Aneta Swat

Ignacio Dolado

Ana Igea

Gonzalo Gomez-Lopez

David G Pisano

Ana Cuadrado

Angel R Nebreda
email: angel.nebreda@irbbarcelona.org

Expression and functional validation of new

Keywords: p38 MAPK, Ras oncogene, gene expression, EGFR Biochemical

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

p38α mitogen-activated protein kinase (MAPK) is an ubiquitously expressed serine/threonine protein kinase, which is activated by many extracellular stimuli and coordinates numerous cellular processes [START_REF] Cuenda | p38 MAP-kinases pathway regulation, function and role in human diseases[END_REF][START_REF] Cuadrado | Mechanisms and functions of p38 MAPK signalling[END_REF]. p38α can also negatively regulate malignant cell transformation, which seems to be accounted for by its inhibitory role on cell cycle progression and its stimulatory effect on apoptosis and differentiation [START_REF] Bulavin | p38 MAP kinase's emerging role as a tumor suppressor[END_REF][START_REF] Wagner | Signal integration by JNK and p38 MAPK pathways in cancer development[END_REF].

Intriguingly, despite the recognized importance of p38α in the regulation of gene expression [START_REF] Crump | Stress-activated MAP kinases in chromatin and transcriptional complexes[END_REF][START_REF] Perdiguero | Transcriptional regulation by the p38 MAPK signaling pathway in mammalian cells[END_REF][START_REF] Ambrosino | TEF-1 and C/EBPbeta are major p38α MAPK-regulated transcription factors in proliferating cardiomyocytes[END_REF][START_REF] Adam | Computational identification of a p38SAPK-regulated transcription factor network required for tumor cell quiescence[END_REF][START_REF] Ferreiro | Whole genome analysis of p38 SAPK-mediated gene expression upon stress[END_REF], little is known about how this p38α function impinges on tumorigenesis. In fact, most of the known tumor suppressor mechanisms coordinated by p38α occur at the posttranslational level. These include the post-translational regulation of ERK1/2 pathway activity, protein stability of cyclin D1, subcellular localization/stability of Cdc25 phosphatases, EGF receptor (EGFR) intracellular fate, and upregulation of cell cycle inhibitors such as p21 Cip1 or p27 Kip1 [START_REF] Bulavin | p38 MAP kinase's emerging role as a tumor suppressor[END_REF][START_REF] Wagner | Signal integration by JNK and p38 MAPK pathways in cancer development[END_REF]. There are a few examples on the regulation of gene expression by p38α with potential relevance for malignant transformation, most notably the transcriptional control of cyclin D1 [10] or the cell cycle inhibitor p16 INK4a [START_REF] Bulavin | Inactivation of the Wip1 phosphatase inhibits mammary tumorigenesis through p38 MAPK-mediated activation of the p16(Ink4a)-p19(Arf) pathway[END_REF], and the upregulation of EGFR mRNA in lung tumors of p38α-deficient mice [START_REF] Ventura | p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation[END_REF].

We have investigated how transcriptional regulation contributes to the tumor suppressor effect of p38α, by comparing whole-genome expression profiles of wild-type (WT) and p38αdeficient (p38α-/-) mouse embryo fibroblasts (MEFs) expressing oncogenic H-Ras G12V . Our previous work showed that p38α deficiency facilitates H-Ras G12V -induced transformation in these cells [START_REF] Alfonso | Proteomic analysis of p38alpha mitogen-activated protein kinase-regulated changes in membrane fractions of RAS-transformed fibroblasts[END_REF][14][START_REF] Swat | Cell density-dependent inhibition of epidermal growth factor receptor signaling by p38alpha mitogen-activated protein kinase via Sprouty2 downregulation[END_REF]. Altogether, we have identified a group of genes that are regulated by p38α in the context of malignant cell transformation, and have also functionally validated 9 of these genes in anchorage-independent cell growth assays. Interestingly, we have found that around 10% of the genes whose expression is negatively regulated by p38α can potentially enhance EGFR signalling. These results provide further evidence for a key role of EGFR pathway down-regulation in tumor suppression by p38α.

MATERIALS AND METHODS

Microarray procedures and data analysis mRNA was extracted from three biological replicates of exponentially proliferating WT and p38α-/-MEFs expressing H-Ras G12V using the Qiagen RNeasy kit according to manufacturer's instructions. RNA quality was checked spectrophotometrically by its 260 to 280 nm absorbance ratio as well as by RT-PCR. RNAs were labelled with Cy5 and Cy3, pooled, hybridized and processed with the Amersham Codelink 20K mouse platform (GeneCore Laboratory, EMBL, Heidelberg).

Microarray dataset normalization was performed via cyclic loess normalization. Differentially expressed genes were obtained by applying linear models with R limma package [START_REF] Smyth | Use of within-array replicate spots for assessing differential expression in microarray experiments[END_REF] (Bioconductor project, http://www.bioconductor.org) and multiple hypotheses testing was accounted for by adjusting the estimated significance level (p value) with the Benjamini & Hochberg False Discovery Rate (FDR) correction. Those genes with FDR<0.05 and FC>1.5 (accounting to a total of 2100 genes) were selected as differentially expressed between H-Ras G12V -expressing p38α-/-and WT MEFs and subsequently discriminated with parametric ANOVA 1-way test (p<0.01) with the GeneSpring 6.2 software (Silicon Genetics). This latter analysis allowed to cut down the number of differentially-expressed genes to a more workable set of 202 genes, which corresponded to those genes deregulated ±1.5-fold between H-Ras G12V -expressing p38α-/-and WT MEFs within the 99% confidence level (p<0.01) and a FDR<0.05.

