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Abstract
Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation 1,4 1,4 1 Xavier Descheˆnes-Simard, Marie-France Gaumont-Leclerc, Ve´ronique Bourdeau, 1 1 2 1 1 Fre´de´ric Lessard, Olga Moiseeva, Vale´rie Forest, Sebastian Igelmann, Fre´de´rick A. Mallette, 3 3 2 2 1,5 Marc K. Saba-El-Leil, Sylvain Meloche, Fred Saad, Anne-Marie Mes-Masson, and Gerardo Ferbeyre 1 2 De´partement de Biochimie, Universite´ de Montre´al, Montre´al, Que´bec H3C 3J7, Canada; CHUM (Centre Hospitalier de l’Universite´ de Montre´al), Universite´ de Montre´al, Montre´al, Que´bec H2L 4M1, Canada; Institut de Recherche en Immunologie et Cance´rologie, Department of Pharmacology, Program in Molecular Biology, Universite´ de Montre´al, Montre´al, Que´bec H3C 3J7, Canada Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAi- mediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence. [Keywords: ERK; benign prostatic hyperplasia; oncogenic ras; proteasome; senescence] Supplemental material is available for this article. Received August 20, 2012; revised version accepted March 25, 2013. Normal mammalian cells respond to oncogenic threats to a noninflammatory cell death process and eventually by triggering intrinsic tumor suppression mechanisms the engulfing of apoptotic cells by the immune system that curtail cell cycle progression and execute cell death (Leist and Jaattela 2001). The effector mechanism of or permanent cell cycle arrest (Lowe et al. 2004). These senescence remains unknown, although these cells seem outcomes were originally discovered by studying the to be also programmed to interact with the immune response of normal cells to Myc overexpression, which system, secreting large amounts of cytokines (Coppe et al. induces apoptosis (Evan et al. 1992), or Ras overexpres- 2010) and being the target of immune-mediated clearance sion, which induces a terminal cell cycle arrest known as (Xue et al. 2007; Kang et al. 2011). cellular senescence (Serrano et al. 1997). Subsequently, Ras-induced senescence depends on the concerted ac- INK4a these mechanisms have been demonstrated in animal tion of the p53, p16 /RB, and PML tumor suppressor models expressing these oncogenes (Braig et al. 2005; pathways (Serrano et al. 1997; Ferbeyre et al. 2000). The Chen et al. 2005; Murphy et al. 2008; DeNicola et al. activation of p53 by ras and other oncogenes involves the 2011). Apoptosis is a programmed response where pro- DNA damage response (DDR) (Bartkova et al. 2006; Di teolytic enzymes digest selective cellular targets, leading Micco et al. 2006; Mallette et al. 2007), a consequence of DNA damage triggered by oncogenic activity. This DNA damage could be the result of a replication stress induced by aberrant activation of replication forks or the increased These authors contributed equally to this work. Corresponding author production of mitochondrial reactive oxygen species (ROS) E-mail [email protected] (Mallette and Ferbeyre 2007). However there is still a gap Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.203984.112. between our current view of oncogene signaling and the 900 GENES & DEVELOPMENT 27:900–915 2013 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/13; www.genesdev.org Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Senescence-associated protein degradation molecular events leading to DNA damage. In addition, at Ser380 did not decrease upon inhibition of ERK2 (Fig. senescence can occur in the absence of DNA damage. For 1A), suggesting that another kinase activated by RasV12 example, in normal fibroblasts expressing oncogenic ras, can catalyze this event or that the remaining levels of the inactivation of the DDR was not sufficient to bypass phospho-ERK in cells expressing shERK are sufficient to senescence (Mallette et al. 2007), and in mice, there is no do it. ERK2 knockdown in cells expressing RasV12 evidence linking DNA damage to Ras-induced senescence inhibited the induction of senescence-associated b-galac- (Efeyan et al. 2009). Other effects of oncogenic activity tosidase (SA-b-Gal) (Fig. 1C), PML bodies, and DNA have been proposed as mediators of senescence and may damage foci (Supplemental Fig. S1C–G). Oncogenic ras INK4a compensate for or cooperate with the DDR. These include engaged the p53/p21, p16 /RB, and p38MAPK path- ARF p19 expression (Ferbeyre et al. 2002), autophagy (Young ways in primary cells, and this was efficiently prevented et al. 2009), mitochondrial dysfunction (Moiseeva et al. by knockdown of ERK2 (Fig. 1D; Supplemental Fig. S1H,I). 2009), cytokines (Coppe et al. 2010), PML bodies (Vernier The induction of several senescence-associated cytokine et al. 2011), and heterochromatin formation (Narita et al. genes by RasV12 was also efficiently blocked by ERK2 2003). However, it remains puzzling why constitutive knockdown (Supplemental Fig. S1J–L). The inhibition of growth factor signaling pathways and the Ras/ERK path- Ras-induced senescence by several shERKs was accom- way trigger these proliferation barriers in normal cells. panied by the adoption of the distinctive cell morphology Here we performed an unbiased screen using shRNA of small cells growing sometimes on top of each other libraries to discover genes required for oncogenic ras to (Fig. 1C), a complete rescue of the proliferation arrest (Fig. regulate senescence in human normal fibroblasts. We 1E), a stimulation of DNA synthesis as measured by BrdU identified the ERK/MAPK as essential mediators of incorporation and KI-67 staining (Fig. 1F), and the expres- S10 senescence and surprisingly found that attenuating ERK sion of mitotic markers such as phospho-H3 or phospho- S28 expression in human or mouse primary fibroblasts allowed H3 (Fig. 1D). Moreover, the high levels of ROS known to their transformation by oncogenic ras.Wealso found that contribute to DNA damage during Ras-induced senescence aberrant Ras/ERK signaling led to a proteasome-dependent (Moiseeva et al. 2009) were decreased in cells depleted of protein degradation process targeting proteins required for ERK2 (Fig. 1G). Taken together, the results indicate that cell cycle progression, cell migration, mitochondrial func- reducing ERK levels shuts down the senescence tumor tions, RNA metabolism, and cell signaling. These findings suppression response to oncogenic ras in normal human were validated in cells with short telomeres and in human fibroblasts. prostate benign neoplasms where we found expression of To assess the generality of these findings, we next senescence markers, high levels of phospho-ERK, and low studied the induction of senescence by oncogenic ras in levels of STAT3, one of the targets of the senescence- primary human mammary epithelial cells (HMECs). In- associated protein degradation (SAPD) process. troduction of oncogenic ras by retroviral gene transfer in these cells induced a senescent phenotype, characterized by induction of PML bodies, DNA damage foci, and cell Results cycle arrest (Supplemental Fig. S2A–D). We also noticed that in cultures of HMECs expressing RasV12, some cells ERK/MAPK are required for Ras-induced