(2012) 31, 3547–3560 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE PEA-15 potentiates H-Ras-mediated epithelial transformation through D

FJ Sulzmaier1,2, MKG Valmiki1,2, DA Nelson3, MJ Caliva1,4, D Geerts5, ML Matter4, EP White3 and JW Ramos1,2

1Cancer Biology Program, University of Hawaii Center, University of Hawaii at Manoa, Honolulu, HI, USA; 2Department of Molecular Biosciences and Bioengineering, University of Hawai’i at Manoa, Honolulu, HI, USA; 3The Cancer Institute of New Jersey, New Brunswick, NJ, USA; 4Department of Cell and Molecular Biology, John A Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI, USA and 5Department of Pediatric Oncology/Hematology, Sophia Children’s Hospital, Rotterdam, The Netherlands

The small GTPase H-Ras is a proto-oncogene that Introduction activates a variety of different pathways including the extracellular-signal-regulated kinase (ERK)/mitogen- The small GTPase Ras is frequently mutated in cancer activated kinase pathway. H-Ras is mutated in and contributes to transformation and tumor progres- many human , and these mutations cause the sion (Dunn et al., 2005; Tan et al., 2005). Physiologi- protein to be constitutively active. Phosphoprotein cally, Ras is involved in a variety of signaling pathways enriched in astrocytes, 15 kDa (PEA-15) blocks ERK- regulating different outcomes (Cordova-Alarcon et al., dependent transcription and inhibits proliferation 2005). Guanosine-triphosphate-loaded Ras activates by sequestering ERK in the cytoplasm. We therefore many pathways, including the mitogen-activated protein investigated whether PEA-15 influences H-Ras-mediated kinase/extracellular-signal-regulated kinase (MAPK/ transformation. We found that PEA-15 does not block H- ERK) pathway, the PI3 kinase pathway and the Ras-activated proliferation when H-Ras is constitutively RalGDS pathway, all of which have known roles in active. We show instead that in H-Ras-transformed mouse cancer via diverse effects both on a cellular and kidney epithelial cells, co-expression of PEA-15 resulted physiological level. For example, Ras/MAPK/ERK in enhanced soft agar colony growth and increased tumor signaling modulates transcription, cell cycle progression, growth in vivo. Overexpression of both H-Ras and PEA- proliferation and differentiation, as well as senescence 15 resulted in accelerated G1/S cell cycle transition and and cell death (Ramos, 2008; Castaneda et al., 2010; increased of the ERK signaling pathway. PEA- Ferro and Trabalzini, 2010). 15 mediated these effects through activation of its binding Mutations in Ras such as K-Ras, N-Ras or partner (PLD1). Inhibition of PLD1 or H-Ras can be found in various tumors and are often interference with PEA-15/PLD1 binding blocked PEA- associated with poor prognosis (Cordova-Alarcon et al., 15’s ability to increase ERK activation. Our findings 2005; Zeng et al., 2010). Mutated H-Ras is associated reveal a novel mechanism by which PEA-15 positively with elevated expression of key cell cycle regulatory regulates Ras/ERK signaling and increases the prolifera- , such as cyclin D1 and cdk4, in in vivo models tion of H-Ras-transformed epithelial cells through of oral squamous-cell carcinoma of the head and neck enhanced PLD1 expression and activation. Thus, our region (Sathyan et al., 2007). Cordova-Alarcon et al. work provides a surprising mechanism by which PEA-15 (2005) showed that mutational activation of H-Ras augments H-Ras-driven transformation. These data reveal accelerates G1/S cell cycle transition in a human cervical that PEA-15 not only suppresses ERK signaling and cancer-derived cell line and has an important tumorigenesis but also alternatively enhances tumorigenesis role in the development of cervical tumors. Oncogenic in the context of active Ras. H-Ras also transforms NIH3T3 fibroblasts, at least in Oncogene (2012) 31, 3547–3560; doi:10.1038/onc.2011.514; part, through upregulation of cyclin D1 due to an published online 21 November 2011 overexpression of Kruppel-like factor 5 (Nandan et al., Keywords: PEA-15; PLD; cell cycle; H-Ras; cell 2004). Interestingly, mutations in H-Ras not only affect transformation gene transcription but also contribute to tumor cell transformation by inducing anti-apoptotic signals. In immortal baby mouse kidney epithelial cells (iBMK), a constitutively activated form of H-Ras inhibited pacli- Correspondence: Dr JW Ramos, Cancer Biology Program, University taxel-induced accumulation of the proapoptotic BH3- of Hawaii Cancer Center, University of Hawaii at Manoa, Biosciences only protein BIM, thereby preventing BIM-dependent Building, Room 241C, 651 Ilalo Street, Honolulu, HI 96813, USA. . In this model, H-Ras to ERK signaling E-mail: [email protected] Received 28 March 2011; revised 18 September 2011; accepted 9 October resulted in phosphorylation of BIM, leading to its 2011; published online 21 November 2011 proteasomal degradation (Tan et al., 2005). PEA-15 enhances H-Ras-mediated cell transformation FJ Sulzmaier et al 3548 The small -containing protein PLD1 mutant preventing binding to PEA-15 blocked phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) activation of the ERK/MAPK pathway. We have thus is a molecular scaffold that regulates several pathways identified a novel mechanism by which PEA-15 can including the Ras/MAPK/ERK signaling cascade positively regulate Ras/ERK/MAPK signaling and (Ramos, 2008; Revet et al., 2008). It has been shown increase the proliferation of H-Ras-transformed epithe- to bind to ERK and prevent ERK-mediated gene lial cells. The enhancement of Ras signaling was in part transcription (Formstecher et al., 2001; Whitehurst due to increased activity of PLD1 resulting from PEA-15 et al., 2004). This can result in a downregulation of cell expression. This is the first evidence to link PEA-15 proliferation, as shown in murine T cells (Pastorino regulation of Ras signaling to effects on PLD. et al., 2010). Interaction of PEA-15 with specific partners is controlled by its phosphorylation and . These factors therefore deter- Results mine the effects of PEA-15 on cellular signaling processes (Renganathan et al., 2005). Phospholipase PEA-15 enhances H-Ras-mediated epithelial cell D (PLD) binds to PEA-15 and is a widely expressed transformation in vitro in an adhesion-independent manner that catalyzes the of phosphatidyl- Expression of oncogenic H-Ras leads to a transformed to phosphatidic (PA) and choline (Wang in iBMK cells, resulting in adhesion- et al., 1999; Yang et al., 2001). Although the independent colony formation in soft agar assays physiological function of this interaction remains (Degenhardt and White, 2006). PEA-15 negatively regulates elusive, PEA-15 stabilizes PLD expression and may H-Ras to ERK signaling and blocks H-Ras-mediated activate it by a yet undefined mechanism (Zhang et al., proliferation signals by sequestering ERK in the 2000). PLD has been shown to modulate the Ras–ERK cytoplasm (Formstecher et al., 2001; Pastorino et al., pathway and regulate the cell cycle and cell prolifera- 2010). The ability of cells to grow in suspension is a tion (Yang et al., 2001; Donaldson, 2009). PLD activity hallmark of transformation. We therefore analyzed is crucial for Ras activation in NIH3T3 cells, where whether co-expression of PEA-15 would abolish PLD-produced PA is necessary for the recruitment of H-Ras-mediated transformation in iBMK cells and son of sevenless (SOS), a direct activator of Ras, to the inhibit Ras-induced growth in soft agar assays. Surprisingly, (Zhao et al., 2007). In addition, in Rat-1 stable co-expression of PEA-15 in the presence of active fibroblasts PA can associate with Raf-1 and target it to H-Ras enhanced soft agar colony growth. Cells stably the membrane. This Ras-independent recruitment is overexpressing both H-Ras and PEA-15 formed important for the subsequent activation of Raf-1 by significantly (Po0.0003) more colonies in soft agar Ras in these cells and is required for further down- compared with cells expressing H-Ras alone (Figure 1a). stream signaling (Rizzo et al., 2000). Elevated PLD Neither the control cell lines nor the cell lines stably activity is found in several tumors, including breast, expressing PEA-15 alone were able to form soft agar gastric and renal (Uchida et al., 1997, 1999; colonies. Co-expression of PEA-15 in cells expressing Noh et al., 2000; Zhao et al.,2000).PLDhasalsobeen the constitutively active form of H-Ras not only shown to have transforming properties in fibroblasts augmented the number of soft agar colonies formed, with aberrant activity and may con- but also increased the diameter of the colonies (Figures tribute to tumor progression in this context (Lu et al., 1b and c). We verified the overexpression of PEA-15 2000; Joseph et al.,2001).Furthermore,inv-Raf and H-Ras after stable transfection by immunoblot transformed NIH3T3 cells, increased PLD activity was (Figure 1d). able to overcome Raf-induced cell cycle arrest (Frankel et al.,1999;Josephet al., 2002). PEA-15 promotes G1- to S-phase transition and does not In this study, we analyzed the effects of PEA-15 alter anoikis expression on H-Ras-transformed mouse kidney epithe- The Ras/MAPK/ERK signaling cascade has been lial cells (iBMK). Surprisingly, we found that PEA-15 shown to be involved in the regulation of cell cycle does not block H-Ras proliferation signals as previously progression both in physiological and pathological described, but in this cellular context enhanced H-Ras- conditions (Cordova-Alarcon et al., 2005; Sathyan driven transformation independent of apoptosis. Co- et al., 2007; Viparelli et al., 2008). We therefore analyzed expression of H-Ras and PEA-15 resulted in enhanced whether the increased colony formation in soft agar was tumor formation in vivo and increased colony formation due to influences of PEA-15 expression on cell cycle in soft agar assays in vitro. Analysis of the cell cycle in regulation. Interestingly, suspended iBMK cells stably iBMK cells revealed an accelerated G1/S cell cycle expressing both H-Ras and PEA-15 had fewer cells at progression when both proteins were expressed. In this the G0/G1 phase and significantly increased cells in the context activity of the Ras/MAPK/ERK signaling S and mitotic phases compared with H-Ras expression pathway as well as the expression of major cell cycle alone. Cell lines stably expressing PEA-15 alone showed regulatory proteins were elevated. At the same time, a decreased number of cells in G0/G1 compared with the expression of both H-Ras and PEA-15 caused an control cell line, but no significant differences could be increase in PLD1 expression and activity compared observed in S and M phases (Figures 2a and b). with the expression of H-Ras alone. Finally, treatment Previous reports indicate that PEA-15 can enhance of these cells with inhibitors of PLD1 or expression of a tumorigenesis by inhibiting apoptosis (Formisano et al., 2005).

