SUMO-modified nuclear cyclin D1 bypasses Ras-induced senescence

Oncogene-induced senescence represents a key tumor suppressive mechanism. Here, we show that Ras oncogene-induced senescence can be mediated by the recently identified haploinsufficient tumor suppressor apoptosis-stimulating protein of p53 (ASPP) 2 through a novel and p53/p19^Arf/p21^waf1/cip1-independent pathway. ASPP2 suppresses Ras-induced small ubiquitin-like modifier (SUMO)-modified nuclear cyclin D1 and inhibits retinoblastoma protein (Rb) phosphorylation. The lysine residue, K33, of cyclin D1 is a key site for this newly identified regulation. In agreement with the fact that its nuclear localization is required for its oncogenic activity, we show that nuclear cyclin D1 is far more potent than wild-type (WT) cyclin D1 in bypassing Ras-induced senescence. Thus, this study identifies SUMO modification as a positive regulator of nuclear cyclin D1, and reveals a new way by which cell cycle entry and senescence are regulated.     

Id-1 delays senescence but does not immortalize keratinocytes

Defining the molecular basis responsible for regulating the proliferative potential of keratinocytes has important implications for normal homeostasis and neoplasia of the skin. Under current culture conditions, neonatal foreskin-derived human keratinocytes possess a relatively short replicative lifespan. Recently it was reported that forced overexpression of the helix-loop-helix protein Id-1 was capable of immortalizing keratinocytes, secondary to activation of telomerase activity and suppression of p16/Rb-mediated growth arrest pathways. To investigate the relationship between Id-1, telomerase activity, telomere length, p16, Rb cell cycle regulators, and senescence, whole populations of keratinocytes were infected with a retrovirus to induce overexpression of Id-1. In these unselected cultures, enhanced Id-1 levels clearly extended the lifespan of keratinocytes, but Id-1 did not prevent the onset of replicative senescence. Under these experimental conditions, Id-1 expression did not trigger induction of telomerase activity, and there was progressive shortening of the telomeres that was accompanied by elevated p16 levels and prevalence of active Rb. The ability of Id-1 to postpone, but not prevent, senescence may be related to partial inhibition of p16 expression, as the Id-1-overexpressing cultures displayed a decreased capacity for 12-O-tetradecanoylphorbol-13-acetate-mediated p16 induction. Thus, while no immortalization was observed, Id-1 could delay the onset of replicative senescence in unselected human keratinocyte populations.     

PEA-15 is inhibited by adenovirus E1A and plays a role in ERK nuclear export and Ras-induced senescence

Oncogenic ras activates multiple signaling pathways to enforce cell proliferation in tumor cells. The ERK1/2 mitogen-activated protein kinase pathway is required for the transforming effects of ras, and its activation is often sufficient to convey mitogenic stimulation. However, in some settings oncogenic ras triggers a permanent cell cycle arrest with features of cellular senescence. How the Ras/ERK1/2 pathway activates different cellular programs is not well understood. Here we show that ERK1/2 localize predominantly in the cytoplasm during ras-induced senescence. This cytoplasmic localization seems to be dependent on an active nuclear export mechanism and can be rescued by the viral oncoprotein E1A. Consistent with this hypothesis, we showed that E1A dramatically down-regulated the expression of the ERK1/2 nuclear export factor PEA-15. Also, RNA interference against PEA-15 restored the nuclear localization of phospho-ERK1/2 in Ras-expressing primary murine embryo fibroblasts and stimulated their escape from senescence. Because senescence prevents the transforming effect of oncogenic ras, our results suggest a tumor suppressor function for PEA-15 that operates by means of controlling the localization of phospho-ERK1/2.     