senescence spontaneously escaped from senescence and started pro- in human fibroblasts liferating as small cells. All of these cells turned out to In order to identify genes that contribute to oncogene- express very low levels of RasGTP and phospho-ERK induced senescence (OIS), we performed RNAi screening (Supplemental Fig. S2E), consistent with the requirement looking for shRNAs able to bypass Ras-induced senes- for strong ERK/MAPK kinase signaling to sustain Ras- cence in human fibroblasts (Supplemental Fig. S1A). induced senescence. Then, we studied the effect of ERK2 shRNAs targeting ERK2 (MAPK1)and HMGB1 were re- knockdown on Ras-induced senescence in HMECs. As covered from the screening. We confirmed the senescence described for human fibroblasts, shERK2 (Fig. 2A) reduced bypass using several shRNAs against ERK1 and ERK2 that the expression of ERK-dependent targets (Fig. 2B) and were all capable of inhibiting RasV12-induced senescence restored cell proliferation (Fig. 2C) and the expression of S10 S28 (Supplemental Fig. S1B). We found a good correlation the mitotic markers phospho-H3 and phospho-H3 . between the degree of total ERK inhibition and the bypass shERK2 allowed RB phosphorylation in HMECs express- of senescence (Supplemental Fig. S1B). More important, ing oncogenic ras, and the expression of E2F target genes since several shRNAs against ERK1 or ERK2 bypassed such as MCM6 (Fig. 2D). shERK2 also prevented the induc- Ras-induced senescence, it is very unlikely that off-target tion of p53 target genes and cytokines by oncogenic ras in effects of shRNAs were responsible for the bypass. To HMECs (Fig. 2E). The senescent phenotype was bypassed further characterize the consequences of ERK inhibition by shERK2 with a frequency several times higher than the for RAS signaling, we then used one shRNA that efficiently spontaneous escape from senescence mentioned above inhibited ERK2, the more abundant ERK isoform in fibro- (Fig. 2F,G). blasts. This shRNA did not inhibit the members of the To demonstrate that reducing ERK levels in a genetic pathway upstream of ERK (RAS, RAF, and MEK) (Fig. 1A) model also prevents Ras-induced senescence, we used but efficiently inhibited the expression of several tran- mouse embryonic fibroblasts (MEFs) from Erk2 knockout scriptional targets of the ERK pathway (Fig. 1B; Pratilas mice (Voisin et al. 2010). In wild-type MEFs, oncogenic et al. 2009). Interestingly, the phosphorylation of p90RSK Ras induced Erk1/2 phosphorylation (Fig. 2H) and the GENES & DEVELOPMENT 901 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Descheˆnes-Simard et al. Figure 1. ERK/MAPK inhibition bypasses Ras-induced senescence. (A) Immunoblots for proteins in the ERK pathway using extracts from fibroblasts expressing H-RasV12 (R) or an empty vector (V) and shRNA against ERK2 (shERK) or a nontargeting shRNA (shCTR) obtained from cells 14 d after infection. (B) Quantitative PCR (qPCR) for ERK2 mRNA and mRNAs encoded by ERK-stimulated genes in cells as in A.(C) SA-b-Gal of cells as in A. Data were quantified from 100 cell counts in triplicate and are presented as the mean percentage of positive cells 6 standard deviation (SD). (D) Immunoblots for cell cycle-regulated proteins in cells as in A.(E) Growth curves started with cells as in A. Data are presented as mean 6 SD of triplicates. (F) Quantitation of BrdU incorporation (2 h of incubation with 10 mM BrdU) and KI-67 staining in cells as in A. Data were quantified from 100 cell counts in triplicate and are presented as the mean of positive cells 6 SD. (***) P < 0.0005, two-sample t-test. (G) Superoxide levels in cells as before, measured by flow cytometry (FACS) after staining with 1 mM fluorescent probe dihydroethidium (DHE) during 1 h. The results are expressed as the percentage of maximum cell number. The maximum cell number is the number of cells for the most-represented fluorescence intensity in the cell population of a condition and is expressed as 100%. All experiments were performed a minimum of three times. 902 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Figure 2. ERK/MAP kinases play a general role in cellular senescence. (A–G) Role of ERK/MAPK in H-RasV12-induced senescence in primary HMECs. (A) Immunoblots to confirm ERK knockdown and expression levels of H-RasV12 in extracts obtained 14 d after infection. (B) qPCR for ERK target genes to confirm the biological effect of ERK knockdown in cells expressing the indicated vectors. (C) qPCR for KI-67, a proliferation marker, in cells as in A.(D) Immunoblots for proteins in the RB pathway and mitosis markers in cells as in A.(E) qPCR for senescence markers in cells as in A.(F) Morphology of HMECs expressing H-RasV12-ER and shRNA against ERK2 (shERK) or a nontargeting shRNA (shCTR). Note the large size and vacuolated cytoplasm of senescent cells in contrast with the small growing cells that escape from senescence (arrows) due to low phospho-ERK levels. (G) Quantification of the bypass from senescence between cells with shControl (shCTR) and shERK2. Error bars represent SD. (**) P < 0.005, two-sample t-test. (H–P) Genetic inactivation of Erk2 bypasses Ras-induced senescence in MEFs. (H) Immunoblots for the indicated proteins in wild-type and Erk2 MEFs expressing H-RasV12, a vector control, or H-RasV12 + E1A 14 d after infection. (I) qPCR for Erk target genes in cells as in H.(J) SA-b-Gal markers in cells as in H. Data were quantified from 100 cell counts in triplicate and are presented as the mean percentage of positive cells 6 SD. (K) Growth curves started with MEFs from wild-type and Erk2 animals expressing H-RasV12 or a vector control 14 d after infection. Data are presented as the mean 6 SD of triplicates. (L–N) qPCR for the indicated genes in cells as in H.(O) S15 / Immunoblots for the indicated senescence markers in cells as in H.(P) Immunoblots against p53 and p53 from Erk2 MEFs treated for 24 h with doxorubicin (300 ng/mL) or vehicle. Experiments were performed n $ 3. Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Descheˆnes-Simard et al. expression of Erk target genes (Fig. 2I). The oncoprotein formation in soft agar (Fig. 3A, top) and inhibited the E1A, able to bypass Ras-induced senescence (Serrano et al. process of contact inhibition, as seen in a focus assay (Fig. 1997), reduced Erk1/2 activation and also the expression of 3A, middle). These two assays are strong indications of the Erk target genes (Fig. 2H,I). In Erk2 MEFs, oncogenic transformation of normal cells into cancer cells. Consis- Ras induced Erk1 but not Erk2 phosphorylation (Fig. 2H). tent with these ex vivo models, primary human fibroblasts This reduction in overall Erk activity was translated in expressing hTERT, RasV12, and shERK2 formed tumors in a reduced induction of Erk target genes (Fig. 2I). More nude mice (Fig. 3A [bottom], E). Upon histological exam- important, oncogenic Ras failed to induce growth arrest, ination, these tumors were characterized by large nuclei, p53, and senescence in Erk2 MEFs, in clear contrast to abundant mitotic images, many blood vessels, and very its effects in wild-type MEFs (Fig. 2J–O). In fact, Erk2 