appeaed to completely aogate the effects of ODC oveexpession. These esults suppot what has een seen in othe tansfomation systems in that oth the Raf/MAPK pathway and the PI3K/Akt pathway play an essential ole in the tansfomation of nomal muine keatinocytes to an invasive phenotype following aeant expession of H-Ras and ODC. ODC oveexpession and activation of Raf/ERK pathway sufficient to induce invasiveness in pimay keatinocytes To detemine the Ras effecto pathways that epesent the minimal essential contiutions that coopeate with elevated levels of ODC to yield invasiveness in epidemal cells, we used the at tacheal xenotansplant assay to evaluate invasiveness in vivo using pimay keatinocytes. Pimay keatinocytes wee isolated fom K6/ODC tansgenic mouse skin (Ke/ODC) and fom thei nomal littemates in ode to otain nomal epidemal cells that diffe only in thei expession of ODC. Both nomal and Ke/ODC wee infected with etovius poducing an activated RasV12 o specific patial loss-of-function Ras effecto mutants with single aminoacid sustitutions in thei effecto loop domain esulting in pefeential activation of one of the thee effecto pathways, Raf, RalGDS, o PI3K (White et al., 1995). Results Dependence of invasive ehavio of ODC/Ras keatinocytes on MAPK and PI3K signaling Pevious in vivo studies have shown that ODC oveexpession coopeates with v-ha-as to convet nomal pimay keatinocytes to invasive malignant cells (Smith et al., 1997; Gilmou et al., 2001). Both the PI3K/Akt and Raf/ERK pathways have een shown to play a ole in the invasive potential of seveal tumo cell types (We et al., 1998; Ulku et al., 2003; Bedogni et al., 2004; Campell and De, 2004). An in vito assay was pefomed using Matigel-coated invasion chames to assess the ole of these pathways in the invasive ehavio of pimay keatinocytes isolated fom the skin of K6/ODC tansgenic mice and thei nomal littemates following etovial infection with v-ha-as. Use of keatinocytes fom K6/ODC tansgenic mice povided epidemal cells that expess high levels of ODC since the keatin 6 pomote that dives the expession of the ODC tansgene is upegulated upon cultuing. Thee was little invasiveness of pimay keatinocytes isolated fom eithe K6/ODC tansgenic o nomal littemate skin. As expected, v-ha-as-infection yielded invasive keatinocytes, ut oth ODC oveexpession and as Nume of Invading Cells Nom Ras Nom Ras+PD Nom Ras+LY Tans Ras Tans Ras+PD Tans Ras+LY Figue 1 Inceased invasiveness of Ras-infected keatinocytes inhiited y PD98059 and LY Gowth facto-educed Matigel invasion chames wee plated with keatinocytes fom K6/ODC tansgenic mice (Tans) o thei nomal littemates (Nom) to assess the invasiveness of each type of keatinocyte. All chames wee etovially infected with v-ha-ras and some cells wee teated with PD98059 (PD) o LY (LY) at the stat of the seum gadient. Afte 24 h insets wee fixed in fomalin, stained with cystal violet, mounted and cells on the undeside of the filte counted. Values ae means7s.d., and ae epesentative of thee sepaate expeiments with thee chames pe teatment goup.

3 It has een peviously demonstated that the V12H- Ras T35S mutant inds and activates Raf, ut not PI3K o Ral-GEF; the V12H-Ras E37G mutant inds and activates only Ral-GDS; and the V12H-Ras Y40C mutant inds and activates only PI3K (White et al., 1995). Thus, we efe to these mutant oncogenic Ras vaiants as S35, G37, and C40, espectively. Specific activation of the Raf/ ERK, PI3K, o RalGDS pathway was confimed fo keatinocytes etovially infected with these Ras mutants (data not shown). Pevious epots have shown that activation of Ras induces ODC activity (Sistonen et al., 1987; Shantz and Pegg, 1998; Shantz, 2004). Wheeas putescine levels damatically inceased appoximately 500-fold in Ke/ ODC compaed to nomal keatinocytes in which putescine levels wee almost nondetectale, spemidine levels inceased only two-fold with often no change in spemine levels (Figue 2). In compaison to the damatic incease in ODC activity and putescine levels in Ke/ODC compaed to nomal keatinocytes, infection with wild-type V12H-Ras induced ODC activity and putescine levels to a less extent. Moeove, thee was no significant effect of the Ras mutants on ODC activity o polyamine levels in Ke/ODC o nomal keatinocytes (Figue 2). Following etovial infection with eithe the wildtype V12H-Ras o one of the V12H-Ras effecto mutants, oth nomal and ODC oveexpessing keatinocytes wee inoculated in pepaed de-epithelialized at tacheas and implanted sucutaneously into athymic nude mice fo 5 weeks to assay the degee of invasion of the keatinocytes though the tacheal wall. Although all keatinocyte goups successfully e-epithelialized within the lumen of the tacheas, the nomal keatinocytes that wee not Ras infected and nomal keatinocytes infected with the fully activated RasV12 o the S35, C40, o G37 Ras effecto mutants showed no o only mild invasiveness in this in vivo assay (Tale 