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Sample records for ras signaling pathway

  1. Mutant K-RAS Promotes Invasion and Metastasis in Pancreatic Cancer Through GTPase Signaling Pathways

    PubMed Central

    Padavano, Julianna; Henkhaus, Rebecca S; Chen, Hwudaurw; Skovan, Bethany A; Cui, Haiyan; Ignatenko, Natalia A

    2015-01-01

    Pancreatic ductal adenocarcinoma is one of the most aggressive malignancies, characterized by the local invasion into surrounding tissues and early metastasis to distant organs. Oncogenic mutations of the K-RAS gene occur in more than 90% of human pancreatic cancers. The goal of this study was to investigate the functional significance and downstream effectors of mutant K-RAS oncogene in the pancreatic cancer invasion and metastasis. We applied the homologous recombination technique to stably disrupt K-RAS oncogene in the human pancreatic cell line MiaPaCa-2, which carries the mutant K-RASG12C oncogene in both alleles. Using in vitro assays, we found that clones with disrupted mutant K-RAS gene exhibited low RAS activity, reduced growth rates, increased sensitivity to the apoptosis inducing agents, and suppressed motility and invasiveness. In vivo assays showed that clones with decreased RAS activity had reduced tumor formation ability in mouse xenograft model and increased survival rates in the mouse orthotopic pancreatic cancer model. We further examined molecular pathways downstream of mutant K-RAS and identified RhoA GTP activating protein 5, caveolin-1, and RAS-like small GTPase A (RalA) as key effector molecules, which control mutant K-RAS-dependent migration and invasion in MiaPaCa-2 cells. Our study provides rational for targeting RhoA and RalA GTPase signaling pathways for inhibition of pancreatic cancer metastasis. PMID:26512205

  2. The Ras-Erk-ETS-Signaling Pathway Is a Drug Target for Longevity.

    PubMed

    Slack, Cathy; Alic, Nazif; Foley, Andrea; Cabecinha, Melissa; Hoddinott, Matthew P; Partridge, Linda

    2015-07-01

    Identifying the molecular mechanisms that underlie aging and their pharmacological manipulation are key aims for improving lifelong human health. Here, we identify a critical role for Ras-Erk-ETS signaling in aging in Drosophila. We show that inhibition of Ras is sufficient for lifespan extension downstream of reduced insulin/IGF-1 (IIS) signaling. Moreover, direct reduction of Ras or Erk activity leads to increased lifespan. We identify the E-twenty six (ETS) transcriptional repressor, Anterior open (Aop), as central to lifespan extension caused by reduced IIS or Ras attenuation. Importantly, we demonstrate that adult-onset administration of the drug trametinib, a highly specific inhibitor of Ras-Erk-ETS signaling, can extend lifespan. This discovery of the Ras-Erk-ETS pathway as a pharmacological target for animal aging, together with the high degree of evolutionary conservation of the pathway, suggests that inhibition of Ras-Erk-ETS signaling may provide an effective target for anti-aging interventions in mammals. PMID:26119340

  3. Disorders of dysregulated signal traffic through the RAS-MAPK pathway: phenotypic spectrum and molecular mechanisms

    PubMed Central

    Tartaglia, Marco; Gelb, Bruce D.

    2010-01-01

    RAS GTPases control a major signaling network implicated in several cellular functions, including cell fate determination, proliferation, survival, differentiation, migration, and senescence. Within this network, signal flow through the RAF-MEK-ERK pathway, the first identified mitogen-associated protein kinase (MAPK) cascade, mediates early and late developmental processes controlling morphology determination, organogenesis, synaptic plasticity and growth. Signaling through the RAS-MAPK cascade is tightly controlled, and its enhanced activation represents a well-known event in oncogenesis. Unexpectedly, in the past few years, inherited dysregulation of this pathway has been recognized as the cause underlying a group of clinically related disorders sharing facial dysmorphism, cardiac defects, reduced postnatal growth, ectodermal anomalies, variable cognitive deficits and susceptibility to certain malignancies as major features. These disorders are caused by heterozygosity for mutations in genes encoding RAS proteins, regulators of RAS function, modulators of RAS interaction with effectors or downstream signal transducers. Here, we provide an overview of the phenotypic spectrum associated with germline mutations perturbing RAS-MAPK signaling, the unpredicted molecular mechanisms converging towards the dysregulation of this signaling cascade, and major genotype-phenotype correlations. PMID:20958325

  4. A novel Ras GTPase (Ras3) regulates conidiation, multi-stress tolerance and virulence by acting upstream of Hog1 signaling pathway in Beauveria bassiana.

    PubMed

    Guan, Yi; Wang, Ding-Yi; Ying, Sheng-Hua; Feng, Ming-Guang

    2015-09-01

    Two Ras ATPases (Ras1 and Ras2) are well known to regulate antagonistically or cooperatively various cellular events in many fungi. Here we show the significance of a novel Ras homolog (Ras3) for Beauveria bassiana. Ras3 possesses five domains and two GTP/GDP switches typical for Ras family and was proven to localize to plasma membrane despite the position change of a membrane-targeting cysteine in C-terminal CAAX motif. Deletion of ras3 altered temporal transcription pattern of ras1 instead of ras2. Compared with wild-type, Δras3 grew significantly faster in a rich medium but slower in some minimal media, and produced far fewer conidia with impaired quality, which was evident with slower germination, attenuated virulence, reduced thermotolerance and decreased UV-B resistance. Moreover, Δras3 was much more sensitive to the oxidative stress of menadione than of H2O2 and to the stress of high osmolarity than of cell wall perturbation during growth. The high sensitivity of Δras3 to menadione was concurrent with reductions in both gene transcripts and total activity of superoxide dismutases. Intriguingly, the high osmosensitivity was concurrent with not only reduced transcripts of a critical transcription factor (Msn2) and most signaling proteins in the high-osmolarity-glycerol pathway of Δras3 but nearly undetectable phosphorylation signal of Hog1 hallmarking the pathway. All the changes were restored by ras3 complementation. Taken together, Ras3 is involved in the Hog1 pathway required for osmoregulation and hence can positively regulate conidiation, germination, multi-stress tolerance and virulence linked to the biological control potential of the filamentous insect pathogen. PMID:26162967

  5. Peroxiredoxin II promotes hepatic tumorigenesis through cooperation with Ras/Forkhead box M1 signaling pathway.

    PubMed

    Park, Y-H; Kim, S-U; Kwon, T-H; Kim, J-M; Song, I-S; Shin, H-J; Lee, B-K; Bang, D-H; Lee, S-J; Lee, D-S; Chang, K-T; Kim, B-Y; Yu, D-Y

    2016-07-01

    The current study was carried out to define the involvement of Peroxiredoxin (Prx) II in progression of hepatocellular carcinoma (HCC) and the underlying molecular mechanism(s). Expression and function of Prx II in HCC was determined using H-ras(G12V)-transformed HCC cells (H-ras(G12V)-HCC cells) and the tumor livers from H-ras(G12V)-transgenic (Tg) mice and HCC patients. Prx II was upregulated in H-ras(G12V)-HCC cells and H-ras(G12V)-Tg mouse tumor livers, the expression pattern of which highly similar to that of forkhead Box M1 (FoxM1). Moreover, either knockdown of FoxM1 or site-directed mutagenesis of FoxM1-binding site of Prx II promoter significantly reduced Prx II levels in H-ras(G12V)-HCC cells, indicating FoxM1 as a direct transcription factor of Prx II in HCC. Interestingly, the null mutation of Prx II markedly decreased the number and size of tumors in H-ras(G12V)-Tg livers. Consistent with this, knockdown of Prx II in H-ras(G12V)-HCC cells reduced the expression of cyclin D1, cell proliferation, anchorage-independent growth and tumor formation in athymic nude mice, whereas overexpression of Prx II increased or aggravated the tumor phenotypes. Importantly, the expression of Prx II was correlated with that of FoxM1 in HCC patients. The activation of extracellular signal-related kinase (ERK) pathway and the expression of FoxM1 and cyclin D1 were highly dependent on Prx II in H-ras(G12V)-HCC cells and H-ras(G12V)-Tg livers. Prx II is FoxM1-dependently-expressed antioxidant in HCC and function as an enhancer of Ras(G12V) oncogenic potential in hepatic tumorigenesis through activation of ERK/FoxM1/cyclin D1 cascade. PMID:26500057

  6. The effect of aquaporin 5 overexpression on the Ras signaling pathway

    SciTech Connect

    Woo, Janghee; Lee, Juna; Kim, Myoung Sook; Jang, Se Jin; Sidransky, David; Moon, Chulso

    2008-03-07

    Human aquaporin 5 (AQP5) has been shown to be overexpressed in multiple cancers, such as pancreatic cancer and colon cancer. Furthermore, it has been reported that ectopic expression of AQP5 leads to many phenotypic changes characteristic of transformation. However, the biochemical mechanism leading to transformation in AQP5-overexpressing cells has not been clearly elucidated. In this report, the overexpression of AQP5 in NIH3T3 cells demonstrated a significant effect on Ras activity and, thus, cell proliferation. Furthermore, this influence was shown to be mediated by phosphorylation of the PKA consensus site of AQP5. This is the first evidence demonstrating an association between AQP5 and a signaling pathway, namely the Ras signal transduction pathway, which may be the basis of the oncogenic properties seen in AQP-overexpressing cells.

  7. BRAF vs RAS oncogenes: are mutations of the same pathway equal? differential signalling and therapeutic implications

    PubMed Central

    Oikonomou, Eftychia; Koustas, Evangelos; Goulielmaki, Maria; Pintzas, Alexander

    2014-01-01

    As the increased knowledge of tumour heterogeneity and genetic alterations progresses, it exemplifies the need for further personalized medicine in modern cancer management. Here, the similarities but also the differential effects of RAS and BRAF oncogenic signalling are examined and further implications in personalized cancer diagnosis and therapy are discussed. Redundant mechanisms mediated by the two oncogenes as well as differential regulation of signalling pathways and gene expression by RAS as compared to BRAF are addressed. The implications of RAS vs BRAF differential functions, in relevant tumour types including colorectal cancer, melanoma, lung cancer are discussed. Current therapeutic findings and future viewpoints concerning the exploitation of RAS-BRAF-pathway alterations for the development of novel therapeutics and efficient rational combinations, as well as companion tests for relevant markers of response will be evaluated. The concept that drug-resistant cells may also display drug dependency, such that altered dosing may prevent the emergence of lethal drug resistance posed a major therapy hindrance. PMID:25361007

  8. GHF-1/Pit-1 functions as a cell-specific integrator of Ras signaling by targeting the Ras pathway to a composite Ets-1/GHF-1 response element.

    PubMed

    Bradford, A P; Conrad, K E; Tran, P H; Ostrowski, M C; Gutierrez-Hartmann, A

    1996-10-01

    Activation of the rat prolactin (rPRL) promoter by Ras is a prototypical example of tissue-specific transcriptional regulation in a highly differentiated cell type. Using a series of site-specific mutations and deletions of the proximal rPRL promoter we have mapped the major Ras/Raf response element (RRE) to a composite Ets-1/GHF-1 binding site located between positions -217 and -190. Mutation of either the Ets-1 or GHF-1 binding sites inhibits Ras and Raf activation of the rPRL promoter, and insertion of this RRE into the rat growth hormone promoter confers Ras responsiveness. We show that Ets-1 is expressed in GH4 cells and, consistent with their functional synergistic interaction, both Ets-1 and GHF-1 are able to bind specifically to this bipartite RRE. We confirm that Ets-1 or a related Ets factor is the nuclear target of the Ras pathway leading to activation of the rPRL promoter and demonstrate that Elk-1 and Net do not mediate the Ras response. Thus, the pituitary-specific POU homeodomain transcription factor, GHF-1, serves as a cell-specific signal integrator by functionally interacting with an Ets-1-like factor, at uniquely juxtaposed binding sites, thereby targeting an otherwise ubiquitous Ras signaling pathway to a select subset of cell-specific GHF-1-dependent genes. PMID:8798730

  9. H-Ras Increases Urokinase Expression and Cell Invasion in Genetically Modified Human Astrocytes Through Ras/Raf/MEK Signaling Pathway

    PubMed Central

    ZHAO, YUNGE; XIAO, AIZHEN; DIPIERRO, CHARLES G.; ABDEL-FATTAH, RANA; AMOS, SAMSON; REDPATH, GERARD T.; CARPENTER, JOAN E.; PIEPER, RUSSELL O.; HUSSAINI, ISA M.

    2008-01-01

    Previous study reported that the activation of Ras pathway cooperated with E6/E7-mediated inactivation of p53/pRb to transform immortalized normal human astrocytes (NHA/hTERT) into intracranial tumors strongly resembling human astrocytomas. The mechanism of how H-Ras contributes to astrocytoma formation is unclear. Using genetically modified NHA cells (E6/E7/hTERT and E6/E7/hTERT/Ras cells) as models, we investigated the mechanism of Ras-induced tumorigenesis. The overexpression of constitutively active H-RasV12 in E6/E7/hTERT cells robustly increased the levels of urokinase plasminogen activator (uPA) mRNA, protein, activity and invasive capacity of the E6/E7/hTERT/Ras cells. However, the expressions of MMP-9 and MMP-2 did not significantly change in the E6/E7/hTERT and E6/E7/hTERT/Ras cells. Furthermore, E6/E7/hTERT/Ras cells also displayed higher level of uPA activity and were more invasive than E6/E7/hTERT cells in 3D culture, and formed an intracranial tumor mass in a NOD-SCID mouse model. uPA specific inhibitor (B428) and uPA neutralizing antibody decreased uPA activity and invasion in E6/E7/hTERT/Ras cells. uPA-deficient U-1242 glioblastoma cells were less invasive in vitro and exhibited reduced tumor growth and infiltration into normal brain in xenograft mouse model. Inhibitors of Ras (FTA), Raf (Bay 54−9085) and MEK (UO126), but not of phosphatidylinositol 3-kinase (PI3K) (LY294002) and of protein kinase C (BIM) pathways, inhibited uPA activity and cell invasion. Our results suggest that H-Ras increased uPA expression and activity via the Ras/Raf/MEK signaling pathway leading to enhanced cell invasion and this may contribute to increased invasive growth properties of astrocytomas. PMID:18383343

  10. Epidermal growth factor and Ras regulate gene expression in GH4 pituitary cells by separate, antagonistic signal transduction pathways.

    PubMed Central

    Pickett, C A; Gutierrez-Hartmann, A

    1995-01-01

    regions on the proximal rPRL promoter. One region maps between -255 and -212, near the Ras response element, and a second maps between -125 and -54. The latter region appears to involve footprint 2, a previously identified repressor site on the rPRL promoter. Neither footprint 1 nor 3, known GHF-1 binding sites, appears to be crucial to RGF-mediated rPRL promoter activation. The results of these studies indicate that in GH4 neuroendocrine cells, rPRL gene regulation by EGF is mediated by a signal transduction pathway that is separate and antagonistic to the Ras pathway. Hence, the functional role of the Ras/Raf/MAP kinase pathway in mediating transcriptional responses to EGF and other receptor tyrosine kinase may differ in highly specialized cell types. PMID:8524243

  11. Manumycin inhibits ras signal transduction pathway and induces apoptosis in COLO320-DM human colon tumourcells

    PubMed Central

    Paolo, A Di; Danesi, R; Nardini, D; Bocci, G; Innocenti, F; Fogli, S; Barachini, S; Marchetti, A; Bevilacqua, G; Tacca, M Del

    2000-01-01

    The aim of the present study was to assess the cytotoxicity of manumycin, a specific inhibitor of farnesyl:protein transferase, as well as its effects on protein isoprenylation and kinase-dependent signal transduction in COLO320-DM human colon adenocarcinoma which harbours a wild-type K- ras gene. Immunoblot analysis of isolated cell membranes and total cellular lysates of COLO320-DM cells demonstrated that manumycin dose-dependently reduced p21 ras farnesylation with a 50% inhibitory concentration (IC50) of 2.51 ± 0.11 μM and 2.68 ± 0.20 μM, respectively, while the geranylgeranylation of p21 rhoA and p21 rap1 was not affected. Manumycin dose-dependently inhibited (IC50= 2.40 ± 0.67 μM) the phosphorylation of the mitogen-activated protein kinase/extracellular-regulated kinase 2 (p42MAPK/ERK2), the main cytoplasmic effector of p21 ras, as well as COLO320-DM cell growth (IC50= 3.58 ± 0.27 μM) without affecting the biosynthesis of cholesterol. Mevalonic acid (MVA, 100 μM), a substrate of the isoprenoid synthesis, was unable to protect COLO320-DM cells from manumycin cytotoxicity. Finally, manumycin 1–25 μM for 24–72 h induced oligonucleosomal fragmentation in a dose- and time-dependent manner and MVA did not protect COLO320-DM cells from undergoing DNA cleavage. The present findings indicate that the inhibition of p21 ras processing and signal transduction by manumycin is associated with marked inhibition of cell proliferation and apoptosis in colon cancer cells and the effect on cell growth does not require the presence of a mutated ras gene for maximal expression of chemotherapeutic activity. © 2000 Cancer Research Campaign PMID:10732765

  12. Noncanonical control of C. elegans germline apoptosis by the insulin/IGF-1 and Ras/MAPK signaling pathways.

    PubMed

    Perrin, A J; Gunda, M; Yu, B; Yen, K; Ito, S; Forster, S; Tissenbaum, H A; Derry, W B

    2013-01-01

    The insulin/IGF-1 pathway controls a number of physiological processes in the nematode worm Caenorhabditis elegans, including development, aging and stress response. We previously found that the Akt/PKB ortholog AKT-1 dampens the apoptotic response to genotoxic stress in the germline by negatively regulating the p53-like transcription factor CEP-1. Here, we report unexpected rearrangements to the insulin/IGF-1 pathway, whereby the insulin-like receptor DAF-2 and 3-phosphoinositide-dependent protein kinase PDK-1 oppose AKT-1 to promote DNA damage-induced apoptosis. While DNA damage does not affect phosphorylation at the PDK-1 site Thr350/Thr308 of AKT-1, it increased phosphorylation at Ser517/Ser473. Although ablation of daf-2 or pdk-1 completely suppressed akt-1-dependent apoptosis, the transcriptional activation of CEP-1 was unaffected, suggesting that daf-2 and pdk-1 act independently or downstream of cep-1 and akt-1. Ablation of the akt-1 paralog akt-2 or the downstream target of the insulin/IGF-1 pathway daf-16 (a FOXO transcription factor) restored sensitivity to damage-induced apoptosis in daf-2 and pdk-1 mutants. In addition, daf-2 and pdk-1 mutants have reduced levels of phospho-MPK-1/ERK in their germ cells, indicating that the insulin/IGF-1 pathway promotes Ras signaling in the germline. Ablation of the Ras effector gla-3, a negative regulator of mpk-1, restored sensitivity to apoptosis in daf-2 mutants, suggesting that gla-3 acts downstream of daf-2. In addition, the hypersensitivity of let-60/Ras gain-of-function mutants to damage-induced apoptosis was suppressed to wild-type levels by ablation of daf-2. Thus, insulin/IGF-1 signaling selectively engages AKT-2/DAF-16 to promote DNA damage-induced germ cell apoptosis downstream of CEP-1 through the Ras pathway. PMID:22935616

  13. Targeting oncogenic Ras signaling in hematologic malignancies

    PubMed Central

    Ward, Ashley F.; Braun, Benjamin S.