low levels of phospho-ERK in the tumor cells (Supple- MEFs had a response to oncogenic ras similar to that of mental Fig. S3B). These low levels of ERK were confirmed wild-type MEFs expressing E1A (Fig. 2J). We also looked at by immunoblots from tumor cells extracts (Supplemental ARF the expression of the tumor suppressor p19 in Erk2-null Fig. S3C) or in cell lines established from the tumors cells expressing RasV12. We found that RasV12 still (Supplemental Fig. S3D). We also found that adding c-MYC ARF induced p19 in these cells (Fig. 2O), but obviously, this to the cells expressing hTERT, RasV12, and shERK2 was not sufficient to trigger senescence. We also found that further enhanced their transformation (Fig. 3A,E). When p53 was stabilized and phosphorylated at Ser15 after tested for their ability to grow on a monolayer of normal treatment of Erk2 MEFs with doxorubicin, indicating fibroblasts, cells recovered from these tumors formed as that their resistance to Ras-induced senescence was not many colonies in focus assays as the parent populations the result of an accidental loss of p53 (Fig. 2P). (Supplemental Fig. S3E). In conjunction, these results Finally, we studied whether inhibition of ERK activity, suggest that cells expressing oncogenic ras and shERK2 not levels, was also sufficient to bypass RasV12-induced did not undergo additional genetic changes in vivo that senescence. We used the MEK inhibitors U0126 and would have further enhanced their transformed pheno- AZD6244, which inhibited ERK phosphorylation induced type. As shown before for the senescence bypass, a differ- by RasV12 in human fibroblasts in a dose-dependent ent anti-ERK shRNA cooperated with oncogenic ras to manner (Supplemental Fig. S2F) without altering ERK transform primary human fibroblasts or HMECs (Supple- mRNA levels (Supplemental Fig. S2G). MEK inhibitors mental Fig. S3F–I). also prevented the induction of ERK target genes (Sup- Finally, we observed that oncogenic ras was able to plemental Fig. S2H,I) and restored RB phosphorylation transform primary MEFs from Erk2 knockout animals and the expression of E2F targets in cells expressing without the need to express any other cooperating onco- RasV12 (Supplemental Fig. S2F). In agreement with pre- gene (Fig. 3B–E). As shown for human fibroblasts, the vious results (Lin et al. 1998), MEK inhibitors blocked ability of cells transformed by oncogenic Ras in Erk2 Ras-induced senescence (Supplemental Fig. S2J), growth knockout MEFs to form colonies in soft agar was the same arrest (Supplemental Fig. S2K–M), and the induction of before and after forming tumors in mice, suggesting that the senescence genes CDKN1A and CDKN2A (Supple- no other genetic modifications occurred in vivo to further mental Fig. S2N). We thus conclude that reducing ERK transform these cells (Supplemental Fig. S3J,K). As shown activity by different manipulations in both human and before (Serrano et al. 1997), oncogenic ras cooperated mouse primary cells compromises Ras-induced senes- with E1A to transform primary rodent cells (Fig. 3B–E). In cence, preventing the induction of several tumor sup- E1A-expressing MEFs, both phospho-Erk1 and phospho- pressor pathways. Next, we asked whether oncogenic ras Erk2 were reduced (Fig. 2H). The same was noticed in was capable of transforming cells where reduced ERK human cells (Supplemental Fig. S3A,C,D). Since decreasing levels prevented the activation of tumor suppressors. ERK activity is sufficient to bypass Ras-induced senescence and promote transformation, these results suggest a novel mechanism by which E1A cooperates with oncogenic ras ERK/MAPK knockdown facilitates Ras-dependent to transform primary cells. Taken together, these results transformation of primary human cells reveal a tumor suppressor function of the ERK kinases in To investigate the effect of ERK inhibition in ras-dependent normal cells and dissociation between the transforming transformation, we first used the experimental system functions of ras, which do not require high ERK activity, where RasV12 cooperates with hTERT and the SV40 early and its ability to induce senescence, which requires high region to transform normal human fibroblasts (Hahn ERK activity. et al. 2002). We reasoned that ERK2 inhibition could be genetically equivalent to the SV40 early region, which Selective ERK-dependent protein degradation also inhibits the activation of p53 and RB by RasV12 characterizes cellular senescence (Hahn et al. 2002). We first prepared IMR90 cells express- ing hTERT using a lentiviral vector. Then, we introduced Next, we addressed the mechanism by which the strength vectors expressing shERK2 and RasV12 or the corre- of ERK activation induces the stress signaling pathways sponding control vectors and confirmed the knockdown leading to senescence. We used vectors able to drive either of ERK in these cells and the expression of oncogenic ras high or low expression levels of RasV12. As expected, high (Supplemental Fig. S3A). We found that ERK inhibition levels of RasV12 led to higher phosphorylation of ERK1/2 S383 S910 by shERK2 in RasV12-expressing cells enabled colony and some ERK targets such as ELK and FAK 904 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Senescence-associated protein degradation Figure 3. ERK/MAPK inhibition promotes Ras-induced transformation. (A, top) Soft agar assay with HeLa cells or IMR90 fibroblasts expressing the indicated vectors. Representative GFP-positive colonies are shown. A focus-forming assay (middle) and tumor formation in nude mice (bottom) were performed with cells as above. Numbers of colonies in soft agar and focus-forming assays are expressed as the mean 6 SD of triplicates. (B) Soft agar assay with wild-type and Erk2 MEFs expressing the indicated vectors. (C) Quantification of B using the CyQuant GR dye. (RFU) Relative fluorescence units (a measure of growth in soft agar). Data are presented as mean 6 SD of triplicates. (D) Tumor formation in nude mice of wild-type (WT) and Erk2 MEFs expressing the indicated vectors. (E) Quantification of tumor formation in nude mice. The number of injections that generated tumors and the time taken by the tumors to reach the threshold of significance (0.2 cm )are shown. (Supplemental Fig. S4A). Low levels of RasV12 slightly and also failed to induce growth arrest, the DDR, and stimulated ERK phosphorylation and p53 target gene ex- senescence (Supplemental Fig. S4C–H). We used these pression (Supplemental Fig. S4A,B) but did not engage the vectors and a battery of phospho-specific antibodies RB tumor suppressor pathway (Supplemental Fig. S4B) (Kinexus) to profile the state of ERK phosphorylation GENES & DEVELOPMENT 905 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Descheˆnes-Simard et al. targets depending on the strength of Ras signaling. We inhibitor MG132 in primary fibroblasts expressing RasV12 found, as anticipated, that some ERK targets were highly (Fig. 4E). In addition, in RasV12-expressing cells treated phosphorylated in cells expressing high levels of RasV12. with MG132, the levels of ubiquitinated HSP70 and However, we also found