Oncogene PEA-15 enhances H-Ras-mediated cell transformation FJ Sulzmaier et al 3549 *** *** *** *** 200 400 m μ 300

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PEA-15 2826 a10 17 16 14 a6 a5 a HR-PEAPEA-15 H-Ras Control H-Ras a HR-PEA 26 Figure 1 PEA-15 enhances H-Ras-mediated epithelial cell transformation in vitro in an adhesion-independent manner. (a) The number of colonies formed by stably transfected iBMK cells in soft agar colony formation assays was analyzed after 14 days of incubation. (b) The colony diameter of colonies formed by stably transfected iBMK cells in soft agar after 14 days of incubation was measured. Statistically significant differences are marked with asterisks (***Po0.001). (c) Bright-field images shown depict average size and morphology of colonies formed by stably transfected cells (H-Ras and HR–PEA) in soft agar colony formation assays. White scale bars represent 200 mm. (d) Immunoblot analysis of iBMK cell lines stably transfected with H-Ras and/or PEA-15. were probed for H-Ras and PEA-15 expression.

Therefore, we investigated whether stable PEA-15 No significant differences were observed with cdc25c expression in iBMK cells would lead to protection from and cdc2. anoikis. In suspension, PEA-15 expression did not alter To exclude effects selected for during the generation anoikis significantly compared with control cell lines, as and culture of the stable transfected cell lines, we shown by the percentage of dead cells. The stable analyzed the expression of two key cell cycle regulatory expression of H-Ras significantly decreased the number proteins downstream of ERK, namely cyclin D and of cells undergoing anoikis. However, no significant cdk4, in a transient model. iBMK cells only carrying the differences were observed between cells stably expres- control vector were transiently transfected with H-Ras sing H-Ras alone and cells expressing both H-Ras and alone or H-Ras and increasing amounts of PEA-15. PEA-15 (Figure 2c). Expression of the transfected constructs was verified by immunoblotting. The findings in the transient model confirmed the results from the experiments with stable PEA-15 alters expression of cell cycle regulatory proteins transfected iBMK cells. Compared with control cells, The observed differences in the cell cycle of stably transient transfection with H-Ras increased the expres- transfected iBMK cell lines in suspension led us to sion of cyclin D but not the level of cdk4 (Figure 3b). analyze the expression levels of ERK-regulated cell cycle Transient co-expression of increasing amounts of PEA- proteins in these cells. As previously reported, H-Ras 15 elevated cyclin D expression levels and in addition enhanced cyclin D expression levels compared with induced increased cdk4 expression (Figure 3b). control cell lines (Lovec et al., 1994; Lavoie et al., 1996; To further elucidate the mechanism by which PEA-15 Nandan et al., 2004). Cells stably expressing both H-Ras alters the expression of cell cycle regulatory proteins in and PEA-15 had a slight increase in cyclin D expression the H-Ras-transformed iBMK background, we analyzed levels compared with cells expressing H-Ras alone. the mRNA expression levels of the affected proteins PEA-15 expression alone slightly induced cyclin D1 cyclin D and cdk4. Transient co-expression of PEA-15 expression compared with control cells (Figure 3a). and H-Ras significantly increased cyclin D transcription The cyclin-dependent kinase cdk4 is an important (Figure 3c). Transient transfection of H-Ras alone did binding partner for cyclin D and is necessary for G1–S not alter cyclin D mRNA expression compared with transition (Connell-Crowley et al., 1997). As described control cells. In contrast, cdk4 mRNA expression was previously, H-Ras transiently decreases cellular cdk4 not affected by the transient transfection of PEA-15. expression (Lazarov et al., 2002; Figure 3a). Moreover, Transient H-Ras expression decreased the amount of PEA-15 expression alone slightly lowered cdk4 levels cdk4 mRNA transcription both when transfected alone when cells were in suspension. Interestingly, PEA-15 and in combination with PEA-15 (Figure 3c). Moreover, was able to maintain cdk4 expression when co- co-transfection of PEA-15 with H-Ras did not augment expressed with H-Ras in suspended cells (Figure 3a). the effect of H-Ras on cdk4.