1, Figue 3a and c). Keatinocytes in these tansplants wee confined to the luminal suface (level 0) o to the supeficial lamina popia (level 1). Futhemoe, wheeas Ke/ODC failed to invade though the tacheal wall, Ke/ODC infected with RasV12 deeply penetated the tacheal wall (Figue 3) as peviously shown (Gilmou et al., 2001). Tale 1 summaizes that in 11/20 (55%) tacheas tansplanted with RasV12-infected Ke/ODC, cells ODC and Raf/ERK tigge keatinocyte invasion invaded into the adventitia (level 3), and an additional fou tacheas (20% of the tansplants) contained tansplanted cells that eached the pas memanacea without invading the adventitia (level 2). The invading cells initially epopulated the luminal epithelium ut invaded all layes of the tacheal wall, esulting in a distotion of the tachea as the cells enveloped oth the a ODC Activity nmols polyamine / mg DNA noninf. S35 WT C40 G37 Nom-non Nom-WT Ke/ODC-non Ke/ODC-WT Putescine Spemidine Spemine Figue 2 Effect of RasV12 and Ras effecto mutants on ODC activity and polyamine levels. Pimay keatinocytes isolated fom the skin of K6/ODC tansgenic mice (Ke/ODC) and thei nomal littemates (Ke/Nom) wee eithe noninfected (Non) o etovially infected with RasV12-WT (WT) o the Ras effecto mutants, RasV12-S35 (S35), RasV12-C40 (C40), o RasV12-G37 (G37). (a) Ke/Nom lysates wee assayed fo ODC enzyme activity. Thee was no significant diffeence in ODC specific activity fo Ke/ODC that wee noninfected o Ras-infected, with an aveage value of 95 nmol 14 CO 2 pe h pe mg potein. () Cell lysates wee assayed fo levels of putescine, spemidine, and spemine, and values ae given fo oth Ke/Nom and Ke/ODC that wee eithe noninfected o infected with RasV12-WT. Thee was no significant effect of etovial infection with the Ras effecto mutants on polyamine levels in Ke/Nom o Ke/ODC lysates compaed to that in noninfected keatinocytes. ODC enzyme activity was expessed as nmol 14 CO 2 pe h pe mg potein and polyamine levels wee expessed as nmol pe mg DNA. Values ae given as the mean7s.d. with at least thee samples pe goup Tale 1 Level of invasion of tacheal tansplants epopulated with keatinocytes infected with RasV12 and diffeent RasV12 effecto mutants Level of invasion No Ras infection RasVI2 RasV12-S35 (Raf/ERK) RasV12 G37 (Ra1GDS) RasV12-C40 (PI3K/Akt) Nom (8) ODC (16) Nom (14) ODC (20) Nom (6) ODC (13) Nom (11) ODC (18) Nom (9) ODC (17) De-epitlelialized tacheas west epopulated with keatinocytes fom K6/ODC tansgenic mice and thei nomal litemates, some of which wee etovially infected with RasV12, RasV12-S35, RasV12-G37, o RasV12-C40. The invasion of the tacheal wall was classified accoding to the level of penetation of the cells eing tested, with level 0 epesenting no invasion and level 3indicating cell invasion though the entie tacheal wall. The total nume of tacheal tansplants/keatinocyte goup is shown in paentheses.

4 1546 ODC and Raf/ERK tigge keatinocyte invasion a Ke/Nom: RasV12 Ke/ODC: RasV12 c d Ke/Nom: RasV12-S35 Ke/ODC: RasV12-S35 e f Ke/ODC: RasV12-G37 Ke/ODC: RasV12-C40 Figue 3 Activation of Raf/ERK pathway and ODC oveexpession sufficient fo invasiveness in keatinocytes. De-epithelialized at tacheas wee inoculated with keatinocytes isolated fom K6/ODC tansgenic mice (Ke/ODC) (, d f) and thei nomal littemates (Ke/Nom) (a and c) and etovially infected with eithe RasV12 (a and ), RasV12-S35 (c and d), RasV12-G37 (e), o RasV12-C40 (f). At 5 weeks afte tansplantation, the epopulated at tacheas wee emoved, fixed, and coss sections stained with hematoxylin and eosin. Lu, lumen of the tachea;, catilage ing of the tachea;, Teflon tuing adjacent to tachea. Black aows point to asement memane of e-epithelialized tacheal lumen; ed aow points to papilloma-like gowth in the noninvasive, ut hypeplastic epithelium in (e). catilage ing as well as the Teflon tuing used to suppot the tacheal tansplants (Figue 3). In contast, all G37-infected Ke/ODC tacheal tansplants and 88% of the C40-infected Ke/ODC tansplants demonstated no o mild invasiveness (level 1) in tacheal xenotansplants (Tale 1, Figue 3e and f). Only S35 Ras-infected Ke/ODC showed invasiveness simila to that seen with RasV12-infected Ke/ODC tansplants, with 8/13(62%) exhiiting level 3invasion pattens (Tale 1, Figue 3d). These esults demonstate that ODC coopeates with activation of the Raf pathway ut not with activation of the RalGDS o PI3K pathways to tansfom pimay keatinocytes to invasive cells. Enhanced activation of MAP kinase activity y polyamines Keatinocytes infected with RasV12 o Ras effecto mutants wee analysed fo phosphotansfease activity associated with immunopecipitated MAP kinase. Using myelin asic potein (MBP) as a sustate, RasV12-WT and S35 mutant Ras-infected keatinocytes demonstated the highest MAP kinase activity (Figue 4a). Ke/ODC not infected with RasV12-WT o mutant Ras possessed highe aseline MAP kinase activity compaed to nomal keatinocytes. In addition, ODC oveexpession appeaed to enhance the MAP kinase activity in keatinocytes expessing eithe the RasV12-WT o