unexpectedly that some phos- STAT3 were increased (Fig. 4F-G). Together, the results pho-ERK targets were less phosphorylated (Fig. 4A). In- are consistent with a model of selective protein degra- S727 triguingly, the reduction in phosphorylation of STAT3 dation triggered by aberrant ERK signaling during Ras- S789 and Caldesmon in cells expressing high levels of Ras induced senescence. However, not all phosphoproteins was also observed at the level of total protein (Supple- found unstable in our proteomic analysis are expected to mental Fig. S4I). As protein phosphorylation and protein be depleted in senescent cells, since their reduction will degradation are often linked (Hunter 2007), we thought depend on the extent of phosphorylation of each protein that one mechanism connecting high ERK signaling to pool and compensatory biosynthesis and the degradation senescence might involve the degradation of phosphory- rate for each protein in normal conditions. In addition, lated proteins due to aberrant ERK signaling. although most phosphoproteins contain candidate ERK To support the model that increased ERK signaling phosphorylation sites, others do not, suggesting that other leads to proteasome-dependent protein degradation in kinases may be engaged by ERK in the process. The case senescent cells, we used large-scale proteomics to look of c-MYC illustrates this point. c-MYC is phosphorylated for phosphoproteins stabilized by the proteasome inhib- by ERK at Ser62, and this phosphorylation facilitates itor MG132 in senescent cells. We identified an enrich- phosphorylation at Thr58 by GSK3; it is this latter event ment of nearly 3000 phosphopeptides from 1018 proteins. that targets c-MYC to the proteasome (Yeh et al. 2004). In Most of the phosphorylation sites identified consisted of agreement with this model, the GSK3 inhibitor CHIR99021 serine/threonine adjacent to a proline, consistent with inhibited the ubiquitination of c-MYC in Ras-expressing ERK/MAPK phosphorylation sites (Fig. 4B; Supplemental cells (Fig. 4H). Nevertheless, the levels of c-MYC, HSP27, Table SI). We also identified stabilized phosphopeptides HSP70, KAP1, RSL1D1, and STAT3 were restored by where the phosphorylated residues were not part of the shERK2 in Ras-expressing IMR90 cells (Fig. 5A) or ERK consensus site that may be targets of ERK-regulated HMECs (Fig. 5B), indicating that ERK signaling, directly kinases. Motif analysis of the phosphopeptides revealed or indirectly, triggers their degradation. In MEFs, we also phosphorylation motifs for proline-directed kinases but observed a decrease in levels of Hsp70, Kap1, and Stat3, also basophilic and acidophilic kinases (Supplemental but not c-Myc, in response to RasV12, and this down- Fig. S5). We analyzed the proteomics data using a FatiGO regulation did not occur in Erk2 MEFs (Fig. 5C). In all single enrichment from the bioinformatics platform cell types, the ERK-dependent decrease in c-MYC, HSP70, Babelomics 4.3. We found that in senescent cells, phos- KAP1, RSL1D1, and STAT3 could not be explained by phoproteins that control the response to growth factor a reduction in their mRNA levels (Supplemental Fig. S7A– stimulation and tumor progression are unstable. Hence, C). However, HSP27 levels were reduced at the mRNA the degradation pattern may favor senescence and inhibit level by an ERK-independent mechanism, explaining why transformation (Fig. 4C). In Supplemental Table SI, we its levels could not be restored by MG132 (Supplemental present a summary of the proteomics data with refer- Fig. S7A,B). Of note, RasV12 induced RSL1D1 mRNA ences to the implication of the proteins in cell senescence levels in human cells, perhaps as a feedback response to and cancer pathways, and in Supplemental Figure S6, we the reduction in protein levels (Supplemental Fig. S7A,B). present the most significant functional categories af- To investigate whether protein degradation is dependent fected by the degradation process. For example, the pro- on ERK levels only or ERK signaling, we used MEK teins FBXL11 (He et al. 2008), HSP70 (Gabai et al. 2009), inhibitors. Both AZD6244 and U0126 restored the levels RSL1D1 (Ma et al. 2008), and TBX2 (Martin et al. 2012) of selected proteins in RasV12-expressing cells (Fig. 5D) have been previously linked to the control of senescence, without corresponding changes in mRNA levels (Supple- validating our results. In addition, the presence of multi- mental Fig. S7D–I), consistent with their ability to bypass ple proteins linked to pseudopods, cell migration, and RasV12-induced senescence (Supplemental Fig. S2F–N). RNA metabolism (Supplemental Table SI) suggests that Cell senescence can also be induced by other stresses, these pathways are targeted by a SAPD process. such as radiation and short telomeres (replicative senes- We characterized in detail the degradation of c-MYC, cence). These situations commonly trigger a persistent HSP27, HSP70, KAP1, RSL1D1, and STAT3. The total DDR (d’Adda di Fagagna et al. 2003), mitochondrial dys- level of these proteins was down-regulated in senescent function, accumulation of ROS (Passos et al. 2007), and cells, and all but HSP27 were restored by treatment with a constitutive ERK activation (Satyanarayana et al. 2004). the proteasome inhibitor MG132, consistent with the We thus reasoned that sustained ERK activation may anticipated role of the proteasome in SAPD (Fig. 4D). This accelerate protein degradation during replicative senes- proteasome-dependent degradation seems to be selective cence and contribute to the establishment and mainte- because total levels of ubiquitinated proteins did not nance of this phenotype. We cultured young normal change significantly in cells expressing RasV12 (Fig. 4D). human fibroblasts until their replicative senescence in We also evaluated the half-life of several of these proteins the presence of two concentrations of the MEK inhibitors after inhibition of protein synthesis with cycloheximide. AZD6244 and U0126. We confirmed that senescent (old) As expected, c-MYC, HSP70, KAP1, RSL1D1, and STAT3 fibroblasts displayed a constitutive high ERK activity, but had a reduced half-life that was restored by the proteasome MEK inhibitors reduced this trait almost to young levels 906 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Figure 4. Selective and proteasome-dependent protein degradation characterizes Ras-induced senescence. (A) Summary of proteomic data obtained from cell lysates of IMR90 fibroblasts expressing a low level (LR) or high level (HR) of oncogenic ras. Cells were harvested 14 d after infection. Data show phosphorylation levels of 14 ERK targets measured by Western blot with phospho-specific antibodies by Kinexus (n = 1). The difference in the relative protein amount in cells that express HR is presented as a percentage of the relative protein amount in LR-expressing cells set as a reference (control). (B) Frequencies of phosphorylation motifs in phosphopeptides stabilized by MG132. Ras senescent cells (10 d after infection with H-RasV12) were treated for 18 h with DMSO (control) or 20 mM MG132. Then, cells were harvested, and