Oncogene PEA-15 enhances H-Ras-mediated cell transformation FJ Sulzmaier et al 3550 HR-PEA (26) H-Ras (a) 200 200 150 G1 M 150 G1 M 50 A S A S 100 100 *

cell counts 50 50 * 0 0 0 200 400 600 0 200 400 600 25 PEA15 (17) Control 200 200 % of cells 150 G1 M 150 G1 M A S A S 100 100 0 G0/G1 S M cell counts 50 50 HR-PEA H-Ras 0 0 0 200 400 600 0 200 400 600

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0 a6 a5 a 28 26 a10 17 16 14 H-RasHR-PEA PEA-15 Control Figure 2 PEA-15 promotes G1- to S-phase transition and does not alter cell anoikis. (a) Histograms of flow cytometric analysis of DNA content in stably transfected iBMK cells related to their stage in the cell cycle (a, apoptotic; G1, synthesis or mitotic phase) after 48 h culture in suspension. (b) Quantitative distribution of H-Ras or H-Ras and PEA-15 stably co-expressing cells (HR–PEA) in G0/ G1, S and M cell cycle phases after 48 h incubation in suspension. Each bar represents the average percentage of three different clones; measurements were done in triplicates. (c) Percentage of dead cells in stably transfected iBMK cell lines after incubation in suspension for 48 h shown as the percentage of cells in sub-G1 phase after staining with propidium iodide. Measurements were done in triplicates. Statistically significant differences are marked with asterisks (*Po0.05, ***Po0.001).

PEA-15 increases activation and expression levels of In parallel to our previous reports, we observed ERK/MAPK pathway members differences in the phosphorylation of the cytoplasmic The Ras–MAPK cascade is closely integrated into the ERK target RSK2 in the different iBMK cell lines regulation of the cell cycle and can influence the (Vaidyanathan et al., 2007). Suspended cells stably co- expression and activity of cell cycle regulatory proteins expressing H-Ras and PEA-15 showed enhanced RSK2 (Viparelli et al., 2008). In addition, PEA-15 has been phosphorylation compared with cells expressing H-Ras shown to regulate the ERK/MAPK signaling pathway alone (Figure 3a). However, PEA-15 overexpression (Formstecher et al., 2001; Pastorino et al., 2010). alone also augmented RSK2 phosphorylation compared Intriguingly, PEA-15 also potentiates Ras activation of with control cell lines in suspension. PEA-15 expression ERK by an adhesion-independent mechanism (Ramos not only increased phosphorylation of RSK2, but also et al., 2000). We therefore analyzed whether the changes caused a modest increase in RSK2 expression when cells in cyclin D and cdk4 expression coincide with increased were in suspension. ERK signaling in the stably transfected iBMK cell lines. Elk-1 is a nuclear target of ERK in Ras–MAPK We determined the activation and expression levels of signaling. We previously reported that PEA-15 can ERK, its cytoplasmic target RSK2 and the nuclear ERK restrict ERK to the cytoplasm and prevent phosphoryla- target Elk-1. PEA-15 and H-Ras individually as well as tion of nuclear targets in astrocytes and T in combination were able to activate ERK equally in (Formstecher et al., 2001; Pastorino et al., 2010). As adherent cells as reported previously (not shown; Ramos expected, expression of oncogenic H-Ras alone in- et al., 2000; Haling et al., 2010). Interestingly, cells in creased phosphorylation of Elk-1 in suspended stably suspension stably expressing both H-Ras and PEA-15 transfected iBMK cells (Figure 3a). Surprisingly, co- showed higher ERK activation compared with cell lines expression of PEA-15 and H-Ras further enhanced the expressing H-Ras or PEA-15 alone (Figure 3a). Expres- phosphorylation of Elk-1 in these cells. No phosphor- sion levels of ERK were unaltered in all iBMK cell lines ylation was detected when only PEA-15 was expressed. (Figure 3a). Transiently transfected cells in suspension showed similar results (Figure 3b). Transfection of H-Ras alone induced phosphorylation of ERK, and PEA-15 induces phosphorylation of ERK in the absence of co-transfection of PEA-15 further enhanced this effect. growth factors Indeed, the more PEA-15 transfected, the greater the PEA-15 can potentiate H-Ras activation of ERK increase in phosphorylated ERK (pERK;Figure 3b). (Ramos et al., 2000). PEA-15 can also increase

Oncogene PEA-15 enhances H-Ras-mediated cell transformation FJ Sulzmaier et al 3551 - - PEA-15 - + + + + H-Ras cyclin D H-RasHR-PEA PEA-15 Control H-RasHR-PEA PEA-15 Control cdk4 cyclin D pERK pERK cdk4 ERK ERK cdc2 pRSK2 (T573) H-Ras cdc25c RSK2 PEA-15 Tubulin pELK1(S383)

cyclin D cdk4 5 30 *** 4 20 3 pIND-PEA15 Raf 2 pIND pIND-PEA15 PEA15 10 +-+ --Pon 1 pERK

% mRNA Expression 0 0 ERK H-Ras - + + - + + PEA15 PEA-15 - - + - - + Figure 3 PEA-15 alters expression of cell cycle regulatory proteins and increases activation and expression levels of MAPK pathway members. (a) Western blot analysis of cell cycle regulatory proteins (left panel) or MAPK pathway members (right panel) in stably transfected iBMK cell lines kept in suspension for 48 h. (b) Western blot analysis of cell cycle regulatory proteins and MAPK pathway members in transiently transfected iBMK cells kept in suspension for 18 h. (c) mRNA expression analysis of cyclin D and cdk4 in transiently transfected iBMK cells kept in suspension for 18 h. Shown are the relative mRNA expression levels compared with the housekeeping gene. Statistically significant differences are marked with asterisks (***Po0.001). (d) Immunoblot of CHOK1 cells transiently transfected with ponasterone-inducible PEA-15 (pIND-PEA-15), vector control (pIND), PEA-15 or Raf. Cells were serum starved and then treated with ponasterone or vehicle control and immunoblotted for expression of pERK, total ERK and PEA-15. phosphorylation of ERK, in part, by inhibiting a prevent phosphorylation of nuclear ERK targets, as feedback loop that inactivates the Ras/MAPK pathway described previously in other cell types (Formstecher (Haling et al., 2010). In contrast, our results showed that et al., 2001; Whitehurst et al., 2004; Pastorino et al., transfection of PEA-15 in iBMK cells expressing active 2010). PEA-15 loses its ability to bind to ERK upon H-Ras increases phosphorylation of ERK (Figures 3a phosphorylation at serine 116 (Ser116; Renganathan and b). We therefore sought to determine whether PEA-15 et al., 2005). We therefore analyzed the phosphorylation is capable of activating ERK independent of prior status of PEA-15 at Ser116 in PEA-15-expressing iBMK activation of the Ras/Raf signaling cascade. We cell lines. All three independent cell lines stably co- analyzed the effects of inducible PEA-15 expression in expressing H-Ras and PEA-15 showed phosphorylation transiently transfected CHO cells in which Ras/MAPK of PEA-15 at Ser116 (Figure 4a). Minimal phosphoryla- signaling was shut down through starvation. Starvation tion at this site was observed in cells expressing PEA-15 of the cells resulted in the expected shut down of Ras– alone. The presence of PEA-15 in all cell lines was MAPK signaling as seen in the absence of pERK in the confirmed by immunoblotting for total PEA-15. vector control. Starvation of the cells did not alter the phos- To confirm that the phosphorylation at Ser116 resulted in phorylation of ERK when a constitutively active form a loss of the ability to sequester ERK in the cytoplasm, we of Raf was transfected or PEA-15 was constitutively analyzed the cellular localization of ERK by immunofluor- overexpressed. Importantly, induction of PEA-15 by escence staining of the stably transfected iBMK cells. In cells ponasterone in the serum-starved cells resulted in ERK expressing PEA-15 alone, ERK was localized predominantly activation to a similar level as the constitutively active in the cytoplasm as previously reported (Figure 4b). The controls (Figure 3d). Minimal activation of ERK was expression of H-Ras alone led to an increased translocation observed in the absence of ponasterone induction likely of ERK into the nucleus. PEA-15 co-expression with H-Ras due to low uninduced expression of PEA-15 in these cells. did not alter the translocation. In these cells, ERK was found both in the cytoplasm and the nucleus, with a slightly PEA-15 is phosphorylated at Ser116 and does not higher concentration of nuclear ERK (Figure 4b). sequester ERK in the cytoplasm when co-expressed with constitutively active H-Ras Ser116 phosphorylation of PEA-15 is necessary for its The elevated level of phosphorylated Elk-1 in cells enhancing effect on H-Ras-induced activation of ERK stably co-expressing H-Ras and PEA-15 led us to the The finding that PEA-15 is phosphorylated at Ser116 conclusion that in these cells PEA-15 is not able to when stably co-transfected with H-Ras into iBMK cells

