ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially
- Chiara Vantaggiato†1, 2,
- Ivan Formentini†1,
- Attilio Bondanza1,
- Chiara Bonini1,
- Luigi Naldini1 and
- Riccardo Brambilla1Email author
© Vantaggiato and Formentini et al.; licensee BioMed Central Ltd. 2006
Received: 11 January 2005
Accepted: 6 April 2006
Published: 28 June 2006
The mitogen-activated protein (MAP) kinases p44ERK1 and p42ERK2 are crucial components of the regulatory machinery underlying normal and malignant cell proliferation. A currently accepted model maintains that ERK1 and ERK2 are regulated similarly and contribute to intracellular signaling by phosphorylating a largely common subset of substrates, both in the cytosol and in the nucleus.
Here, we show that ablation of ERK1 in mouse embryo fibroblasts and NIH 3T3 cells by gene targeting and RNA interference results in an enhancement of ERK2-dependent signaling and in a significant growth advantage. By contrast, knockdown of ERK2 almost completely abolishes normal and Ras-dependent cell proliferation. Ectopic expression of ERK1 but not of ERK2 in NIH 3T3 cells inhibits oncogenic Ras-mediated proliferation and colony formation. These phenotypes are independent of the kinase activity of ERK1, as expression of a catalytically inactive form of ERK1 is equally effective. Finally, ectopic expression of ERK1 but not ERK2 is sufficient to attenuate Ras-dependent tumor formation in nude mice.
These results reveal an unexpected interplay between ERK1 and ERK2 in transducing Ras-dependent cell signaling and proliferation. Whereas ERK2 seems to have a positive role in controlling normal and Ras-dependent cell proliferation, ERK1 probably affects the overall signaling output of the cell by antagonizing ERK2 activity.
The small GTPase Ras, its relatives and their effectors are central to the signaling networks that are involved in a variety of regulatory processes in the cell, from proliferation and tumorigenesis to development and synaptic plasticity [1–3]. The signaling cascade involving the Raf, MEK (mitogen-activated protein (MAP) or extracellular signal-regulated (ERK) kinase) and ERK families of kinases is among the best characterized pathways downstream of Ras. This signaling module couples receptor-mediated activation of Ras to cytoplasmic and nuclear events, leading to phosphorylation of key structural and regulatory components [4–8].
Approximately 15% of human cancers contain activating mutations in one of the Ras genes [1, 9]. This figure under-represents the actual involvement of Ras pathways in tumorigenesis, however, as other downstream signaling components, such as B-Raf, are frequently found in their oncogenic form in tumors in which Ras is not itself mutated . Importantly, though, induction of missense activating mutations or deletions in regulatory domains might not be the only mechanism leading to deregulation of the Ras-ERK pathway and malignancy. Although there is no evidence so far to suggest that either MEK1/2 or ERK1/2 proteins can become oncogenic in spontaneous tumors, their activity is massively upregulated in several human cancers . For instance, in human leukemia samples, both MEKs and ERKs are often hyperphosphorylated and activated, suggesting a causal relationship between stimulation of the Ras-ERK pathway and tumorigenesis and providing a conceptual framework for potential therapeutic targeting (as reviewed in ).
One important aspect of the regulation of the Ras-ERK cascade is the specific, non-redundant role of protein isoforms in this pathway. Gene-targeted and transgenic mouse lines have proved invaluable in determining specific phenotypes associated with most signaling components in the pathway, including lines defective in one of all three Ras proteins (K-ras, N-ras and H-ras), the Raf isoforms c-Raf-1, Raf-A and Raf-B, the MEKs MEK1 and MEK2, the Ras GTPase-activating proteins GAP-1 and NF1, the Ras guanine nucleotide-releasing factors RasGRF1 and RasGRF2, and the adaptor proteins Sos1, Grb2 and Shc [1, 4, 13–24]. Moreover, for some components of the pathway, such as c-Raf-1 and B-Raf, significant structural differences are the basis not only of their differential regulation, but possibly also of their oncogenic potential .