EPEC-activated ERK1/2 participate in inflammatory response but not tight junction barrier disruption

Enteropathogenic Escherichia coli (EPEC) alters many functions of the host intestinal epithelia. Inflammation is initiated by activation of nuclear factor (NF)-kappaB, and paracellular permeability is enhanced via a Ca2+- and myosin light-chain kinase (MLCK)-dependent pathway. The aims of this study were to identify signaling pathways by which EPEC triggers inflammation and to determine whether these pathways parallel or diverge from those that alter permeability. EPEC-induced phosphorylation and degradation of the primary inhibitor of NF-kappaB (IkappaBalpha) were tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta independent. In contrast to Salmonella typhimurium, EPEC-stimulated IkappaBalpha degradation and IL-8 expression did not require Ca2+. Instead, extracellular signal-regulated kinase (ERK)-1/2 was significantly and rapidly activated. ERK1/2 inhibitors attenuated IkappaBalpha degradation and IL-8 expression. Although ERK1/2 can activate MLCK, its inhibition had no impact on EPEC disruption of the tight junction barrier. In conclusion, EPEC-induced inflammation 1) is TNF-alpha and IL-1beta receptor independent, 2) utilizes pathways differently from S. typhimurium, 3) requires ERK1/2, and 4) employs signals that are distinct from those that alter permeability. This is the first time that EPEC-activated signaling cascades have been linked to independent functional consequences.     

PI3K is required for insulin-stimulated but not EGF-stimulated ERK1/2 activation

The Ras/Raf/extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway is known to cross-talk with other signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, the role of PI3K in ERK-1/2 activation induced by tyrosine kinase receptors was not fully understood. Here, we report that two structurally distinct PI3K inhibitors, wortmannin and LY294002, inhibited insulin-induced activation of ERK1/2 but had no effect on EGF-induced activation of ERK1/2 in hepatocellular carcinoma BEL-7402 and SMMC-7721 cells, breast cancer MCF-7 cells, and prostate cancer LNCaP cells. Although protein kinase C could act as a mediator between PI3K and ERK1/2, protein kinase C inhibitor chelerythrine chloride did not inhibit insulin-induced ERK1/2 activation. Both insulin- and EGF-induced ERK1/2 activation are strictly dependent on Ras activation, however, wortmannin only inhibited insulin-induced, but not EGF-induced Ras activation. These results indicate that PI3K plays different roles in the activation of Ras/ERK1/2 signaling by insulin and EGF, and that insulin-stimulated, but not EGF-stimulated, ERK1/2 and Akt signalings diverge at PI3K.     

Bcl-2 promotes premature senescence induced by oncogenic Ras

The expression of the apoptosis inhibitory protein, Bcl-2, is increased in naturally senescing human fibroblasts and upon induction of their senescence-like growth arrest by oxidative stress, implying its role in maintaining their extended viability. Oncogenic Ras(V12) protein induces signaling cascades that result in the premature senescence of primary fibroblast cells, which are insensitive to oncogene-dependent apoptosis. Here we show that constitutive expression of Bcl-2 accelerates selected features of the Ras-induced senescence program in primary human fibroblasts. Yet, Bcl-2 also inhibits fibroblast apoptosis induced by exogenous H(2)O(2), while both signals induce an increased endogenous Bcl-2 expression in these cells. Together, these data suggest a context-dependent phenotypic function of Bcl-2 in the regulation of overlapping cell fate specification programs, with potential implications for both physiology and multistep tumorigenesis.     

Escape from premature senescence is not sufficient for oncogenic transformation by Ras

Resistance of primary cells to transformation by oncogenic Ras has been attributed to the induction of replicative growth arrest. This irreversible 'fail-safe mechanism' resembles senescence and requires induction by Ras of p19ARF and p53 (refs 3-5). Mutation of either p19ARF or p53 alleviates Ras-induced senescence and facilitates oncogenic transformation by Ras. Here we report that, whereas Rb and p107 are each dispensable for Ras-induced replicative arrest, simultaneous ablation of both genes disrupts Ras-induced senescence and results in unrestrained proliferation. This occurs despite activation by Ras of the p19ARF /p53 pathway, identifying pRb and p107 as essential mediators of Ras-induced antiproliferative p19ARF/p53 signalling. Unexpectedly, in contrast to p19ARF or p53 deficiency, loss of Rb/p107 function does not result in oncogenic transformation by Ras, as Ras-expressing Rb-/-/p107-/- fibroblasts fail to grow anchorage-independently in vitro and are not tumorigenic in vivo. These results demonstrate that in the absence of both Rb and p107 cells are resistant to p19ARF/p53-dependent protection against Ras-induced proliferation, and uncouple escape from Ras-induced premature senescence from oncogenic transformation.     