Oncogene PEA-15 enhances H-Ras-mediated cell transformation FJ Sulzmaier et al 3552 DAPI ERK1/2 merge

HR-PEA PEA-15 (14) a10 26 28 PEA-15

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8 n.s. * - + + + H-Ras 6 - - + - PEA-15 WT - - - + PEA-15 S116A 4 pERK 2 ERK pERK expression 0 α-Tubulin control H-Ras WT S116A HR-PEA Figure 4 PEA-15 is phosphorylated at Ser116 and does not sequester ERK in the cytoplasm when co-expressed with constitutively active H-Ras. (a) Western blot analysis of PEA-15 phosphorylation at Ser116 and total PEA-15 expression in stably transfected iBMK cells. (b) Immunofluorescence staining of ERK1/2 in stably transfected iBMK cell lines plated on fibronectin showing the cellular localization of ERK1/2 in these cells. Nuclei are visualized by 4,6-diamidino-2-phenylindole (DAPI) staining. Both signals are also shown in a merged image. White scale bars represent 20 mm. Ser116 phosphorylation of PEA-15 is necessary for its enhancing effect on H-Ras-induced activation of ERK. (c) Western blot analysis of pERK and total ERK in iBMK cells transiently expressing combinations of H-Ras, PEA-15 wild type (WT) or a S116A mutant of PEA-15 that cannot be phosphorylated at Ser116. Cells were kept in suspension for 18 h. Phosphorylation of ERK was normalized to total ERK expression. Shown are the mean and s.e.m of three independent experiments. Statistically significant differences are marked with asterisks (*Po0.05).

made us further analyze the importance of this iBMK cells, expression of H-Ras doubled the amount of phosphorylation for the enhancement of H-Ras- PLD1 compared with control cells (Figure 5a). mediated cell transformation. We analyzed the ability Co-expression of both PEA-15 and H-Ras further of a constitutively unphosphorylated PEA-15 mutant enhanced this effect and resulted in a more than 3-fold (S116A) to increase H-Ras-induced activation of ERK. higher expression compared with the control cell line This mutant has an amino-acid substitution at the and 1.5-times higher than in cells expressing only H-Ras. Ser116 phosphorylation site that prevents phosphoryla- Transient overexpression of PEA-15 and H-Ras also tion. In a transient system, we transfected iBMK cells affected PLD1 activity. H-Ras and PEA-15 both with H-Ras in combination with wild-type PEA-15 or individually as well as in combination increased the the Ser116 phosphorylation-deficient (S116A) mutant. PLD1 activity of iBMK cells compared with control As expected transfection of H-Ras increased activation cells (Figure 5b). Co-expression of PEA-15 and H-Ras of ERK. PEA-15 co-transfection further enhanced this resulted in higher PLD1 activity increases than H-Ras effect. Interestingly, co-transfection of S116A failed to expression alone. show significant enhancement of the H-Ras-induced pERK elevation (Figure 4c). Levels of activated ERK were still higher than in control cells, but not PEA-15 co-localizes with PLD1 significantly increased compared with H-Ras-only- PEA-15’s ability to regulate PLD1 expression levels or expressing cells. activity has been shown to be dependent on the interaction of these two proteins, and targeting this interaction can impair PLD1 functions (Zhang et al., PEA-15 increases PLD1 expression and activity 2000; Viparelli et al., 2008). We analyzed the expression PEA-15 does not appear to block a feedback loop that pattern of PLD1 and PEA-15 in iBMK cells stably negatively regulates ERK in iBMK cells and so an expressing PEA-15 or H-Ras and PEA-15 to analyze alternative mechanism must explain the increased ERK H-Ras-mediated effects on the localization of these activity. PEA-15 also binds to and stabilizes PLD1, and proteins and to determine their co-localization. Immu- can lead to increased PLD activity (Zhang et al., 2000). nofluorescence images showed a similar pattern of PLD1 is involved in regulating both Ras–MAPK expression of both proteins (Figure 5c). High levels of signaling and cell cycle progression (Yang et al., 2001; PLD1 and PEA-15 could be found in the nuclear and Donaldson, 2009). PLD1 therefore was a promising cytoplasmic regions surrounding the nucleus. The candidate to mediate PEA-15’s effects on ERK activity proteins co-localize in those regions (yellow color) and and cyclin D/cdk4 expression. In transiently transfected that is independent from H-Ras expression. Upon

Oncogene PEA-15 enhances H-Ras-mediated cell transformation FJ Sulzmaier et al 3553 4

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Figure 5 PEA-15 increases expression and activity of PLD1 and co-localizes with it. (a) Western blot analysis of PLD1 expression in transiently transfected iBMK cells kept in suspension for 18 h. Shown are western blot (lower panel) and quantified expression levels normalized to a-tubulin. (b) PLD1 activity in transiently transfected iBMK cells after incubation in suspension for 18 h. Shown is the percentage of PLD1 activity compared with control cells. (c) Immunofluorescence staining of stably transfected iBMK cells plated on fibronectin-coated glass coverslips. Binding of specific primary antibodies against PLD1 or PEA-15 was visualized using fluorescence- labeled secondary antibodies (A488, green and Alexa 647, red). Slides were analyzed using a Leica TCS-SP5 confocal microscope. White scale bars represent 20 mm. Co-localization is shown in yellow. overexpression of H-Ras, both PEA-15 and PLD1 can we analyzed the phosphorylation status of ERK by be found in patches in the in addition to immunoblotting. Transient expression of either PEA-15 the nuclear and perinuclear localization. Within these or H-Ras resulted in elevated levels of pERK compared spots, both proteins co-localize as seen in the magnified with control cells (Figure 6a). Co-expression of both images (Figure 5c, right panel). proteins increased ERK activation even further than either protein individually. Treatment with either PLD1 inhibitor VU 0155069 or n-butanol impaired pERK in Inhibition of PLD1 reduces PEA-15-induced increase in cells transiently transfected with PEA-15, H-Ras or both ERK activation proteins. The greatest effects were observed in cells To verify that PEA-15’s influence on ERK activation is expressing both PEA-15 and H-Ras, where the inhibitor mediated through PLD1, we inhibited PLD1 and treatment resulted in about 60% less ERK activity. Both analyzed the effects on PEA-15-mediated activation of inhibitors had similar effects on the treated cell lines ERK. PLD was either inhibited with n-butanol or a (Figure 6a). To exclude cytotoxic effects as the main small- inhibitor (VU 0155069) that has been reason for the decreased ERK activation, we analyzed described previously to selectively inhibit enzyme func- the impacts of the PLD1 inhibitor VU 0155069 on the tion of the PLD1 isoform (Scott et al., 2009). To cell viability. Staining of VU-treated iBMK cells with evaluate the effects of this treatment on ERK activation, propidium iodide showed only a slight decrease in cell