protein extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for phosphoproteomics (n = 2, in triplicate each time). Phosphopeptides enriched in cells treated with MG132 were analyzed with the Motif-X software tool. Three motif families were identified (acidic, basic, and proline-directed), and almost half the phosphopeptides with an enriched motif have a proline-directed motif. (C) FatiGO single-enrichment analysis of phosphopeptides enriched in Ras- senescent cells treated MG132 with the Babelomics 4.3 platform. This platform was used to identify gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome terms that were significantly enriched (Supplemental Fig. S5B). Then, these terms and their associated peptides were grouped in the indicated general categories. These categories were also classified according to their predicted ability to induce senescence or limit transformation when they are decreased. (D) Immunoblots for the indicated proteins and total ubiquitinylated proteins from fibroblasts expressing oncogenic ras or an empty vector (10 d after infection) and treated for 18 h with 20 mM MG132 or DMSO as control (n = 3). (E) Protein stability assays for the indicated proteins in cells as in D. Cells were treated with DMSO, 10 mg/mL cycloheximide, or 20 mM MG132 for the indicated times. The relative protein quantity was evaluated by immunoblotting and quantification with Adobe Photoshop CS4 or Image Lab 4.0 (n $ 2). (F,G) Immunoprecipitation of HSP70 or STAT3 in extracts from IMR90 cells expressing H-RasV12 or an empty vector 10 d after infection and treated for 18 h with 20 mM MG132 and immunoblotted against mono- and polyubiquitinylated conjugates (n $ 2). (H) Immunoprecipitation of c-MYC in extracts from cells expressing H-RasV12 or an empty vector (10 d after infection) and treated for 18 h with 20 mM MG132 and/or GSK3 inhibitor CHIR99021 (3 mM) and immunoblotted against mono- and polyubiquitinylated conjugates. Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Descheˆnes-Simard et al. Figure 5. Role of ERK kinases in SAPD. (A) Immunoblots showing the levels of indicated proteins in IMR90 cells expressing H-RasV12 (R) or a vector control (V) and shERK2 or a control shRNA (shCTR) 14 d after infection (n $ 3). (B) Immunoblots for proteins and conditions as in A but for HMECs (n = 3). (C) Immunoblots showing the levels of the indicated proteins in wild-type or Erk2 MEFs expressing oncogenic ras or a vector control 14 d after infection (n = 3). (D) Immunoblots for the indicated proteins in IMR90 cells with a vector control (V) or H-RasV12 (R) and treated with the indicated chemicals. The treatments started immediately after infection, and the medium was changed every 2 d. Cells were harvested 10 d after infection (n = 2). (Fig. 6A). This inhibition of phospho-ERK levels translated cells, and some of them were found slightly increased in a decrease of p53 phosphorylation at Ser15 and an (Supplemental Fig. S8I). The half-life of all of these pro- increase in RB phosphorylation, indicating a cell cycle teins was decreased in senescent cells (Fig. 6J) and re- pattern of young healthy cells (Fig. 6A). In addition, the stored by MG132 (Supplemental Fig. S8J). Culturing cells levels of ERK target genes LIF and DUSP6 increased with with MEK inhibitors prevented the proteasome-depen- replicative senescence and were decreased by MEK inhib- dent down-regulation of these proteins (Fig. 6K), and the itors (Fig. 6B,C). MEK inhibitors improved cell proliferation mRNAs for all of them but HSP27 were not changed and the life span of normal human fibroblasts according to (Supplemental Fig. S8K). Altogether, the results indicate several criteria. First, they increased the expression of the that replicative senescence and OIS involve a proteasome- proliferation markers KI-67 and MCM6 (Fig. 6D). Second, dependent degradation of multiple proteins (SAPD), which they increased the number of passages upon serial culture of can potentially explain the activation of many stress normal human fibroblasts (Fig. 6E). Third, in a proliferation signaling pathways in senescent cells and their resistance assay performed over 14 d, cells from late-passage cultures to proliferating stimuli (Fig. 6L). The mechanistic con- proliferated faster if they were previously treated with MEK nections between the SAPD and the stresses that char- inhibitors (Fig. 6F). Finally, since the loss of proliferation acterize senescence suggest multiple positive feedback potential of normal human fibroblasts upon serial culturing loops in the process that may contribute to the robustness is due to cellular senescence, we measured the senescence of senescence as a tumor suppressor mechanism. markers IL6 (part of the senescence-associated secretory If SAPD is important for senescence, it should be possible phenotype) and SA-b-Gal. MEK inhibitors reduced signifi- to induce senescence by mimicking the process and bypass cantly the accumulation of both markers in late-passage senescence by inhibiting it. We tested this idea first by cultures (Fig. 6G,H). Taken together, our data indicate that using shRNAs against HSP27, HSP70, KAP1, RSL1D1, ERK signaling is required for both replicative senescence and STAT3 (Supplemental Fig. S9A,B), and, as expected, and OIS and raise the question of whether the SAPD the individual knockdown of these proteins triggers phenotype is also relevant for replicative senescence. cellular senescence in both primary fibroblasts and To investigate whether the proteins that we found IMR90 cells transformed by hTERT, RasV12, and shERK2 down-regulated in Ras-induced senescence were also re- (Fig. 7A; Supplemental Fig. S9A,B). Unfortunately, we duced during replicative senescence, we measured their could not rescue senescence with proteasome inhibitors levels in old and young cultures by immunoblotting. because they stabilize multiple tumor suppressors upon Again, we characterized these cells for a variety of cell long-term incubation, leading to cell death. On the other cycle and senescence markers (Supplemental Fig. S8A–H). hand, ectopic expression of the viral oncoproteins E1A We found that c-MYC, HSP27, HSP70, KAP1, RSL1D1, and E6/E7 (Fig. 7B-D) restored cell proliferation and and STAT3 were all found down-regulated in replicative inhibited senescence (Fig. 7E; Supplemental Fig. S9C–E). senescence, and their levels were restored by the protea- The expression of all tested SAPD targets—c-MYC, some inhibitor MG132 (Fig. 6I). The mRNAs coding for HSP27, HSP70, KAP1, RSL1D1, and STAT3—was restored these proteins were not down-regulated in senescent by these viral oncoproteins (Fig. 7F), while the expression 908 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Senescence-associated protein degradation Figure 6. ERK and the SAPD during replicative senescence. (A) Immunoblots for the indicated proteins in young (population doublings [PDL] = 21.5) and old (PDL $ 40) IMR90 cells treated with the indicated concentrations of MEK inhibitors or DMSO as control for 27 d. Fresh medium and inhibitors were added every 2 d. (B–D) qPCR for mRNAs encoded by ERK-stimulated genes and proliferation markers in cells as in A.