Stimulation of oncogenic metabotropic glutamate receptor 1 in melanoma cells activates ERK1/2 via PKCepsilon

Metabotropic glutamate receptor 1 (Grm1, formerly mGluR1) is a G protein coupled receptor (GPCR) normally expressed and functional in the central nervous system. Studies of our transgenic mouse melanoma model (TG-3) revealed that ectopic expression of Grm1 in melanocytes is sufficient to induce melanoma development in vivo [P.M. Pollock, K. Cohen-Solal, R. Sood, J. Namkoong, J.J. Martino, A. Koganti, H. Zhu, C. Robbins, I. Makalowska, S.S. Shin, Y. Marin, K.G. Roberts, L.M. Yudt, A. Chen, J. Cheng, A. Incao, H.W. Pinkett, C.L. Graham, K. Dunn, S.M. Crespo-Carbone, K.R. Mackason, K.B. Ryan, D. Sinsimer, J. Goydos, K.R. Reuhl, M. Eckhaus, P.S. Meltzer, W.J. Pavan, J.M. Trent, S. Chen, Nat. Genet. 34 (2003) 108-112.]. We have established and characterized several cell lines in vitro from independent mouse melanoma tumors [Y.E. Marín, J. Namkoong, S.S. Shin, J. Raines, K. Degenhardt, E. White, S. Chen, Neuropharmacol. 49 (2005) 70-79.]. These cell lines are useful tools in the studies of signaling events that may be mediated by Grm1 in transformed melanocytes. Here we show that stimulation of Grm1 by l-quisqualate, a group I metabotropic glutamate receptor agonist, results in inositol triphosphate (IP3) accumulation, and the activation of ERK1/2 in these cell lines. IP3 accumulation and ERK1/2 activation were inhibited by pretreatment of the tumor cells with a Grm1-specific antagonist (LY367385) or by dominant negative mutants of Grm1, demonstrating the specificity of these events. We also show that ERK1/2 activation by Grm1 was PKC-dependent, but cAMP and PKA-independent. PKCepsilon was shown to play a pivotal role in Grm1-mediated ERK1/2 phosphorylation. Insights into the signaling cascades mediated by Grm1 in melanoma cells may aid in the identification of key molecular targets for the future design of combined therapies for melanoma.     

Attenuation of TORC1 signaling delays replicative and oncogenic RAS-induced senescence

Numerous stimuli, including oncogenic signaling, DNA damage or eroded telomeres trigger proliferative arrest, termed cellular senescence. Accumulating evidence suggests that cellular senescence is a potent barrier to tumorigenesis in vivo, however oncogene induced senescence can also promote cellular transformation.¹ Kang TW, Yevsa T, Woller N, Hoenicke L, Wuestefeld T, Dauch D, et al. Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature 2011; 479:547 - 51; http://dx.doi.org/10.1038/nature10599; PMID: 22080947 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar]^,2 Rodier F, Campisi J. Four faces of cellular senescence. J Cell Biol 2011; 192:547 - 56; http://dx.doi.org/10.1083/jcb.201009094; PMID: 21321098 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] Several oncogenes, whose overexpression results in cellular senescence, converge on the TOR (target of rapamycin) pathway. We therefore examined whether attenuation of TOR results in delay or reversal of cellular senescence. By using primary human fibroblasts undergoing either replicative or oncogenic RAS-induced senescence, we demonstrated that senescence can be delayed, and some aspects of senescence can be reversed by inhibition of TOR, using either the TOR inhibitor rapamycin or by depletion of TORC1 (TOR Complex 1). Depletion of TORC2 fails to affect the course of replicative or RAS-induced senescence. Overexpression of REDD1 (Regulated in DNA Damage Response and Development), a negative regulator of TORC1, delays the onset of replicative senescence. These results indicate that TORC1 is an integral component of the signaling pathway that mediates cellular senescence.     