Oncogene PEA-15 enhances H-Ras-mediated cell transformation FJ Sulzmaier et al 3554 control 0.4 VU n-Butanol --++--++H-Ras 0.3 - + - + - + - + PEA-15 --- -++++VU 0.2 pERK 0.1 tERK

pERK expression α-Tubulin 0 --++H-Ras - + - + PEA-15

50 120 150 * 100 40 control A 80 D 100 30 VU 60 20 40 50 % live cells Cell counts * 20 10 Soft Agar Colonies 0 0 0 100 101 102 103 104 control VU H-Ras HR-PEA PI Figure 6 Inhibition of PLD1 suppresses PEA-15’s effect on ERK phosphorylation, but only slightly impairs cell viability. (a) Normalized expression of pERK in transiently transfected cell lines treated with 1-butanol, VU 0155069 or the carrier control EtOH. Expression levels were determined by western blotting; levels of pERK are normalized to the expression of total ERK and a-tubulin. Shown are the mean expression levels from three independent experiments. Western blot of the phosphorylation status of ERK in transiently transfected iBMK cells treated with the PLD1-specific inhibitor VU 0155069 (50 mM, 30 min) or with the carrier control EtOH. (b) Histogram of flow cytometric analysis of viability of iBMK cells treated with VU 0155069 (black line) compared with cells treated with the carrier control EtOH (gray, solid) based on their propidium iodide (PI) uptake. Relative fluorescence emission of PI corresponds to live (A) or dead (D) cells. Right panel: percentage of live cells in iBMK cell population after incubation with VU 0155069 or carrier control. Measurements were done in triplicates. (c) Inhibition of PLD1 inhibits soft agar colony formation. iBMK cells stably expressing H-Ras or H-Ras and PEA-15 were preincubated for 30 min with 50 mM VU 0155069 or carrier control and then plated in soft agar. Formed colonies were counted after 14 days. Experiment was done in duplicates and repeated twice. Statistically significant differences are marked with an asterisk (*Po0.05).

viability. Compared with control cells the inhibitor (Viparelli et al., 2008). These findings in addition to our treatment resulted only in about 8% loss of live cells finding that PLD1 co-localizes with PEA-15 in iBMK (Figure 6b). The histogram shows similar curves for cells raised the question whether binding of the two control cells (gray) or VU-treated cells (black) with proteins is necessary for the observed enhancement of similar populations of dead cells (D) and cells alive (A) ERK activity in H-Ras-expressing iBMK cells. The after 30 min of inhibitor treatment. PEA-15 binding region of PLD1 has already been mapped to a C-terminal domain D4 (AA 712-1074) and the residues 762–801 within this domain have been Inhibition of PLD1 inhibits soft agar colony formation identified as the shortest PEA-15-binding segment As shown before, oncogenic H-Ras is able to transform (Figure 7a; Zhang et al., 2000; Doti et al., 2010). Here, iBMK cells (Degenhardt and White, 2006). We analyzed we used a PLD1 construct only consisting of the D4 whether PLD1 activity is necessary for this transforma- domain (Figure 7b) and lacking catalytic activity tion. We determined the effect of PLD1 inhibitor on soft (PLD1-D4). When overexpressed, this construct com- agar colony formation using stably transfected iBMK petes with the endogenous PLD1 and keeps it from cells. As previously shown, cells expressing H-Ras were binding to PEA-15. We analyzed whether transient able to form colonies independent from adhesion to a overexpression of this mutant in addition to transient , and PEA-15 co-expression greatly magnified expression of H-Ras and PEA-15 would affect the this effect (Figure 6c). Pretreatment of cells with the previously observed changes in pERK. Without over- PLD1-specific inhibitor VU 0155069 significantly in- expression of mutant PLD1-D4, H-Ras expression led hibited the formation of colonies in cells expressing to increased ERK activation and PEA-15 enhanced this PEA-15 and H-Ras. effect as expected (Figure 7a). Interestingly, co-expres- sion of PLD1-D4 prevented the increase in pERK upon Binding of PEA-15 to PLD1 is required for PEA-15’s H-Ras and PEA-15 co-transfection. pERK levels in effect on H-Ras-mediated enhancement of ERK activity these cells are not significantly different from cells only PEA-15 can bind to PLD1, and it has been shown that expressing H-Ras. There was no statistically significant disruption of the PEA-15–PLD1 interaction can impair effect of the PLD-D4 mutant on H-Ras-only-transfected downstream PLD1 effects such as PKC-a activation cells (Figure 7a).

Oncogene PEA-15 enhances H-Ras-mediated cell transformation FJ Sulzmaier et al 3555 8 w/o PLD1-D4 w/ PLD1-D4 This elevated PEA-15 expression is peculiar to clear cell RCC, as the unrelated renal Wilms’ tumor, or Ewing sarcoma, shows only low PEA-15 expression, which is 6 not significantly correlated to important clinical para- meters (results not shown). PLD1 expression is not 4 significantly different in clear cell RCC and normal kidney tissue (Figure 8c). However, PLD1 expression is lower in Wilms’ tumor and Ewing sarcoma (results not

pERK expression 2 shown). Our data was further confirmed by results obtained with two other tumor series in the public 0 domain (results not shown): Li-39 (for Ewing) and control H-Ras HR-PEA15 EXPO-261 (for kidney).

pERK Discussion ERK

α Our findings reveal a novel mechanism by which PEA-15 -Tubulin can enhance H-Ras/MAPK signaling and contribute to H-Ras-mediated transformation of iBMK cells. In this epithelial cell line, co-expression of H-Ras and 1 PX PH CI CII LOOP CIII CIV CT 1074 PLD1 PEA-15 increased expression of cell cycle regulatory 712 CIII CIV CT 1074 PLD1 D4 proteins cyclin D and cdk4, as well as activity of the PEA15 binding ERK/MAPK pathway in an adhesion-independent manner. This resulted in accelerated G1/S cell cycle Figure 7 Binding of PEA-15 to PLD1 is required for PEA-15’s effect on H-Ras-mediated enhancement of ERK activity. transition and a higher tumor formation rate both (a) Western blot analysis of pERK and total ERK in iBMK cells in vitro and in vivo. PEA-15 acted in part through PLD1, transiently expressing combinations of H-Ras, PEA-15 and the whose expression level and activity were elevated in cells PEA-15- mutant of PLD1 (PLD1-D4). Cells were overexpressing PEA-15. Inhibition of PLD1 catalytic kept in suspension for 18 h. pERK levels are normalized to the total ERK expression. Shown are the mean and s.e.m of three activity or interference with PEA-15 binding to PLD1 independent experiments. (b) Structure of PLD1 and its PEA-15- abolished the enhancing effect of PEA-15 on ERK containing the D4 domain used in the experiment. activation. This suggests that PEA-15 influences H-Ras- Boxes show functional domains; the PEA-15-binding site is mediated cell transformation through increased PLD1 highlighted. CT, C terminus; CI–CIV, conserved regions; PH, signaling. pleckstrin domain; PX, phox homology domain. Previous reports have shown the ability of H-Ras to contribute to cell transformation. Ras has been reported in various cell types to affect transcription, drive cell PEA-15 promotes earlier formation of tumors in vivo cycling and proliferation or to block apoptosis and To elucidate whether the observed effects of PEA-15 on anoikis (Macaluso et al., 2002; Cordova-Alarcon et al., H-Ras-mediated cell transformation are in vitro artifacts 2005; Viparelli et al., 2008). Degenhardt and White or also apply in vivo, we analyzed how PEA-15 (2006) described that overexpression of constitutively expression alters active H-Ras transformation in the active H-Ras (G12V) is enough to transform iBMK presence of a tumor microenvironment in nude mice. No cells. H-Ras promotes the proteasomal degradation of tumors developed when the control cell line or the cell the proapoptotic Bcl-2 family protein BIM through the line stably expressing PEA-15 alone was injected. The Raf/MAPK pathway in these cells (Tan et al., 2005). injection of cells stably expressing H-Ras led to the Conversely, PEA-15 has generally been described to formation of tumors in mice. Importantly, we observed block Ras/MAPK-mediated gene transcription and to on average larger tumors when injecting H-Ras and inhibit proliferation by sequestering ERK in the PEA-15 co-expressing cells compared with an injection cytoplasm (Formstecher et al., 2001; Pastorino et al., of cells expressing H-Ras alone. Furthermore, tumors 2010). Moreover, in ovarian cancer cells PEA-15 has resulting from injection of H-Ras and PEA-15 been shown to exert tumor-suppressive effects, at least co-expressing cells formed earlier than those arising in part, through induction of ERK-dependent autop- from H-Ras expression alone (Figure 8a). hagy. However, the mechanism of PEA-15’s involve- ment is not fully understood and it is unclear whether PEA-15 is significantly overexpressed in human clear cell H-Ras signaling is involved in this regulation RCC tissue samples (Bartholomeusz et al., 2008). To further investigate the in vivo relevance of our In contrast to reports suggesting a tumor-suppressor findings, we analyzed the expression levels of PEA-15 function of PEA-15, our findings confirm that PEA-15 and PLD1 in human tumor tissue samples and normal expression can also potentiate ERK activation as we kidney tissue. PEA-15 expression was significantly have previously described (Ramos et al., 2000). How- higher in clear cell renal cell carcinoma (RCC) ever, oncogenic H-Ras expression remains necessary for compared with normal kidney tissue (Figures 8b and c). full transformation, as overexpression of PEA-15 alone