Gene functions were manually annotated on the basis of exhaustive searches in PubMed and Gene Ontology-based applications such as the FatiGO and Source Batch databases. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Gene set enrichment analysis (GSEA)

GSEA was performed with the EGFR-associated set of 19-genes identified in this study versus microarray data from [START_REF] Jimeno | Coordinated epidermal growth factor receptor pathway gene overexpression predicts epidermal growth factor receptor inhibitor sensitivity in pancreatic cancer[END_REF] as reference. Affymetrix arrays (GEO ID: GSE9599) were normalized using Robust Multi-Array Average (RMA). Whole microarray genes were ranked based on limma moderated t statistic. After Kolmogorov-Smirnoff testing, the gene set was considered statistically significant if FDR <0.25, a widely accepted cut-off for the identification of biologically relevant gene sets [START_REF] Subramanian | Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles[END_REF].

mRNA isolation and RT-PCR analysis

Total RNA was extracted from MEFs or mouse tumors using the RNeasy Kit (Qiagen) according to the manufacturer´s instructions. RNA concentration was determined by measuring the absorbance at 260 nm and the quality was monitored by the ratio of absorbance at 260/280 nm and by 1% agarose gel electrophoresis. cDNA was prepared using M-MLV reverse transcription (Invitrogen) and used as the template for PCR amplification. PCR primers were designed with the Vector NTI 8 software (InforMax) and used at a final concentration of 160 nM (Supplementary Table 1). RT-PCR conditions were as follows: reverse transcription at 37ºC for 90 min, denaturation at 95ºC for 2 min, followed by 30-35 cycles of denaturation at 95ºC for 45 s, annealing at 55ºC for 45 s and elongation at 72ºC for 60 s. A final step of elongation at 72ºC for 10 min was performed. The products of the RT-PCR reaction were analyzed by 1% agarose gel electrophoresis, stained with ethidium bromide and imaged in the Gel Doc 2000 system (Bio-Rad).

Quantitative Real-Time PCR (qRT-PCR) analysis was performed as previously described [START_REF] Swat | Cell density-dependent inhibition of epidermal growth factor receptor signaling by p38alpha mitogen-activated protein kinase via Sprouty2 downregulation[END_REF]. Primer sequences are listed in Supplementary Table 2.

Cell culture and reagents

Primary WT and p38α-/-MEFs were immortalized using the 3T3 protocol, maintained as previously described [14] and used as the starting biological material in the experiments described. The term MEFs was used throughout the manuscript to refer to spontaneously immortalized cells or derivatives of them (i.e. H-Ras G12V transformed). MEFs expressing 4hydroxytamoxifen (OHT)-inducible H-Ras G12V have been previously described [14]. HEK293 human epithelial cells were maintained in the same medium as MEFs and used for transient transfection. 293T cells were used to generate retroviruses to stably infect MEFs and were provided by M. Serrano (CNIO, Madrid).

Expression constructs and retroviral infections

pBabe-puro-H-Ras G12V and pWZL-hygro-based constructs containing the Gstm2, Cd9, Mmp3, Egln3, Pmp22, Aig1, Ctsh, Nck2 and Rtn4 mouse cDNAs were used for the generation of retroviruses and subsequent MEF transduction as previously described [14]. Cloning details, cDNA origin, amplification primers and restriction sites are provided in Supplementary Table 3. Myc-tagged versions of pWZL-hygro-Gstm2 and pWZL-hygro-Cd9 were prepared by sitedirected mutagenesis on the non-tagged constructs (QuickChange, Stratagene) and verified by sequencing. The retroviral construct MSCV-hygro-p38α [START_REF] Ambrosino | Negative feedback regulation of MKK6 mRNA stability by p38alpha mitogen-activated protein kinase[END_REF] was used to re-express p38α in p38α-/-MEFs for the rescue experiments.

Immunoprecipitation

Control and H-Ras G12V -expressing WT MEFs were transduced with Cd9 or an empty vector and then EGFR was immunoprecipitated. Subconfluent 10-cm cell culture plates were washed twice with ice-cold PBS, scraped on ice and harvested by centrifugation at 400xg and 4ºC for 10 min. The cell pellets were lysed in 100-600 µl of modified RIPA lysis buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl 2 , 1 mM EGTA, 1% TritonX-100, 1% DOC, 0.1% SDS, 20 mM NaF, 0.1 mM sodium orthovanadate, 1 mM PMSF, 2.5 mM benzamidine, 2 µM microcystin and 10 µg/ml leupeptin and aprotinin). Whole cell extracts (500 µg) were immunoprecipitated with anti-EGFR antibody (Santa Cruz Biotechnology) overnight at 4ºC. Immunoprecipitates were incubated with protein G-sepharose (Amersham) for 1 h at 4ºC, washed 3 times, analyzed by SDS-PAGE and visualized with anti-EGFR antibody (Santa Cruz Biotechnology).