Surprisingly, relatively little is known about possible specific roles for the two major ERK isoforms, ERK1 (p44) and ERK2 (p42). These two proteins are co-expressed in virtually all tissues but with a remarkably variable relative abundance, ERK2 being the predominant isoform in brain and hematopoietic cells [12, 26, 27]. Given the extensive aminoacid identity between the two molecules and their apparently similar spatio-temporal regulation, the current working model regards them essentially as interchangeable. Nevertheless, important recent evidence suggests that there could be quantitative differences in ERK1 and ERK2 dynamics and that these could have a significant role in their regulation. ERK1-deficient mice are viable, with no obvious compensatory upregulation of ERK2 protein levels but showing a deficit in thymocyte maturation . A recent T-cell-specific knockout of ERK2 further supports a crucial role for MAP-kinase signaling in the immune system . On the other hand, global ERK2-deficient mice die early in development, showing that ERK1 cannot compensate in the embryo for ERK2 [30–32].
One possible interpretation of these data is that although ERK2 is essential for transduction of signals, ERK1 could instead have an accessory role, possibly enabling a fine tuning of ERK2 activity. Two related lines of evidence strongly support the idea that ERK1 acts in a complex manner, at least in certain circumstances, by attenuating ERK2 activity. First, both in fibroblasts and in neurons derived from ERK1-deficient mice, stimulus-dependent activation of ERK2 (but not its basal activity) was found to be significantly upregulated, as revealed by the increased level of ERK2 phosphorylation and immediate-early gene transcription [28, 33]. Second, enhancement of ERK2-dependent signaling in the nervous system of the ERK1 mutant mice has been linked to improvement of certain forms of learning and memory .
To investigate whether such mechanisms are also implicated in the control of cell proliferation, we examined ERK activation and growth rates both in genetically altered mouse fibroblasts and using RNA interference (RNAi) technology [34–36].
Results and discussion
Enhancement of ERK2 signaling in ERK1 mutant fibroblasts provides a significant growth advantage
Our own previous work  has shown that in primary neurons of the central nervous system, neurotransmitter stimulation results in a significant hyperactivation of ERK2 in the absence of ERK1. On the basis of these findings, we proposed a competition model between ERK1 and ERK2 in their interaction with the upstream kinase MEK. According to this model, we speculated that in the absence of ERK1, the pool of ERK2 molecules could be more efficiently activated, resulting in an increased downstream transmission of the signal .
Specific knockdowns of ERK1 and ERK2 demonstrate a differential role for the two kinases in cell signaling
Differential MEK-ERK1 and MEK-ERK2 interactions
One of the assumptions of the competition model is that in activated cells MEK-ERK complexes should preferentially contain ERK2. Moreover, in the absence of ERK1 we would expect to observe a significant increase in MEK-ERK2 interactions. To investigate this possibility and to provide a direct support for the model, we performed immunoprecipitation studies with a specific antibody recognizing both MEK isoforms and then determined the composition of ERK1 and ERK2 in the complex with two distinct antisera. As indicated in Figure 3b, in the absence of ERK1, binding of ERK2 to MEK appears slightly but significantly increased. Quantification of three experiments in Figure 3c demonstrates that ERK2 levels associated with MEK in the absence of ERK1 are 70% higher than in the control extracts. We detected a much smaller change in ERK1 levels in ERK2 KD cells (20%), however. This could possibly be due to a combination of various factors: the presence of some detectable residual ERK2 protein in the MEK complex from ERK2 KD cells; a lower expression level of ERK1 in comparison with ERK2; or a potentially lower affinity of ERK1 for MEK1/2.
ERK1 knockdown in NIH 3T3 cells facilitates growth, whereas ERK2 knockdown inhibits it
Cell-cycle progression is highly regulated in multicellular organisms, and the loss of any regulatory mechanism could result in tumor formation. Cancer cells can grow in multiple layers and in anchorage-independent conditions, showing less ordered growth and reduced cell-cell contact inhibition. To determine the role of ERK1 and ERK2 in cell growth and Ras-mediated transformation, we made knockdowns of both ERK isoforms in NIH 3T3 cells, either in a wild-type or in oncogenic H-RasQ61L background, and performed a colony-formation assay, a common test for cell transformation. In this assay, cells transformed with oncogenes such as Ras produce colonies of larger size than cells transfected with vector alone . Importantly, this test does not rely on stable transfectants, as selection and scoring are done within 10 days of transfection.