Oncogene PEA-15 enhances H-Ras-mediated cell transformation FJ Sulzmaier et al 3556 1000 2 2.0

800 Control PEA-15 (14) 1.5 PEA-15 (16) 600 -7 H-Ras P = 1.9 x 10 400 HR-PEA (12) 1.0 HR-PEA (26) 200 0.5 Expression Units Tumor volume in mm 0 0 0 20 40 60 80 Kidney ccRCC Time in days

P = 0.35 2000 Plasma membrane -4 250 1800 P = 8.1 x 10

1600 -3 P = 4.9 x 10 200 PC PA PA PC Sos 1400 PLD H-Ras Raf PLD 1200 150 12GTP 1000 P 800 100 P PEA15 P P PEA15 600 3 ERK PEA15 400 50 cdk4 200 cyclinD PLD1 mRNA expression PEA15 mRNA expression 0 Ewing (37) ccRCC (10) Kidney (12) Wilm’s (27) 0 ccRCC (10) Kidney (12) S

G2 G1 M

Figure 8 PEA-15 co-expression promotes earlier H-Ras tumor formation in nude mice in vivo; PEA-15 expression is elevated in human clear cell RCC (ccRCC) samples. (a) Tumor formation in nude mice was followed after injection of iBMK cells stably expressing H-Ras, PEA-15, HR–PEA or control vector. Graphs represent changes in tumor volume from up to 60 days. Mice were euthanized once the tumor burden was too great. (b) Differential expression analysis of PEA-15 levels in human kidney samples and human ccRCC samples showing the relative protein expression units. Data were extracted from the Oncomine cancer microarray database. As a corrected measure of significance t-statistics with false discovery rates were used. (c) PEA-15 and PLD1 expression in different tumor types and normal kidney. The data shown are from the Francesconi-37 Ewing sarcoma series (for Ewing), or from the Kort-79 mixed kidney series (for all other samples). Statistical analysis was performed using the non-parametric Kruskal–Wallis test; Po0.05 was considered significant. The error bars represent the s.e.m. (d) Model of a proposed mechanism by which PEA-15 can increase H-Ras-driven activation of ERK. Overexpression of PEA-15 in iBMK cells overexpressing constitutively active H-Ras causes higher PLD1 activity either through higher PLD1 expression levels or a yet undefined activation mechanism. Subsequently, PLD1 produces increased amounts of the signaling molecule PA. PA then increases recruitment of the guanine nucleotide exchange factor SOS (1) and/or the kinase Raf (2) to the plasma membrane. H-Ras is constitutively bound to the plasma membrane and is immediately able to induce higher signaling rates upon guanosine triphosphate loading. This leads to the observed downstream effects, such as increased ERK activity and cell cycle progression. In this scenario, PEA-15 is phosphorylated at Ser116. Unphosphorylated PEA-15 (3) is able to bind ERK and sequester it in the cytoplasm as previously reported.

did not lead to a transformed phenotype. PEA-15 loop in which ERK phosphorylates fibroblast receptor overexpression in iBMK cells prevents ERK transloca- substrate 2a to shut down Ras signals in fibroblasts. We tion to the nucleus as previously reported (Formstecher report an alternative mechanism by which PEA-15 can et al., 2001). However, when co-transfected with H-Ras, activate ERK through direct activation of PLD1, which we could not observe this effect. ERK is not sequestered leads to activation of Ras/MAPK signaling upstream of in the cytoplasm and can translocate into the nucleus. ERK. We found that even under serum-starved condi- This is likely due to phosphorylation of PEA-15 at tions lacking any initial Ras signaling, PEA-15 is still Ser116, a phosphorylation that decreases PEA-15’s capable of activating ERK to a similar level as Ras. binding to ERK (Renganathan et al., 2005). A The PEA-15 binding partner PLD1 had a part in this phosphorylation-deficient mutant of PEA-15 was not context. PLD1 has been described previously to be able to further enhance H-Ras-driven enhancement of involved in the regulation of many signaling pathways ERK activity. Thus, phosphorylation at Ser116 is including the Ras–ERK pathway, thereby driving cell required for PEA-15 effects on H-Ras/ERK MAP cycling and proliferation (Yang et al., 2001; Donaldson, kinase signaling. 2009). PLD1 is activated by H-Ras and has been proven Ras–ERK signaling is regulated through a system of to be crucial for the transformation of rat fibroblasts by positive and negative feedback loops (Ramos, 2008). H-Ras (Buchanan et al., 2005; Shi et al., 2007). PLD1 Haling et al. (2010) reported that PEA-15 can increase has been reported to regulate Ras–ERK signaling ERK activation by interrupting a negative feedback both upstream and downstream of Ras through the