5 a IP: Anti-MAPK IB: Anti-p-MBP IB Noninfected RasV12-WT RasV12-C40 RasV12-G37 RasV12-S35 Ke/Nom Noninfected RasV12-WT RasV12-C40 RasV12-G37 RasV12-S35 Ke/ODC βactin Figue 4 Elevated levels of ODC stimulate ERK kinase activity in keatinocytes. (a) Cell lysates wee pepaed fom pimay keatinocytes isolated fom K6/ODC tansgenic mice (Ke/ODC) o thei nomal littemates (Ke/Nom), some of which wee etovially infected with RasV12-WT, RasV12-C40, RasV12-G37, RasV12-S35. Lysates wee assayed fo phosphotansfease activity associated with immunopecipitated ERK using the sustate, myelin asic potein (MPB), followed y immunolot analysis (IB) fo phosphoylated MBP sustate using an antiphospho-mbp antiody. () Immunolot analysis (IB) in which lysates wee poed fo Ras and -actin potein as a loading contol. Ras ODC and Raf/ERK tigge keatinocyte invasion a Ral-GTP Total Ral β-actin p-akt Akt Nomal Ke/ODC Non WT WT+LY Non WT Nomal Non WT WT +LY Ke/ODC Non WT WT +LY 1547 ulin any of the mutant Ras vaiants, with the highest MAP kinase activity exhiited in WT Ras and S35 Rasinfected Ke/ODC (Figue 4a). Total Ras potein was inceased in all Ras-infected cells (Figue 4). c Non Nomal C40 S35 Non Ke/ODC C40 S35 Elevated ODC activates PI3K/Akt ut not RalGDS Since oveexpession of ODC coopeated with only the S35 Ras mutant and not the C40 (PI3K/Akt effecto) o G37 (RalGDS effecto) mutants in the tacheal xenotansplant invasion assay, a possile mechanism y which inceased levels of polyamines tansfom keatinocytes is y activating eithe the PI3K/Akt o RalGDS signaling pathways. A pull-down assay using the Ral- GTP inding fagment fom the Rg1 Ral GEF was used to monito the level of activated GTP-ound Ral A. When compaed with noninfected nomal keatinocytes o Ke/ODC, RasV12-WT-expessing keatinocytes showed inceased levels of Ral-GTP (Figue 5a). Howeve, oveexpession of ODC had no effect on the level of Ral-GTP (Figue 5a) in pimay cultues of keatinocytes. As expected, the PI3K inhiito LY had no effect on the Ral GEF pathway. Westen lotting of the phosphoylated fom of Akt, a downsteam taget of PI3K, evealed a fou- to fivefold incease in the level of phospho-akt in Ke/ODC oth efoe and afte infection with RasV12- WT compaed to nomal littemate keatinocytes (Figue 5). In contast, although RasV12-WT infection inceased levels of Ras two-fold in oth nomal and ODC tansgenic keatinocytes (data not shown), phospho-akt was minimally inceased (1.7-fold) in Rasinfected keatinocytes when nomalized to the level of total Akt and potein. Levels of phosphoylated Akt wee deceased elow the level found in nomal keatinocytes following teatment with LY294002, a PI3K inhiito (Figue 5). Assays fo kinase activity associated with immunopecipitated Akt using Bad as a p-bad Ras β-actin Figue 5 ODC oveexpession activates Akt signaling ut not Ral. Pimay keatinocytes isolated fom K6/ODC tansgenic mice (Ke/ODC) o thei nomal littemates (Nomal) wee etovially infected with RasV12-WT (WT), RasV12-S35 (S35), o RasV12- C40 (C40) o mock infected (Non). Some cells wee teated with 50 mm LY fo 30 min efoe havest to inhiit the PI3K pathway. (a) Ral-GTP was pecipitated, and activated Ral-A fom the pull-down was detected y immunolot analysis using a Ral-A antiody. Lysates wee also analysed fo total Ral-A and -actin as loading contols. () Immunolot analysis of keatinocyte lysates in which lots wee poed with antiodies specific fo the phosphoylated fom of Akt o total Akt and epoed fo Ras and tuulin potein as a loading contol. (c) Lysates wee assayed fo kinase activity associated with immunopecipitated Akt using the sustate, Bad, and anti-phospho-bad as antiody to detect the phosphoylated sustate in an immunolot analysis. Banding patten is epesentative of immunolots fom at least thee independent expeiments. sustate evealed moe Akt kinase activity in Ke/ODC compaed to keatinocytes isolated fom nomal littemate skin (Figue 5c). As expected, Bad phosphoylation was inceased in C40-infected keatinocytes (Figue 5c) and RasV12-WT-infected keatinocytes (data not shown) and to a lesse extent in S35-infected keatinocytes (Figue 5c). Howeve, the kinase activity associated with Akt was elevated six- to eight-fold moe