    2012-01-01

    Ras proteins are critical nodes in cellular signaling that integrate inputs from activated cell surface receptors and other stimuli to modulate cell fate through a complex network of effector pathways. Oncogenic RAS mutations are found in ∼ 25% of human cancers and are highly prevalent in hematopoietic malignancies. Because of their structural and biochemical properties, oncogenic Ras proteins are exceedingly difficult targets for rational drug discovery, and no mechanism-based therapies exist for cancers with RAS mutations. This article reviews the properties of normal and oncogenic Ras proteins, the prevalence and likely pathogenic role of NRAS, KRAS, and NF1 mutations in hematopoietic malignancies, relevant animal models of these cancers, and implications for drug discovery. Because hematologic malignancies are experimentally tractable, they are especially valuable platforms for addressing the fundamental question of how to reverse the adverse biochemical output of oncogenic Ras in cancer. PMID:22898602

  14. Combination of a Selective HSP90α/β Inhibitor and a RAS-RAF-MEK-ERK Signaling Pathway Inhibitor Triggers Synergistic Cytotoxicity in Multiple Myeloma Cells

    PubMed Central

    Mimura, Naoya; Minami, Jiro; Ohguchi, Hiroto; Yoshida, Yasuhiro; Sagawa, Morihiko; Gorgun, Gullu; Cirstea, Diana; Cottini, Francesca; Jakubikova, Jana; Tai, Yu-Tzu; Chauhan, Dharminder; Richardson, Paul G.; Munshi, Nikhil; Ando, Kiyoshi; Utsugi, Teruhiro; Hideshima, Teru; Anderson, Kenneth C.

    2015-01-01

    Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/β inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/β inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM. PMID:26630652

  15. Combination of a Selective HSP90α/β Inhibitor and a RAS-RAF-MEK-ERK Signaling Pathway Inhibitor Triggers Synergistic Cytotoxicity in Multiple Myeloma Cells.

    PubMed

    Suzuki, Rikio; Kikuchi, Shohei; Harada, Takeshi; Mimura, Naoya; Minami, Jiro; Ohguchi, Hiroto; Yoshida, Yasuhiro; Sagawa, Morihiko; Gorgun, Gullu; Cirstea, Diana; Cottini, Francesca; Jakubikova, Jana; Tai, Yu-Tzu; Chauhan, Dharminder; Richardson, Paul G; Munshi, Nikhil; Ando, Kiyoshi; Utsugi, Teruhiro; Hideshima, Teru; Anderson, Kenneth C

    2015-01-01

    Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/β inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/β inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM. PMID:26630652

  16. Comparative Transcriptome Analysis Reveals Novel Roles of the Ras and Cyclic AMP Signaling Pathways in Environmental Stress Response and Antifungal Drug Sensitivity in Cryptococcus neoformans ▿ †

    PubMed Central

    Maeng, Shinae; Ko, Young-Joon; Kim, Gyu-Bum; Jung, Kwang-Woo; Floyd, Anna; Heitman, Joseph; Bahn, Yong-Sun

    2010-01-01

    The cyclic AMP (cAMP) pathway plays a central role in the growth, differentiation, and virulence of pathogenic fungi, including Cryptococcus neoformans. Three upstream signaling regulators of adenylyl cyclase (Cac1), Ras, Aca1, and Gpa1, have been demonstrated to control the cAMP pathway in C. neoformans, but their functional relationship remains elusive. We performed a genome-wide transcriptome analysis with a DNA microarray using the ras1Δ, gpa1Δ, cac1Δ, aca1Δ, and pka1Δ pka2Δ mutants. The aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka2Δ mutants displayed similar transcriptome patterns, whereas the ras1Δ mutant exhibited transcriptome patterns distinct from those of the wild type and the cAMP mutants. Interestingly, a number of environmental stress response genes are modulated differentially in the ras1Δ and cAMP mutants. In fact, the Ras signaling pathway was found to be involved in osmotic and genotoxic stress responses and the maintenance of cell wall integrity via the Cdc24-dependent signaling pathway. Notably, the Ras and cAMP mutants exhibited hypersensitivity to a polyene drug, amphotericin B, without showing effects on ergosterol biosynthesis, which suggested a novel method of antifungal combination therapy. Among the cAMP-dependent gene products that we characterized, two small heat shock proteins, Hsp12 and Hsp122, were found to be involved in the polyene antifungal drug susceptibility of C. neoformans. PMID:20097740

  17. DA-Raf–dependent inhibition of the Ras-ERK signaling pathway in type 2 alveolar epithelial cells controls alveolar formation

    PubMed Central

    Watanabe-Takano, Haruko; Takano, Kazunori; Sakamoto, Akemi; Matsumoto, Kenji; Tokuhisa, Takeshi; Endo, Takeshi; Hatano, Masahiko

    2014-01-01

    Alveolar formation is coupled to the spatiotemporally regulated differentiation of alveolar myofibroblasts (AMYFs), which contribute to the morphological changes of interalveolar walls. Although the Ras-ERK signaling pathway is one of the key regulators for alveolar formation in developing lungs, the intrinsic molecular and cellular mechanisms underlying its role remain largely unknown. By analyzing the Ras-ERK signaling pathway during postnatal development of lungs, we have identified a critical role of DA-Raf1 (DA-Raf)—a dominant-negative antagonist for the Ras-ERK signaling pathway—in alveolar formation. DA-Raf–deficient mice displayed alveolar dysgenesis as a result of the blockade of AMYF differentiation. DA-Raf is predominantly expressed in type 2 alveolar epithelial cells (AEC2s) in developing lungs, and DA-Raf–dependent MEK1/2 inhibition in AEC2s suppresses expression of tissue inhibitor of matalloprotienase 4 (TIMP4), which prevents a subsequent proteolytic cascade matrix metalloproteinase (MMP)14–MMP2. Furthermore, MMP14–MMP2 proteolytic cascade regulates AMYF differentiation and alveolar formation. Therefore, DA-Raf–dependent inhibition of the Ras-ERK signaling pathway in AEC2s is required for alveolar formation via triggering MMP2 activation followed by AMYF differentiation. These findings reveal a pivotal role of the Ras-ERK signaling pathway in the dynamic regulation of alveolar development. PMID:24843139

  18. The Ras/PKA signaling pathway may control RNA polymerase II elongation via the Spt4p/Spt5p complex in Saccharomyces cerevisiae.

    PubMed Central

    Howard, Susie C; Hester, Arelis; Herman, Paul K

    2003-01-01

    The Ras signaling pathway in Saccharomyces cerevisiae controls cell growth via the cAMP-dependent protein kinase, PKA. Recent work has indicated that these effects on growth are due, in part, to the regulation of activities associated with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. However, the precise target of these Ras effects has remained unknown. This study suggests that Ras/PKA activity regulates the elongation step of the RNA polymerase II transcription process. Several lines of evidence indicate that Spt5p in the Spt4p/Spt5p elongation factor is the likely target of this control. First, the growth of spt4 and spt5 mutants was found to be very sensitive to changes in Ras/PKA signaling activity. Second, mutants with elevated levels of Ras activity shared a number of specific phenotypes with spt5 mutants and vice versa. Finally, Spt5p was efficiently phosphorylated by PKA in vitro. Altogether, the data suggest that the Ras/PKA pathway might be directly targeting a component of the elongating polymerase complex and that this regulation is important for the normal control of yeast cell growth. These data point out the interesting possibility that signal transduction pathways might directly influence the elongation step of RNA polymerase II transcription. PMID:14668364

  19. Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes.

    PubMed Central

    van den Berghe, N; Ouwens, D M; Maassen, J A; van Mackelenbergh, M G; Sips, H C; Krans, H M

    1994-01-01

    The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21ras, are involved in insulin-mediated glucose transport. We did this by examining the time- and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at approximately 30 min. The activation of p21ras was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-[14C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF. Although these results cannot exclude the possibility that p21ras and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport. Images PMID:7511205

  20. Involvement of the Ras/extracellular signal-regulated kinase signalling pathway in the regulation of ERCC-1 mRNA levels by insulin.

    PubMed Central

    Lee-Kwon, W; Park, D; Bernier, M

    1998-01-01

    Expression of DNA repair enzymes, which includes ERCC-1, might be under the control of hormonal and growth factor stimulation. In the present study it was observed that insulin increased ERCC-1 mRNA levels both in Chinese hamster ovary cells overexpressing human insulin receptors (HIRc cells) and in fully differentiated 3T3-L1 adipocytes. To investigate the mechanisms underlying the increase in ERCC-1 gene expression in HIRc cells, we used a variety of pharmacological tools known to inhibit distinct signalling pathways. None of these inhibitors affected the amount of ERCC-1 mRNA in unstimulated cells. The pretreatment of cells with two chemically unrelated phosphatidylinositol 3'-kinase inhibitors, wortmannin and LY294002, failed to block the doubling of ERCC-1 mRNA content by insulin. Similarly, inhibition of pp70 S6 kinase by rapamycin had no apparent effects on this insulin response. In contrast, altering the p21(ras)-dependent pathway with either manumycin, an inhibitor of Ras farnesylation, or PD98059, an inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase, suppressed the induction of ERCC-1 mRNA by insulin (P<0.001). Furthermore inhibition of RNA and protein synthesis negatively regulated the expression of this insulin-regulated gene (P<0.005). These results suggest that insulin enhances ERCC-1 mRNA levels by the activation of the Ras-ERK-dependent pathway without the involvement of the phosphatidylinositol 3'-kinase/pp70 S6 kinase. PMID:9531502

  1. Regulation of SREBPs by Sphingomyelin in Adipocytes via a Caveolin and Ras-ERK-MAPK-CREB Signaling Pathway.

    PubMed

    Makdissy, Nehman; Haddad, Katia; Mouawad, Charbel; Popa, Iuliana; Younsi, Mohamed; Valet, Philippe; Brunaud, Laurent; Ziegler, Olivier; Quilliot, Didier

    2015-01-01

    Sterol response element binding protein (SREBP) is a key transcription factor in insulin and glucose metabolism. We previously demonstrated that elevated levels of membrane sphingomyelin (SM) were related to peroxisome proliferator-activated receptor-γ (PPARγ), which is a known target gene of SREBP-1 in adipocytes. However, the role of SM in SREBP expression in adipocytes remains unknown. In human abdominal adipose tissue from obese women with various concentrations of fasting plasma insulin, SREBP-1 proteins decreased in parallel with increases in membrane SM levels. An inverse correlation was found between the membrane SM content and the levels of SREBP-1c/ERK/Ras/PPARγ/CREB proteins. For the first time, we demonstrate the effects of SM and its signaling pathway in 3T3-F442A adipocytes. These cells were enriched or unenriched with SM in a range of concentrations similar to those observed in obese subjects by adding exogenous natural SMs (having different acyl chain lengths) or by inhibiting neutral sphingomyelinase. SM accumulated in caveolae of the plasma membrane within 24 h and then in the intracellular space. SM enrichment decreased SREBP-1 through the inhibition of extracellular signal-regulated protein kinase (ERK) but not JNK or p38 mitogen-activated protein kinase (MAPK). Ras/Raf-1/MEK1/2 and KSR proteins, which are upstream mediators of ERK, were down-regulated, whereas SREBP-2/caveolin and cholesterol were up-regulated. In SM-unmodulated adipocytes treated with DL-1-Phenyl-2-Palmitoylamino-3-morpholino-1-propanol (PPMP), where the ceramide level increased, the expression levels of SREBPs and ERK were modulated in an opposite direction relative to the SM-enriched cells. SM inhibited the insulin-induced expression of SREBP-1. Rosiglitazone, which is an anti-diabetic agent and potent activator of PPARγ, reversed the effects of SM on SREBP-1, PPARγ and CREB. Taken together, these findings provide novel insights indicating that excess membrane SM might

  2. Regulation of SREBPs by Sphingomyelin in Adipocytes via a Caveolin and Ras-ERK-MAPK-CREB Signaling Pathway

    PubMed Central

    Makdissy, Nehman; Popa, Iuliana; Younsi, Mohamed; Valet, Philippe; Brunaud, Laurent; Ziegler, Olivier; Quilliot, Didier

    2015-01-01

    Sterol response element binding protein (SREBP) is a key transcription factor in insulin and glucose metabolism. We previously demonstrated that elevated levels of membrane sphingomyelin (SM) were related to peroxisome proliferator–activated receptor-γ (PPARγ), which is a known target gene of SREBP-1 in adipocytes. However, the role of SM in SREBP expression in adipocytes remains unknown. In human abdominal adipose tissue from obese women with various concentrations of fasting plasma insulin, SREBP-1 proteins decreased in parallel with increases in membrane SM levels. An inverse correlation was found between the membrane SM content and the levels of SREBP-1c/ERK/Ras/PPARγ/CREB proteins. For the first time, we demonstrate the effects of SM and its signaling pathway in 3T3-F442A adipocytes. These cells were enriched or unenriched with SM in a range of concentrations similar to those observed in obese subjects by adding exogenous natural SMs (having different acyl chain lengths) or by inhibiting neutral sphingomyelinase. SM accumulated in caveolae of the plasma membrane within 24 h and then in the intracellular space. SM enrichment decreased SREBP-1 through the inhibition of extracellular signal-regulated protein kinase (ERK) but not JNK or p38 mitogen-activated protein kinase (MAPK). Ras/Raf-1/MEK1/2 and KSR proteins, which are upstream mediators of ERK, were down-regulated, whereas SREBP-2/caveolin and cholesterol were up-regulated. In SM-unmodulated adipocytes treated with DL-1-Phenyl-2-Palmitoylamino-3-morpholino-1-propanol (PPMP), where the ceramide level increased, the expression levels of SREBPs and ERK were modulated in an opposite direction relative to the SM-enriched cells. SM inhibited the insulin-induced expression of SREBP-1. Rosiglitazone, which is an anti-diabetic agent and potent activator of PPARγ, reversed the effects of SM on SREBP-1, PPARγ and CREB. Taken together, these findings provide novel insights indicating that excess membrane SM

  3. Comparative proteomic analysis of compartmentalised Ras signalling

    PubMed Central

    Hernandez-Valladares, Maria; Prior, Ian A.