Oncogene PEA-15 enhances H-Ras-mediated cell transformation FJ Sulzmaier et al 3557 generation of PA. PA is crucial for epidermal growth nucleotide exchange factor SOS recruited to the plasma factor-induced activation of Ras because of its recruit- membrane as described by Hancock (2007). H-Ras is ment of the guanine nucleotide exchange factor SOS to bound to the plasma membrane and activated upon the plasma membrane (Hancock, 2007). However, PA guanosine triphosphate loading by SOS (Figure 8d, no. 1). seems to also exert effects downstream of Ras where it is Alternatively, increased PA can result in a higher necessary to allow Ras binding to Raf-1 (Rizzo et al., recruitment of the kinase Raf to the membrane 2000). Both studies show that PLD1 is an important and enhance Ras–Raf binding (Figure 8d, no. 2) as in Ras–MAPK signaling and acts as a positive previously shown by Rizzo et al. (2000). Both mechan- regulator. isms can lead to the observed downstream effects and We observed higher levels of PLD1 upon over- result in elevated ERK activity. Although both path- expression of PEA-15 in iBMK cells. This may have ways are possible, an enhancement of Ras–MAPK been due to a stabilization of PLD1 levels by PEA-15 as signaling at the Raf level seems more likely in our study. previously described (Zhang et al., 2000). In iBMK cells, Transfected oncogenic H-Ras G12V already is consti- PEA-15 and PLD1 show co-localization. PEA-15 co- tutively active, making further enhancement of the localizes with PLD1 in the nucleus and in perinuclear signaling cascade by PEA-15 upstream of H-Ras less regions, and upon H-Ras co-expression also in regions likely. In the presence of oncogenic H-Ras, PEA-15 is at the cell membrane. Additionally, overexpression of phosphorylated at Ser116 and does not restrict ERK to PEA-15 or H-Ras increased PLD1 activity. Possibly due the cytoplasm. Note that unphosphorylated PEA-15 has to the increased levels of protein, PLD1 activity was been previously shown to negatively regulate Ras– higher when both H-Ras and PEA-15 were expressed as MAPK signaling by preventing ERK translocation into compared with overexpression of H-Ras alone. the nucleus (Figure 8d, no. 3; Formstecher et al., 2001; To assess the involvement of PLD1 in H-Ras- Pastorino et al., 2010). mediated transformation in iBMK cells, we used two On the basis of these results we propose a novel different inhibitor approaches. We blocked PLD1 function of PEA-15 in enhancing H-Ras-driven trans- catalytic activity with the commonly used but less formation. This effect was also observed in an in vivo specific inhibitor n-butanol, and also looked at the mouse model, where PEA-15 co-expression promoted effects of a PLD1-specific inhibitor VU 0155069. This earlier and larger tumor formation. H-Ras-activating potent small-molecule inhibitor has been described to mutations are common among various kinds of human selectively inhibit the PLD1 isoform (Scott et al., 2009). malignancies, and about 30% of human cancers show We analyzed phosphorylation of ERK as a major mutations in the Ras gene that cause the gene product to transforming signaling pathway in these cells (Tan et al., be constitutively active (Dunn et al., 2005). Addition- 2005) and used it as a readout after interfering with ally, other common mutations in tumors, such as PLD1 catalytic activity. Treatment of iBMK cells with activating epidermal growth factor receptor mutations, inhibitors of PLD1 hydrolytic activity blocked ERK can induce increased H-Ras activity as shown in human phosphorylation upon both H-Ras and H-Ras/PEA-15 RCCs (Barrett et al., 2009). Interestingly, these tumors co-overexpression, resulting in up to a 60% decrease in also show elevated PEA-15 expression levels compared ERK activity. PLD1 inhibition in this experiment only with normal kidney tissues, mainly in clear cell RCC slightly reduced the viability of the cells. A loss of PLD1 (Figures 8b and c). Renal Wilms’ tumor or Ewing catalytic activity also resulted in inhibition of soft agar sarcoma, tumors in which no Ras mutations are growth. Neither H-Ras nor H-Ras/PEA-15 co-expres- involved in the , show only low PEA-15 sing cells formed colonies after pretreatment with the expression (Figure 8c; Waber et al., 1993; Radig et al., specific PLD1 inhibitor. We further analyzed the 1998). involvement of PLD1 by putting special focus on its These findings together with our results in mouse binding to PEA-15. We used the PLD1-D4 domain as a kidney epithelial cells indicate that PEA-15 can potenti- mutant construct that specifically keeps functional ate tumorigenesis in the context of oncogenic Ras endogenous PLD1 from binding to PEA-15. This signaling. Thus, PEA-15 can either enhance or impair allowed us to analyze the importance of this protein tumorigenesis depending on the signaling pathways interaction for the activation of ERK in the iBMK active in the specific tumor cell. system. Co-expression of mutant PLD1-D4 blocked the increase of pERK upon H-Ras and PEA-15 co- expression, further confirming the necessity of func- tional PLD1 and an intact PLD1–PEA-15 interaction Materials and methods for PEA-150s ability to increase H-Ras mediated ERK activation. Cell lines and reagents On the basis of our findings and previous reports, Parent iBMK cells were developed in the laboratory of Eileen White and described previously (Tan et al., 2005). Stable PEA-15 could act at least in part through two different iBMK cells expressing PEA-15, H-Ras, H-Ras and PEA-15 ways to enhance H-Ras-mediated cell transformation (HR–PEA) or the vector control were derived by electropora- through PLD1 (Figure 8d). Overexpression of PEA-15 tion with pcDNA1.H-rasV12 (Lin et al., 1995), pIRESpuro3- results in higher PLD1 activity, which leads to increased PEA-15 or pcDNA3.1 (Tan et al., 2005), followed by selection amounts of the signaling molecule PA. Elevated with G418 (vector control, H-Ras) or puromycin (PEA-15 and signaling through PA can enhance levels of the guanine HR–PEA). For every type, three independent cell lines were

Oncogene PEA-15 enhances H-Ras-mediated cell transformation FJ Sulzmaier et al 3558 generated and analyzed. Cells were grown in DMEM (Thermo Each cell line was examined in triplicate, and for each Fisher Scientific Inc., Waltham, MA, USA) supplemented with measurement approximately 20 000 cells were analyzed. 4mM L-glutamine, 10% fetal bovine serum, nonessential amino , penicillin/streptomycin and 800 mg/ml G418 or 10 mg/ml puromycin. The successful transfection was further mRNA expression analysis confirmed by western blotting. In addition, iBMK cells iBMK cells carrying the vector control pcDNA3.1 were carrying the vector control pcDNA3.1 were transiently transiently transfected with 2 mg of H-Ras, PEA-15 or both transfected with the constructs mentioned above using constructs. At 48 h after transfection, cells were harvested, Lipofectamine (Invitrogen, Carlsbad, CA, USA) as described plated on agarose-coated dishes (to prevent adherence) and by the manufacturer’s protocol. For suspension cultures, cells incubated for 18 h. Samples were lysed with Qiagen RLT lysis were harvested and plated on cell culture dishes coated with buffer (Qiagen Inc., Valencia, CA, USA). The lysates were 3% agarose for the indicated amount of time. homogenized using QIAShredder columns (Qiagen). RNA was Antibodies used were specific for H-Ras (C-20), RSK2 extracted using Qiagen’s RNeasy Mini kit according to the (C-19), cdk4 (C-22), cdc25c (C-20), a-tubulin (DM1A), actin manufacturer’s protocol. RNA integrity was assessed on RNA (C-11), PEA-15 (H-80; Santa Cruz Biotechnology, Santa Cruz, Nano 6000 chips and 2100 Agilent Bioanalyzer (Agilent CA, USA); cdc2, (eBioscience, San Diego, CA, USA); cyclin D Technologies, Santa Clara, CA, USA). RNA was converted (DCS6), p44/42 MAPK (Erk1/2), phospho-p44/42 MAPK to complementary DNA (cDNA) using the High Capacity (Erk1/2; Thr202/ Tyr204), phospho p90RSK (Thr573), PLD1 cDNA RT Kit with RNase Inhibitor (Life Technologies (, Danvers, MA, USA); phospho-Elk1 (S383; Corporation, Carlsbad, CA, USA). cDNA levels were New England Biolabs, Ipswich, MA, USA); phospho PED/ quantified using quantitative PCR on an HT 7900 FAST PEA 15 (pS116; Invitrogen); PLD1 (N-term; Epitomics, real-Time PCR System (Applied Biosystems, Foster City, CA, Burlingame, CA, USA); and PEA-15 (from Dr Herve´ USA). Quantitative PCR was performed on a 96-well plates in Chneiweiss, Institut National de la Sante´et de la Recherche triplicates for the two genes of interest (CCND1 and cdk4) and Medicale´, Paris, France). Ponasterone was purchased from -actin as housekeeping gene. Primer assays for genes of Sigma-Aldrich Corp. (St Louis, MO, USA) and VU 0155069 interest and housekeeping gene (CCND1 and cdk4 QuantiTect from Tocris Bioscience (Ellisville, MO, USA). SYBRGreen assays) were purchased from Qiagen.