Immunoblotting, transformation and tumorigenicity assays

Immunoblot analysis was performed as previously described [14] using the following primary antibodies: c-Myc (9E10, Santa Cruz Biotechnology), p38 MAPK (C20-G, Santa Cruz Biotechnology), tubulin (clone DM1A, Sigma), Ras (clone 18, Sigma), phospho-ERK (9101, Cell Signaling Technology), phospho-AKT (9271, Cell Signaling Technology), EGFR (1005, sc-03, Santa Cruz Biotechnology), P-EGFR (Y1068, Cell Signaling), Egln3 (Novus Biologicals) and Nck2 (Upstate Biotechnology).

Cell proliferation (MTT) and anchorage-independent growth (soft agar) assays were performed as previously reported [14]. Briefly, soft agar assays were performed in duplicates with a top layer of 0.35% agar and a bottom layer of 0.5% agar. Cell colonies were stained with 0.1% crystal violet (Sigma) at the end of the experiment and counted if bigger than 0.2 mm. Colony numbers normally displayed a standard deviation of ±500 colonies. Pictures of representative fields were taken at 40x magnification with a Leica MZ6 microscope coupled to RS Photometrics camera.

Statistical analysis

Unless otherwise indicated, all results are expressed as a mean ± standard deviation (S.D.) for at least 3 independent experiments. Statistical analysis was performed using Student's t test with a statistically significant p value (p<0.01).

RESULTS

Gene expression profiling of H-Ras G12V -transformed WT and p38α-/-MEFs

In order to identify new targets of p38α in the regulation of oncogene-induced transformation, RNA was extracted from biological triplicates of exponentially proliferating H-Ras G12Vtransformed WT and p38α-/-MEFs, labelled and analyzed with the Amersham Codelink® mouse 20K platform. Statistical filtering, retaining genes with an increased or decreased expression greater than ±1.5 fold, a false discovery rate (FDR) < 0.05 and an adjusted p-value < 0.01, revealed 202 genes that were significantly deregulated between WT and p38α-/-MEFs (Supplementary Table 4). Among them, 139 genes (69%) were up-regulated and 63 genes (31%) were down-regulated in H-Ras G12V -p38α-/-MEFs (Figure 1). Genes were subsequently clustered according to their biological function using Gene Ontology (GO)-based approaches and subcategorized (Figure 1 and Supplementary Table 5). A flowchart showing all the individual steps undertaken is shown in Supplementary Figure S1.

Gene expression validation by semiquantitative RT-PCR in cultured MEFs

To validate microarray gene expression differences, 31 out of the 202 genes potentially regulated by p38α were selected for semi-quantitative RT-PCR analysis, which was performed on new mRNA preparations extracted from the two transformed MEF lines growing exponentially. Gene selection was based on the different mRNA expression levels in H-Ras G12V -p38α-/-versus H-Ras G12V -WT MEFs, as well as on the putative involvement of each gene in Ras-induced transformation or p38α signalling. Out of the 31 genes selected, 19 were up-regulated and the other 12 were down-regulated in H-Ras G12V -p38α-/-cells (Supplementary Table 6).

Two independent cellular systems were used to dissect the relative contribution of p38α and H-Ras G12V to the expression of these genes. The first involved established WT and p38α-/-MEFs constitutively expressing H-Ras G12V for at least two weeks [START_REF] Alfonso | Proteomic analysis of p38alpha mitogen-activated protein kinase-regulated changes in membrane fractions of RAS-transformed fibroblasts[END_REF]14], which allowed us to evaluate the steady-state gene expression levels in each cell line. The second consisted of WT and p38α-/-MEFs expressing an OHT-inducible form of H-Ras G12V (ER-HRas G12V ) [14]. This system allowed us to monitor gene expression in both cell lines in the absence of H-Ras G12V signalling as well as early (24 h) after its activation with OHT (Figure 2A).

We found that the majority of the genes analyzed by semi-quantitative RT-PCR were deregulated between stable H-Ras G12V -expressing WT and p38α-/-MEFs (Figures 2B and2C, two right lanes) in a way that mirrored the microarray results in more than 90% of the cases (note microarray fold change column to the right). However, the differential expression levels of Ralgds, Rtn4 and Ctsl in H-Ras G12V -WT versus H-Ras G12V -p38α-/-MEFs were less clear than expected from the microarray results. This might be related to sensitivity limitations of the semi-quantitative RT-PCR technique, at least for Rtn4 (+2.2) and Ctsl (+2.4), since their moderate upregulation was detectable by qRT-PCR (see below Figures 3 and4A). Of note, we could not detect expression of Stac2, Rom1, Dpep1, Traf1 and Lzts2.

Concerning the remaining 26 genes, analysis of ER-HRas G12V -expressing WT and p38α-/-MEFs revealed that the expression of half of them was dependent on p38α (Figure 2B, first and third lines marked with asterisks), whereas the other half were additionally influenced by H-Ras G12V signalling (Figure 2C, second and fourth lanes marked with asterisks). Interestingly, the effect of H-Ras G12V on gene expression in the latter case was already clear 24 h after the onset of H-Ras G12V signalling and was usually maintained during the transformation process, in MEFs stably expressing H-Ras G12V . However, the relative mRNA abundance of some genes in H-Ras G12V -expressing WT versus p38α-/-MEFs was attenuated (Ralgds, Ctsl) or enhanced (Cd9, Ctsh) with time, arguing for a kinetic component in the regulation of gene expression by p38α and H-Ras G12V . This could in principle be due either to specific changes in p38α or H-Ras G12V signalling or to non-specific alterations or selective pressure related to the transformed phenotype. For example Cxcl1, Nck2 and Tnfrsf23 showed opposite expression patterns in early transformed versus established cell lines, whereas Rtn4 and Snfl1k showed opposite regulation in non-transformed versus H-Ras G12V -expressing cells. Interestingly, p38α reconstitution into p38α-/-MEFs rescued the expression levels of putative p38α-regulated genes to about WT levels in both non-transformed (Figure 2D) and transformed cells (Supplementary Figure S2). Taken together, the results suggest that, at least the selected genes that were analysed are indeed regulated by p38α, although some of them can be additionally modulated by the cellular context (e.g. non-transformed versus transformed cells and early versus late transformation stages).