Representative plates of each transfection are shown in Figure 4a. The summary data shown in Figure 4b clearly indicate that ERK2 knockdown negatively affects both normal and Ras-mediated cell growth. Although loss of ERK1 caused a significant increase in the growth of wild-type cells, however, the effect of the ablation of this MAP kinase on H-RasQ61L-dependent proliferation was surprisingly marginal, and unexpectedly in the direction of a small decrease rather than an increase. These data confirm that ERK1 can negatively modulate normal cell growth in NIH 3T3 cells. It seems, however, that loss of ERK1 in ERK1 KD cells cannot further increase Ras-mediated cell transformation but rather causes a small but consistent reduction in the colonies produced by this potent oncogene. Although this fact suggests that overexpression of oncogenic Ras could determine a ceiling effect in the rate of cell growth, it also leaves open the possibility that in such conditions of abnormally high signaling activation, ERK1 might still have a role qualitatively similar to that of ERK2 and could be positively engaged in the generation of cell-proliferation responses.
Ectopic expression of ERK1 but not of ERK2 results in the inhibition of Ras-dependent cell growth
Therefore, we next asked whether ectopic expression of these kinases might interfere with growth of transformed cells, by examining the growth of double transfectants containing oncogenic Ras and one of the wild-type or mutant kinases described above. The underlying idea, taken from previous genetic studies in Drosophila and in mouse, was that the possible effect of a 'modulator' of cell growth might be manifested in a sensitized background, here provided by RasQ61L. Surprisingly, expression of ERK1 kinase resulted in a significant reversion of the cell-proliferation effect caused by oncogenic Ras, whereas neither ERK2 nor p38 seemed to affect the overall growth rate (Figure 5c,d).
These data indicate that ERK1 protein expression counteracts Ras-dependent cell transformation. Importantly, this effect seems to be largely independent of ERK1's kinase activity but rather is due to protein-protein interactions, as the ERK1K72R mutant was almost as effective as wild-type ERK1. Strikingly, overexpression of ERK2 has little effect on cell proliferation, suggesting that levels of this protein are not rate-limiting, at least in this cell type.
Ectopic expression of ERK1 attenuates Ras-dependent growth in transformation assays
As observed in Figure 5, expression of a kinase-defective mutant of ERK1 was found to be very effective in inhibiting cell proliferation in NIH 3T3. This observation is consistent with the MEK-ERK competition model, as one of the predictions of this model is that a kinase-defective form of ERK1 should efficiently displace the endogenous ERK2 protein from MEK and therefore significantly reduce the overall signaling output. We also speculated, however, that a kinase-defective mutant of ERK2 should act as inhibitor of endogenous ERK2 and that its effect could possibly be even more pronounced than that caused by ERK1. To test this hypothesis we generated a kinase-dead ERK2 mutant, ERK2K52R, and compared its effect in the colony formation assay with that of the ERK1K72R mutant . As shown in Figure 6b, both ERK kinase-defective mutants were very effective in reducing oncogenic Ras-mediated colony formation, but ERK2K52R caused an almost complete inhibition whereas ERK1K72R reduced growth to only 40% of the total.
These data further support the idea that ERK1 and ERK2 compete for binding to MEK and therefore that their level of expression is crucial to the fine tuning of output signaling. Importantly, similar results were obtained with a different in vitro proliferation test, the soft agar assay (data not shown).
ERK1 attenuates Ras-dependent tumor formation in nude mice
To study tumor growth we used male, 4- to 6-week-old athymic nude mice. Cells of each clone were injected subcutaneously into each flank of the nude mice, using five animals for each clone. Nude mice were examined daily and tumor size was recorded from day 4 to 9, as indicated in Figure 7b. Although both RasQ61L-transformed cells and cells double-transfected with ERK2 or p38 produced large tumors, cells double-transfected with either ERK1 or ERK1K72R produced significantly smaller ones.
The experiment was terminated 10 days after injection and tumors were removed and weighed. Representative explant sizes are shown in Figure 7c and the mean weight data of two experiments are shown in the Figure 7d. Although mice injected with control vector cells did not develop tumors, all the other animals did. Tumors from mice injected with NIH 3T3 cells cotransfected with RasQ61L and ERK1 or ERK1K72R were, however, significantly smaller (means of 0.32 g and 0.29 g, respectively) than those obtained from mice injected with RasQ61L (1.2 g, p < 0.001 for both comparisons). No significant differences were seen when instead comparing tumors from mice injected with RasQ61L alone and double transfectants with ERK2 or p38 (1.04 g and 0.98 g on average, respectively).