6 1548 ODC and Raf/ERK tigge keatinocyte invasion in Ke/ODC infected with eithe the C40 Ras mutant (Figue 5) o RasV12-WT (data not shown) compaed to that with nomal keatinocytes. To investigate the activation status of Akt in tacheal xenotansplants epopulated with keatinocytes expessing high levels of ODC o infected with Ras mutants, tacheal coss sections wee examined y immunohistochemisty to detect phospho-akt. Thee wee no stained cells o vey weak staining in tacheas epopulated with eithe nomal keatinocytes (Figue 6a) o S35-infected keatinocytes (Figue 6d). In contast, phosphoylated, activated Akt was eadily detected in the cytoplasm of ODC-oveexpessing keatinocytes (Figue 6 and c) that had e-epithelialized the lumen of the tacheas and appeaed to e confined to supaasal layes of the newly fomed epithelium (Figue 6c). Staining was limited to epithelial cells, suggesting that the suounding stomal cells did not possess activated Akt. Moeove, activated phospho- Akt was detected in invasive S35-infected Ke/ODC cells (Figue 6e and f). Thus, elevated levels of ODC activity and polyamines lead to activation of Akt in pimay keatinocytes. Although the antiapoptotic activity of Akt is wellknown, it also egulates othe aspects of cellula function, including migation, glucose metaolism, and potein synthesis. Since Akt pomotes potein synthesis via inceasing the phosphoylation of its downsteam taget, mammalian taget of apamycin (mtor) (Bjonsti and Houghton, 2004), we examined whethe the phosphoylation and activation of the eukayotic initiation, facto 4E-inding potein 1 (4E-BP1), and p70 S6 kinase (p70s6k) and its susequent phosphoylation of S6 iosomal potein (S6p) ae inceased in Ke/ODC. As seen in Figue 7a, oveexpession of ODC in pimay keatinocytes inceased the phosphoylation of p70s6k to the same degee as seum stimulation in the nomal keatinocytes. Both the PI3K inhiito LY and the mtor inhiito apamycin makedly inhiited the phosphoylation of Th-389 of p70s6k in oth nomal keatinocytes and Ke/ODC. Similaly, phosphoylation of S6 iosomal potein and of 4eBP1 was significantly inceased in ODC-oveexpessing keatinocytes compaed to contol keatinocytes (Figue 7). Teatment of cells with the inhiitos LY o apamycin confimed the Akt and mtordependence of p70s6k, S6 iosomal potein, and 4eBP1 phosphoylation esulting fom inceased ODC activity (Figue 7). These esults show that elevated levels of polyamines in pimay muine keatinocytes activate oth the Raf/ERK and Akt/mTOR pathway, ut not the RalGDS signaling pathway. a c Lu Lu Lu d e f Lu Lu Figue 6 Immunohistochemical detection of activated Akt in ODC oveexpessing keatinocytes in tacheal xenotansplants. Phosphoylated, activated Akt was detected y immunohistochemisty in tacheal tansplant coss sections epopulated with (a) Ke/Nom; ( and c) Ke/ODC; (d) Ke/Nom infected with RasV12-S35; (e and f) Ke/ODC infected with RasV12-S35. Note that (c) and (f) ae highe magnifications of the views in () and (e), espectively; in (e), aow points to stained keatinocytes that have invaded past the tacheal wall to epopulate aound the Teflon tuing placed next to the implanted tacheas. Lu, lumen of the tachea;, catilage ing of the tachea;, Teflon tuing adjacent to tachea.

7 a Nomal LY Rapamycin Ke/ODC LY Rapamycin ODC and Raf/ERK tigge keatinocyte invasion Min Seum Stimulation P-Th389 p70s6k p70s6k β-actin 1549 Nomal Ke/ODC LY Rapamycin P-S6p S6p P-4E-BP1 4E-BP1 Figue 7 ODC oveexpession inceases phosphoylation of mtor-egulated poteins in keatinocytes. Immunolot analysis of lysates of K6/ODC and nomal littemate keatinocytes that wee cultued in seum-fee medium ovenight and then teated 30 min with 50 mm LY o 10 nm apamycin efoe havest. Some cells in (a) wee also seum stimulated fo 10 min efoe havest and then cell lysates poed fo phosphoylated p70s6k, total p70s6k, o -actin; () keatinocytes emained on seum-fee medium without seum stimulation and lysates wee poed fo phosphoylated S6 iosomal potein (P-S6p), total S6p, phosphoylated 4E- BP1, o total 4E-BP1. Polyamine activation of Rho and Rac Cell migation, which is an integal pat of tumo invasion, depends on the actions of the GTPases, Rho and Rac (Buidge and Wenneeg, 2004). When activated (GTP-ound), Rho and Rac inteact with a vaiety of effectos to tigge signaling pathways that egulate cell polifeation, cell adhesion, and tumoigenesis (Buidge and Wenneeg, 2004). To study the effect of elevated levels of ODC activity and polyamines on the activity of Rho and Rac, pull-down assays using eithe Rhotekin (fo Rho) (Ren et al., 1999) o GST- PAK (fo Rac) (Benad et al., 1999) wee used to measue the levels of active, GTP ound poteins. Infection of muine keatinocytes with RasV12-WT douled the levels of Rho-GTP as well as Rac-GTP with no change in the total poteins (Figue 8 and data not shown). Although thee was no diffeence in the total potein levels of RhoA, activated GTP-ound Rho was inceased thee-fold in ODC oveexpessing keatinocytes (Figue 8a). Seum stimulation fo 10 min did not futhe incease the level of GTP-ound Rho in nomal o ODC oveexpessing keatinocytes. Similaly, the level of activated GTP-ound Rac was inceased fivefold in Ke/ODC compaed to nomal littemate keatinocytes (Figue 8). Discussion Pevious studies have shown that elevated levels of ODC ae essential fo the pomotion of skin tumoigenesis in oth UV-iadiated (Ahmad et al., 2001; Tang et al., 2004) and cacinogen-initiated mouse skin (O Bien et al., 1997) and in skin expessing an oncogene such as a mutated as o MEK1 (Smith et al., 1998; Feith a Minutes