Gene expression validation by qRT-PCR in cultured cells and mouse tumors

In order to evaluate the relevance of the observed gene expression differences between WT and p38α-/-MEFs for in vivo tumorigenesis, selected genes were further analyzed by qRT-PCR in subcutaneous tumors obtained from the injection of H-Ras G12V -expressing WT and p38α-/-MEFs in nude mice [14], as well as in lung tumors induced by K-Ras G12V expression in WT and p38α-/-mice [START_REF] Ventura | p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation[END_REF]. We found that all the genes analyzed were similarly deregulated in a p38α-dependent manner in transformed MEFs, MEF-derived subcutaneous tumors and K-Ras G12V -induced lung tumors, in accordance with the original results obtained from the microarray analysis (Figure 3). Although the microarray-based profiling and qRT-PCR analysis did not result in identical quantitative changes, gene expression fold changes were qualitatively conserved in the different tumor models tested (Figure 3 and Supplementary Table 7). These results link the p38α-mediated modulation of particular mRNAs with the ability of p38α to regulate tumorigenesis.

Functional validation of genes deregulated between H-Ras G12V -transformed WT and p38α-/-MEFs

Next, we selected 9 out of the 26 genes mentioned above, according to their level of deregulation in H-Ras G12V -expressing p38α-/-MEFs and their putative connection with the process of cell transformation, to perform functional studies (Table 1). Three genes encoded membrane (Aig1) or extracellular matrix (Mmp3, Ctsh) proteins and were selected due to their acute deregulation in H-Ras G12V -expressing p38α-/-versus WT MEFs. Little is known about Aig1 (androgen-induced 1) in the context of malignant transformation. In contrast, Mmp3 (matrix metallopeptidase 3 or stromelysin-1) and Ctsh (Cathepsin H) are known to be involved in tumor progression (Table 1). Four other genes encoded the tyrosine kinase signalling-modulating proteins Cd9 and Nck2, the proliferation-regulating protein Pmp22 and the pleiotropic protein Rtn4, and were all upregulated in the more transformed H-Ras G12V -p38α-/-MEFs, suggesting that enhanced expression of these genes might contribute to transformation. Accordingly, some of them have been described to be upregulated in tumors and potentially associated with tumor progression (Table 1). Finally, two genes that were down-regulated in H-Ras G12V -p38α-/-MEFs, namely Gstm2 and Egln3, were also considered good candidates to test functionally due to their reported roles in oxidative stress and apoptosis, respectively (Table 1).

We have previously reported that the absence of p38α enhances H-Ras G12V -induced anchorage-independent growth [14]. Thus, genes found up-regulated in H-Ras G12V -p38α-/-MEFs were over-expressed in H-Ras G12V -WT MEFs (Table 1) to test if they could enhance the ability of transformed cells to grow in soft agar. Proliferation rates were also measured in parallel, and over-expression was confirmed by immunoblotting or RT-PCR (Supplementary Figure S3A), depending on the availability of antibodies. Interestingly, Mmp3, Cd9 and Rtn4 enhanced the ability of H-Ras G12V -WT MEFs both to proliferate and to grow anchorageindependently (Table 1 and Supplementary Figures S3B, S3C andS4). Similarly, Nck2 stimulated the proliferation of H-Ras G12V -WT MEFs to p38α-/-levels, although it did not affect H-Ras G12V -induced soft agar growth (Table 1 and Supplementary Figures S3B andS4). These results suggest that p38α might regulate different traits of the cancer cell (i.e. proliferation and anchorage-independent growth) by specifically targeting the expression of distinct genes in each case. Overall, it appears that the down-regulation of these four genes by p38α may contribute to its inhibitory effect on H-Ras G12V -induced transformation of MEFs. In contrast, no conclusions could be drawn on Aig1 since we did not manage to express this gene (data not shown), whereas Pmp22 unexpectedly decreased, rather than enhanced, the transformed phenotype of H-Ras G12V -WT MEFs (Supplementary Figures S3D andS4). This would agree with the pro-apoptotic and anti-proliferative functions ascribed to Pmp22 (Table 1), but raises the question of whether the endogenous Pmp22 is actually functional in p38α-/-MEFs, since Pmp22 over-expression in these cells (where it is already upregulated) attenuates their transformed phenotype as well (Supplementary Figure S3D).