These data on tumorigenicity in vivo fully confirm the results of the in vitro functional assay, indicating a potential anti-oncogenic role of ERK1 in its ability to reduce Ras-mediated cell transformation and tumor formation, at least in this experimental system.
The recent generation of mice with targeted mutations of the ERK1 and ERK2 genes has provided an excellent opportunity to determine the level of redundancy and overlap in function between these two major MAP kinase isoforms. ERK1 mutant mice have a strikingly milder phenotype than ERK2 mutants, which die early in development [28, 30–32]. At the molecular level, one of the most intriguing features of ERK1-deficient cells is the enhanced stimulus-dependent activation of ERK2 in the absence of any compensatory increase in ERK2 protein levels. We believe that this effect is largely due to a competition between ERK1 and ERK2 in binding to the upstream kinase MEK. In a previous report  we supported this claim by showing that the kinase-defective ERK1K72R mutant is just as effective as wild-type ERK1 in rescuing the phenotype in ERK1-deficient MEFs . The aim of the present work was to extend those preliminary findings and explore the possibility that ERK1 acts, at least in certain cellular settings, as a negative modulator of cell proliferation, by interfering with Ras-ERK2-dependent signaling. First we examined growth rates of MEFs obtained from ERK1-deficient animals. In the absence of ERK1, MEFs proliferate faster than control cells. Accordingly, whereas ERK1 knockdown by RNAi in both MEFs and NIH 3T3 cells facilitates cell growth, ERK2 silencing causes severe cell-proliferation deficits. By contrast, a mild over-expression of ERK1 is sufficient to slow down cell growth mediated by oncogenic Ras, and this concomitantly impairs ERK2 activation. This inhibitory effect of ERK1, which cannot be achieved by ectopically expressing ERK2, also affects the ability of oncogenic Ras to transform NIH 3T3 cells in functional assays such as colony formation and to cause tumors in nude mice. ERK1 seems to act by displacing ERK2 from the upstream kinase MEK rather than by regulating downstream effectors, as a kinase-inactive form of ERK1 is equally effective in causing the observed phenotypes. This hypothesis is also directly supported by the observation that in ERK1-deficient fibroblasts, the amount of MEK-ERK2 complexes is significantly increased.
The hypothesis that signaling molecules can act as titrating agents, sequestering central components of the signaling machinery, has already gained some experimental support. For instance, Rap1, a Ras-related small GTPase, was originally identified as a suppressor of K-Ras-mediated transformation because when overexpressed in NIH 3T3 cells it binds with high affinity to Raf-1 and therefore traps this downstream Ras effector in an inactive complex ( and reviewed in [48, 49]). Moreover, a provocative paper suggested in 2001  that wild-type K-ras protein could itself act as a tumor suppressor of oncogenic K-Ras. In addition, certain B-Raf kinase-defective mutants can be highly oncogenic because they interact with wild-type c-Raf-1 and thus activate MEKs with greater activity .
Our observation that ERK1 can interfere with ERK2-dependent signaling follows on from these pieces of evidence, but a few specific features of our results should be highlighted. First, no mutation in the ERK1 protein seems to be required to exert the effect on ERK2 phosphorylation, as can be seen by simply overexpressing wild-type ERK1. Second, no enzymatic activity is strictly necessary, as indicated by the effect of ERK1K72R. Importantly, the homologous kinase-dead mutant of ERK2, ERK2K52R, is even more effective in interfering with endogenous ERK signaling, further supporting the competition model. Third, complete ablation of ERK1 from cells is sufficient to provide a significant growth advantage, in both primary fibroblasts and cultured NIH 3T3 cells, although this manipulation is ineffective in promoting oncogenic Ras-mediated transformation. Fourth, overexpression of ERK1 does not affect normal growth but only that dependent on oncogenic Ras. All these observations need to be explained by a detailed cellular and biochemical model of ERK regulation, which goes beyond the present work.