Seum: Rho-GTP Total Rho β-actin Rac-GTP Total Rac β-actin Ke/Nom Ke/ODC N T N T Ras: Figue 8 Activation of Rho and Rac in ODC oveexpessing keatinocytes. Lysates wee pepaed fom pimay keatinocytes isolated fom the skin of K6/ODC tansgenic mice (Ke/ODC) o thei nomal littemates (Ke/Nom). Cells wee cultued in seumfee media ovenight and some wee seum-stimulated pio to havest. (a) Immunolot analysis of pull-down assay fo Rho-GTP. Rho-A antiody was used to detect activated Rho-GTP fom a pull-down assay using eads covalently ound to Rhotekin and also total Rho-A in cell lysates. Immunolots wee epoed with antiody to detect -actin potein as a loading contol. () Pulldown assay of Rac-GTP using Rac antiody to detect activated Rac and total Rac in non-seum-stimulated Ke/Nom (N) o Ke/ ODC (T), some of which wee etovially infected with RasV12- WT. The anding patten is epesentative of pull-down assays fom at least thee independent expeiments.

8 1550 ODC and Raf/ERK tigge keatinocyte invasion et al., 2005). Howeve, aeant expession of Ras efficiently dives tumoigenesis though multiple downsteam effectos. This study demonstates that only the Raf/ERK signaling pathway is needed to coopeate with inceased polyamine levels to tansfom nomal keatinocytes to a malignant, invasive phenotype. Moeove, elevated levels of ODC and polyamines activate the PI3K signaling pathway and the Rho/Rac pathway independently of a mutated as, thus seving to augment the tumoigenic effects of Ras. The majoity of epithelial tumos exhiit aeant expession of ODC and inceased levels of polyamines. Elevated polyamine levels have een shown to stimulate cell polifeation (Gilmou et al., 1999), migation (Ray et al., 2003), and angiogenesis (Lan et al., 2000, 2005). Howeve, the molecula mechanism y which polyamines stimulate these pocesses and pomote tumoigenesis is not well undestood. As polyamines ae stong polycationic molecules that eadily ind a vaiety of macomolecules, it is not supising that they influence many iological pathways. Wheeas studies using inhiitos of polyamine iosynthetic enzymes have shown that polyamines ae essential fo nomal cellula function and suvival, this study is unique in focusing on effects of elevated levels of ODC and polyamines in a nomal epithelial cell type when expessed with an oncogenic signal. Invasiveness in nomal keatinocytes equies activation of the Raf/MEK/ERK signaling pathway since expession of eithe the RasV12-WT o the RasV12-S35 mutant was equied fo invasion using the tacheal in vivo assay. Although ODC oveexpessing keatinocytes demonstate some activation of the Raf/MEK/ERK pathway, ou studies show that it is not sufficient to cause invasiveness in keatinocytes expessing eithe the RasV12-C40 (activation of PI3K/Akt) o the RasV12-G37 (RalGDS activation) mutants. That ODC oveexpession does not activate the RalGDS signaling pathway o coopeate with the RasV12-G37 mutant indicates that activation of the RalGDS pathway is not necessay in the convesion of nomal keatinocytes to an invasive phenotype. Moeove, since RasV12-WT infected keatinocytes ae not invasive without inceased ODC activity, ODC-activation of the Ras effectos, Raf/MEK/ERK and PI3K/Akt, does not appea to e sufficient to convet nomal keatinocytes to invasive tumo cells. Ou data suggest that inceased levels of polyamines must activate effecto pathways in addition to effectos activated y RasV12- WT to induce invasive ehavio in nomal keatinocytes. The MAP kinase pathway is clealy citical fo tumoigenesis in muine keatinocytes (Tautani et al., 2003; Scholl et al., 2004), and the Raf-MAP kinase pathway is activated in almost a thid of human tumos as detemined y the phosphoylation status of the MAPKs ERK1 and ERK2 (Hoshino et al., 1999). Howeve, wheeas the Raf-MAP kinase pathway is sufficient to cause tansfomation of NIH 3T3 mouse fiolasts (Shields et al., 2000), this kinase cascade alone is not sufficient to cause full in vito tansfomation of a vaiety of odent o human epithelial cells (Oldham et al., 1996; Hamad et al., 2002; Ulku et al., 2003; Collette et al., 2004). The Raf/MAPK pathway has een demonstated to e the pimay oncogenic effecto of Ras in numeous in vivo studies, including multiple tansgenic mouse skin models (Tautani et al., 2003; Ulku et al., 2003; Bedogni et al., 2004; Scholl et al., 2004; Feith et al., 2005) and following etovial infusion of Ras effecto mutants into the cental mammay duct (McFalin et al., 2003). Although pevious studies have epoted the fomation of enign papillomas following tansgenic oveexpession of Raf o MEK, we have demonstated that neithe fully activated Ras no activation of the Raf pathway confes an invasive phenotype on nomal keatinocytes without oveexpession of ODC. Activation of Ras o moe than one of its effecto pathways has een shown to induce ODC activity in fiolastic NIH 3T3 cells (Shantz, 2004). Howeve, this study is the fist to chaacteize the