Attenuation of TORC1 signaling delays replicative and oncogenic RAS-induced senescence

Numerous stimuli, including oncogenic signaling, DNA damage or eroded telomeres trigger proliferative arrest, termed cellular senescence. Accumulating evidence suggests that cellular senescence is a potent barrier to tumorigenesis in vivo, however oncogene induced senescence can also promote cellular transformation.^1,2 Several oncogenes, whose overexpression results in cellular senescence, converge on the TOR (target of rapamycin) pathway. We therefore examined whether attenuation of TOR results in delay or reversal of cellular senescence. By using primary human fibroblasts undergoing either replicative or oncogenic RAS-induced senescence, we demonstrated that senescence can be delayed, and some aspects of senescence can be reversed by inhibition of TOR, using either the TOR inhibitor rapamycin or by depletion of TORC1 (TOR Complex 1). Depletion of TORC2 fails to affect the course of replicative or RAS-induced senescence. Overexpression of REDD1 (Regulated in DNA Damage Response and Development), a negative regulator of TORC1, delays the onset of replicative senescence. These results indicate that TORC1 is an integral component of the signaling pathway that mediates cellular senescence.     

MEK1 mutations,but not ERK2 mutations,occur in melanomas and colon carcinomas,but none in thyroid carcinomas

Mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in the pathogenesis of melanoma, colon cancer and thyroid cancer, which commonly harbor RAS and BRAF mutations. However, mutations in exon 2 of MEK1 and exon 7 of ERK2 have not been investigated in these cancers although they are occasionally found in some other cancers or cell lines. In this study, we performed mutational analysis to search for these mutations in 185 samples, including 167 tumor samples and 18 cell lines of melanoma, colon cancer, and thyroid cancer. We found one MEK1 mutation in 1 of 37(3%) melanoma tumor samples and another MEK1 mutation in 1 of 45 (2.2%) colon cancer samples. We did not find any MEK1 mutation in 99 thyroid tumor samples and 12 thyroid cancer cell lines. We also did not find any ERK2 mutation in melanoma, colon cancer, and thyroid cancer. We thus report for the first time a low prevalence of MEK1 mutations in melanoma and colon cancer. Both of the two mutants have been demonstrated to be activating in the MAPK signaling pathway and may therefore provide potential target for effective therapy in cases of melanomas and colon cancer harboring these mutations.     

IL-2 activation of NK cells: involvement of MKK1/2/ERK but not p38 kinase pathway

IL-2 stimulates extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in various immune cell populations. The functional roles that these kinases play are still unclear. In this study, we examined whether MAPK kinase (MKK)/ERK and p38 MAPK pathways are necessary for IL-2 to activate NK cells. Using freshly isolated human NK cells, we established that an intact MKK/ERK pathway is necessary for IL-2 to activate NK cells to express at least four known biological responses: LAK generation, IFN-gamma secretion, and CD25 and CD69 expression. IL-2 induced ERK activation within 5 min. Treatment of NK cells with a specific inhibitor of MKK1/2, PD98059, during the IL-2 stimulation blocked in a dose-dependent manner each of four sequelae, with inhibition of lymphokine-activated killing induction being least sensitive to MKK/ERK pathway blockade. Activation of p38 MAPK by IL-2 was not detected in NK cells. In contrast to what was observed by others in T lymphocytes, SB203850, a specific inhibitor of p38 MAPK, did not inhibit IL-2-activated NK functions. This data indicate that p38 MAPK activation was not required for IL-2 to activate NK cells for the four functions examined. These results reveal selective signaling differences between NK cells and T lymphocytes; in NK cells, the MKK/ERK pathway and not p38 MAPK plays a critical positive regulatory role during activation by IL-2.     