(E) PDL of normal human fibroblasts in the presence of the indicated concentrations of MEK inhibitors or vehicle during 80 d. The experiment was started with 1 3 10 middle-age (PDL = 34) IMR90 cells for each condition. (F) Relative growth of late-passage human fibroblasts (PLD $ 41) after 60 d of treatments with MEK inhibitors or vehicle evaluated by a crystal violet assay. (G) qPCR for IL6 in cells as in A.(H) SA-b-Gal of cells as before after 40 d of treatments. Data were quantified from 100 cell counts in triplicate and are presented as the mean percentage of positive cells 6 SD (shown in the bottom right of every panel). (I) Immunoblots for the indicated proteins in young (Y) (PDL = 21.5) and old (O) (PDL = 40) IMR90 cells treated with 20 mM MG132 or vehicle (n = 2). (J) Relative decrease of half-lives for the indicated proteins calculated from cycloheximide stability assays as presented in Figure 4E. (K) Immunoblots for the indicated proteins in cells as in A.(L) High-strength ERK signals or short telomeres lead to protein degradation, which in turn activates multiple stress responses that characterize cellular senescence. Note that the process could be self-sustained by multiple positive feedback loops. Lower levels of ERK signaling are permissive for Ras-dependent transformation in cooperation with other signals stimulated by oncogenic ras. GENES & DEVELOPMENT 909 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Descheˆnes-Simard et al. Figure 7. Inactivation and stabilization of targets of SAPD (A) SA-b-Gal of wild-type IMR90 cells or transformed IMR90 cells (hTERT, H-RasV12, and shERK2) expressing the indicated shRNA expression vectors fixed 10 d after infection. Data were quantified from 100 cell counts in triplicate and are presented as the mean percentage of positive cells 6 SD. These experiments were done in triplicate and at least two times. (B–D) qPCR for the viral oncoproteins E6, E7, and E1A expressed from retroviral vectors in IMR90 cells. (E) SA-b-Gal of IMR90 fibroblasts expressing an empty vector, the human papillomavirus E6/E7 oncoproteins, or the adenovirus E1A oncoproteins together with H-RasV12 or an empty vector. SA-b-Gal activity was measured 14 d after infection. Data were quantified from 100 cell counts in triplicate and are presented as the mean percentage of positive cells 6 SD. (F) Immunoblots for cell cycle-regulated proteins and SAPD targets using extracts from cells as in E. of their mRNAs was not generally increased (Supplemen- which can be considered senescent lesions (Choi et al. tal Fig. S9F–H), indicating a close correlation between 2000; Vernier et al. 2011), we found very high levels of INK4a SAPD inhibition and bypass of senescence. phospho-ERK as well as the senescence markers p16 and PML in epithelial cells of prostate acini (Fig. 8A,B; High levels of ERK/MAPK activation characterize Supplemental Fig. S10A,B). In prostate carcinomas, ERK benign prostate tumors and predict a better outcome activation was never seen as high as in BPH (Fig. 8A,B vs. in malignant tumors C). Moreover, in epithelial cells, nuclear phospho-ERK To confirm the biological and clinical importance of levels had an inverse correlation with the Gleason pattern senescence stimulated by hyperactivation of ERK and (P < 0.05) (Supplemental Fig. S10C) and a positive correla- the SAPD process, we next investigated several prostate tion with the presence of regions of BPH in the same tumors. In cases of benign prostatic hyperplasia (BPH), prostate (P < 0.05) (Supplemental Fig. S10D). We also found 910 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Senescence-associated protein degradation Figure 8. Phospho-ERK and STAT3 in the normal prostate and BPH. (A) Phospho-ERK staining in samples from patients with BPH or normal controls. White arrows point to the stroma, and black arrows point to epithelial cells. (B) Quantification of phospho-ERK data in normal and BPH patients according to three degrees of staining intensity (none, 0; moderate, 1; and high, 2). The differences between normal tissues and BPH were evaluated with the nonparametric Mann-Whitney U-test. (C)Degreesofphospho-ERK staining (0,1,and 2) inprostate cancer. The survival Kaplan-Meier curves describe the time for biochemical relapse (BCR) in patients with different levels of staining. (D) Degrees of phospho-ERK staining (0, 1, and 2) in normal tissue adjacent to prostate cancer and survival Kaplan-Meier curves as in C.(E) STAT3 staining in samples from patients with BPH (n = 43) or normal controls (n = 3) or cancer patients (n = 20). The percentage of patients with a STAT3 staining of four different degrees of intensity (none, 0; low, 1; moderate, 2; and high, 3) is shown in the right panel. The differences between normal and tumor tissues versus BPH were evaluated with the nonparametric Mann-Whitney U-test, P = 0.0003. a negative correlation between phospho-ERK staining in Consistent with the results shown above characterizing the nucleus of the tumor cells or in the tissue adjacent to BPH as senescent benign lesions with high levels of the tumor and patients’ biochemical relapse (measured by phospho-ERK, STAT3 was found down-regulated in BPH their prostate-specific antigen [PSA] levels; P < 0.06) (Fig. when compared with normal tissues, while most malig- 8C,D). Consistent with our results, ERK activation is rare nant tumors displayed high levels of STAT3 (Fig. 8E). in human pancreatic cancers, where ras mutations occur in ;90% of the cases (Yip-Schneider et al. 2001), and Discussion correlations between ERK activation and good prognosis were previously reported in prostate (Malik et al. 2002) and We show here that the outcome of the stimulation of the breast cancer patients (Svensson et al. 2005). ERK/MAP kinases depends on the expression levels and We also stained sections of normal prostate, BPH, and activity of ERK1/2. In normal cells, high levels of stimu- prostate cancer for one of the targets of SAPD, STAT3. lation led to cellular senescence. In contrast, reducing ERK GENES & DEVELOPMENT 911 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Descheˆnes-Simard et al. levels and, as a consequence, its activity rescued cells ever, not all phosphoproteins stabilized by proteasome from senescence and facilitated cell transformation by inhibitors in senescent cells were recognized ERK targets oncogenic ras. Our results are consistent with previous or contained ERK phosphorylation sites. This suggests data showing that MEK inhibitors can bypass Ras-in- that other kinases downstream from ERK or the DDR duced senescence (Lin et al. 1998) and that shRNAs may provide signals for phosphorylation-dependent pro- against MEK increased tumor formation in Myc-expressing tein degradation, leading to senescence. In addition, an cells (Bric et al. 2009). Collectively, these studies reveal overall reduction of phosphatase activity in senescent a tumor suppressor role for the ERK/MAP kinase path- cells due to ROS may also contribute to phosphorylation- way, which depends on the strength of its activation, and dependent