effect of activation of Ras o its effecto pathways on ODC activity in pimay epithelial cells. Moeove, wheeas tansgenic oveexpession of MEK led to inceased ODC activity in esulting skin papillomas, MEK oveexpession did not induce ODC activity in nontumo eaing skin (Feith et al., 2005). We have found that endogenous ODC activity and putescine levels wee slightly elevated in nontansgenic, nomal keatinocytes following the etovial intoduction of RasWT ut not the Ras patial loss-of-function mutants that activated the PI3-kinase o Raf/MEK/ERK pathways. Although induction of ODC activity is necessay fo Ras o RhoA-tansfomation of NIH 3T3 fiolasts (Shantz and Pegg, 1998), ou data indicate that RasV12-WT-induction of ODC activity is not sufficient in itself to convet nomal keatinocytes to malignant invasive cells. All togethe, these data indicate that convesion of a nomal keatinocyte to an invasive tumo cell equies activation of the Raf/MAPK signaling pathway and a theshold level of ODC activity and/o a selective susceptiility of a supopulation of keatinocytes o stem cells within the skin. Similaly, seveal components of the PI3K/Akt pathway ae dysegulated in a wide spectum of human cance (Cantley and Neel, 1999; Vivanco and Sawyes, 2002). We show that elevated polyamine levels in pimay cultues of keatinocytes lead to activation of Akt as evidenced y inceased phosphoylation of Akt and Akt-associated kinase activity. This is seen in vivo as well since ODC-oveexpessing keatinocytes expess high levels of phospho-akt in epopulated luminal epithelium of tacheal xenotansplants. In contast, nomal keatinocytes and keatinocytes infected with the S35 Ras mutant, specific fo activation of the Raf pathway, expess elatively low levels of phosphoylated Akt potein in tacheal xenotansplants. Activation of the PI3K/Akt pathway tigges a cascade of esponses that egulate cell polifeation, suvival, and potein synthesis, all of which dive tumo pogession (Vivanco and Sawyes, 2002). Akt phosphoylation leads to pofound effects on the egulation of cell metaolism and the initiation of potein synthesis y phosphoylating and activating the mtor. As a

9 senso of nutient status, mtor is egulated y mitogens, nutients, and enegy levels, and it mediates cell gowth and polifeation though the egulation, via phosphoylation, of its downsteam tagets, p70 iosomal S6 kinase and 4E-BP1 (Bjonsti and Houghton, 2004). We have shown that inceased ODC enzyme activity in keatinocytes inceases the phosphoylation status and activation of the p70 S6 kinase and its sustate S6 iosomal potein. Paadoxically, we have found that shot-tem teatment with the specific inhiito of ODC, DFMO, initially enhances the phosphoylation of these tanslational poteins, ut that futhe DFMO teatment ove 5 days inhiits the phosphoylation (unpulished esults). Since DFMO has een shown to inhiit seine/theonine phosphatase 2A activity and, thus, incease phosphoylation of apoptotic poteins such as Bad (Ray et al., 2005), it is possile that a DFMO-induction of a phosphatase may esult in the initial lack of a DFMO-evesal of mtor sustate phosphoylation. Futue studies will exploe whethe the mtor signaling pathway esponds to vaying fluctuations in polyamine pools within specific cellula compatments o is activated in esponse to inceased polyamine iosynthesis. Simila to Ras, the Rho-family GTPases (e.g. RhoA, Rac1, and Cdc42) function as molecula switches y cycling etween an active guanosine tiphosphate-ound state and an inactive guanosine diphosphate-ound state. Both Rho and Rac paticipate in many cellula pocesses involving actin cytoskeleton eoganization such as cell cell adhesion and cell motility (Van Aelst and D Souza-Schoey, 1997; Benitah et al., 2004; Jaffe and Chenoff, 2004), and they have een implicated in multiple steps duing tansfomation, including tumo invasiveness (Boettne and Van Aelst, 2002). RhoA activation induces cell contactility and stess fie fomation (Ridley and Hall, 1992), and Rac1 stimulates actin polymeization and the fomation of lamellipodia in cultued cells (Ridley et al., 1992; Buidge and Wenneeg, 2004). Rac-dependent focal complexes develop into Rho-dependent focal adhesions that povide the cell with anchoage ehind the lamellipodium as the cell migates fowad (Rottne et al., 1999). We have shown that ODC oveexpession activates oth Rho and Rac in nomal keatinocytes. Ou data ae consistent with pevious studies using polyamine depleted cells that show the equiement fo activation of oth Rac1 and RhoA fo optimal intestinal epithelial cell migation (Ray et al., 2002, 2003), and that suggest that polyamines ae equied fo Rac1 activation and the sequential activation of RhoA. Ou studies also show that RasV12-WT-infection o ODC oveexpession in pimay muine keatinocytes esults in activation of oth RhoA and Rac1. Howeve, Ras tansfomation has een epoted to yield damatic cell type-dependent diffeences in the levels