Regulation of epidermal growth factor-induced connexin 43 gap junction communication by big mitogen-activated protein kinase1/ERK5 but not ERK1/2 kinase activation

The gap junction protein, Cx43, plays a pivotal role in coupling cells electrically and metabolically, and the putative phosphorylation sites that modulate its function are reflected as changes in gap junction communication. Growth factor stimulation has been correlated with a decrease in gap junction communication and a parallel activation of ERK1/2; the inhibition of epidermal growth factor (EGF)-induced Cx43 gap junction uncoupling was observed by using the MEK1/2 inhibitor, PD98059. Because 1) BMK1/ERK5, another MAPK family member also activated by growth factors, possesses a phosphorylation motif similar to ERK1/2, and 2) it has been reported that PD98059 can inhibit not only MEK1/2-ERK1/2 but also MEK5-BMK1 activation, we investigated whether BMK1 can regulate EGF-induced Cx43 gap junction uncoupling and phosphorylation, comparing this to the role of ERK1/2 on Cx43 function and phosphorylation induced by EGF. Selective activation or inactivation of ERK1/2 by using a constitutively active form or a dominant negative form of MEK1 did not regulate Cx43 gap junction coupling. In contrast, we found that BMK1, selectively activated by constitutively active MEK5alpha, induced gap junction uncoupling, and the inhibition of BMK1 activation by transfection of dominant negative BMK1 prevented EGF-induced gap junction uncoupling. Activated BMK1 selectively phosphorylates Cx43 on Ser-255 in vitro and in vivo, but not on S279/S282, which are reported as the consensus phosphorylation sites for MAPK. Furthermore, by co-immunoprecipitation, we found that BMK1 directly associates with Cx43 in vivo. These data indicate that BMK1 is more important than ERK1/2 in EGF-mediated Cx43 gap junction uncoupling by association and Cx43 Ser- 255 phosphorylation.     

LPS-Induced G-CSF Expression in Macrophages Is Mediated by ERK2, but Not ERK1

Granulocyte colony-stimulating factor (G-CSF) selectively stimulates proliferation and differentiation of neutrophil progenitors which play important roles in host defense against infectious agents. However, persistent G-CSF production often leads to neutrophilia and excessive inflammatory reactions. There is therefore a need to understand the mechanism regulating G-CSF expression. In this study, we showed that U0126, a MEK1/2 inhibitor, decreases lipopolysaccharide (LPS)-stimulated G-CSF promoter activity, mRNA expression and protein secretion. Using short hairpin RNA knockdown, we demonstrated that ERK2, and not ERK1, involves in LPS-induced G-CSF expression, but not LPS-regulated expression of TNF-α. Reporter assays showed that ERK2 and C/EBPβ synergistically activate G-CSF promoter activity. Further chromatin immunoprecipitation (ChIP) assays revealed that U0126 inhibits LPS-induced binding of NF-κB (p50/p65) and C/EBPβ to the G-CSF promoter, but not their nuclear protein levels. Knockdown of ERK2 inhibits LPS-induced accessibility of the G-CSF promoter region to DNase I, suggesting that chromatin remodeling may occur. These findings clarify that ERK2, rather than ERK1, mediates LPS-induced G-CSF expression in macrophages by remodeling chromatin, and stimulates C/EBPβ-dependent activation of the G-CSF promoter. This study provides a potential target for regulating G-CSF expression.     

Nox1 redox signaling mediates oncogenic Ras-induced disruption of stress fibers and focal adhesions by down-regulating Rho