    2015-01-01

    Ras proteins are membrane bound signalling hubs that operate from both the cell surface and endomembrane compartments. However, the extent to which intracellular pools of Ras can contribute to cell signalling is debated. To address this, we have performed a global screen of compartmentalised Ras signalling. We find that whilst ER/Golgi- and endosomal-Ras only generate weak outputs, Ras localised to the mitochondria or Golgi significantly and distinctly influence both the abundance and phosphorylation of a wide range of proteins analysed. Our data reveal that ~80% of phosphosites exhibiting large (≥1.5-fold) changes compared to control can be modulated by organellar Ras signalling. The majority of compartmentalised Ras-specific responses are predicted to influence gene expression, RNA splicing and cell proliferation. Our analysis reinforces the concept that compartmentalisation influences Ras signalling and provides detailed insight into the widespread modulation of responses downstream of endomembranous Ras signalling. PMID:26620772

  4. RalA, a GTPase targeted by miR-181a, promotes transformation and progression by activating the Ras-related signaling pathway in chronic myelogenous leukemia

    PubMed Central

    Luo, Xiaochuang; Yang, Juhua; Li, Yumin; Li, Tianfu; Wang, Ruirui; Fei, Jia

    2016-01-01

    BCR/ABL is a well-known activator of multiple signaling pathways. RalA, a Ras downstream signaling molecule and a small GTPase, plays an important role in Bcr-Abl-induced leukemogenesis but the exact mechanism remains elusive. Here, we show that RalA GTPase activity is commonly high in chronic myelogenous leukemia (CML) cell lines and patient samples. Overexpression of RalA results in malignant transformation and progression, and induces resistance to imatinib (IM) in BaF3 and K562 cell lines. RalA reduced survival and led to IM resistance in a xenografted mouse model. Ablation of RalA by either siRNA or miR-181a, a RalA targeting microRNA, attenuated the malignant phenotypes in K562 cells. RBC8, a selective Ral inhibitor, enhanced the inhibitory effects of IM in K562, KCL22 and BaF3-P210 cells. Interestingly, the phospho-specific protein microarray assay revealed that multiple phosphorylation signal proteins were decreased by RalA inhibition, including SAPK, JNK, SRC, VEGFR2, P38 MAPK, c-Kit, JunB, and Keratin18. Among them, P38 MAPK and SAPK/JNK are Ras downstream signaling kinases. Taken together, RalA GTPase might be an important oncogene activating the Ras-related signaling pathway in CML. PMID:26967392

  5. Differentiation of central nervous system neuronal cells by fibroblast-derived growth factor requires at least two signaling pathways: roles for Ras and Src.

    PubMed Central

    Kuo, W L; Chung, K C; Rosner, M R

    1997-01-01

    To evaluate the role of mitogen-activated protein (MAP) kinase and other signaling pathways in neuronal cell differentiation by basic fibroblast-derived growth factor (bFGF), we used a conditionally immortalized cell line from rat hippocampal neurons (H19-7). Previous studies have shown that activation of MAP kinase kinase (MEK) is insufficient to induce neuronal differentiation of H19-7 cells. To test the requirement for MEK and MAP kinase (ERK1 and ERK2), H19-7 cells were treated with the MEK inhibitor PD098059. Although the MEK inhibitor blocked the induction of differentiation by constitutively activated Raf, the H19-7 cells still underwent differentiation by bFGF. These results suggest that an alternative pathway is utilized by bFGF for differentiation of the hippocampal neuronal cells. Expression in the H19-7 cells of a dominant-negative Ras (N17-Ras) or Raf (C4-Raf) blocked differentiation by bFGF, suggesting that Ras and probably Raf are required. Expression of dominant-negative Src (pcSrc295Arg) or microinjection of an anti-Src antibody blocked differentiation by bFGF in H19-7 cells, indicating that bFGF also signals through a Src kinase-mediated pathway. Although neither constitutively activated MEK (MEK-2E) nor v-Src was sufficient individually to differentiate the H19-7 cells, coexpression of constitutively activated MEK and v-Src induced neurite outgrowth. These results suggest that (i) activation of MAP kinase (ERK1 and ERK2) is neither necessary nor sufficient for differentiation by bFGF; (ii) activation of Src kinases is necessary but not sufficient for differentiation by bFGF; and (iii) differentiation of H19-7 neuronal cells by bFGF requires at least two signaling pathways activated by Ras and Src. PMID:9234720

  6. Involvement of deregulated epiregulin expression in tumorigenesis in vivo through activated Ki-Ras signaling pathway in human colon cancer cells.

    PubMed

    Baba, I; Shirasawa, S; Iwamoto, R; Okumura, K; Tsunoda, T; Nishioka, M; Fukuyama, K; Yamamoto, K; Mekada, E; Sasazuki, T

    2000-12-15

    To identify the genes located downstream of the activated Ki-Ras signaling pathways in human colon cancer cells, a PCR-based cDNA subtraction library was constructed between HCT116 cells and HCT116-derived activated Ki-ras-disrupted cells (HKe3). One of the genes in HCT116 that was evidently up-regulated was epiregulin, a member of the epidermal growth factor family that is expressed in many kinds of human cancer cells. HKe3-stable transfectants expressing activated Ki-Ras regained over-expression of epiregulin. To further elucidate the biochemical structure and significance of epiregulin expression in tumorigenesis, HKe3-stable transfectants expressing epiregulin (e3-pSE cells) were established. Epiregulin existed as highly glycosylated membrane-bound forms, and TPA rapidly induced ectodomain shedding of epiregulin. Furthermore, the conditioned medium of e3-pSE cells showed more DNA synthesis for 32D cells expressing epidermal growth factor receptor (DER) cells than that of HKe3. Although anchorage-independent growth in soft agar was not observed for e3-pSE cells, tumorigenicity in nude mice was observed evidently, and their growth rate was correlated with each amount of exogenous epiregulin expression. These results suggested that activated Ki-Ras will be one of the factors contributing to the overexpression of epiregulin in human colon cancer cells, and that epiregulin will play a critical role in human tumorigenesis in vivo. PMID:11156386

  7. Ras trafficking, localization and compartmentalized signalling

    PubMed Central

    Prior, Ian A.; Hancock, John F.

    2012-01-01

    Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes. PMID:21924373

  8. RasGRP3, a Ras activator, contributes to signaling and the tumorigenic phenotype in human melanoma

    PubMed Central

    Li, Luowei; Kedei, Noemi; Tóth, Zsuzsanna E.; Czap, Alexandra; Velasquez, Julia F.; Mihova, Daniela; Michalowski, Aleksandra M.; Yuspa, Stuart H.; Blumberg, Peter M.

    2014-01-01

    RasGRP3, an activator for H-Ras, R-Ras and Rap1/2, has emerged as an important mediator of signaling downstream from receptor coupled phosphoinositide turnover in B and T cells. Here, we report that RasGRP3 showed a high level of expression in multiple human melanoma cell lines as well as in a subset of human melanoma tissue samples. Suppression of endogenous RasGRP3 expression in these melanoma cell lines reduced Ras-GTP formation as well as c-Met expression and Akt phosphorylation downstream from HGF or EGF stimulation. RasGRP3 suppression also inhibited cell proliferation and reduced both colony formation in soft agar and xenograft tumor growth in immunodeficient mice, demonstrating the importance of RasGRP3 for the transformed phenotype of the melanoma cells. Reciprocally, overexpression of RasGRP3 in human primary melanocytes altered cellular morphology, markedly enhanced cell proliferation, and rendered the cells tumorigenic in a mouse xenograft model. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. The identification of the role of RasGRP3 in melanoma highlights its importance, as a Ras activator, in the phosphoinositide signaling pathway in human melanoma and provides a new potential therapeutic target. PMID:21602881

  9. Differential utilization of Ras signaling pathways by macrophage colony-stimulating factor (CSF) and granulocyte-macrophage CSF receptors during macrophage differentiation.

    PubMed

    Guidez, F; Li, A C; Horvai, A; Welch, J S; Glass, C K

    1998-07-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) independently stimulate the proliferation and differentiation of macrophages from bone marrow progenitor cells. Although the GM-CSF and M-CSF receptors are unrelated, both couple to Ras-dependent signal transduction pathways, suggesting that these pathways might account for common actions of GM-CSF and M-CSF on the expression of macrophage-specific genes. To test this hypothesis, we have investigated the mechanisms by which GM-CSF and M-CSF regulate the expression of the macrophage scavenger receptor A (SR-A) gene. We demonstrate that induction of the SR-A gene by M-CSF is dependent on AP-1 and cooperating Ets domain transcription factors that bind to sites in an M-CSF-dependent enhancer located 4.1 to 4.5 kb upstream of the transcriptional start site. In contrast, regulation by GM-CSF requires a separate enhancer located 4.5 to 4.8 kb upstream of the transcriptional start site that confers both immediate-early and sustained transcriptional responses. Results of a combination of DNA binding experiments and functional assays suggest that immediate transcriptional responses are mediated by DNA binding proteins that are constitutively bound to the GM-CSF enhancer and are activated by Ras. At 12 to 24 h after GM-CSF treatment, the GM-CSF enhancer becomes further occupied by additional DNA binding proteins that may contribute to sustained transcriptional responses. In concert, these studies indicate that GM-CSF and M-CSF differentially utilize Ras-dependent signal transduction pathways to regulate scavenger receptor gene expression, consistent with the distinct functional properties of M-CSF- and GM-CSF-derived macrophages. PMID:9632769

  10. Quadruple wild-type (WT) GIST: defining the subset of GIST that lacks abnormalities of KIT, PDGFRA, SDH, or RAS signaling pathways

    PubMed Central

    Pantaleo, Maria A; Nannini, Margherita; Corless, Christopher L; Heinrich, Michael C

    2015-01-01

    A subset of GISTs lack mutations in the KIT/PDGFRA or RAS pathways and yet retain an intact succinate dehydrogensase (SDH) complex. We propose that these KIT/PDGFRA/SDH/RAS-P WT GIST tumors be designated as quadruple wild-type (WT) GIST. Further molecular and clinicophatological characterization of quadruple WT GIST will help to determine their prognosis as well as assist in the optimization of medical management, including clinical test of novel therapies. PMID:25165019

  11. Quadruple wild-type (WT) GIST: defining the subset of GIST that lacks abnormalities of KIT, PDGFRA, SDH, or RAS signaling pathways.

    PubMed

    Pantaleo, Maria A; Nannini, Margherita; Corless, Christopher L; Heinrich, Michael C

    2015-01-01

    A subset of GISTs lack mutations in the KIT/PDGFRA or RAS pathways and yet retain an intact succinate dehydrogensase (SDH) complex. We propose that these KIT/PDGFRA/SDH/RAS-P WT GIST tumors be designated as quadruple wild-type (WT) GIST. Further molecular and clinicophatological characterization of quadruple WT GIST will help to determine their prognosis as well as assist in the optimization of medical management, including clinical test of novel therapies. PMID:25165019

  12. Adenosine dialdehyde suppresses MMP-9-mediated invasion of cancer cells by blocking the Ras/Raf-1/ERK/AP-1 signaling pathway.

    PubMed

    Kim, Ji Hye; Kim, Jong Heon; Kim, Seung Cheol; Yi, Young-Su; Yang, Woo Seok; Yang, Yanyan; Kim, Han Gyung; Lee, Jae Yong; Kim, Kyung-Hee; Yoo, Byong Chul; Hong, Sungyoul; Cho, Jae Youl

    2013-11-01

    Adenosine dialdehyde (AdOx) inhibits transmethylation by the accumulation of S-adenosylhomocysteine (SAH), a negative feedback inhibitor of methylation, through the suppression of SAH hydrolase (SAHH). In this study, we aimed to determine the regulatory effect of AdOx on cancer invasion by using three different cell lines: MDA-MB-231, MCF-7, and U87. The invasive capacity of these cells in the presence (MCF-7) or absence (MDA-MB-231 and U87) of phorbal 12-myristate 13-acetate (PMA) was strongly decreased by AdOx treatment. Furthermore, the expression, secretion, and activation of matrix metalloproteinase (MMP)-9, a critical enzyme regulating cell invasion, in these cells were diminished by AdOx treatment. AdOx strongly suppressed AP-1-mediated luciferase activity and, in parallel, reduced the translocation of c-Fos and c-Jun into the nucleus. AdOx was shown to block a series of upstream AP-1 activation signaling complexes composed of extracellular signal-related kinase (ERK), mitogen-activated protein ERK kinase (MEK)1/2, Raf-1, and Ras, as assessed by measuring the levels of the phosphorylated and membrane-translocated forms. Furthermore, we found that suppression of SAHH by siRNA and 3-deazaadenosine, knock down of isoprenylcysteine carboxyl methyltransferase (ICMT), and treatment with SAH showed inhibitory patterns similar to those of AdOx. Therefore, our data suggest that AdOx is capable of targeting the methylation reaction regulated by SAHH and ICMT and subsequently downregulating MMP-9 expression and decreasing invasion of cancer cells through inhibition of the Ras/Raf-1/ERK/AP-1 pathway. PMID:23994169

  13. Ras Homolog Enriched in Brain (Rheb) Enhances Apoptotic Signaling*

    PubMed Central

    Karassek, Sascha; Berghaus, Carsten; Schwarten, Melanie; Goemans, Christoph G.; Ohse, Nadine; Kock, Gerd; Jockers, Katharina; Neumann, Sebastian; Gottfried, Sebastian; Herrmann, Christian; Heumann, Rolf; Stoll, Raphael

    2010-01-01

    Rheb is a homolog of Ras GTPase that regulates cell growth, proliferation, and regeneration via mammalian target of rapamycin (mTOR). Because of the well established potential of activated Ras to promote survival, we sought to investigate the ability of Rheb signaling to phenocopy Ras. We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNFα, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner. Knocking down endogenous Rheb or applying rapamycin led to partial protection, identifying Rheb as a mediator of cell death. Ras and c-Raf kinase opposed the apoptotic effects induced by UV light or TNFα but did not prevent Rheb-mediated apoptosis. To gain structural insight into the signaling mechanisms, we determined the structure of Rheb-GDP by NMR. The complex adopts the typical canonical fold of RasGTPases and displays the characteristic GDP-dependent picosecond to nanosecond backbone dynamics of the switch I and switch II regions. NMR revealed Ras effector-like binding of activated Rheb to the c-Raf-Ras-binding domain (RBD), but the affinity was 1000-fold lower than the Ras/RBD interaction, suggesting a lack of functional interaction. shRNA-mediated knockdown of apoptosis signal-regulating kinase 1 (ASK-1) strongly reduced UV or TNFα-induced apoptosis and suppressed enhancement by Rheb overexpression. In conclusion, Rheb-mTOR activation not only promotes normal cell growth but also enhances apoptosis in response to diverse toxic stimuli via an ASK-1-mediated mechanism. Pharmacological regulation of the Rheb/mTORC1 pathway using rapamycin should take the presence of cellular stress into consideration, as this may have clinical implications. PMID:20685651

  14. Molecular Pathways: Targeting the Dependence of Mutant RAS Cancers on the DNA Damage Response

    PubMed Central

    Grabocka, Elda; Commisso, Cosimo; Bar-Sagi, Dafna

    2014-01-01

    Of the genes mutated in cancer, RAS remains the most elusive to target. Recent technological advances and discoveries have greatly expanded our knowledge of the biology of oncogenic Ras and its role in cancer. As such, it has become apparent that a property that intimately accompanies RAS-driven tumorigenesis is the dependence of RAS mutant cells on a number of non-oncogenic signaling pathways. These dependencies arise as a means of adaptation to Ras-driven intracellular stresses and represent unique vulnerabilities of mutant RAS cancers. A number of studies have highlighted the dependence of mutant RAS cancers on the DNA damage response and identified the molecular pathways that mediate this process including signaling from wild-type Ras isoforms, ATR/Chk1, and DNA damage repair pathways. Here we review these findings, and discuss the combinatorial use of DNA damaging chemotherapy with blockade of wild-type H- and N-Ras signaling by farnesyltransferase inhibitors, Chk1 inhibitors, or small molecule targeting DNA damage repair as potential strategies through which the dependence of RAS cancers on the DNA damage response can be harnessed for therapeutic intervention. PMID:25424849

  15. RasGRP1 overexpression in T-ALL increases basal nucleotide exchange on Ras rendering the Ras/PI3K/Akt pathway responsive to protumorigenic cytokines.