Western blotting Immunofluorescence Cells were cultured as described. After 48 h of incubation, cells Each iBMK cell line was grown on glass coverslips coated with were lysed with ice-cold M2 buffer as previously described 10 mg/ml fibronectin under regular cell culture conditions as (Ramos et al., 2000). Cell lysate protein was resolved by SDS– described above. When about 50% confluent, cells were fixed in polyacrylamide gel electrophoresis. Expression and phosphor- 2% paraformaldehyde, permeabilized in 0.1% Triton X-100 in ylation of proteins were determined by western blotting using phosphate-buffered saline and blocked with 1% normal goat specific antibodies. Binding of primary antibodies was detected serum. Cells were stained with primary antibody against ERK1/ using either horseradish peroxidase-conjugated secondary 2 (p44/42; Cell Signaling), PEA-15 (Santa Cruz Biotechnology, antibodies (Cell Signaling) or IRDye 680 goat anti-mouse no. sc-166678) or PLD1 (Epitomics, no. 2315-1). Alexa Fluor and IRDye 800 goat anti-rabbit antibodies (Li-Cor Biosciences, 488, 594 and 647 secondary antibodies (Invitrogen) were used to Lincoln, NE, USA). Bands were visualized by autoradiography fluorescently label bound primary. Coverslips were mounted on after incubation with enhanced chemiluminescence reagents slides using 40,6-diamidino-2-phenylindole containing mounting (PerkinElmer, Waltham, MA, USA) or detected using an medium (Vector Labs, Burlingame, CA, USA) or Fluoromount Odyssey Infrared Imaging System (Li-Cor Biosciences, Lin- G (no. 17984-25, Electron Microscopy Sciences, Hatfield, PA, coln, NE, USA). Scanned blots were cropped to improve clarity USA). Images were acquired on a Leica TCS-SP5 confocal and conciseness of presentation using Adobe Photoshop CS microscope (Leica Microsystems, Buffalo Grove, IL, USA) (Adobe Systems Inc., San Jose, CA, USA). using a  62 oil immersion objective or a Zeiss Axiovert 100 M fluorescent microscope using a  100 oil-immersion objective Soft agar colony formation assay and Zeiss AvioVision acquisition software (Carl Zeiss Micro- Equal cell numbers (1  104) of each stably transfected iBMK Imaging Inc., Thornwood, NY, USA). cell line were cultured for 14 days suspended in Dulbecco’s modified Eagle’s Medium (DMEM) in a 0.3 % agar solution on agar (0.6% in DMEM)-coated culture plates. Formed colonies PLD1 activity assay in the soft agar assay were counted and analyzed using a Bio- Transiently transfected iBMK cells carrying the vector control Rad imager and Quantity One software v4.6 (Bio-Rad, Hercules, pcDNA3.1 were lysed by three freeze and thaw cycles in ice- CA, USA). For the inhibitor assay, cells were preincubated with cold Tris (50 mM, pH 8.0). PLD activity was measured using the Amplex Red Phospholipase D Assay Kit (Invitrogen) 50 mM VU 0155069 for 30 min and an equal inhibitor concentra- tion was added to the soft agar solution. following the manufacturer’s protocol.

Cell cycle analysis and viability assay Tumor formation in nude mice Equal numbers of iBMK cells (2 Â 105) from each cell line were Tumor formation in nude mice was performed as previously plated on agarose-coated dishes to keep the cells in suspension. described (Streit et al., 1999). In brief, equal cell numbers of Cells were cultured for 48 h as described above. Subsequently, each iBMK cell line were injected subcutaneously in the cells were fixed in 70 % EtOH and stained in permeabilization abdominal region of 6-week-old nude mice (Taconic, German- buffer containing 100 mg/ml propidium iodide, 50 mg/ml town, NY, USA). We used five mice for each iBMK cell line RNAase and 0.2% Triton X. For the viability assay, cells and followed tumor growth for 60 days. Tumor growth were cultured as indicated and stained with propidium iodide. was monitored weekly by measurements with a 6-inch dial Cells were analyzed using a FASCcan flow cytometer and caliper (General Tools Mfg Co., New York, NY, USA). Mice CellQuest software (Becton Dickinson, San Jose, CA, USA). were killed once the tumor had reached 500 mm2 size. All

Oncogene PEA-15 enhances H-Ras-mediated cell transformation FJ Sulzmaier et al 3559 experiments were approved by the Institutional Review Board Center—University of Amsterdam. R2 can be accessed at of Rutgers, The State University of New Jersey. http://r2.amc.nl. Statistical analysis Analysis of microarray expression arrays in the public domain All results are shown as mean±s.e.m. Statistical significance CEL data from the public Affymetrix (Affymetrix Inc., Santa was determined using one-way analysis of variance, followed Clara, CA, USA) data sets (Barrett et al., 2009) for the by Bonferroni’s multiple comparison test for all data sets, Francesconi-37 (GSE12102), Kort-79 (GSE11024), Li-39 except for the human tumor expression analysis, where the (GSE6120) and Tumor Kidney EXPO-261 (GSE2109) series non-parametric Kruskal–Wallis test was used. P-values o0.05 were downloaded from the NCBI Omnibus were considered significant. (GEO) site and analyzed as described in (Revet et al., 2008). Annotations and clinical data for these series are available from http://www.ncbi.nlm.nih.gov/geo/. Affymetrix probe sets Conflict of interest were selected using the R2 bioinformatic platform (see below). All gene transcript levels were determined from data image The authors declare no conflict of interest. files using GeneChip operating software (MAS5.0 and GCOS1.0; Affymetrix). Samples were scaled by setting the average intensity of the middle 96% of all probe set signals to a Acknowledgements fixed value of 100 for every sample in the data set, allowing comparisons between microarrays. The TranscriptView We thank Dr Maarit Tiirikainen and the University of Hawaii genomic analysis and visualization tool was used to check Cancer Center Genomics Shared Resource laboratory for their whether the probe set selected had an antisense position in an assistance with the mRNA expression analysis. We also thank exon of the gene (http://bioinfo.amc.uva.nl/human-genetics/ Anna Knapinska, Shirley Young-Robbins and Marci Take- transcriptview/). The probe sets selected for a gene showed the moto for their excellent technical assistance. This work was highest expression in samples containing a present call for that supported by the National Institutes of Health National gene: 200788_s_at (PEA-15) and 226636_at (PLD1). All Cancer Institute (R01CA93849 to JWR) and National Institute essential conclusions remain the same for the additional valid of General Medicine (R01GM088266 to JWR) and the Victoria probe sets 200787_s_at (for PEA-15) and 177_at or 205203_at S and Bradley Geist Foundation (to JWR). MLM was (for PLD1). All analyses were performed using R2; an supported by NIH National Center for Reasearch Resources Affymetrix analysis and visualization platform developed in Grant RR016453. DAN and EPW were supported by a grant the Department of Human Genetics at the Academic Medical from the National Institutes of Health (R37CA53370 to EPW).

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