In a complementary set of experiments, we over-expressed in H-Ras G12V -p38α-/-MEFs three genes found up-regulated in H-Ras G12V -WT cells (Table 1) and tested if they could inhibit transformation. We found that Egln3 attenuated the proliferation rate and the ability to grow in soft-agar of H-Ras G12V -p38α-/-MEFs to about H-Ras G12V -WT levels (Supplementary Figure S5A, S5B and 5C), in agreement with its lower expression levels in p38α-/-MEFs (Supplementary Table 6) and its reported pro-apoptotic activity (Table 1). In contrast, Ctsh affected neither proliferation nor soft agar colony formation in H-Ras G12V -p38α-/-MEFs (Table 1 and Supplementary Figures S5A andS5B). This is in agreement with the putative matrix remodelling function of Ctsh, which is likely to regulate tumor cell invasion rather than cell proliferation or anchorage-independent growth. Of note, Gstm2 did not significantly affect H-Ras G12V -p38α-/-MEFs , but enhanced the proliferation (Supplementary Figure S5D) and soft agar growth [14] of H-Ras G12V -WT MEFs. This apparent paradox could be explained if one considers that Gstm2 over-expression interferes with the activation and subsequent proapoptotic function of p38α in H-Ras G12V Gstm2 binding to ASK1, an upstream activator of p38α, which in turn blocks the activation of the pathway by H-Ras G12V -induced reactive oxygen species (ROS). Thus, cells overexpressing H-Ras G12V and Gstm2 bypass the inhibitory apoptotic response mediated by p38α, allowing the accumulation of high levels of ROS, which enhances malignant transformation. Since Gstm2 over-expression targets p38α, it is not surprising that it has no effect on H-Ras G12V -p38α-/-MEFs.

Regulation of EGFR signalling by transcriptional targets of p38α in malignant transformation

Experiments using mouse models of cancer suggests that EGFR transcription could be targeted by p38α for tumorigenesis regulation in vivo [START_REF] Ventura | p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation[END_REF], in agreement with studies using cultured cells [START_REF] Frey | p38 kinase regulates epidermal growth factor receptor downregulation and cellular migration[END_REF][START_REF] Winograd-Katz | Cisplatin induces PKB/Akt activation and p38(MAPK) phosphorylation of the EGF receptor[END_REF][START_REF] Zwang | p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy[END_REF]. Moreover, EGFR activity is enhanced in p38α-deficient MEFs and contributes to the more transformed phenotype of H-Ras G12V -p38α-/-MEFs [START_REF] Swat | Cell density-dependent inhibition of epidermal growth factor receptor signaling by p38alpha mitogen-activated protein kinase via Sprouty2 downregulation[END_REF]. We found a set of 19 genes that were deregulated between H-Ras G12V -expressing WT and p38α-/-MEFs and were linked to EGFR signalling (Table 2). Most of these genes encode proteins that positively regulate EGFR signalling and were up-regulated in H-Ras G12V -p38α-/-MEFs, indicating negative regulation by p38α. These include the plasma membrane protein Cd9 that stimulates EGFR activity by facilitating the interaction of the receptor with its ligands, the mitogenic EGFR ligand Areg previously associated with malignant cell transformation, the transcription factor Klf5 that stimulates EGFR expression, the Src-family kinase Hck that participates in EGFR recycling and signal relay as well as in cell survival, and the tyrosine kinase substrate Eps8 that amplifies EGFR signalling following mitogenic stimulation (Table 2). Validation by qRT-PCR confirmed the differential expression of these genes in H-Ras G12V -p38α-/-versus H-Ras G12V -WT MEFs (Figure 4A), as predicted from the microarray results.

To strengthen the implication of the 19-gene dataset in the regulation of EGFR signalling, we performed Gene set enrichment analysis (GSEA). We found that our set of p38α-regulated genes aligned with the EGFR-associated gene signature (GEO ID:GSE9599) of a study correlating the sensitivity of human pancreatic tumor cell lines to the EGFR inhibitor erlotinib with the expression of an EGFR-associated gene network that sustains high levels of EGFR activity [START_REF] Jimeno | Coordinated epidermal growth factor receptor pathway gene overexpression predicts epidermal growth factor receptor inhibitor sensitivity in pancreatic cancer[END_REF]. Interestingly, GSEA revealed that our 19-gene cluster was significantly enriched in erlotinib-sensitive pancreatic tumors (FDR q-value = 0.165) (Supplementary Figure S6) correlating with high EGFR activity. This result supports the importance of EGFR signalling inhibition by p38α in the regulation of cell transformation.

At the mechanistic level, we confirmed that Cd9 over-expression in H-Ras G12V -WT MEFs not only enhanced their transformed phenotype to H-Ras G12V -p38α-/-levels (Supplementary Figures S3B andS3C), but also activated EGFR kinase activity, as indicated by higher levels of phospho-EGFR Y1068 (Figure 4B, left) and its downstream signalling pathways ERK1/2 and AKT (Figure 4B, right). This argues that p38α negatively regulates cell transformation, at least in part, by inhibiting EGFR via down-regulation of several genes that stimulate its activity or downstream signal relay. Of note, the interplay between EGFR and p38α in cell transformation might not be limited to a cluster of EGFR-regulating genes, but also involve direct regulation of the EGFR mRNA by p38α. We found that the EGFR mRNA was upregulated in the absence of p38α in three different cellular systems: H-Ras G12V -transformed MEFs growing exponentially, subcutaneous xenografts derived from H-Ras G12V -expressing MEFs and K-Ras V12 -induced lung tumors in mice (Figure 4C).