    PubMed

    Ksionda, O; Melton, A A; Bache, J; Tenhagen, M; Bakker, J; Harvey, R; Winter, S S; Rubio, I; Roose, J P

    2016-07-14

    Ras GTPases are activated by RasGEFs and inactivated by RasGAPs, which stimulate the hydrolysis of RasGTP to inactive RasGDP. GTPase-impairing somatic mutations in RAS genes, such as KRAS(G12D), are among the most common oncogenic events in metastatic cancer. A different type of cancer Ras signal, driven by overexpression of the RasGEF RasGRP1 (Ras guanine nucleotide-releasing protein 1), was recently implicated in pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients and murine models, in which RasGRP1 T-ALLs expand in response to treatment with interleukins (ILs) 2, 7 and 9. Here, we demonstrate that IL-2/7/9 stimulation activates Erk and Akt pathways downstream of Ras in RasGRP1 T-ALL but not in normal thymocytes. In normal lymphocytes, RasGRP1 is recruited to the membrane by diacylglycerol (DAG) in a phospholipase C-γ (PLCγ)-dependent manner. Surprisingly, we find that leukemic RasGRP1-triggered Ras-Akt signals do not depend on acute activation of PLCγ to generate DAG but rely on baseline DAG levels instead. In agreement, using three distinct assays that measure different aspects of the RasGTP/GDP cycle, we established that overexpression of RasGRP1 in T-ALLs results in a constitutively high GTP-loading rate of Ras, which is constantly counterbalanced by hydrolysis of RasGTP. KRAS(G12D) T-ALLs do not show constitutive GTP loading of Ras. Thus, we reveal an entirely novel type of leukemogenic Ras signals that is based on a RasGRP1-driven increased in flux through the RasGTP/GDP cycle, which is mechanistically very different from KRAS(G12D) signals. Our studies highlight the dynamic balance between RasGEF and RasGAP in these T-ALLs and put forth a new model in which IL-2/7/9 decrease RasGAP activity. PMID:26549032

  16. Aspafilioside B induces G2/M cell cycle arrest and apoptosis by up-regulating H-Ras and N-Ras via ERK and p38 MAPK signaling pathways in human hepatoma HepG2 cells.

    PubMed

    Liu, Wei; Ning, Rui; Chen, Rui-Ni; Huang, Xue-Feng; Dai, Qin-Sheng; Hu, Jin-Hua; Wang, Yu-Wen; Wu, Li-Li; Xiong, Jing; Hu, Gang; Guo, Qing-Long; Yang, Jian; Wang, Hao

    2016-05-01

    We recently establish that aspafilioside B, a steroidal saponin extracted from Asparagus filicinus, is an active cytotoxic component. However, its antitumor activity is till unknown. In this study, the anticancer effect of aspafilioside B against HCC cells and the underlying mechanisms were investigated. Our results showed that aspafilioside B inhibited the growth and proliferation of HCC cell lines. Further study revealed that aspafilioside B could significantly induce G2 phase cell cycle arrest and apoptosis, accompanying the accumulation of reactive oxygen species (ROS), but blocking ROS generation with N-acetyl-l-cysteine (NAC) could not prevent G2/M arrest and apoptosis. Additionally, treatment with aspafilioside B induced phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAP kinase. Moreover, both ERK inhibitor PD98059 and p38 inhibitor SB203580 almost abolished the G2/M phase arrest and apoptosis induced by aspafilioside B, and reversed the expression of cell cycle- and apoptosis-related proteins. We also found that aspafilioside B treatment increased both Ras and Raf activation, and transfection of cells with H-Ras and N-Ras shRNA almost attenuated aspafilioside B-induced G2 phase arrest and apoptosis as well as the ERK and p38 activation. Finally, in vivo, aspafilioside B suppressed tumor growth in mouse xenograft models, and the mechanism was the same as in vitro study. Collectively, these findings indicated that aspafilioside B may up-regulate H-Ras and N-Ras, causing c-Raf phosphorylation, and lead to ERK and p38 activation, which consequently induced the G2 phase arrest and apoptosis. This study provides the evidence that aspafilioside B is a promising therapeutic agent against HCC. PMID:25683703

  17. Relapsed neuroblastomas show frequent RAS-MAPK pathway mutations

    PubMed Central

    Eleveld, Thomas F.; Oldridge, Derek A.; Bernard, Virginie; Koster, Jan; Daage, Leo Colmet; Diskin, Sharon J.; Schild, Linda; Bentahar, Nadia Bessoltane; Bellini, Angela; Chicard, Mathieu; Lapouble, Eve; Combaret, Valérie; Legoix-Né, Patricia; Michon, Jean; Pugh, Trevor J.; Hart, Lori S.; Rader, JulieAnn; Attiyeh, Edward F.; Wei, Jun S.; Zhang, Shile; Naranjo, Arlene; Gastier-Foster, Julie M.; Hogarty, Michael D.; Asgharzadeh, Shahab; Smith, Malcolm A.; Guidry Auvil, Jaime M.; Watkins, Thomas B. K.; Zwijnenburg, Danny A.; Ebus, Marli E.; van Sluis, Peter; Hakkert, Anne; van Wezel, Esther; van der Schoot, C. Ellen; Westerhout, Ellen M.; Schulte, Johannes H.; Tytgat, Godelieve A.; Dolman, M. Emmy M.; Janoueix-Lerosey, Isabelle; Gerhard, Daniela S.; Caron, Huib N.; Delattre, Olivier; Khan, Javed; Versteeg, Rogier; Schleiermacher, Gudrun; Molenaar, Jan J.; Maris, John M.

    2016-01-01

    The majority of neuroblastoma patients have tumors that initially respond to chemotherapy, but a large proportion of patients will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole genome sequencing of 23 paired diagnostic and relapsed neuroblastomas showed clonal evolution from the diagnostic tumor with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK signaling pathway. Seven events were detected only in the relapse tumor while the others showed clonal enrichment. In neuroblastoma cell lines we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18, 61%) and these lesions predicted for sensitivity to MEK inhibition in vitro and in vivo. Our findings provide the rationale for genetic characterization of relapse neuroblastoma and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease. PMID:26121087

  18. The Differential Effects of Wild-Type and Mutated K-Ras on MST2 Signaling Are Determined by K-Ras Activation Kinetics

    PubMed Central

    Romano, David; Maccario, Helene; Doherty, Carolanne; Quinn, Niall P.

    2013-01-01

    K-Ras is frequently mutated in human cancers. Mutant (mt) K-Ras can stimulate both oncogenic transformation and apoptosis through activation of extracellular signal-regulated kinase (ERK) and AKT pathways and the MST2 pathway, respectively. The biological outcome is determined by the balance and cross talk between these pathways. In colorectal cancer (CRC), a K-Ras mutation is negatively correlated with MST2 expression, as mt K-Ras can induce apoptosis by activating the MST2 pathway. However, wild-type (wt) K-Ras can prevent the activation of the MST2 pathway upon growth factor stimulation and enable transformation by mt K-Ras in CRC cells that express MST2. Here we have investigated the mechanism by which wt and mt K-Ras differentially regulate the MST2 pathway and MST2-dependent apoptosis. The ability of K-Ras to activate MST2 and MST2-dependent apoptosis is determined by the differential activation kinetics of mt K-Ras and wt K-Ras. Chronic activation of K-Ras by mutation or overexpression of Ras exchange factors results in the activation of MST2 and LATS1, increased MST2-LATS1 complex formation, and apoptosis. In contrast, transient K-Ras activation upon epidermal growth factor (EGF) stimulation prevents the formation of the MST2-LATS1 complex in an AKT-dependent manner. Our data suggest that the close relationship between Ras prosurvival and proapoptotic signaling is coordinated via the differential regulation of the MST2-LATS1 interaction by transient and chronic stimuli. PMID:23459937

  19. CD4+ T-Cell Decline after the Interruption of Antiretroviral Therapy in ACTG A5170 Is Predicted by Differential Expression of Genes in the Ras Signaling Pathway*

    PubMed Central

    Wang, Zhining; Su, Zhaohui; Nau, Martin E.; Krambrink, Amy; Skiest, Daniel J.; Margolis, David M.

    2008-01-01

    Abstract Patterns of expressed genes examined in cryopreserved peripheral blood mononuclear cells (PBMCs) of seropositive persons electing to stop antiretroviral therapy in the AIDS Clinical Trials Group Study A5170 were scrutinized to identify markers capable of predicting the likelihood of CD4+ T-cell depletion after cessation of antiretroviral therapy (ART). A5170 was a multicenter, 96-week, prospective study of HIV-infected patients with immunological preservation on ART who elected to interrupt therapy. Study entry required that the CD4 count was greater than 350 cells/mm3 within 6 months of ART initiation. Median nadir CD4 count of enrollees was 436 cells/mm3. Two cohorts, matched for clinical characteristics, were selected from A5170. Twenty-four patients with an absolute CD4 cell decline of less that 20% at week 24 (good outcome group) and 24 with a CD4 cell decline of >20% (poor outcome group) were studied. The good outcome group had a decline in CD4+ T-cell count that was 50% less than the poor outcome group. Significance analysis of microarrays identified differential gene expression (DE) in the two groups in data obtained from Affymetrix Human FOCUS GeneChips. DE was significantly higher in the poor outcome group than in the good outcome group. Prediction analysis of microarrays (PAM-R) identified genes that classified persons as to progression with greater than 80% accuracy at therapy interruption (TI) as well as at 24 weeks after TI. Gene set enrichment analysis (GSEA) identified a set of genes in the Ras signaling pathway, associated with the downregulation of apoptosis, as significantly upregulated in the good outcome group at cessation of ART. These observations identify specific host cell processes associated with differential outcome in this cohort after TI. PMID:18724805

  20. Regulation of H-Ras-driven MAPK signaling, transformation and tumorigenesis, but not PI3K signaling and tumor progression, by plasma membrane microdomains.

    PubMed

    Michael, J V; Wurtzel, J G T; Goldfinger, L E

    2016-01-01

    markedly suppressed tumor growth by H-Ras and H-Ras-tR, indicating that sustained PI3K signaling is a critical pathway for H-Ras-driven tumor progression, independent of microdomains. PMID:27239960

  1. Ras Family Small GTPase-mediated Neuroprotective Signaling in Stroke

    PubMed Central

    Shi, Geng-Xian; Andres, Douglas A.; Cai, Weikang

    2012-01-01

    Selective neuronal cell death is one of the major causes of neuronal damage following stroke, and cerebral cells naturally mobilize diverse survival signaling pathways to protect against ischemia. Importantly, therapeutic strategies designed to improve endogenous anti-apoptotic signaling appear to hold great promise in stroke treatment. While a variety of complex mechanisms have been implicated in the pathogenesis of stroke, the overall mechanisms governing the balance between cell survival and death are not well-defined. Ras family small GTPases are activated following ischemic insults, and in turn, serve as intrinsic switches to regulate neuronal survival and regeneration. Their ability to integrate diverse intracellular signal transduction pathways makes them critical regulators and potential therapeutic targets for neuronal recovery after stroke. This article highlights the contribution of Ras family GTPases to neuroprotective signaling cascades, including mitogen-activated protein kinase (MAPK) family protein kinase- and AKT/PKB-dependent signaling pathways as well as the regulation of cAMP response element binding (CREB), Forkhead box O (FoxO) and hypoxia-inducible factor 1(HIF1) transcription factors, in stroke. PMID:21521171

  2. Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway

    PubMed Central

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li-Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-01-01

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors. PMID:26080442

  3. Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

    DOE PAGESBeta

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li -Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-06-16

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referredmore » to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.« less

  4. Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

    SciTech Connect

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li -Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-06-16

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.

  5. A novel quinoline, MT477: suppresses cell signaling through Ras molecular pathway, inhibits PKC activity, and demonstrates in vivo anti-tumor activity against human carcinoma cell lines.

    PubMed

    Jasinski, Piotr; Welsh, Brandon; Galvez, Jorge; Land, David; Zwolak, Pawel; Ghandi, Lori; Terai, Kaoru; Dudek, Arkadiusz Z

    2008-06-01

    MT477 is a novel thiopyrano[2,3-c]quinoline that has been identified using molecular topology screening as a potential anticancer drug with a high activity against protein kinase C (PKC) isoforms. The objective of the present study was to determine the mechanism of action of MT477 and its activity against human cancer cell lines. MT477 interfered with PKC activity as well as phosphorylation of Ras and ERK1/2 in H226 human lung carcinoma cells. It also induced poly-caspase-dependent apoptosis. MT477 had a dose-dependent (0.006 to 0.2 mM) inhibitory effect on cellular proliferation of H226, MCF-7, U87, LNCaP, A431 and A549 cancer cell lines as determined by in vitro proliferation assays. Two murine xenograft models of human A431 and H226 lung carcinoma were used to evaluate tumor response to intraperitoneal administration of MT477 (33 microg/kg, 100 microg/kg, and 1 mg/kg). Tumor growth was inhibited by 24.5% in A431 and 43.67% in H226 xenografts following MT477 treatment, compared to vehicle controls (p < 0.05). In conclusion, our empirical findings are consistent with molecular modeling of MT477's activity against PKC. We also found, however, that its mechanism of action occurs through suppressing Ras signaling, indicating that its effects on apoptosis and tumor growth in vivo may be mediated by Ras as well as PKC. We propose, therefore, that MT477 warrants further development as an anticancer drug. PMID:17957339

  6. SUMO wrestling with Ras

    PubMed Central

    Zhang, Haibo; Luo, Ji

    2016-01-01

    ABSTRACT This review discusses our current understanding of the small ubiquitin-like modifier (SUMO) pathway and how it functionally intersects with Ras signaling in cancer. The Ras family of small GTPases are frequently mutated in cancer. The role of the SUMO pathway in cancer and in Ras signaling is currently not well understood. Recent studies have shown that the SUMO pathway can both regulate Ras/MAPK pathway activity directly and support Ras-driven oncogenesis through the regulation of proteins that are not direct Ras effectors. We recently discovered that in Ras mutant cancer cells, the SUMOylation status of a subset of proteins is altered and one such protein, KAP1, is required for Ras-driven transformation. A better understanding of the functional interaction between the SUMO and Ras pathways could lead to new insights into the mechanism of Ras-driven oncogenesis. PMID:27057691

  7. Inhibition of the Ras-Net (Elk-3) pathway by a novel pyrazole that affects microtubules.

    PubMed

    Wasylyk, Christine; Zheng, Hong; Castell, Christelle; Debussche, Laurent; Multon, Marie-Christine; Wasylyk, Bohdan

    2008-03-01

    Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment. PMID:18316589

  8. ASPP1 and ASPP2 bind active RAS, potentiate RAS signalling and enhance p53 activity in cancer cells

    PubMed Central

    Wang, Y; Godin-Heymann, N; Dan Wang, X; Bergamaschi, D; Llanos, S; Lu, X

    2013-01-01

    RAS mutations occur frequently in human cancer and activated RAS signalling contributes to tumour development and progression. Apart from its oncogenic effects on cell growth, active RAS has tumour-suppressive functions via its ability to induce cellular senescence and apoptosis. RAS is known to induce p53-dependent cell cycle arrest, yet its effect on p53-dependent apoptosis remains unclear. We report here that apoptosis-stimulating protein of p53 (ASPP) 1 and 2, two activators of p53, preferentially bind active RAS via their N-terminal RAS-association domains (RAD). Additionally, ASPP2 colocalises with and contributes to RAS cellular membrane localisation and potentiates RAS signalling. In cancer cells, ASPP1 and ASPP2 cooperate with oncogenic RAS to enhance the transcription and apoptotic function of p53. Thus, loss of ASPP1 and ASPP2 in human cancer cells may contribute to the full transforming property of RAS oncogene. PMID:23392125

  9. Copy number variants including RAS pathway genes-How much RASopathy is in the phenotype?

    PubMed

    Lissewski, Christina; Kant, Sarina G; Stark, Zornitza; Schanze, Ina; Zenker, Martin

    2015-11-01

    The RASopathies comprise a group of clinically overlapping developmental syndromes the common pathogenetic basis of which is dysregulated signal flow through the RAS-MAPK pathway. Mutations in several components or modifiers of the pathway have been identified in Noonan syndrome and related disorders. Over the past years copy number variants (CNVs) encompassing RAS pathway genes (PTPN11, RAF1, MEK2, or SHOC2) have been reported in children with developmental syndromes. These observations raised speculations that the associated phenotypes represent RASopathies, implying that the increased or reduced expression of the respective RAS pathway component and a consecutive dysregulation of RAS pathway signalling is responsible for the clinical picture. Herein, we present two individuals and three of their relatives harboring duplications of either 3p25.2 including the RAF1 locus or 19p13.3 including the MEK2 locus. Duplication carriers exhibited variable clinical phenotypes including non-specific facial dysmorphism, short stature, and learning difficulties. A careful review of the literature supported the impression that phenotypes associated with CNVs including RAS pathway genes commonly share non-specific symptoms with RASopathies, while the characteristic "gestalt" is lacking. Considering the known molecular pathogenesis of RASopathies, it is questionable that a modest increase in the expression of a functionally normal signaling component can mimic the effects of a qualitatively abnormal (hyperactive) mutant protein. We thus argue that current empirical and biological evidence is still insufficient to allow the conclusion that an altered copy number of a RAS pathway component is indeed the mechanism that is critical for the phenotype associated with CNVs including RASopathy genes. PMID:25974318

  10. Prostaglandin E2 Blocks Menadione-Induced Apoptosis through the Ras/Raf/Erk Signaling Pathway in Promonocytic Leukemia Cell Lines

    PubMed Central

    Yeo, Hyun-Seok; Shehzad, Adeeb; Lee, Young Sup

    2012-01-01

    Altered oxidative stress has long been observed in cancer cells, and this biochemical property of cancer cells represents a specific vulnerability that can be exploited for therapeutic benefit. The major role of an elevated oxidative stress for the efficacy of molecular targeted drugs is under investigation. Menadione is considered an attractive model for the study of oxidative stress, which can induce apoptosis in human leukemia HL-60 cell lines. Prostaglandin E2 (PGE2) via its receptors not only promotes cell survival but also reverses apoptosis and promotes cancer progression. Here, we present evidence for the biological role of PGE2 as a protective agent of oxidative stress-induced apoptosis in monocytic cells. Pretreatment of HL-60 cells with PGE2 markedly ameliorated the menadione-induced apoptosis and inhibited the degradation of PARP and lamin B. The EP2 receptor antagonist AH6809 abrogated the inhibitory effect of PGE2, suggesting the role of the EP2/cAMP system. The PKA inhibitor H89 also reversed apoptosis and decreased the PKA activity that was elevated 10-fold by PGE2. The treatment of HL-60 cells with NAC or zinc chloride showed a similar protective effect as with PGE2 on menadione-treated cells. Furthermore, PGE2 activated the Ras/Raf/MEK pathway, which in turn initiated ERK activation, and ultimately protected menadione-induced apoptosis. These results imply that PGE2 via cell survival pathways may protect oxidative stress-induced apoptosis in monocytic cells. This study warrants further pre-clinical investigation as well as application towards leukemia clinics. PMID:22450688

  11. The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis

    SciTech Connect

    Tsujita, Maristela; Batista, Wagner L.; Ogata, Fernando T.; Monteiro, Hugo P. Arai, Roberto J.