protein degradation and likely explains why anticipate that molecular mechanisms controlling ERK SAPD is also observed in cells with short telomeres, kinase levels and activity are critical for tumor suppres- which are characterized by mitochondrial dysfunction sion and are likely targets of the transformation process. and oxidative stress (Passos et al. 2007; Sahin and Most functions of the ERK/MAP kinase pathway de- Depinho 2010). pend on the ability of the ERK kinases to phosphorylate The increase in phospho-ERK levels during replicative multiple target proteins. How, then, do these kinases senescence and the increase in the life span of normal stimulate or inhibit cell proliferation, depending on the human fibroblasts after using inhibitors of the ERK strength of their activity? An unbiased proteomic study pathway suggest a role for ERK signaling in the activation comparing the levels and phosphorylation of several ERK of the senescence program by short telomeres. We found targets in conditions of high or moderate activity led to the that the phosphorylation of p53 at Ser15, a hallmark of identification of a selective and ERK-dependent protein the DDR, was prevented by MEK inhibitors. This obser- degradation process in senescent cells that we named vation is consistent with results showing that inhibition SAPD. SAPD provides a direct mechanism to explain the of the DDR can restore cell cycle progression despite anti-proliferative prosenescence functions of the ERK/ telomere shortening (d’Adda di Fagagna et al. 2003). In MAP kinase pathway because phosphorylation and pro- some contexts, preventing telomere shortening by ex- tein degradation are tightly linked (Hunter 2007). During pressing telomerase can delay aging in mice (Tomas- normal signaling, this process will only reduce a minor Loba et al. 2008; Jaskelioff et al. 2011). Since MEK fraction of every ERK target protein because only a minor inhibitors were able to extend the replicative life span fraction of each protein is phosphorylated (Olsen et al. of normal human fibroblasts, which is normally limited 2010). However, aberrant ERK signaling can effectively by short telomeres, they could be tested to mitigate deplete some ERK targets because the fraction of phos- signals from short telomeres during aging or age-related phorylation will be dramatically increased (Supplemental diseases. Fig. S11). SAPD also explains the inability of cells with Clinical studies in a variety of cancers indicate that in activated oncogenes to proliferate despite activation of some tumors, phospho-ERK levels are remarkably low. growth signaling pathways. Analysis of individual SAPD The most compelling example of phospho-ERK down- targets likely explains many traits of senescence, includ- regulation was described in pancreatic tumors where ras ing the cell cycle arrest, DDR, telomere dysfunction, and mutations occur frequently (Yip-Schneider et al. 2001). cell motility defects (Supplemental Table SI). For example, Presumably, pancreatic carcinogenesis selects early for the down-regulation of c-MYC or HSP70 was reported to events that down-regulate ERK levels/activity to avoid be sufficient to trigger senescence (Guney et al. 2006; Wu senescence and other anti-proliferative consequences of et al. 2007; Gabai et al. 2009), and the down-regulation of high ERK activity. Interestingly, patients with pancreatic STAT3 and several mitochondrial import proteins could tumors with high ERK levels had better survival and account for the mitochondrial dysfunction associated responded better to treatment (Chadha et al. 2006), with Ras-induced senescence (Gough et al. 2009; Moiseeva suggesting that some of the tumor suppressor functions et al. 2009). Interestingly, both c-Myc and STAT3 coop- of the ERK pathway could be reactivated in cancer erate with oncogenic ras to transform primary cells (Land patients. Likewise, in mammary carcinomas, phospho- et al. 1983; Gough et al. 2009) and are targets of ERK- ERK levels correlated with good prognosis and a less dependent protein degradation. Remarkably, the oncopro- aggressive phenotype (Milde-Langosch et al. 2005; Svensson tein E1A cooperates with ras for transformation, but it is et al. 2005), and similar correlations were found in brain not clear how E1A may block p53 functions or stabilize tumors as well (Mawrin et al. 2003, 2005). We extend c-MYC. We show now that by reducing overall ERK these observations here by showing that phospho-ERK phosphorylation, perhaps via the induction of ERK-spe- levels were remarkably low in the most aggressive pros- cific phosphatases (Callejas-Valera et al. 2008), E1A can tate tumors. Also, patients with tumors having high cooperate with RasV12 as ERK knockdown or the SV40 phospho-ERK levels had a better prognosis. In agreement early region. with our findings, it has been reported that advanced The mechanistic links between ERK activity and pro- prostate cancer correlates with low phospho-ERK and tein degradation will require further studies. However, high AKT levels (Malik et al. 2002). It has been reported as most phosphopeptides stabilized by proteasome inhibi- well that, in some tumors, phospho-ERK levels are very tors in Ras senescent cells contained serine or threonine high and that MEK inhibitors may have therapeutic value residues adjacent to proline (Fig. 4B; Supplemental Table (Sebolt-Leopold 2008). We anticipate that such tumors SI), a characteristic of ERK phosphorylation sites. How- contain genetic or epigenetic lesions that inactivate the 912 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Senescence-associated protein degradation protein degradation mechanism acting downstream from For xenografts, BALB/c 6-wk-old nude mice (Charles River) were injected subcutaneously in both flanks with 10 cells ERK to mediate tumor suppression. resuspended in 250 mL of PBS mixed with 250 mL of Matrigel Astonishingly, we found a dramatic increase in phos- (BD Biosciences) at 4°C. Tumor formation was evaluated over pho-ERK staining along with the senescence markers a period of 60 d. Tumors >0.2 cm were counted, and mice were INK4a PML and p16 in BPH. Senescent cells have been also euthanized before the end point of the experiment when tumor observed in benign human nevi, which, like BPH, rarely volume reached 2 cm . progress into malignant melanomas (Michaloglou et al. Cell lines, reagents, plasmids, quantitative PCR (qPCR), cell 2005). On the other hand, in some mouse models of growth analysis, soft agar, focus-forming assay, and protein prostate cancer, premalignant lesions (prostatic intraepi- analysis (immunoblotting and immunofluorescence) are de- thelial neoplasias [PINs]) display some markers of senes- scribed in the Supplemental Material. cence in association with less aggressive but detectable tumor progression (Chen et al. 2005). It will be very Immunohistochemistry important to characterize which traits associated with We used samples from seven patients diagnosed with BPH and senescence determine the reversibility of the cell cycle five different tissue microarrays (TMAs) (Diallo et al. 2007). The arrest and the