DISCUSSION

Cancer is a genetic disease characterized by the simultaneous disruption of several cellular traits required for organism homeostasis. At the molecular level, the combination of deregulated cell proliferation and reduced apoptosis is thought to set the founding for neoplastic progression. Accordingly, the ability of p38α to engage cell cycle arrest and apoptosis has been shown to play an important tumor suppressor role [START_REF] Bulavin | p38 MAP kinase's emerging role as a tumor suppressor[END_REF][START_REF] Wagner | Signal integration by JNK and p38 MAPK pathways in cancer development[END_REF]. p38α counteracts the transforming activity of oncogenic Ras in several cellular and animal models through various mechanisms, most of them occurring at the post-translational level [START_REF] Bulavin | Inactivation of the Wip1 phosphatase inhibits mammary tumorigenesis through p38 MAPK-mediated activation of the p16(Ink4a)-p19(Arf) pathway[END_REF][START_REF] Ventura | p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation[END_REF]14,[START_REF] Nicke | Involvement of MINK, a Ste20 family kinase, in Ras oncogene-induced growth arrest in human ovarian surface epithelial cells[END_REF]. However, cancer progression is normally accompanied by profound alterations in gene expression, which prompted us to investigate transcriptional targets of p38α in the regulation of tumorigenesis. We have found a number of genes not previously connected to p38α signalling that are likely to mediate its negative effect on H-Ras G12V -induced transformation. Intriguingly, our analysis has also identified various p38α-induced genes that are related to cell migration, invasion and angiogenesis, which may belong to a family of p38α effectors mediating some of its less characterized pro-oncogenic functions [START_REF] Dolado | Regulation of tumorigenesis by p38α MAP kinase[END_REF]. In fact, emerging evidence suggest that p38α may have oncogenic rather than tumor suppressive functions in some advanced human tumors [START_REF] Wagner | Signal integration by JNK and p38 MAPK pathways in cancer development[END_REF]. Further studies are needed to understand the cellular contexts in which p38α can either suppress or promote tumor formation. We report here a set of p38α-regulated genes that are associated with tumor suppressor functions of p38α in different tumor models, and may be useful to identify human cancers where p38α negatively regulates tumor formation.

New p38α effectors in the regulation of cell survival and proliferation

Down-regulation of p38α enhances H-Ras G12V -induced transformation of MEFs, as indicated by their increased proliferation and survival, refringent morphology, loss of contact inhibition and anchorage-independent growth [14]. Not surprisingly, we have found that genes stimulating cell proliferation and survival are negatively regulated by p38α including Ralgds, a Ras effector that promotes cell survival [START_REF] Gonzalez-Garcia | RalGDS is required for tumor formation in a model of skin carcinogenesis[END_REF], and Tnfrsf23, a putative tumor necrosis factor receptor decoy that inhibits apoptosis [START_REF] Bridgham | Alternatively spliced variants of Gallus gallus TNFRSF23 are expressed in the ovary and differentially regulated by cell signaling pathways[END_REF]. Conversely, apoptosis-stimulating genes such as Egln3 [START_REF] Lee | Neuronal apoptosis linked to EglN3 prolyl hydroxylase and familial pheochromocytoma genes: developmental culling and cancer[END_REF] are up-regulated in the less transformed H-Ras G12V -WT MEFs, in line with the importance of the apoptotic activity of p38α for tumor suppression [14]. Of note, p38α seems to regulate Egln3 expression at both transcriptional and post-translational levels [START_REF] Khurana | Regulation of the ring finger E3 ligase Siah2 by p38 MAPK[END_REF]. It is possible that other genes found in our study are also post-transcriptionally regulated by p38α, for instance through the well known p38α-MK2-mediated stabilization of mRNAs containing AU-rich 3'UTRs [START_REF] Kotlyarov | Protein kinases as substrates for stress-activated protein kinases[END_REF].

Potential membrane targets of p38α in cell transformation

Apart from survival and proliferation, p38α can also regulate other traits of the cancer cell, such as cell morphology, contact inhibition and anchorage-independent growth, which are likely regulated by signals relied from plasma membrane proteins [14,[START_REF] Swat | Cell density-dependent inhibition of epidermal growth factor receptor signaling by p38alpha mitogen-activated protein kinase via Sprouty2 downregulation[END_REF]. Previous attempts to identify proteins that were differentially expressed in the membranes of H-Ras G12Vexpressing WT and p38α-/-MEFs had limited success [START_REF] Alfonso | Proteomic analysis of p38alpha mitogen-activated protein kinase-regulated changes in membrane fractions of RAS-transformed fibroblasts[END_REF]. Interestingly, we now report several p38α-regulated genes encoding integral-to-membrane or membrane-associated proteins that may be involved in such processes (Supplementary Table 5). These include Ezr, encoding the cytoskeleton protein Ezrin known to be important for the regulation of cell-cell contact inhibition and tumorigenesis [START_REF] Quang | Ezrin function is required for ROCK-mediated fibroblast transformation by the Net and Dbl oncogenes[END_REF], as well as various genes encoding transmembrane proteins such as tetraspanin Tspan13 or protocadherins Pcdh7 and Pcdh10. Of note, certain protocadherins (i.e. Fat1) are emerging as important regulators of contact inhibition [START_REF] Tanoue | Mammalian Fat1 cadherin regulates actin dynamics and cell-cell contact[END_REF], a process where p38α plays a key role [START_REF] Swat | Cell density-dependent inhibition of epidermal growth factor receptor signaling by p38alpha mitogen-activated protein kinase via Sprouty2 downregulation[END_REF]. Future studies should unveil whether these proteins may link p38α signalling with the regulation of membrane-mediated cellular processes associated to malignant transformation. A striking finding of our studies has been the identification of 19 genes (Table 2) that impinge on EGFR signalling and whose expression seems to be co-ordinately regulated by p38α to inhibit malignant transformation. These include proteins that enhance EGFR expression (Klf5), activation (Hck, Cd9, Prkca, Areg) and downstream signalling (Nck2, Limk2, Mmp3) or that regulate EGFR intracellular trafficking (Ctsl, Lamp2, Arfgap3, Cdc42ep5). Interestingly, we have shown that Cd9 over-expression not only stimulates EGFR signalling in H-Ras G12V -WT cells but also enhances transformation to H-Ras G12V -p38α-/-levels. The ability of p38α to down-regulate EGFR signalling at various levels may also contribute to the inhibitory role of p38α in Ras-induced transformation, since Ras has been shown to require EGFR signalling for full transformation [START_REF] Gangarosa | A raf-independent epidermal growth factor receptor autocrine loop is necessary for Ras transformation of rat intestinal epithelial cells[END_REF][START_REF] Sibilia | The EGF receptor provides an essential survival signal for SOS-dependent skin tumor development[END_REF].