Generation of reactive oxygen species (ROS) by Ras oncogene-induced NADPH oxidase (Nox) 1 is required for Ras transformation phenotypes including anchorage-independent growth, morphological transformation, and tumorigenesity, but the signaling mechanism downstream of Nox1 remains elusive. Rho is known to be a critical regulator of actin stress fiber formation. Nonetheless, Rho was reported to no longer couple to loss of actin stress fibers in Ras-transformed Swiss3T3 cells despite the elevation of Rho activity. In this study, however, we demonstrate that Rho is inactivated in K-Ras-transformed normal rat kidney cells, and that abrogation of Nox1-generated ROS by Nox1 small interference RNAs or diphenyleneiodonium restores Rho activation, suggesting that Nox1-generated oxidants mediate down-regulation of the Rho activity. This down-regulation involves oxidative inactivation of the low molecular weight protein-tyrosine phosphatase by Nox1-generated ROS and a subsequent elevation in the tyrosine-phosphorylated active form of p190RhoGAP, the direct target of the phosphatase. Furthermore, the decreased Rho activity leads to disruption of both actin stress fibers and focal adhesions in Ras-transformed cells. As for Rac1, Rac1 also appears to participate in the down-regulation of Rho via Nox1. Our discovery defines a mediating role of Nox1-redox signaling for Ras oncogene-induced actin cytoskeletal changes.     

Physiological changes in GRK2 regulate CCL2-induced signaling to ERK1/2 and Akt but not to MEK1/2 and calcium

G protein-coupled receptor (GPCR) kinase 2 (GRK2) regulates G protein-coupled receptor signaling via agonist-induced receptor phosphorylation and desensitization. GRK2 can also modulate cellular activation by interacting with downstream signaling molecules. The intracellular GRK2 level changes during inflammatory conditions. We investigated how IL-1β-induced changes in endogenous GRK2 expression influence chemokine receptor signaling in primary astrocytes. Culturing astrocytes with IL-1β for 24 h induced a 2–3-fold increase in GRK2 and decreased C–C chemokine ligand 2 (CCL2)-induced ERK1/2 activation. Conversely, the 45% decrease in GRK2 expression in astrocytes from GRK2+/− animals resulted in a more pronounced CCL2-induced ERK1/2 phosphorylation. Increased GRK2 inhibited CCL2-induced Akt phosphorylation at Thr308 and Ser473 as well as pPDK-1 translocation. In contrast, altered GRK2 levels did not change the CCL2-induced increase in intracellular calcium or MEK1/2 phosphorylation. These data suggest that altered GRK2 expression modulates chemokine signaling downstream of the receptor. We found that GRK2 kinase activity was not required to decrease chemokine-induced ERK1/2 phosphorylation, whereas regulation of CCL2-induced Akt phosphorylation did require an active GRK2 kinase domain. Collectively, these data suggest that changes in endogenous GRK2 expression in primary astrocytes regulate chemokine receptor signaling to ERK1/2 and to PDK-1-Akt downstream of receptor coupling via kinase-dependent and kinase-independent mechanisms, respectively.     

Arsenite inhibits Ras-dependent activation of ERK but activates ERK in the presence of oncogenic Ras in baboon vascular smooth muscle cells

Exposure to arsenical compounds enhances the risk of atherosclerosis. The reason is unknown but it might be because an effect of arsenite (As3+) on plaque smooth muscle cells (SMCs) activation of extracellular signal-regulated kinase (ERK), a crucial mediator of SMC function. We found that arsenite inhibits the activation of ERK by platelet-derived growth factor-BB (PDGF-BB). This inhibitory effect depends on the time of arsenite exposure, is reversible, and is attenuated by preincubation of SMCs with the antioxidant N-acetyl-cysteine. These observations are consistent with the assumption that oxidative stress is involved. The blockade of ERK by arsenite may be mediated by an inhibition of Ras as arsenite prevents GTP-loading of Ras in response to PDGF-BB. Moreover, the Ras blockade by arsenite is not specific for PDGF-BB because it was also observed following stimulation of SMCs with EGF. To address the role of Ras, we expressed constitutively active, GTP-bound Ha-Ras (V12Ras). Unexpectedly, in V12Ras expressing-SMCs, arsenite stimulates ERK, but still decreases ERK activity in the presence of PDGF-BB. Our data suggest that arsenite inhibits the Ras/ERK pathway in SMCs, and that arsenite may activate ERK in Ras-transformed cells by mechanisms different from those employed by growth factors.