    2008-05-16

    p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras{sup C118S}) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG.

  12. The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis.

    PubMed

    Tsujita, Maristela; Batista, Wagner L; Ogata, Fernando T; Stern, Arnold; Monteiro, Hugo P; Arai, Roberto J

    2008-05-16

    p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras(C118S)) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG. PMID:18325324

  13. MicroRNA-122 confers sorafenib resistance to hepatocellular carcinoma cells by targeting IGF-1R to regulate RAS/RAF/ERK signaling pathways.

    PubMed

    Xu, Yanmin; Huang, Ji; Ma, Leina; Shan, Juanjuan; Shen, Junjie; Yang, Zhi; Liu, Limei; Luo, Yongli; Yao, Chao; Qian, Cheng

    2016-02-28

    Sorafenib is the first-line treatment for advanced hepatocellular carcinoma (HCC), but the clinical response to sorafenib is seriously limited by drug resistance. In this study, we investigated the molecular mechanisms of sorafenib resistance in HCC cells. Our miRNA microarray data indicate that liver-specific miR-122 expression was significantly reduced in sorafenib-resistant cells. Overexpression of miR-122 made drug-tolerant cells sensitive to sorafenib and induced apoptosis. Insulin-like growth factor 1 receptor (IGF-1R) was validated as a target of miR-122 and was repressed by this miRNA. miR-122-induced apoptosis was repressed by the IGF-1R activator IGFI or IGFII. Conversely, the IGF-1R inhibitor PPP or NVP-AEW541 in combination with sorafenib significantly induced cell apoptosis and disrupted tolerance to drugs in vitro. These results indicated that activation of IGF-1R by ectopic down-regulation of miR-122 counteracted the effects of sorafenib-induced apoptosis, thus conferring sorafenib resistance. Further study revealed that activation of IGF-1R by miR-122 down-regulation contributed to activation of RAS/RAF/ERK signaling, which was associated with drug resistance. Our data imply that an intimate correlation between miR-122 and IGF-1R abnormal expression is a critical determinant of sorafenib tolerance. PMID:26655273

  14. Abnormal Ras signaling in Costello syndrome (CS) negatively regulates enamel formation

    PubMed Central

    Goodwin, Alice F.; Tidyman, William E.; Jheon, Andrew H.; Sharir, Amnon; Zheng, Xu; Charles, Cyril; Fagin, James A.; McMahon, Martin; Diekwisch, Thomas G.H.; Ganss, Bernhard; Rauen, Katherine A.; Klein, Ophir D.

    2014-01-01

    RASopathies are syndromes caused by gain-of-function mutations in the Ras signaling pathway. One of these conditions, Costello syndrome (CS), is typically caused by an activating de novo germline mutation in HRAS and is characterized by a wide range of cardiac, musculoskeletal, dermatological and developmental abnormalities. We report that a majority of individuals with CS have hypo-mineralization of enamel, the outer covering of teeth, and that similar defects are present in a CS mouse model. Comprehensive analysis of the mouse model revealed that ameloblasts, the cells that generate enamel, lacked polarity, and the ameloblast progenitor cells were hyperproliferative. Ras signals through two main effector cascades, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways. To determine through which pathway Ras affects enamel formation, inhibitors targeting either PI3K or MEK 1 and 2 (MEK 1/2), kinases in the MAPK pathway, were utilized. MEK1/2 inhibition rescued the hypo-mineralized enamel, normalized the ameloblast polarity defect and restored normal progenitor cell proliferation. In contrast, PI3K inhibition only corrected the progenitor cell proliferation phenotype. We demonstrate for the first time the central role of Ras signaling in enamel formation in CS individuals and present the mouse incisor as a model system to dissect the roles of the Ras effector pathways in vivo. PMID:24057668

  15. Ras and Rap Signaling in Synaptic Plasticity and Mental Disorders

    PubMed Central

    Stornetta, Ruth L.; Zhu, J. Julius

    2011-01-01

    The Ras family GTPases (Ras, Rap1, and Rap2) and their downstream mitogen-activated protein kinases (ERK, JNK, and p38MAPK) and PI3K signaling cascades control various physiological processes. In neuronal cells, recent studies have shown that these parallel cascades signal distinct forms of AMPA-sensitive glutamate receptor trafficking during experience-dependent synaptic plasticity and adaptive behavior. Interestingly, both hypo- and hyper-activation of Ras/Rap signaling impair the capacity of synaptic plasticity, underscoring the importance of a “happy-medium” dynamic regulation of the signaling. Moreover, accumulating reports have linked various genetic defects that either up- or down-regulate Ras/Rap signaling with a number of mental disorders associated with learning disability (e.g., Alzheimer’s disease, Angelman syndrome, autism, cardio-facio-cutaneous syndrome, Coffin-Lowry syndrome, Costello syndrome, Cowden and Bannayan-Riley-Ruvalcaba syndromes, fragile X syndrome, neurofibromatosis type 1, Noonan syndrome, schizophrenia, tuberous sclerosis, and X-linked mental retardation), highlighting the necessity of happy-medium dynamic regulation of Ras/Rap signaling in learning behavior. Thus, the recent advances in understanding of neuronal Ras/Rap signaling provide a useful guide for developing novel treatments for mental diseases. PMID:20431046

  16. Ras Conformational Ensembles, Allostery, and Signaling.

    PubMed

    Lu, Shaoyong; Jang, Hyunbum; Muratcioglu, Serena; Gursoy, Attila; Keskin, Ozlem; Nussinov, Ruth; Zhang, Jian

    2016-06-01

    Ras proteins are classical members of small GTPases that function as molecular switches by alternating between inactive GDP-bound and active GTP-bound states. Ras activation is regulated by guanine nucleotide exchange factors that catalyze the exchange of GDP by GTP, and inactivation is terminated by GTPase-activating proteins that accelerate the intrinsic GTP hydrolysis rate by orders of magnitude. In this review, we focus on data that have accumulated over the past few years pertaining to the conformational ensembles and the allosteric regulation of Ras proteins and their interpretation from our conformational landscape standpoint. The Ras ensemble embodies all states, including the ligand-bound conformations, the activated (or inactivated) allosteric modulated states, post-translationally modified states, mutational states, transition states, and nonfunctional states serving as a reservoir for emerging functions. The ensemble is shifted by distinct mutational events, cofactors, post-translational modifications, and different membrane compositions. A better understanding of Ras biology can contribute to therapeutic strategies. PMID:26815308

  17. Genomic profiling of malignant phyllodes tumors reveals aberrations in FGFR1 and PI-3 kinase/RAS signaling pathways and provides insights into intratumoral heterogeneity.

    PubMed

    Liu, Su-Yang; Joseph, Nancy M; Ravindranathan, Ajay; Stohr, Bradley A; Greenland, Nancy Y; Vohra, Poonam; Hosfield, Elizabeth; Yeh, Iwei; Talevich, Eric; Onodera, Courtney; Van Ziffle, Jessica A; Grenert, James P; Bastian, Boris C; Chen, Yunn-Yi; Krings, Gregor

    2016-09-01

    Malignant phyllodes tumors of the breast are poorly understood rare neoplasms with potential for aggressive behavior. Few efficacious treatment options exist for progressed or metastatic disease. The molecular features of malignant phyllodes tumors are poorly defined, and a deeper understanding of the genetics of these tumors may shed light on pathogenesis and progression and potentially identify novel treatment approaches. We sequenced 510 cancer-related genes in 10 malignant phyllodes tumors, including 5 tumors with liposarcomatous differentiation and 1 with myxoid chondrosarcoma-like differentiation. Intratumoral heterogeneity was assessed by sequencing two separate areas in 7 tumors, including non-heterologous and heterologous components of tumors with heterologous differentiation. Activating hotspot mutations in FGFR1 were identified in 2 tumors. Additional recurrently mutated genes included TERT promoter (6/10), TP53 (4/10), PIK3CA (3/10), MED12 (3/10), SETD2 (2/10) and KMT2D (2/10). Together, genomic aberrations in FGFR/EGFR PI-3 kinase and RAS pathways were identified in 8 (80%) tumors and included mutually exclusive and potentially actionable activating FGFR1, PIK3CA and BRAF V600E mutations, inactivating TSC2 mutation, EGFR amplification and PTEN loss. Seven (70%) malignant phyllodes tumors harbored TERT aberrations (six promoter mutations, one amplification). For comparison, TERT promoter mutations were identified by Sanger sequencing in 33% borderline (n=12) and no (0%, n=8) benign phyllodes tumors (P=0.391 and P=0.013 vs malignant tumors, respectively). Genetic features specific to liposarcoma, including CDK4/MDM2 amplification, were not identified. Copy number analysis revealed intratumoral heterogeneity and evidence for divergent tumor evolution in malignant phyllodes tumors with and without heterologous differentiation. Tumors with liposarcomatous differentiation revealed more chromosomal aberrations in non-heterologous components compared with

  18. Extracellular matrix-modulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function.

    PubMed

    Dragojlovic-Munther, Michelle; Martinez-Agosto, Julian A

    2013-12-15

    Maintenance of hematopoietic progenitors ensures a continuous supply of blood cells during the lifespan of an organism. Thus, understanding the molecular basis for progenitor maintenance is a continued focus of investigation. A large pool of undifferentiated blood progenitors are maintained in the Drosophila hematopoietic organ, the larval lymph gland, by a complex network of signaling pathways that are mediated by niche-, progenitor-, or differentiated hemocyte-derived signals. In this study we examined the function of the Drosophila fibroblast growth factor receptor (FGFR), Heartless, a critical regulator of early lymph gland progenitor specification in the late embryo, during larval lymph gland hematopoiesis. Activation of Heartless signaling in hemocyte progenitors by its two ligands, Pyramus and Thisbe, is both required and sufficient to induce progenitor differentiation and formation of the plasmatocyte-rich lymph gland cortical zone. We identify two transcriptional regulators that function downstream of Heartless signaling in lymph gland progenitors, the ETS protein, Pointed, and the Friend-of-GATA (FOG) protein, U-shaped, which are required for this Heartless-induced differentiation response. Furthermore, cross-talk of Heartless and target of rapamycin signaling in hemocyte progenitors is required for lamellocyte differentiation downstream of Thisbe-mediated Heartless activation. Finally, we identify the Drosophila heparan sulfate proteoglycan, Trol, as a critical negative regulator of Heartless ligand signaling in the lymph gland, demonstrating that sequestration of differentiation signals by the extracellular matrix is a unique mechanism employed in blood progenitor maintenance that is of potential relevance to many other stem cell niches. PMID:23603494

  19. Extracellular matrix-modulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function

    PubMed Central

    Dragojlovic-Munther, Michelle; Martinez-Agosto, Julian A

    2014-01-01

    Maintenance of hematopoietic progenitors ensures a continuous supply of blood cells during the lifespan of an organism. Thus, understanding the molecular basis for progenitor maintenance is a continued focus of investigation. A large pool of undifferentiated blood progenitors are maintained in the Drosophila hematopoietic organ, the larval lymph gland, by a complex network of signaling pathways that are mediated by niche-, progenitor-, or differentiated hemocyte-derived signals. In this study we examined the function of the Drosophila fibroblast growth factor receptor (FGFR), Heartless, a critical regulator of early lymph gland progenitor specification in the late embryo, during larval lymph gland hematopoiesis. Activation of Heartless signaling in hemocyte progenitors by its two ligands, Pyramus and Thisbe, is both required and sufficient to induce progenitor differentiation and formation of the plasmatocyte-rich lymph gland cortical zone. We identify two transcriptional regulators that function downstream of Heartless signaling in lymph gland progenitors, the ETS protein, Pointed, and the Friend-of-GATA (FOG) protein, U-shaped, which are required for this Heartless-induced differentiation response. Furthermore, cross-talk of Heartless and target of rapamycin signaling in hemocyte progenitors is required for lamellocyte differentiation downstream of Thisbe-mediated Heartless activation. Finally, we identify the Drosophila heparan sulfate proteoglycan, Trol, as a critical negative regulator of Heartless ligand signaling in the lymph gland, demonstrating that sequestration of differentiation signals by the extracellular matrix is a unique mechanism employed in blood progenitor maintenance that is of potential relevance to many other stem cell niches. PMID:23603494

  20. Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes

    PubMed Central

    Cailotto, Frederic; Bianchi, Arnaud; Sebillaud, Sylvie; Venkatesan, Narayanan; Moulin, David; Jouzeau, Jean-Yves; Netter, Patrick

    2007-01-01

    ANK is a multipass transmembrane protein transporter thought to play a role in the export of intracellular inorganic pyrophosphate and so to contribute to the pathophysiology of chondrocalcinosis. As transforming growth factor-beta-1 (TGF-β1) was shown to favor calcium pyrophosphate dihydrate deposition, we investigated the contribution of ANK to the production of extracellular inorganic pyrophosphate (ePPi) by chondrocytes and the signaling pathways involved in the regulation of Ank expression by TGF-β1. Chondrocytes were exposed to 10 ng/mL of TGF-β1, and Ank expression was measured by quantitative polymerase chain reaction and Western blot. ePPi was quantified in cell supernatants. RNA silencing was used to define the respective roles of Ank and PC-1 in TGF-β1-induced ePPi generation. Finally, selective kinase inhibitors and dominant-negative/overexpression plasmid strategies were used to explore the contribution of several signaling pathways to Ank induction by TGF-β1. TGF-β1 strongly increased Ank expression at the mRNA and protein levels, as well as ePPi production. Using small interfering RNA technology, we showed that Ank contributed approximately 60% and PC-1 nearly 20% to TGF-β1-induced ePPi generation. Induction of Ank by TGF-β1 required activation of the extracellular signal-regulated kinase (ERK) pathway but not of p38-mitogen-activated protein kinase or of protein kinase A. In line with the general protein kinase C (PKC) inhibitor calphostin C, Gö6976 (a Ca2+-dependent PKC inhibitor) diminished TGF-β1-induced Ank expression by 60%, whereas a 10% inhibition was observed with rottlerin (a PKCδ inhibitor). These data suggest a regulatory role for calcium in TGF-β1-induced Ank expression. Finally, we demonstrated that the stimulatory effect of TGF-β1 on Ank expression was inhibited by the suppression of the Ras/Raf-1 pathway, while being enhanced by their constitutive activation. Transient overexpression of Smad 7, an inhibitory Smad, failed

  1. Integrating signals between cAMP and the RAS/RAF/MEK/ERK signalling pathways. Based on the anniversary prize of the Gesellschaft für Biochemie und Molekularbiologie Lecture delivered on 5 July 2003 at the Special FEBS Meeting in Brussels.

    PubMed

    Dumaz, Nicolas; Marais, Richard

    2005-07-01

    One of the hallmarks of cAMP is its ability to inhibit proliferation in many cell types, but stimulate proliferation in others. Clearly cAMP has cell type specific effects and the outcome on proliferation is largely attributed to crosstalk from cAMP to the RAS/RAF/mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway. We review the crosstalk between these two ancient and conserved pathways, describing the molecular mechanisms underlying the interactions between these pathways and discussing their possible biological importance. PMID:16008550

  2. Decoding RAS isoform and codon-specific signalling

    PubMed Central

    Newlaczyl, Anna U.; Hood, Fiona E.; Coulson, Judy M.; Prior, Ian A.