The EGFR pathway as a transcriptional target of p38α in cell transformation

We have recently reported that p38α can inhibit EGFR at the post-translational level upon cell-cell contact [START_REF] Swat | Cell density-dependent inhibition of epidermal growth factor receptor signaling by p38alpha mitogen-activated protein kinase via Sprouty2 downregulation[END_REF], preventing cell overgrowth at high cellular densities. The relative contribution of transcriptional versus post-translational regulation to the overall effect of p38α on EGFR signalling is unclear and may depend on the transformed state of the cell, the cell cycle phase and the scope of the response induced by p38α. For instance, p38α regulates EGFR mRNA levels in proliferating, H-Ras G12V -transformed cells, but no differences were found in EGFR mRNA abundance between contact-inhibited, non-transformed WT and p38α-/-MEFs [START_REF] Swat | Cell density-dependent inhibition of epidermal growth factor receptor signaling by p38alpha mitogen-activated protein kinase via Sprouty2 downregulation[END_REF]. It is possible that the post-translational regulation of EGFR by p38α in contact inhibition is aimed at eliciting a fast response, whereas the long-term inhibition of H-Ras G12V transformation by p38α requires tuning the steady-state expression levels of genes encoding proteins, which in turn impinge on EGFR signalling.

In summary, we have identified and validated in different models of tumorigenesis several p38α targets that are functionally implicated in transformation. A significant number of the genes that are down-regulated by p38α at the mRNA level encode positive regulators of EGFR signalling, suggesting that inhibition of this pathway probably represents an important tumor suppressor function of p38α.

Table 1 Genes differentially expressed between H-Ras G12V -p38α-/-and H-Ras G12V -WT MEFs that were selected for functional validation Cdc42 effector protein (Rho GTPase binding) 5

EGFR degradation [START_REF] Bulavin | Inactivation of the Wip1 phosphatase inhibits mammary tumorigenesis through p38 MAPK-mediated activation of the p16(Ink4a)-p19(Arf) pathway[END_REF] +201.81 Anxa8

(NM_013473)
Annexin A8 EGFR internalization [START_REF] Ventura | p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation[END_REF] +2.04 Lamp2

(NM_010685)
Lysosomal membrane glycoprotein 2

EGFR endocytosis [START_REF] Alfonso | Proteomic analysis of p38alpha mitogen-activated protein kinase-regulated changes in membrane fractions of RAS-transformed fibroblasts[END_REF] Downregulated by p38α Phospholipase C, gamma 2 EGFR signal rely [START_REF] Ambrosino | Negative feedback regulation of MKK6 mRNA stability by p38alpha mitogen-activated protein kinase[END_REF] Upregulated by p38α

-25.18 Cxcl1 (NM_021704) Chemokine (C-X-C motif) ligand 1
EGFR signaling [START_REF] Frey | p38 kinase regulates epidermal growth factor receptor downregulation and cellular migration[END_REF] * microarray-based n-fold change (2 log2ratio ) in H-Ras G12V -p38α-/-versus H-Ras G12V -WT MEFs.

Functional classification of genes differentially expressed between H-Ras G12V -p38α-/-and H-Ras G12V -WT MEFs

Genetic deletion of p38α significantly affected the expression of 202 genes. The number of genes within each functional category, either up-regulated (black) or down-regulated (white) in H-Ras G12V -p38α-/-MEFs, is indicated as a percentage of the total number of genes.

Figure 3 Quantitative RT-PCR (qRT-PCR) analysis of gene expression in various tumor models

qRT-PCR was performed on RNA extracted from exponentially proliferating, H-Ras G12Vexpressing WT and p38α-/-MEFs (light gray), subcutaneous xenografts derived from the same cells grown in nude mice for two weeks (white), or K-Ras G12V -induced lung tumors in WT and p38α-/-mice (dark grey). qRT-PCR results were compared with the respective microarray fold change value (black). Asterisks indicate fold changes greater than 28. All values together with their corresponding standard deviations are listed in Supplementary Table 7.