Certainly, other regulatory mechanisms are likely to be in place and to contribute significantly to the functional differences between ERK1 and ERK2. For instance, the rate of translocation, dephosphorylation and sequestration in the nucleus of the two ERK proteins could be different. Although our experiments with the two kinase-defective mutants suggest that protein-protein interactions in the absence of kinase activity are dominant in the process, it is formally possible that smaller differences in substrate specificity could also partially contribute to specific aspects of cell physiology controlled by the two isoforms. Another important aspect that should be taken into consideration in the future is the potential role of scaffolding complexes in the differential regulation of ERK1, ERK2 and MEK interactions (reviewed in [50, 51]). This possibility was suggested previously by computational studies revealing that an optimal concentration of scaffold proteins relative to their kinase partners is always required to maximize signaling output. Alterations of the ratio of ERK1/ERK2 to MEK and to any scaffold protein within the cell could therefore affect the threshold properties of the system and contribute to some of the non-linear cellular responses [52, 53].
Despite these open questions and the limitations of our experimental approach, we believe we have at least identified a new, possibly important aspect of ERK regulation. We are just starting to uncover a system of previously unappreciated complexity, involving dynamic interactions of MEK1/2, ERK1 and ERK2. Future work at the biophysical and computational level will certainly be required to clarify these important mechanisms.
Materials and methods
To generate plasmids for stable RNAi, oligonucleotides encoding shRNAs against ERK1, ERK2 and related scrambled control sequences were purchased from VBC Genomics (Vienna, Austria), annealed and cloned into BglII and XhoI sites under the control of a constitutive human H1 promoter (pSUPER_Puro) . A set of six 19-mer shRNAs for each MAP kinase gene was designed as described  and tested, and the most efficient ones were chosen. The following oligonucleotides were used (bold text indicates the targeted sequence in the coding region that creates the stem of each shRNA; underlined text indicates the point mutations): for mouse ERK1, 5'-GATCCCCGACCGGATGTTAACCTTCATTCAAGAGATGAAGGTTAACACCGGTCTTTTTGGAAA-3' and 5'-AGCTTTTCCAAAAAGACCGGATGTTAACCTTCATCTCTTGAATGAAGGTTAACATCCGGTCGGG-3', targeting exon 7; for mouse ERK2, 5'-GATCCCCGTACAGAGCTCCAGAAATTTTCAAGAGAAATTTCTGGAGCTCTGTACTTTTTGGAAA-3' and 5'-AGCTTTTCCAAAAAGTACAGAGCTCCAGAAATTTCTCTTGAAAATTTCTGGAGCTCTGTACGGG-3', targeting exon 4; for the ERK1 control, 5'-GATCCCCGACCGGAT AG TAACCTTCATTCAAGAGATGAAGGTTA CT ATCCGGTCTTTTTGGAAC-3' and 5'-TCGAGTTCCAAAAAGACCGGAT AG TAACCTTCATCTCTTGAATGAAGGTTA CT ATCCGGTCGGG-3'; for the ERK2 control, 5'-GATCCCCGTACAGAGC GT CAGAAATTTTCAAGAGAAATTTCTG AC GCTCTGTACTTTTTGGAAC-3' and 5'-TCGAGTTCCAAAAAGTACAGAGC GT CAGAAATTTCTCTTGAAAATTTCTG AC GCTCTGTACGGG-3'.
All lentiviral constructs were built from plasmid pCCLsin.cPPT.PGK.eGFP.WPRE, as described elsewhere . For cloning into LVs, each shRNA-H1/mPGK-Puro cassette was cut out from pSUPER_Puro vectors and cloned into the BamHI/ClaI site of the vector. Enhanced green fluorescent protein (eGFP) was removed by BamHI/SalI digestion, then the vector was blunted and self-ligated.
For ectopic expression in the colony formation assay, mouse ERK1, ERK1K72R, ERK2 and ERK2K52R were epitope-tagged at the amino terminus with hemagglutinin and cloned into the pMex-NeoR expression vector. An oncogenic form of H-Ras (H-RasQ61L) was instead Myc-tagged and also cloned into the pMex-NeoR expression vector.
Viral vector production and titration
VSV-pseudotyped third-generation LVs were produced by transient four-plasmid cotransfection into 293T cells using the calcium phosphate method and purified by ultracentrifugation, with the modification that 1 mM sodium butyrate was added to the cultures for vector collection. Supernatants were collected 48 h after transfection, filtered through a 0.22 mm membrane and concentrated by ultracentrifugation as described .