    2014-01-01

    RAS proteins are key signalling hubs that are oncogenically mutated in 30% of all cancer cases. Three genes encode almost identical isoforms that are ubiquitously expressed, but are not functionally redundant. The network responses associated with each isoform and individual oncogenic mutations remain to be fully characterized. In the present article, we review recent data defining the differences between the RAS isoforms and their most commonly mutated codons and discuss the underlying mechanisms. PMID:25109951

  3. RAS signalling through PI3-Kinase controls cell migration via modulation of Reelin expression

    PubMed Central

    Castellano, Esther; Molina-Arcas, Miriam; Krygowska, Agata Adelajda; East, Philip; Warne, Patricia; Nicol, Alastair; Downward, Julian

    2016-01-01

    RAS signalling through phosphoinositide 3-kinase (PI3-Kinase) has been shown to have an essential role in tumour initiation and maintenance. RAS also regulates cell motility and tumour invasiveness, but the role of direct RAS binding to PI3-Kinase in this remains uncertain. Here, we provide evidence that disruption of RAS interaction with PI3-Kinase p110α decreases cell motility and prevents activation of Rac GTPase. Analysis of gene expression in cells lacking RAS interaction with p110α reveals increased levels of the extracellular matrix glycoprotein Reelin and activation of its downstream pathway resulting in upregulation of E-cadherin expression. Induction of the Reelin/E-cadherin axis is also observed in Kras mutant lung tumours that are regressing due to blockade of RAS interaction with PI3-Kinase. Furthermore, loss of Reelin correlates with decreased survival of lung and breast cancer patients. Reelin thus plays a role in restraining RAS and PI3-kinase promotion of cell motility and potentially tumour metastasis. PMID:27071537

  4. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    SciTech Connect

    Naumov, Inna; Kazanov, Dina; Lisiansky, Victoria; Starr, Alex; Aroch, Ilan; Shapira, Shiran; Kraus, Sarah; Arber, Nadir

    2012-01-15

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  5. RAS/MAPK Activation Drives Resistance to Smo Inhibition, Metastasis, and Tumor Evolution in Shh Pathway-Dependent Tumors.

    PubMed

    Zhao, Xuesong; Ponomaryov, Tatyana; Ornell, Kimberly J; Zhou, Pengcheng; Dabral, Sukriti K; Pak, Ekaterina; Li, Wei; Atwood, Scott X; Whitson, Ramon J; Chang, Anne Lynn S; Li, Jiang; Oro, Anthony E; Chan, Jennifer A; Kelleher, Joseph F; Segal, Rosalind A

    2015-09-01

    Aberrant Shh signaling promotes tumor growth in diverse cancers. The importance of Shh signaling is particularly evident in medulloblastoma and basal cell carcinoma (BCC), where inhibitors targeting the Shh pathway component Smoothened (Smo) show great therapeutic promise. However, the emergence of drug resistance limits long-term efficacy, and the mechanisms of resistance remain poorly understood. Using new medulloblastoma models, we identify two distinct paradigms of resistance to Smo inhibition. Sufu mutations lead to maintenance of the Shh pathway in the presence of Smo inhibitors. Alternatively activation of the RAS-MAPK pathway circumvents Shh pathway dependency, drives tumor growth, and enhances metastatic behavior. Strikingly, in BCC patients treated with Smo inhibitor, squamous cell cancers with RAS/MAPK activation emerged from the antecedent BCC tumors. Together, these findings reveal a critical role of the RAS-MAPK pathway in drug resistance and tumor evolution of Shh pathway-dependent tumors. PMID:26130651

  6. RAS/MAPK activation drives resistance to Smo inhibition, metastasis and tumor evolution in Shh pathway-dependent tumors

    PubMed Central

    Zhao, Xuesong; Ponomaryov, Tatyana; Ornell, Kimberly J.; Zhou, Pengcheng; Dabral, Sukriti K.; Pak, Ekaterina; Li, Wei; Atwood, Scott X.; Whitson, Ramon J.; Chang, Anne Lynn S.; Li, Jiang; Oro, Anthony E.; Chan, Jennifer A.; Kelleher, Joseph F.; Segal, Rosalind A.

    2015-01-01

    Aberrant Shh signaling promotes tumor growth in diverse cancers. The importance of Shh signaling is particularly evident in medulloblastoma and basal cell carcinoma (BCC), where inhibitors targeting the Shh pathway component Smoothened (Smo) show great therapeutic promise. However, the emergence of drug resistance limits long-term efficacy and the mechanisms of resistance remain poorly understood. Using new medulloblastoma models, we identify two distinct paradigms of resistance to Smo inhibition. Sufu mutations lead to maintenance of the Shh pathway in the presence of Smo inhibitors. Alternatively activation of the RAS/MAPK pathway circumvents Shh pathway-dependency, drives tumor growth and enhances metastatic behavior. Strikingly, in BCC patients treated with Smo inhibitor, squamous cell cancers with RAS/MAPK activation emerged from the antecedent BCC tumors. Together these findings reveal a critical role of RAS/MAPK pathway in drug resistance and tumor evolution of Shh pathway-dependent tumors. PMID:26130651

  7. Ras protein/cAMP-dependent protein kinase signaling is negatively regulated by a deubiquitinating enzyme, Ubp3, in yeast.

    PubMed

    Li, Yang; Wang, Yuqi

    2013-04-19

    Ras proteins and cAMP-dependent protein kinase (protein kinase A, PKA) are important components of a nutrient signaling pathway that mediates cellular responses to glucose in yeast. The molecular mechanisms that regulate Ras/PKA-mediated signaling remain to be fully understood. Here, we provide evidence that Ras/PKA signaling is negatively regulated by a deubiquitinating enzyme, Ubp3. Disrupting the activity of Ubp3 leads to hyperactivation of PKA, as evidenced by much enhanced phosphorylation of PKA substrates, decreased accumulation of glycogen, larger cell size, and increased sensitivity to heat shock. Levels of intracellular cAMP and the active forms of Ras proteins are also elevated in the ubp3Δ mutant. Consistent with a possibility that the increased cAMP is responsible for the abnormal signaling behavior of the ubp3Δ mutant, overexpressing PDE2, which encodes a phosphodiesterase that hydrolyzes cAMP, significantly relieves the cell size increase and heat shock sensitivity of the mutant. Further analysis reveals that Ubp3 interacts with a Ras GTPase-accelerating protein, Ira2, and regulates its level of ubiquitination. Together, our data indicate that Ubp3 is a new regulator of the Ras/PKA signaling pathway and suggest that Ubp3 regulates this pathway by controlling the ubiquitination of Ras GTPase-accelerating protein Ira2. PMID:23476013

  8. RasGRP1 opposes proliferative EGFR–SOS1–Ras signals and restricts intestinal epithelial cell growth

    PubMed Central

    Depeille, Philippe; Henricks, Linda M.; van de Ven, Robert A. H.; Lemmens, Ed; Wang, Chih-Yang; Matli, Mary; Werb, Zena; Haigis, Kevin M.; Donner, David; Warren, Robert; Roose, Jeroen P.

    2015-01-01

    The character of EGFR signals can influence cell fate but mechanistic insights into intestinal EGFR-Ras signalling are limited. Here we show that two distinct Ras nucleotide exchange factors, RasGRP1 and SOS1, lie downstream of EGFR but act in functional opposition. RasGRP1 is expressed in intestinal crypts where it restricts epithelial growth. High RasGRP1 expression in colorectal cancer (CRC) patient samples correlates with a better clinical outcome. Biochemically, we find that RasGRP1 creates a negative feedback loop that limits proliferative EGFR–SOS1–Ras signals in CRC cells. Genetic Rasgrp1 depletion from mice with either an activating mutation in KRas or with aberrant Wnt signalling due to a mutation in Apc resulted in both cases in exacerbated Ras–ERK signalling and cell proliferation. The unexpected opposing cell biological effects of EGFR–RasGRP1 and EGFR–SOS1 signals in the same cell shed light on the intricacy of EGFR-Ras signalling in normal epithelium and carcinoma. PMID:26005835

  9. Overexpressed galectin-3 in pancreatic cancer induces cell proliferation and invasion by binding Ras and activating Ras signaling.

    PubMed

    Song, Shumei; Ji, Baoan; Ramachandran, Vijaya; Wang, Huamin; Hafley, Margarete; Logsdon, Craig; Bresalier, Robert S

    2012-01-01

    Pancreatic cancer (PDAC) is a lethal disease with a five-year survival of 3-5%. Mutations in K-Ras are found in nearly all cases, but K-Ras mutations alone are not sufficient for the development of PDAC. Additional factors contribute to activation of Ras signaling and lead to tumor formation. Galectin-3 (Gal-3), a multifunctional β-galactoside-binding protein, is highly expressed in PDAC. We therefore investigated the functional role of Gal-3 in pancreatic cancer progression and its relationship to Ras signaling. Expression of Gal-3 was determined by immunohistochemistry, Q-PCR and immunoblot. Functional studies were performed using pancreatic cell lines genetically engineered to express high or low levels of Gal-3. Ras activity was examined by Raf pull-down assays. Co-immunoprecipitation and immunofluorescence were used to assess protein-protein interactions. In this study, we demonstrate that Gal-3 was highly up-regulated in human tumors and in a mutant K-Ras mouse model of PDAC. Down-regulation of Gal-3 by lentivirus shRNA decreased PDAC cell proliferation and invasion in vitro and reduced tumor volume and size in an orthotopic mouse model. Gal-3 bound Ras and maintained Ras activity; down-regulation of Gal-3 decreased Ras activity as well as Ras down-stream signaling including phosphorylation of ERK and AKT and Ral A activity. Transfection of Gal-3 cDNA into PDAC cells with low-level Gal-3 augmented Ras activity and its down-stream signaling. These results suggest that Gal-3 contributes to pancreatic cancer progression, in part, by binding Ras and activating Ras signaling. Gal-3 may therefore be a potential novel target for this deadly disease. PMID:22900040

  10. Relapsed neuroblastomas show frequent RAS-MAPK pathway mutations.

    PubMed

    Eleveld, Thomas F; Oldridge, Derek A; Bernard, Virginie; Koster, Jan; Daage, Leo Colmet; Diskin, Sharon J; Schild, Linda; Bentahar, Nadia Bessoltane; Bellini, Angela; Chicard, Mathieu; Lapouble, Eve; Combaret, Valérie; Legoix-Né, Patricia; Michon, Jean; Pugh, Trevor J; Hart, Lori S; Rader, JulieAnn; Attiyeh, Edward F; Wei, Jun S; Zhang, Shile; Naranjo, Arlene; Gastier-Foster, Julie M; Hogarty, Michael D; Asgharzadeh, Shahab; Smith, Malcolm A; Guidry Auvil, Jaime M; Watkins, Thomas B K; Zwijnenburg, Danny A; Ebus, Marli E; van Sluis, Peter; Hakkert, Anne; van Wezel, Esther; van der Schoot, C Ellen; Westerhout, Ellen M; Schulte, Johannes H; Tytgat, Godelieve A; Dolman, M Emmy M; Janoueix-Lerosey, Isabelle; Gerhard, Daniela S; Caron, Huib N; Delattre, Olivier; Khan, Javed; Versteeg, Rogier; Schleiermacher, Gudrun; Molenaar, Jan J; Maris, John M

    2015-08-01

    The majority of patients with neuroblastoma have tumors that initially respond to chemotherapy, but a large proportion will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole-genome sequencing of 23 paired diagnostic and relapse neuroblastomas showed clonal evolution from the diagnostic tumor, with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK pathway. Seven of these events were detected only in the relapse tumor, whereas the others showed clonal enrichment. In neuroblastoma cell lines, we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18; 61%), and these lesions predicted sensitivity to MEK inhibition in vitro and in vivo. Our findings provide a rationale for genetic characterization of relapse neuroblastomas and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease. PMID:26121087

  11. Involvement of Ras/Raf/AP-1 in BMP-4 signaling during Xenopus embryonic development.

    PubMed Central

    Xu, R H; Dong, Z; Maeno, M; Kim, J; Suzuki, A; Ueno, N; Sredni, D; Colburn, N H; Kung, H F

    1996-01-01

    Previously, we elucidated the role of bone morphogenetic protein 4 (BMP-4) in the dorsal-ventral patterning of the Xenopus embryo by using a dominant negative mutant of the BMP-4 receptor (DN-BR). The present paper describes the involvement of Ras, Raf, and activator protein 1 (AP-1) in BMP-4 signaling during Xenopus embryonic development. The AP-1 activity was determined by injecting an AP-1-dependent luciferase reporter gene into two-cell-stage Xenopus embryos and measuring the luciferase activity at various developmental stages. We found that injection of BMP-4 mRNA increased AP-1 activity, whereas injection of DN-BR mRNA inhibited AP-1 activity. Similar inhibitory effects were seen with injection of mRNAs encoding dominant negative mutants of c-Ha-Ras, c-Raf, or c-Jun. These results suggest that the endogenous AP-1 activity is regulated by BMP-4/Ras/Raf/Jun signals. We next investigated the effects of Ras/Raf/AP-1 signals on the biological functions of BMP-4. DN-BR-induced dorsalization of the embryo, revealed by the formation of a secondary body axis or dorsalization of the ventral mesoderm explant analyzed by histological and molecular criteria, was significantly reversed by coinjection of [Val12]Ha-Ras, c-Raf, or c-Jun mRNA. Furthermore, the BMP-4-stimulated erythroid differentiation in the ventral mesoderm was substantially inhibited by coinjection with the dominant negative c-Ha-Ras, c-Raf, or c-Jun mutant. Our results suggest the involvement of Ras/Raf/AP-1 in the BMP-4 signaling pathway. Images Fig. 2 Fig. 3 Fig. 4 PMID:8570644

  12. Ras-Mek-Erk Signaling Regulates Nf1 Heterozygous Neointima Formation

    PubMed Central

    Stansfield, Brian K.; Bessler, Waylan K.; Mali, Raghuveer; Mund, Julie A.; Downing, Brandon D.; Kapur, Reuben; Ingram, David A.

    2015-01-01

    Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes neurofibromin, a negative regulator of diverse Ras signaling cascades. Arterial stenosis is a nonneoplastic manifestation of NF1 that predisposes some patients to debilitating morbidity and sudden death. Recent murine studies demonstrate that Nf1 heterozygosity (Nf1+/−) in monocytes/macrophages significantly enhances intimal proliferation after arterial injury. However, the downstream Ras effector pathway responsible for this phenotype is unknown. Based on in vitro assays demonstrating enhanced extracellular signal-related kinase (Erk) signaling in Nf1+/− macrophages and vascular smooth muscle cells and in vivo evidence of Erk amplification without alteration of phosphatidylinositol 3-kinase signaling in Nf1+/− neointimas, we tested the hypothesis that Ras-Erk signaling regulates intimal proliferation in a murine model of NF1 arterial stenosis. By using a well-established in vivo model of inflammatory cell migration and standard cell culture, neurofibromin-deficient macrophages demonstrate enhanced sensitivity to growth factor stimulation in vivo and in vitro, which is significantly diminished in the presence of PD0325901, a specific inhibitor of Ras-Erk signaling in phase 2 clinical trials for cancer. After carotid artery injury, Nf1+/− mice demonstrated increased intimal proliferation compared with wild-type mice. Daily administration of PD0325901 significantly reduced Nf1+/− neointima formation to levels of wild-type mice. These studies identify the Ras-Erk pathway in neurofibromin-deficient macrophages as the aberrant pathway responsible for enhanced neointima formation. PMID:24211110

  13. Ras-Mek-Erk signaling regulates Nf1 heterozygous neointima formation.

    PubMed

    Stansfield, Brian K; Bessler, Waylan K; Mali, Raghuveer; Mund, Julie A; Downing, Brandon D; Kapur, Reuben; Ingram, David A

    2014-01-01

    Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes neurofibromin, a negative regulator of diverse Ras signaling cascades. Arterial stenosis is a nonneoplastic manifestation of NF1 that predisposes some patients to debilitating morbidity and sudden death. Recent murine studies demonstrate that Nf1 heterozygosity (Nf1(+/-)) in monocytes/macrophages significantly enhances intimal proliferation after arterial injury. However, the downstream Ras effector pathway responsible for this phenotype is unknown. Based on in vitro assays demonstrating enhanced extracellular signal-related kinase (Erk) signaling in Nf1(+/-) macrophages and vascular smooth muscle cells and in vivo evidence of Erk amplification without alteration of phosphatidylinositol 3-kinase signaling in Nf1(+/-) neointimas, we tested the hypothesis that Ras-Erk signaling regulates intimal proliferation in a murine model of NF1 arterial stenosis. By using a well-established in vivo model of inflammatory cell migration and standard cell culture, neurofibromin-deficient macrophages demonstrate enhanced sensitivity to growth factor stimulation in vivo and in vitro, which is significantly diminished in the presence of PD0325901, a specific inhibitor of Ras-Erk signaling in phase 2 clinical trials for cancer. After carotid artery injury, Nf1(+/-) mice demonstrated increased intimal proliferation compared with wild-type mice. Daily administration of PD0325901 significantly reduced Nf1(+/-) neointima formation to levels of wild-type mice. These studies identify the Ras-Erk pathway in neurofibromin-deficient macrophages as the aberrant pathway responsible for enhanced neointima formation. PMID:24211110