of Rho-GTP and Rac-GTP. Fo instance, activated Ras-tansfomed MDCK epithelial cells have low Rac activity and upegulated Rho activity (Zondag et al., 2000). Convesely, Ras-tansfomed HB4A mammay epithelial cells have high Rac and low Rho activity, and thee ae ODC and Raf/ERK tigge keatinocyte invasion no changes in Rho and Rac activity in Ras-tansfomed HA1 kidney cells (Sahai et al., 2001). Most studies of cell migation have used fiolasts, and considealy less is known aout epithelial cell migation. Blocking eithe Ras o Rac pathways using dominant-negative mutants inhiits cell motility (Ridley et al., 1995; Keely et al., 1997), highlighting the impotant ole of oth Ras and Rac in cell migation. Although activation of Ras and the Rho-family memes plays an impotant ole in cell motility and invasiveness, the GTPase cosstalk in migation is complex. Ou studies suggest that it is modulated y changes in the intacellula levels of polyamines. Polyamines may activate Rac though PI3K, as Tiam1-mediated Rac activation is dependent on PI3K activity (Sande et al., 1998). Altenatively, uokinase plasminogen activato (upa) is upegulated when ODC is oveexpessed in pemalignant epidemal SP-1 cells (Smith et al., 1997). Oveexpession of upa ecepto has een shown to stimulate cellula migation though the activation of Rac (Vial et al., 2003), and this may e dependent on polyamine-induced upa inding (Stuge et al., 2002). It is likely that the convesion of nomal keatinocytes to an invasive phenotype esults fom not only the equied activation of Raf-MAPK ut also the diect o indiect polyamine-activation of Rho and/o Rac signaling. In summay, we have shown that the activation of the Raf/MAPK pathway is sufficient to coopeate with elevated levels of ODC activity acting, in pat, though activation of the PI3K/Akt and Rho signaling pathways, to confe an invasive phenotype on nomal keatinocytes. Although activation of Raf/MAPK signaling is necessay fo invasion, ou esults indicate that polyamines must also activate signaling pathways in addition to the downsteam effecto pathways of Ras fo convesion of nomal keatinocytes to invasive tumo cells. Moeove, the activation of PI3K/Akt in keatinocytes inceases cell invasion in addition to the well-chaacteized ole that this pathway plays in cell suvival though the inhiition of apoptosis. These esults povide a molecula famewok fo futue eseach studying specific mechanisms of polyamines in pomoting tumoigenesis. Mateials and methods Pimay cultues of epidemal cells Pimay cultues of epidemal cells wee isolated fom 3- to 4- day-old K6/ODC tansgenic newon pups and thei nomal littemates y a typsin flotation pocedue (Yuspa and Hais, 1974; Hennings et al., 1980). K6/ODC tansgenic pups wee distinguished fom thei nomal littemates y PCR genotyping fo the K6/ODC tansgene (Megosh et al., 1995). Cells wee cultued in low-calcium keatinocyte media ((EMEM) w/o calcium (BioWhittake Walkesville, MD, USA) supplemented with 8% chelex-teated fetal ovine seum and 0.05 mm calcium) at 351C with 5% CO 2. In some instances, keatinocytes wee infected with a eplication-defective etovius containing fully activated RasV12-WT; the Ras patial loss-of-function mutants RasV12-C40, RasV12-G37, o RasV12-S35 (White et al., 1995); o the v-ha-as oncogene 1551

10 1552 ODC and Raf/ERK tigge keatinocyte invasion (Roop et al., 1986). Helpe vius-fee etoviuses wee poduced y tansfection of Phoenix ecotopic packaging cells (Seano et al., 1997). Filteed supenatant was titeed using NIH 3T3 cells, and high tite ( CFU) vial supenatant was supplemented with 4 mg/ml polyene to infect feshly isolated keatinocytes within h postplating. No dug selection was used. Efficiency of gene tansfe y this appoach was veified y immunolot analyses. Pimay keatinocytes wee cultued fo 2 5 days efoe havesting. Some keatinocytes wee seum-staved ovenight (16 h) in low-calcium media containing 0.1% ovine seum alumin. To inhiit the Raf/ERK, PI3K, o mtor pathways, keatinocytes wee teated with 20 mm PD98059, 50 mm LY294002, o 10 nm apamycin (Caliochem, La Jolla, CA, USA), espectively, as additions to the conditioned media fo 30 min efoe havesting. Matigel invasion assay Keatinocytes wee plated at cells pe gowth factoeduced Matigel insets (Becton Dickinson, Bedfod, MA, USA), followed y infection of some cells with the v-ha-as etovius the next day (Roop et al., 1986). At 24 h afte infection, a gadient was stated y adding low-calcium seumfee media supplemented with 0.1% BSA to the inteio of the inset and adding low-calcium media supplemented with 5% chelexed FBS to the lowe compatment of the invasion chame. Afte 24 h, cells that had migated though the matix wee fixed in 10% neutal uffeed fomalin, stained with 1% cystal violet and then mounted on a slide and counted unde the micoscope. Each condition was pefomed in tiplicate. Tacheal xenotansplant assay Rat tacheas (Zivic Mille Laoatoies, Pittsugh, PA, USA) wee