The average number of viral vector particles were measured by the HIV-1 gag p24 antigen immunocapture assay (NEN Life Science Products, Boston, USA) and viral titer was determined by real-time PCR .
Cell culture and biochemistry
MEF cultures were prepared from wild-type and knockout embryos at embryonic day 13.5. Cells at early passages were serum starved for 24 h and then stimulated with 20% serum for various times; protein was then extracted and analyzed by SDS-PAGE and western blotting . For the RNase protection assay, RNA extraction and analysis from stimulated cell monolayer was performed as previously described . For cell-proliferation studies in MEFs or NIH 3T3 cells, 1.25 × 105 cells were seeded in each six-well plate on day 1 and maintained in Dulbecco's Modified Eagle Medium (DMEM) at different serum concentrations, with 2 μg/ml puromycin if required. Medium was changed every day. Duplicate samples for each serum concentration and for each genotype were counted every day for 5 days using a Burker counting chamber. For biochemistry, NIH 3T3 cells were transfected with pSUPER_Puro-shRNAs or with hemagglutinin epitope-tagged ERK1, ERK1K72R, ERK2, p38 and Myc epitope-tagged H-RasQ61L cloned into the pMex-NeoR vector by calcium phosphate precipitation . Stable transfectants were obtained after selection in G418 (Geneticin, Gibco-Invitrogen, Carlsbad, USA) or puromycin (Clontech, Mountain View, USA).
NIH 3T3 cells were stably transfected with ERK1 and ERK2 knockdown or control plasmids. Cells were washed with ice-cold phosphate-buffered saline before extraction in 1 ml of lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM EGTA, 1 mM EDTA, 1% Triton X-100, 2 mM MgCl2, 50 mM NaF, 10 μM Na3VO4) containing 0.2 mM phenyl methyl sulfonyl fluoride (PMSF) and 1× COMPLETE cocktail of protein inhibitors (Roche, Basel, Switzerland). Extracts were clarified at 14,000 g for 5 min at 4°C. Equal amounts of each protein lysate (5 mg) were pre-cleared with ExtraCruz-F immunoprecipitation (IP) matrix (Santa Cruz Biotechnology, Santa Cruz, USA) for 30 min at 4°C and incubated for an additional 1 h with polyclonal anti-MEK-1/2 antibodies (sc-436, Santa Cruz) complexed with the IP matrix. Immune complexes were collected and washed three times with lysis buffer, and western blotting was performed with polyclonal anti-ERK1 (sc-94) and anti-ERK2 (sc-153) antibodies (Santa Cruz), which preferentially recognize ERK1 and ERK2, respectively.
Colony formation assay in NIH 3T3 cells
Colony formation assays were performed as previously described . On day 0, NIH 3T3 cells were seeded in 100-mm plates, 1.5 × 105 cells per plate, and were transfected the following day. Two days after transfection, cells were trypsinized and plated on 100-mm plates, 103 cells per plate in DMEM containing 10% bovine calf serum and 500 mg/ml G418 for the selection of neomycin-resistant cells. Each transfection sample was plated into four plates. Medium was changed every 3 days and after 10 days clones were washed with water and fixed in 10% formaldehyde (Sigma-Aldrich, St. Louis, USA) for 10 min. Plates were then washed once with water and stained for 5 min in 0.5% crystal violet (Fluka, Sigma-Aldrich, St. Louis, USA) in 20% methanol and finally washed with water to remove background staining. Images were acquired with a scanner and all colonies larger then 1.5 mm in diameter were counted.
Tumorigenicity assay in nude mice
G418-positive clones selected as described above were used for the tumorigenicity assay . 1 × 106 cells of each clone in log-phase growth were injected subcutaneously into each of two flanks of male 4- to 6-week-old athymic nude mice. For each clone five animals were used. The nude mice were examined daily and tumor sizes were recorded. The experiment was terminated 10 days after injection, animals were sacrificed and tumors were removed and weighed.
We thank Rony Seger, Gimmi Ratto, Jacques Pouysségur, Gilles Pagès and Paul Orban for critical and helpful reading of the manuscript and Mario Amendola and Monica Martignoni for excellent technical support. This work was supported by the Italian Federation for Cancer Research (FIRC) and by the Italian Ministry for the University and Research (MIUR), to RB.
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