  14. Transformation by v-Src: Ras-MAPK and PI3K-mTOR mediate parallel pathways.

    PubMed

    Penuel, E; Martin, G S

    1999-06-01

    An increase in the level of active, GTP-bound Ras is not necessary for transformation of chicken embryo fibroblasts (CEF) by v-Src. This suggests that other Ras-independent pathways contribute to transformation by v-Src. To address the possibility that activation of phosphatidylinositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR/FRAP), represents one of these pathways, we have examined the effect of simultaneous inhibition of the Ras-MAPK and PI3K-mTOR pathways on transformation of CEF by v-Src. Transformation was assessed by the standard parameters of morphological alteration, increased hexose uptake, loss of density inhibition, and anchorage-independent growth. Inhibition of the Ras-MAPK pathway by expression of the dominant-negative Ras mutant HRasN17 or by addition of the MAPK kinase (MEK) inhibitor PD98059 reduced several of these parameters but failed to block transformation. Similarly, inhibition of the PI3K-mTOR pathway by addition of the PI3K inhibitor 2-[4-morpholinyl]-8-phenyl-4H-1-benzopyran-4-one (LY294002) or the mTOR inhibitor rapamycin, although reducing several parameters of transformation, also failed to block transformation. However, simultaneous inhibition of signaling by the Ras-MAPK pathway and the PI3K-mTOR pathway essentially blocked transformation. These data indicate that transformation of CEF by v-Src is mediated by two parallel pathways, the Ras-MAPK pathway and the PI-3K-mTOR pathway, which both contribute to transformation. The possibility that simultaneous activation of other pathways is also required is not excluded. PMID:10359590

  15. Transformation by v-Src: Ras-MAPK and PI3K-mTOR Mediate Parallel Pathways

    PubMed Central

    Penuel, Elicia; Martin, G. Steven

    1999-01-01

    An increase in the level of active, GTP-bound Ras is not necessary for transformation of chicken embryo fibroblasts (CEF) by v-Src. This suggests that other Ras-independent pathways contribute to transformation by v-Src. To address the possibility that activation of phosphatidylinositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR/FRAP), represents one of these pathways, we have examined the effect of simultaneous inhibition of the Ras-MAPK and PI3K-mTOR pathways on transformation of CEF by v-Src. Transformation was assessed by the standard parameters of morphological alteration, increased hexose uptake, loss of density inhibition, and anchorage-independent growth. Inhibition of the Ras-MAPK pathway by expression of the dominant-negative Ras mutant HRasN17 or by addition of the MAPK kinase (MEK) inhibitor PD98059 reduced several of these parameters but failed to block transformation. Similarly, inhibition of the PI3K-mTOR pathway by addition of the PI3K inhibitor 2-[4-morpholinyl]-8-phenyl-4H-1-benzopyran-4-one (LY294002) or the mTOR inhibitor rapamycin, although reducing several parameters of transformation, also failed to block transformation. However, simultaneous inhibition of signaling by the Ras-MAPK pathway and the PI3K-mTOR pathway essentially blocked transformation. These data indicate that transformation of CEF by v-Src is mediated by two parallel pathways, the Ras-MAPK pathway and the PI-3K-mTOR pathway, which both contribute to transformation. The possibility that simultaneous activation of other pathways is also required is not excluded. PMID:10359590

  16. Ras, Rac1, and phosphatidylinositol-3-kinase (PI3K) signaling in nitric oxide induced endothelial cell migration.

    PubMed

    Eller-Borges, Roberta; Batista, Wagner L; da Costa, Paulo E; Tokikawa, Rita; Curcio, Marli F; Strumillo, Scheilla T; Sartori, Adriano; Moraes, Miriam S; de Oliveira, Graciele A; Taha, Murched O; Fonseca, Fábio V; Stern, Arnold; Monteiro, Hugo P

    2015-05-01

    The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras - ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (Ras(N17)) or with the non-nitrosatable mutant of Ras (Ras(C118S)); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK. PMID:25819133

  17. Growth hormone signaling pathways.

    PubMed

    Carter-Su, Christin; Schwartz, Jessica; Argetsinger, Lawrence S

    2016-06-01

    Over 20years ago, our laboratory showed that growth hormone (GH) signals through the GH receptor-associated tyrosine kinase JAK2. We showed that GH binding to its membrane-bound receptor enhances binding of JAK2 to the GHR, activates JAK2, and stimulates tyrosyl phosphorylation of both JAK2 and GHR. The activated JAK2/GHR complex recruits a variety of signaling proteins, thereby initiating multiple signaling pathways and cellular responses. These proteins and pathways include: 1) Stat transcription factors implicated in the expression of multiple genes, including the gene encoding insulin-like growth factor 1; 2) Shc adapter proteins that lead to activation of the grb2-SOS-Ras-Raf-MEK-ERK1,2 pathway; 3) insulin receptor substrate proteins implicated in the phosphatidylinositol-3-kinase and Akt pathway; 4) signal regulatory protein α, a transmembrane scaffold protein that recruits proteins including the tyrosine phosphatase SHP2; and 5) SH2B1, a scaffold protein that can activate JAK2 and enhance GH regulation of the actin cytoskeleton. Our recent work has focused on the function of SH2B1. We have shown that SH2B1β is recruited to and phosphorylated by JAK2 in response to GH. SH2B1 localizes to the plasma membrane, cytoplasm and focal adhesions; it also cycles through the nucleus. SH2B1 regulates the actin cytoskeleton and promotes GH-dependent motility of RAW264.7 macrophages. Mutations in SH2B1 have been found in humans exhibiting severe early-onset childhood obesity and insulin resistance. These mutations impair SH2B1 enhancement of GH-induced macrophage motility. As SH2B1 is expressed ubiquitously and is also recruited to a variety of receptor tyrosine kinases, our results raise the possibility that effects of SH2B1 on the actin cytoskeleton in various cell types, including neurons, may play a role in regulating body weight. PMID:26421979

  18. Targeting RTK Signaling Pathways in Cancer

    PubMed Central

    Regad, Tarik

    2015-01-01

    The RAS/MAP kinase and the RAS/PI3K/AKT pathways play a key role in the regulation of proliferation, differentiation and survival. The induction of these pathways depends on Receptor Tyrosine Kinases (RTKs) that are activated upon ligand binding. In cancer, constitutive and aberrant activations of components of those pathways result in increased proliferation, survival and metastasis. For instance, mutations affecting RTKs, Ras, B-Raf, PI3K and AKT are common in perpetuating the malignancy of several types of cancers and from different tissue origins. Therefore, these signaling pathways became prime targets for cancer therapy. This review aims to provide an overview about the most frequently encountered mutations, the pathogenesis that results from such mutations and the known therapeutic strategies developed to counteract their aberrant functions. PMID:26404379

  19. Small Molecule APY606 Displays Extensive Antitumor Activity in Pancreatic Cancer via Impairing Ras-MAPK Signaling

    PubMed Central

    Guo, Na; Liu, Zuojia; Zhao, Wenjing; Wang, Erkang; Wang, Jin

    2016-01-01

    Pancreatic cancer has been found with abnormal expression or mutation in Ras proteins. Oncogenic Ras activation exploits their extensive signaling reach to affect multiple cellular processes, in which the mitogen-activated protein kinase (MAPK) signaling exerts important roles in tumorigenesis. Therapies targeted Ras are thus of major benefit for pancreatic cancer. Although small molecule APY606 has been successfully picked out by virtual drug screening based on Ras target receptor, its in-depth mechanism remains to be elucidated. We herein assessed the antitumor activity of APY606 against human pancreatic cancer Capan-1 and SW1990 cell lines and explored the effect of Ras-MAPK and apoptosis-related signaling pathway on the activity of APY606. APY606 treatment resulted in a dose- and time-dependent inhibition of cancer cell viability. Additionally, APY606 exhibited strong antitumor activity, as evidenced not only by reduction in tumor cell invasion, migration and mitochondrial membrane potential but also by alteration in several apoptotic indexes. Furthermore, APY606 treatment directly inhibited Ras-GTP and the downstream activation of MAPK, which resulted in the down-regulation of anti-apoptotic protein Bcl-2, leading to the up-regulation of mitochondrial apoptosis pathway-related proteins (Bax, cytosolic Cytochrome c and Caspase 3) and of cyclin-dependent kinase 2 and Cyclin A, E. These data suggest that impairing Ras-MAPK signaling is a novel mechanism of action for APY606 during therapeutic intervention in pancreatic cancer. PMID:27223122

  20. Small Molecule APY606 Displays Extensive Antitumor Activity in Pancreatic Cancer via Impairing Ras-MAPK Signaling.

    PubMed

    Guo, Na; Liu, Zuojia; Zhao, Wenjing; Wang, Erkang; Wang, Jin

    2016-01-01

    Pancreatic cancer has been found with abnormal expression or mutation in Ras proteins. Oncogenic Ras activation exploits their extensive signaling reach to affect multiple cellular processes, in which the mitogen-activated protein kinase (MAPK) signaling exerts important roles in tumorigenesis. Therapies targeted Ras are thus of major benefit for pancreatic cancer. Although small molecule APY606 has been successfully picked out by virtual drug screening based on Ras target receptor, its in-depth mechanism remains to be elucidated. We herein assessed the antitumor activity of APY606 against human pancreatic cancer Capan-1 and SW1990 cell lines and explored the effect of Ras-MAPK and apoptosis-related signaling pathway on the activity of APY606. APY606 treatment resulted in a dose- and time-dependent inhibition of cancer cell viability. Additionally, APY606 exhibited strong antitumor activity, as evidenced not only by reduction in tumor cell invasion, migration and mitochondrial membrane potential but also by alteration in several apoptotic indexes. Furthermore, APY606 treatment directly inhibited Ras-GTP and the downstream activation of MAPK, which resulted in the down-regulation of anti-apoptotic protein Bcl-2, leading to the up-regulation of mitochondrial apoptosis pathway-related proteins (Bax, cytosolic Cytochrome c and Caspase 3) and of cyclin-dependent kinase 2 and Cyclin A, E. These data suggest that impairing Ras-MAPK signaling is a novel mechanism of action for APY606 during therapeutic intervention in pancreatic cancer. PMID:27223122

  1. Targeting the RAS pathway by mitogen-activated protein kinase inhibitors.

    PubMed

    Kiessling, Michael K; Rogler, Gerhard

    2015-01-01

    Targeting of oncogenic driver mutations with small-molecule inhibitors resulted in powerful treatment options for cancer patients in recent years. The RAS (rat sarcoma) pathway is among the most frequently mutated pathways in human cancer. Whereas targeting mutant Kirsten RAS (KRAS) remains difficult, mutant B rapidly accelerated fibrosarcoma (BRAF) kinase is an established drug target in cancer. Now data show that neuroblastoma RAS (NRAS) and even Harvey RAS (HRAS) mutations could be predictive markers for treatment with mitogen-activated protein kinase (MEK) inhibitors. This review discusses recent preclinical and clinical studies of MEK inhibitors in BRAF and RAS mutant cancer. PMID:26691679

  2. Systemic Regulation of RAS/MAPK Signaling by the Serotonin Metabolite 5-HIAA

    PubMed Central

    Schmid, Tobias; Snoek, L. Basten; Fröhli, Erika; van der Bent, M. Leontien; Kammenga, Jan; Hajnal, Alex

    2015-01-01

    Human cancer is caused by the interplay of mutations in oncogenes and tumor suppressor genes and inherited variations in cancer susceptibility genes. While many of the tumor initiating mutations are well characterized, the effect of genetic background variation on disease onset and progression is less understood. We have used C. elegans genetics to identify genetic modifiers of the oncogenic RAS/MAPK signaling pathway. Quantitative trait locus analysis of two highly diverged C. elegans isolates combined with allele swapping experiments identified the polymorphic monoamine oxidase A (MAOA) gene amx-2 as a negative regulator of RAS/MAPK signaling. We further show that the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), which is a product of MAOA catalysis, systemically inhibits RAS/MAPK signaling in different organs of C. elegans. Thus, MAOA activity sets a global threshold for MAPK activation by controlling 5-HIAA levels. To our knowledge, 5-HIAA is the first endogenous small molecule that acts as a systemic inhibitor of RAS/MAPK signaling. PMID:25978500

  3. Basal but not luminal mammary epithelial cells require PI3K/mTOR signaling for Ras-driven overgrowth.

    PubMed

    Plichta, Kristin A; Mathers, Jessica L; Gestl, Shelley A; Glick, Adam B; Gunther, Edward J

    2012-11-15

    The mammary ducts of humans and mice are comprised of two main mammary epithelial cell (MEC) subtypes: a surrounding layer of basal MECs and an inner layer of luminal MECs. Breast cancer subtypes show divergent clinical behavior that may reflect properties inherent in their MEC compartment of origin. How the response to a cancer-initiating genetic event is shaped by MEC subtype remains largely unexplored. Using the mouse mammary gland, we designed organotypic three-dimensional culture models that permit challenge of discrete MEC compartments with the same oncogenic insult. Mammary organoids were prepared from mice engineered for compartment-restricted coexpression of oncogenic H-RAS(G12V) together with a nuclear fluorescent reporter. Monitoring of H-RAS(G12V)-expressing MECs during extended live cell imaging permitted visualization of Ras-driven phenotypes via video microscopy. Challenging either basal or luminal MECs with H-RAS(G12V) drove MEC proliferation and survival, culminating in aberrant organoid overgrowth. In each compartment, Ras activation triggered modes of collective MEC migration and invasion that contrasted with physiologic modes used during growth factor-initiated branching morphogenesis. Although basal and luminal Ras activation produced similar overgrowth phenotypes, inhibitor studies revealed divergent use of Ras effector pathways. Blocking either the phosphoinositide 3-kinase or the mammalian target of rapamycin pathway completely suppressed Ras-driven invasion and overgrowth of basal MECs, but only modestly attenuated Ras-driven phenotypes in luminal MECs. We show that MEC subtype defines signaling pathway dependencies downstream of Ras. Thus, cells-of-origin may critically determine the drug sensitivity profiles of mammary neoplasia. PMID:23010075

  4. Signal Integration by Lipid-Mediated Spatial Cross Talk between Ras Nanoclusters

    PubMed Central

    Zhou, Yong; Liang, Hong; Rodkey, Travis; Ariotti, Nicholas; Parton, Robert G.

    2014-01-01

    Lipid-anchored Ras GTPases form transient, spatially segregated nanoclusters on the plasma membrane that are essential for high-fidelity signal transmission. The lipid composition of Ras nanoclusters, however, has not previously been investigated. High-resolution spatial mapping shows that different Ras nanoclusters have distinct lipid compositions, indicating that Ras proteins engage in isoform-selective lipid sorting and accounting for different signal outputs from different Ras isoforms. Phosphatidylserine is a common constituent of all Ras nanoclusters but is only an obligate structural component of K-Ras nanoclusters. Segregation of K-Ras and H-Ras into spatially and compositionally distinct lipid assemblies is exquisitely sensitive to plasma membrane phosphatidylserine levels. Phosphatidylserine spatial organization is also modified by Ras nanocluster formation. In consequence, Ras nanoclusters engage in remote lipid-mediated communication, whereby activated H-Ras disrupts the assembly and operation of spatially segregated K-Ras nanoclusters. Computational modeling and experimentation reveal that complex effects of caveolin and cortical actin on Ras nanoclustering are similarly mediated through regulation of phosphatidylserine spatiotemporal dynamics. We conclude that phosphatidylserine maintains the lateral segregation of diverse lipid-based assemblies on the plasma membrane and that lateral connectivity between spatially remote lipid assemblies offers important previously unexplored opportunities for signal integration and signal processing. PMID:24366544

  5. Resequencing Analysis of the Candidate Tyrosine Kinase and RAS Pathway Gene Families in Multiple Myeloma

    PubMed Central

    Hucthagowder, Vishwanathan; Meyer, Rekha; Mullins, Chelsea; Nagarajan, Rakesh; DiPersio, John F.; Vij, Ravi; Tomasson, Michael H.; Kulkarni, Shashikant

    2012-01-01

    Multiple myeloma (MM) is an incurable, B-cell malignancy, characterized by the clonal proliferation and accumulation of malignant plasma cells in bone marrow. Despite recent advances in understanding of genomic aberrations, a comprehensive catalogue of clinically actionable mutations in MM is just beginning to emerge. The tyrosine kinase (TK) and RAS oncogenes, which encode important regulators of various signaling pathways, are among the most frequently altered gene families in cancer. To clarify the role of TK and RAS genes in pathogenesis of MM, we performed a systematic, targeted screening of mutations on prioritized RAS and TK genes, in CD138 sorted bone marrow specimens from 42 untreated patients. We identified a total of 24 mutations in KRAS, PIK3CA, INSR, LTK and MERTK genes. In particular, seven novel mutations in addition to known KRAS mutations were observed. Prediction analysis tools, PolyPhen and SIFT were used to assess the functional significance of these novel mutations. Our analysis predicted that these mutations may have a deleterious effect, resulting in functional alteration of proteins involved in the pathogenesis of myeloma. While further investigation is needed to determine the functional consequences of these proteins, mutational testing of the RAS and TK genes in larger myeloma cohorts might be also useful to establish the recurrent nature of these mutations. PMID:22939401