Figure 4 Transcriptional regulation of EGFR signalling by p38α in oncogenesis

(A) EGFR signalling-related genes were validated by qRT-PCR in exponentially proliferating H-Ras G12V -p38α-/-versus H-Ras G12V -WT MEFs and fold changes (white) were plotted along with the microarray fold changes for each gene (black). (B) WT MEFs were transduced with H-Ras G12V , Cd9 or empty vectors (-), as indicated, and whole cell lysates were analyzed by immunoblotting with the indicated antibodies either directly (right) or after immunoprecipitation with EGFR antibodies (left). (C) qRT-PCR analysis of EGFR mRNA in exponentially proliferating H-Ras G12V -expressing p38α-/-versus WT MEFs (black), in tumor xenografts derived from the same MEFs and grown for two weeks in nude mice (grey), and in lung tumors induced by K-Ras G12V expression in p38α-/-mice versus WT mice (white). Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  -expressing cells [14]. Mechanistically, this involves Biochemical Journal Immediate Publication. Published on 12 Jan 2011 as manuscript BJ20101410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101410A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 12 Jan 2011 as manuscript BJ20101410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101410A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 12 Jan 2011 as manuscript BJ20101410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101410 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Swat et al., Figure 1 G12V -p38α-/-MEFs downregulated in HRas G12V -p38α-/-MEFs Biochemical Journal Immediate Publication. Published on 12 Jan 2011 as manuscript BJ20101410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101410 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

  Publication. Published on 12 Jan 2011 as manuscript BJ20101410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101410 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited Publication. Published on 12 Jan 2011 as manuscript BJ20101410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101410 A c c e p t e d M a n u s c r i p t

  Biochemical Journal Immediate Publication. Published on 12 Jan 2011 as manuscript BJ20101410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101410

Table 2

 2 EGFR pathway-modulating genes that are regulated by p38α

	n-fold	Gene symbol	Gene description	Effect	1
	change*	(NCBI Acc.No.)
	+10.41 Hck	Hematopoietic cell kinase	EGFR activation and signal rely [1]
		(NM_010407)
	+7.05 Areg	Amphiregulin	EGFR ligand [2]
		(NM_009769)
	+5.19 Cd9	CD9 antigen	EGFR paracrine activation [3]
		(NM_007657)
	+3.83 Klf5	Kruppel-like factor 5	EGFR transcription [4]
		(NM_009769)
	+2.03 Eps8	EGFR pathway substrate 8	EGFR signaling [5]
		(NM_007945)
	+1.55 Prkca	Protein kinase C, alpha	EGFR activation and signal rely [6]
		(NM_011101)
	+1.84 Limk2	LIM motif-containing protein	EGFR effector [7]
		(NM_010718)	kinase 2
	+3.11 Arfgap3	ADP-ribosylation factor GTPase	EGFR endocytosis [8]
		(NM_025445)	activating protein 3
	+2.36 Nck2	Non-catalytic region of tyrosine	EGFR SH2-domain adaptor protein [9]
		(NM_010879)	kinase adaptor protein 2
	+2.47 Ctsl	Cathepsin L	EGFR recycling [10]
		(NM_009984)
	+2.37 Cdc42ep5
		(NM_026772)

  1 references are provided in Supplementary material

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Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Ras G12V expression. Up and Down indicate mRNAs up-regulated or down-regulated, respectively, in established H-Ras G12V -p38α-/-MEFs according to the microarray analysis; FC, fold change in the microarray analysis. (C) Genes differentially expressed in MEFs by the combined effect of both p38α and H-Ras G12V . Samples as in (B). Asterisks indicate changes caused mainly by H-Ras G12V expression. (D) Changes in gene expression were confirmed by analysing WT, p38α-/-and p38α add-back p38α-/-MEFs. Immunoblot analysis is shown at the bottom. The experiments were performed at least three times with similar results.

	Figure 2 Semi-quantitative RT-PCR validation of genes differentially regulated by p38α
	and/or H-Ras G12V
	(A) Immunoblot analysis of cell extracts from ER-HRas G12V -expressing WT and p38α-/-
	MEFs stimulated with 1 µM OHT for 24 h. (B) WT and p38α-/-MEFs expressing ER-
	HRas G12V were treated with OHT, as indicated. Established H-Ras G12V -expressing p38α-/-and
	WT MEFs were analysed in parallel. Asterisks indicate changes related to p38α deficiency,
	but independently of H-

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

ACKNOWLEDGMENTS

We thank Stephan Tenbaum and Juan Jose Ventura (CNIO, Madrid) for providing mouse lung tumor samples, and Vladimir Benes and Tommi Ivacevic (EMBL, Heidelberg) for help with microarray data processing. A.S. acknowledges a FPU fellowship from the Spanish Ministerio de Ciencia e Innovacion (MICINN). This work was funded by grants from MICINN (BFU2007-60575 and RD06/0020/0083), Fundacion Cientifica de la AECC and Fundacion La Caixa.

No effect No effect * microarray-based n-fold change (2 log2ratio ) in H-Ras G12V -p38α-/-versus H-Ras G12V -WT MEFs. 1 references are provided in Supplementary material 2 effect of gene overexpression on H-Ras G12V -induced cell proliferation (see Suppl. Fig. S3-S5). 3 effect of gene overexpression on H-Ras G12V -induced soft agar growth (see Suppl. Fig. S3, S4).