potential for further tumor progression. first is comprised of 49 normal prostate specimens from autop- The incidence of both prostate cancer and BPH increases sies. The four others contain tissues obtained from 64 patients with age, and both require androgens for growth, but with primary prostate cancer. They are comprised of related unlike PIN, BPH is not a premalignant lesion (Bostwick nonneoplasic tissues adjacent to prostate cancer and cancerous et al. 1992). Comparison of lesions that do not progress, tissues from 64 patients who underwent radical prostatectomy. such as BPH, and lesions that eventually become malig- Regions of normal, intraepithelial neoplasia or cancerous epi- nant tumors, such as PIN (Bostwick 1996), can help us thelial tissue were identified by two pathologists and subse- understand how senescence prevents tumor formation in quently spotted on TMAs (Diallo et al. 2007). Specimens were vivo. One such factor is the presence of PML bodies, obtained from consenting patients, and the institutional ethics which accumulate in an ERK-dependent manner during review committee approved the study. We also purchased TMAs from Biochain Institute (catalog no. Z5070001). They comprised OIS in cell culture and are highly expressed in BPH but 63 cores, with 43 cases of BPH and 16 cases of adenocarcinomas. absent in PIN lesions (Vernier et al. 2011). To circumvent Normal prostate samples were obtained from BioChain Institute. ERK-dependent tumor suppression, some tumors may Tissue sections and TMAs were stained with a mouse mono- select for a reduction of ERK levels/activity, while others T202/Y204 clonal anti-phospho-ERK1/2 (1:175; clone E10, no. 9106, may disable the SAPD mechanism. The relationship Cell Signaling Technology), anti-PML antibody (1:300; clone PG- between ERK levels, senescence, and transformation is INK4a M3, Sc-966, Santa Cruz Biotechnology), and anti-p16 (1:25; also relevant for anti-cancer therapeutics, as drugs that clone F-12, Sc-1661, Santa Cruz Biotechnology). Primary anti- inhibit the pathway may inhibit tumor suppression in body detection was done using the LSAB 2 peroxidase system some contexts, while drugs increasing ERK activity from DAKO. Briefly, tissue samples were deparaffinized, rehy- drated, and treated with 0.3% H O in methanol to eliminate beyond the threshold required for senescence may have 2 2 endogenous peroxidase activity. Antigen epitope retrieval was anti-tumor effects. In conclusion, we describe a plausible performed by heating for 15 min at 95°C in Tris–EDTA buffer (10 mechanism linking hyperactivation of signaling path- mM Tris Base, 1 mM EDTA solution at pH 8.0) for phospho-ERK ways with protein degradation and the cellular defects INK4a and PML or 10 mM citrate buffer (pH 6.0) for p16 . All and malfunctions associated with cellular senescence, subsequent steps were done at room temperature. The sections suggesting that attenuation of excessive or aberrant sig- were blocked with a protein-blocking serum-free reagent (DAKO) naling can have anti-aging effects by preventing cellular and incubated with primary antibody for 60 min followed by a 20- senescence. min treatment with the secondary biotinylated antibody (DAKO) and then incubated for 20 min with streptavidin–peroxidase label (DAKO). Reaction products were developed with diaminobenzidine (DAKO) containing 0.3% H O as a substrate for peroxidase. Materials and methods 2 2 Nuclei were counterstained with Harris hematoxylin (Sigma- Aldrich). Mice and cells All mouse experiments were conducted in accordance with institutional and national guidelines and regulations. Condi- Statistics flox/flox tional Erk2 female mice (Voisin et al. 2010) were crossed with male mice carrying a Cre-expressing transgene under the Statistical analysis was performed using SPSS software 16.0 control of a Sox2 promoter (Hayashi et al. 2002) to generate (SPSS, Inc.). The expression level of phosphorylated ERK1/2 /+ / heterozygous Erk2 animals. Erk2 knockout embryos (Erk2 ) was evaluated on a scale of 0 (for no expression) to 2 (strong were obtained in normal Mendelian frequencies when male expression) in both the epithelial cells (nucleus and cytoplasm) /+ flox/flox Sox2-Cre; Erk2 mice were crossed to Erk2 females. and the stroma of normal prostate samples, normal adjacent Sox2-mediated excision by the Cre recombinase permitted the tissues, prostate tumor tissues, and BPH. The nonparametric rescue of the otherwise embryonic-lethal phenotype caused by Mann-Whitney U-test was used to show significant differences the absence of Erk2 by specifically excising Erk2 floxed alleles in between the normal, normal adjacent, PIN, tumor, and BPH the epiblast and maintaining functional floxed alleles in the groups. Correlations in expression between cell types and/or extraembryonic tissues of the embryo. MEFs were prepared at subcellular expression levels were done using the nonparamet- embryonic day 14.5 (E14.5) as previously reported (Voisin et al. ric Spearman’s rank correlation coefficient or the Pearson x 2010). analysis. GENES & DEVELOPMENT 913 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Descheˆnes-Simard et al. Diallo JS, Aldejmah A, Mouhim AF, Peant B, Fahmy MA, Acknowledgments Koumakpayi IH, Sircar K, Begin LR, Mes-Masson AM, Saad We thank James R. Davie, Elliot Drobetsky, Philippe Roux, F. 2007. NOXA and PUMA expression add to clinical Patrick J. Padisson, Peiqing Sun, Ste´phane Roy, Jacques Landry, markers in predicting biochemical recurrence of prostate and Jason C. Young for critical reading, reagents, and/or support. cancer patients in a survival tree model. Clin Cancer Res 13: We thank Eric Bonneil and the Institut de Recherche en 7044–7052. Immunologie et Cance´rologie (IRIC) proteomic service for the Di Micco R, Fumagalli M, Cicalese A, Piccinin S, Gasparini P, phosphopeptide analysis and identification, and Louise Cournoyer, Luise C, Schurra C, Garre M, Nuciforo PG, Bensimon A, Catherine Me´nard, and Fre´de´rique Badeaux for technical assis- et al. 2006. Oncogene-induced senescence is a DNA damage tance. This work was supported by grants from the Canadian response triggered by DNA hyper-replication. Nature 444: Institute of Health and Research (CIHR) to G.F. and S.M. G.F. is 638–642. a FRSQ senior fellow. 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GENES & DEVELOPMENT 915 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation Xavier Deschênes-Simard, Marie-France Gaumont-Leclerc, Véronique Bourdeau, et al. Genes Dev. 2013, 27: originally published online April 18, 2013 Access the most recent version at doi:10.1101/gad.203984.112 http://genesdev.cshlp.org/content/suppl/2013/04/11/gad.203984.112.DC1 Supplemental Material This article cites 59 articles, 22 of which can be accessed free at: References http://genesdev.cshlp.org/content/27/8/900.full.html#ref-list-1 License Receive free email alerts when new articles cite this article - sign up in the box at the top Email Alerting right corner of the article or click here. Service Copyright © 2013 by Cold Spring Harbor Laboratory Press
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