mounted on Teflon tuing to maintain thei oiginal shape and length, and wee de-epithelialized y epeated feeze/thawing. Pimay keatinocytes wee havested y teatment with 0.25% typsin-0.01% EDTA and esuspended in medium. De-epithelialized tacheas wee inoculated with ml cells (10 7 cells/ml), and the ends sealed with sugical clips (Gilmou et al., 2001). Tacheas wee then sugically implanted s.c. into the dosum of anesthetized athymic nude mice. At 5 weeks afte tansplantation, the tacheas wee emoved, fixed ovenight in 4% paafomaldehyde in PBS, sectioned into 3mm-thick ings, and paaffin emedded. Tacheal coss-sections wee stained with hematoxylin and eosin, and the invasion of the tacheal wall was classified accoding to the level of penetation of the cells eing tested, with level 0 indicating no invasion and level 3indicating cell invasion though the entie tacheal wall. Immunolot analyses Levels of the poteins of inteest in keatinocytes compaed to contol cells wee measued y immunolot analysis as peviously descied (Lan et al., 2000, 2005). The potein content was detemined using the Bio-Rad D/C potein assay kit (Bio-Rad Laoatoies, Hecules, CA, USA). Potein was sepaated y SDS PAGE, tansfeed onto PVDF memanes (Millipoe, Bedfod, MA, USA), and incuated with pimay antiodies to phosphoylated Akt (Se473), phosphoylated GSK3 (Se9), phosphoylated p70 S6 kinase (Th389), phosphoylated S6 iosomal potein (Se235/236), phosphoylated 4E-BP1 (Se65), p70 S6 kinase, S6 iosomal potein (all fom Cell Signaling Technology, Bevely, MA, USA), H-Ras, RhoA, Akt (Santa Cuz Biotechnology, Santa Cuz, CA, USA), o 4E-BP1 (Lin et al., 1994) (a gift fom J Lawence). Susequently, the lots wee incuated with espective hoseadish peoxidase-laeled seconday antiodies (Bio-Rad, Hecules, CA, USA). Filtes wee epoed with antiodies against -actin (Sigma, St Louis, MO, USA) o tuulin ( Reseach Poducts, Camidge, MA, USA) to veify equal loading of potein. Bound antiodies wee visualized using enhanced chemiluminescence (Amesham Biosciences, Piscataway, NJ, USA). All immunolots shown ae epesentative of at least thee independent expeiments. Fo MAP kinase activity assays, keatinocytes wee havested in a uffe consisting of 20 mm MOPS, ph 7.2, 25 mm -glyceol phosphate, 5 mm EGTA, 1 mm sodium othovanadate, 1 mm dithiotheitol, containing 1 mg/ml each of apotinin, leupeptin, pepstatin, 1 mm sodium fluoide and 1mM Pefaloc. MAPK (ERK1/2) was immunopecipitated using anti-mapk antiody coupled to agaose eads (Upstate Biotechnology, Chalottesville, VA, USA) and then assayed fo associated phosphotansfease activity using the sustate, MPB. The phosphoylated MPB sustate was then analysed y Westen immunolot using an antiody specific fo phosphoylated MPB (Upstate Biotechnology, Chalottesville, VA, USA). To assay fo phosphotansfease activity associated with activated Akt1, keatinocytes wee pocessed fo Akt immunopecipitation followed y a kinase assay of the immunopecipitates with the sustate, BAD, y use of an Akt1/PKBa. Immunopecipitation Kinase Assay Kit otained fom Upstate Biotechnology (Chalottesville, VA, USA) and was pefomed accoding to the manufactue s potocol. Phosphoylated-BAD was analysed y Westen immunolot using an antiody specific fo phosphoylated BAD (Upstate Biotechnology, Chalottesville, VA, USA). Pull-down assays fo Ral, Rho, and Rac activation Levels of activated Ral-A-GTP, GTP-Rho, and Rac-GTP wee detemined in keatinocyte lysates using the Ral Activation Assay Kit, Rho Activation Assay Kit, and Rac Activation Assay Kit, espectively, accoding to the manufactue s instuctions (Upstate Biotechnology, Chalottesville, VA, USA). Using agaose eads ound to a GST fusion potein coesponding to esidues of a at Ral inding potein, Ral-GTP was pecipitated, and Ral A-GTP was detected y Westen immunolot analysis using an antiody specific fo Ral-A (Upstate Biotechnology, Chalottesville, VA, USA). Likewise, cell extacts wee incuated with eads coupled to the Rho-inding domain of Rhotekin, which inds specifically to activated RhoA (Rho-GTP). Pull-assays wee analysed y 12.5% SDS PAGE and immunolotting with antiody against RhoA (Santa Cuz Biotechnology, Santa Cuz, CA, USA) alongside coesponding total cell extacts fo nomalization of total RhoA potein levels. A simila appoach was used fo Rac-GTP analysis as that fo Rho- GTP, ut the pull-down assay was pefomed using agaose eads coupled to the Rac inding domain of GST-PAK. Pulldown assays and total cell lysates wee assayed y immunolot analysis using an antiody specific fo Rac (Upstate Biotechnology, Chalottesville, VA, USA). Acknowledgements We thank Ds Alex Mulle and Cheyl Hos fo valuale comments on the manuscipt. We also thank Gwen Gilliad fo histology assistance and Loetta Rossino fo manuscipt pepaation. This wok was suppoted y the NIH gant CA This wok was suppoted y Gant CA to SKG fom the National Cance Institute.

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