  6. Hyperactive Ras/MAPK signaling is critical for tibial nonunion fracture in neurofibromin-deficient mice.

    PubMed

    Sharma, Richa; Wu, Xiaohua; Rhodes, Steven D; Chen, Shi; He, Yongzheng; Yuan, Jin; Li, Jiliang; Yang, Xianlin; Li, Xiaohong; Jiang, Li; Kim, Edward T; Stevenson, David A; Viskochil, David; Xu, Mingjiang; Yang, Feng-Chun

    2013-12-01

    Neurofibromatosis type 1 (NF1) is a common genetic disorder affecting 1 in 3500 individuals. Patients with NF1 are predisposed to debilitating skeletal manifestations, including osteopenia/osteoporosis and long bone pseudarthrosis (nonunion fracture). Hyperactivation of the Ras/mitogen-activated protein kinase (MAPK) pathway in NF1 is known to underlie aberrant proliferation and differentiation in cell lineages, including osteoclast progenitors and mesenchymal stem cells (MSCs) also known as osteoblast progenitors (pro-OBLs). Our current study demonstrates the hyper Ras/MAPK as a critical pathway underlying the pathogenesis of NF1-associated fracture repair deficits. Nf1-deficient pro-OBLs exhibit Ras/MAPK hyperactivation. Introduction of the NF1 GTPase activating-related domain (NF1 GAP-related domain) in vitro is sufficient to rescue hyper Ras activity and enhance osteoblast (OBL) differentiation in Nf1(-/-) pro-OBLs and NF1 human (h) MSCs cultured from NF1 patients with skeletal abnormalities, including pseudarthrosis or scoliosis. Pharmacologic inhibition of mitogen-activated protein kinase kinase (MEK) signaling with PD98059 partially rescues aberrant Erk activation while enhancing OBL differentiation and expression of OBL markers, osterix and osteocalcin, in Nf1-deficient murine pro-OBLs. Similarly, MEK inhibition enhances OBL differentiation of hMSCs. In addition, PD98059 rescues aberrant osteoclast maturation in Nf1 haploinsufficient bone marrow mononuclear cells (BMMNCs). Importantly, MEK inhibitor significantly improves fracture healing in an NF1 murine model, Col2.3Cre;Nf1(flox/-). Collectively, these data indicate the Ras/MAPK cascade as a critical pathway in the pathogenesis of bone loss and pseudarthrosis related to NF1 mutations. These studies provide evidence for targeting the MAPK pathway to improve bone mass and treat pseudarthrosis in NF1. PMID:23863460

  7. Bypassing both surface attachment and surface recognition requirements for appressorium formation by overactive ras signaling in Magnaporthe oryzae.

    PubMed

    Zhou, Xiaoying; Zhao, Xinhua; Xue, Chaoyang; Dai, Yafeng; Xu, Jin-Rong

    2014-09-01

    Magnaporthe oryzae forms a highly specialized infection structure called an appressorium for plant penetration. In M. oryzae and many other plant-pathogenic fungi, surface attachment and surface recognition are two essential requirements for appressorium formation. Development of appressoria in the air has not been reported. In this study, we found that expression of a dominant active MoRAS2(G18V) allele in M. oryzae resulted in the formation of morphologically abnormal appressoria on nonconducive surfaces, in liquid suspensions, and on aerial hyphae without attachment to hard surfaces. Both the Pmk1 mitogen-activated protein kinase cascade and cAMP signaling pathways that regulate surface recognition and appressorium morphogenesis in M. oryzae were overactivated in the MoRAS2(G18V) transformant. In mutants deleted of PMK1 or CPKA, expression of MoRAS2(G18V) had no significant effects on appressorium morphogenesis. Furthermore, expression of dominant MoRAS2 in Colletotrichum graminicola and C. gloeosporioides also caused the formation of appressorium-like structures in aerial hyphae. Overall, our data indicate that MoRas2 functions upstream from both the cAMP-PKA and Pmk1 pathways and overactive Ras signaling leads to improper activation of these two pathways and appressorium formation without surface attachment in appressorium-forming pathogens. PMID:24835254

  8. Defined spatiotemporal features of RAS-ERK signals dictate cell fate in MCF-7 mammary epithelial cells

    PubMed Central

    Herrero, Ana; Casar, Berta; Colón-Bolea, Paula; Agudo-Ibáñez, Lorena; Crespo, Piero

    2016-01-01

    Signals conveyed through the RAS-ERK pathway are essential for the determination of cell fate. It is well established that signal variability is achieved in the different microenvironments in which signals unfold. It is also known that signal duration is critical for decisions concerning cell commitment. However, it is unclear how RAS-ERK signals integrate time and space in order to elicit a given biological response. To investigate this, we used MCF-7 cells, in which EGF-induced transient ERK activation triggers proliferation, whereas sustained ERK activation in response to heregulin leads to adipocytic differentiation. We found that both proliferative and differentiating signals emanate exclusively from plasma membrane–disordered microdomains. Of interest, the EGF signal can be transformed into a differentiating stimulus by HRAS overexpression, which prolongs ERK activation, but only if HRAS localizes at disordered membrane. On the other hand, HRAS signals emanating from the Golgi complex induce apoptosis and can prevent heregulin-induced differentiation. Our results indicate that within the same cellular context, RAS can exert different, even antagonistic, effects, depending on its sublocalization. Thus cell destiny is defined by the ability of a stimulus to activate RAS at the appropriate sublocalization for an adequate period while avoiding switching on opposing RAS signals. PMID:27099370

  9. The Kinase Activity-deficient Isoform of the Protein Araf Antagonizes Ras/Mitogen-activated Protein Kinase (Ras/MAPK) Signaling in the Zebrafish Embryo*

    PubMed Central

    Xiong, Cong; Liu, Xingfeng; Meng, Anming

    2015-01-01

    Raf kinases are important components of the Ras-Raf-Mek-Erk pathway and also cross-talk with other signaling pathways. Araf kinase has been demonstrated to inhibit TGF-β/Smad2 signaling by directly phosphorylating and accelerating degradation of activated Smad2. In this study, we show that the araf gene expresses in zebrafish embryos to produce a shorter transcript variant, araf-tv2, in addition to the full-length variant araf-tv1. araf-tv2 is predicted to encode a C-terminally truncated peptide without the kinase activity domain. Araf-tv2 can physically associate with Araf-tv1 but does not antagonize the inhibitory effect of Araf-tv1 on TGF-β/Smad2 signaling. Instead, Araf-tv2 interacts strongly with Kras and Nras, ultimately blocking MAPK activation by these Ras proteins. In zebrafish embryos, overexpression of araf-tv2 is sufficient to inhibit Fgf/Ras-promoted Erk activation, mesodermal induction, dorsal development, and neuroectodermal posteriorization. Therefore, different isoforms of Araf may participate in similar developmental processes but by regulating different signaling pathways. PMID:26306042

  10. Plant farnesyltransferase can restore yeast Ras signaling and mating

    SciTech Connect

    Yalovsky, S.; Callan, K.L.; Narita, J.O.

    1997-04-01

    Farnesyltransferase (FTase) is a heterodimeric enzyme that modifies a group of proteins, including Ras, in mammals and yeasts. Plant FTase {alpha} and {beta} subunits were cloned from tomato and expressed in the yeast Saccharomyces cerevisiae to assess their functional conservation in farnesylating Ras and a-factor proteins, which are important for cell growth and mating. The tomato FTase {beta} subunit (LeFTB) alone was unable to complement the growth defect of ram1{del} mutant yeast strains in which the chromosomal FTase {beta} subunit gene was deleted, but coexpression of LeFTB with the plant {alpha} subunit gene (LeFTA) restored normal growth, Ras membrane association, and mating. LeFTB contains a novel 66-amino-acid sequence domain whose deletion reduces the efficiency of tomato FTase to restore normal growth to yeast ram1{del} strains. Coexpression of LeFTA and LeFTB in either yeast or insect cells yielded a functional enzyme that correctly farnesylated CaaX-motif-containing peptides. Despite their low degree of sequence homology, yeast and plant FTases shared similar in vivo and in vitro substrate specificities, demonstrating that this enzymatic modification of proteins with intermediates from the isoprenoid biosynthesis pathway is conserved in evolutionarily divergent eukaryotes. 56 refs., 7 figs., 1 tab.

  11. Mitochondrial Activity and Cyr1 Are Key Regulators of Ras1 Activation of C. albicans Virulence Pathways

    PubMed Central

    Grahl, Nora; Demers, Elora G.; Lindsay, Allia K.; Harty, Colleen E.; Willger, Sven D.; Piispanen, Amy E.; Hogan, Deborah A.

    2015-01-01

    Candida albicans is both a major fungal pathogen and a member of the commensal human microflora. The morphological switch from yeast to hyphal growth is associated with disease and many environmental factors are known to influence the yeast-to-hyphae switch. The Ras1-Cyr1-PKA pathway is a major regulator of C. albicans morphogenesis as well as biofilm formation and white-opaque switching. Previous studies have shown that hyphal growth is strongly repressed by mitochondrial inhibitors. Here, we show that mitochondrial inhibitors strongly decreased Ras1 GTP-binding and activity in C. albicans and similar effects were observed in other Candida species. Consistent with there being a connection between respiratory activity and GTP-Ras1 binding, mutants lacking complex I or complex IV grew as yeast in hypha-inducing conditions, had lower levels of GTP-Ras1, and Ras1 GTP-binding was unaffected by respiratory inhibitors. Mitochondria-perturbing agents decreased intracellular ATP concentrations and metabolomics analyses of cells grown with different respiratory inhibitors found consistent perturbation of pyruvate metabolism and the TCA cycle, changes in redox state, increased catabolism of lipids, and decreased sterol content which suggested increased AMP kinase activity. Biochemical and genetic experiments provide strong evidence for a model in which the activation of Ras1 is controlled by ATP levels in an AMP kinase independent manner. The Ras1 GTPase activating protein, Ira2, but not the Ras1 guanine nucleotide exchange factor, Cdc25, was required for the reduction of Ras1-GTP in response to inhibitor-mediated reduction of ATP levels. Furthermore, Cyr1, a well-characterized Ras1 effector, participated in the control of Ras1-GTP binding in response to decreased mitochondrial activity suggesting a revised model for Ras1 and Cyr1 signaling in which Cyr1 and Ras1 influence each other and, together with Ira2, seem to form a master-regulatory complex necessary to integrate

  12. A new genetic model of activity-induced Ras signaling dependent pre-synaptic plasticity in Drosophila

    PubMed Central

    Freeman, Amanda; Bowers, Mallory; Mortimer, Alysia Vrailas; Timmerman, Christina; Roux, Stephanie; Ramaswami, Mani; Sanyal, Subhabrata

    2010-01-01

    Techniques to induce activity-dependent neuronal plasticity in vivo allow the underlying signaling pathways to be studied in their biological context. Here, we demonstrate activity-induced plasticity at neuromuscular synapses of Drosophila double mutant for comatose (an NSF mutant) and Kum (a SERCA mutant), and present an analysis of the underlying signaling pathways. comt; Kum (CK) double mutants exhibit increased locomotor activity under normal culture conditions, concomitant with a larger neuromuscular junction synapse and stably elevated evoked transmitter release. The observed enhancements of synaptic size and transmitter release in CK mutants are completely abrogated by: a) reduced activity of motor neurons; b) attenuation of the Ras/ERK signaling cascade; or c) inhibition of the transcription factors Fos and CREB. all of which restrict synaptic properties to near wild type levels. Together, these results document neural activity-dependent plasticity of motor synapses in CK animals that requires Ras/ERK signaling and normal transcriptional activity of Fos and CREB. Further, novel in vivo reporters of neuronal Ras activation and Fos transcription also confirm increased signaling through a Ras/AP-1 pathway in motor neurons of CK animals, consistent with results from our genetic experiments. Thus, this study: a) provides a robust system in which to study activity-induced synaptic plasticity in vivo; b) establishes a causal link between neural activity, Ras signaling, transcriptional regulation and pre-synaptic plasticity in glutamatergic motor neurons of Drosophila larvae; and c) presents novel, genetically encoded reporters for Ras and AP-1 dependent signaling pathways in Drosophila. PMID:20193670

  13. Ras-dependent and -independent pathways target the mitogen-activated protein kinase network in macrophages.

    PubMed Central

    Büscher, D; Hipskind, R A; Krautwald, S; Reimann, T; Baccarini, M

    1995-01-01

    Mitogen-activated protein kinases (MAPKs) are activated upon a variety of extracellular stimuli in different cells. In macrophages, colony-stimulating factor 1 (CSF-1) stimulates proliferation, while bacterial lipopolysaccharide (LPS) inhibits cell growth and causes differentiation and activation. Both CSF-1 and LPS rapidly activate the MAPK network and induce the phosphorylation of two distinct ternary complex factors (TCFs), TCF/Elk and TCF/SAP. CSF-1, but not LPS, stimulated the formation of p21ras. GTP complexes. Expression of a dominant negative ras mutant reduced, but did not abolish, CSF-1-mediated stimulation of MEK and MAPK. In contrast, activation of the MEK kinase Raf-1 was Ras independent. Treatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 suppressed LPS-mediated, but not CSF-1-mediated, activation of Raf-1, MEK, and MAPK. Similarly, down-regulation or inhibition of protein kinase C blocked MEK and MAPK induction by LPS but not that by CSF-1. Phorbol 12-myristate 13-acetate pretreatment led to the sustained activation of the Raf-1 kinase but not that of MEK and MAPK. Thus, activated Raf-1 alone does not support MEK/MAPK activation in macrophages. Phosphorylation of TCF/Elk but not that of TCF/SAP was blocked by all treatments that interfered with MAPK activation, implying that TCF/SAP was targeted by a MAPK-independent pathway. Therefore, CSF-1 and LPS target the MAPK network by two alternative pathways, both of which induce Raf-1 activation. The mitogenic pathway depends on Ras activity, while the differentiation signal relies on protein kinase C and phosphatidylcholine-specific phospholipase C activation. PMID:7799956

  14. Ras-activated Dsor1 promotes Wnt signaling in Drosophila development.

    PubMed

    Hall, Eric T; Verheyen, Esther M

    2015-12-15

    Wnt/Wingless (Wg) and Ras-MAPK signaling both play fundamental roles in growth and cell fate determination, and when dysregulated, can lead to tumorigenesis. Several conflicting modes of interaction between Ras-MAPK and Wnt signaling have been identified in specific cellular contexts, causing synergistic or antagonistic effects on target genes. We find novel evidence that the Drosophila homolog of the dual specificity kinases MEK1/2 (also known as MAP2K1/2), Downstream of Raf1 (Dsor1), is required for Wnt signaling. Knockdown of Dsor1 results in loss of Wg target gene expression, as well as reductions in stabilized Armadillo (Arm; Drosophila β-catenin). We identify a close physical interaction between Dsor1 and Arm, and find that catalytically inactive Dsor1 causes a reduction in active Arm. These results suggest that Dsor1 normally counteracts the Axin-mediated destruction of Arm. We find that Ras-Dsor1 activity is independent of upstream activation by EGFR, and instead it appears to be activated by the insulin-like growth factor receptor to promote Wg signaling. Taken together, our results suggest that there is a new crosstalk pathway between insulin and Wg signaling that is mediated by Dsor1. PMID:26542023

  15. Statins inhibited the MIP-1α expression via inhibition of Ras/ERK and Ras/Akt pathways in myeloma cells.

    PubMed

    Tsubaki, Masanobu; Mashimo, Kenji; Takeda, Tomoya; Kino, Toshiki; Fujita, Arisa; Itoh, Tatsuki; Imano, Motohiro; Sakaguchi, Katsuhiko; Satou, Takao; Nishida, Shozo

    2016-03-01

    Macrophage inflammatory protein-1alpha (MIP-1α) is detected at high concentrations in patients with multiple myeloma. It is thought to play an important role in the etiology of multiple myeloma and osteolysis. Thus, inhibiting MIP-1α expression may be useful in developing therapeutic treatments for multiple myeloma-induced osteolysis. In this study, we investigated the potential of statins to inhibit mRNA expression and secretion of MIP-1α in mouse myeloma cells (MOPC-31C). We found that statins inhibited the lipopolysaccharide (LPS)-induced MIP-1α mRNA expression and protein secretion in MOPC-31C cells. This inhibition was reversed when farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), intermediates of the mevalonate pathway, were combined with statins. Furthermore, statins reduced the GTP form of Ras, a phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated Akt. Our results indicate that statins inhibit biosynthesis of FPP and GGPP and thereby down regulate signal transduction of Ras/ERK and Ras/Akt pathways. The net effect suppresses LPS-induced MIP-1α mRNA expression and protein secretion in MOPC-31C cells. Thus, statins hold great promise for developing effective therapies against myeloma-induced osteolysis. PMID:26898421

  16. TOR and RAS pathways regulate desiccation tolerance in Saccharomyces cerevisiae

    PubMed Central

    Welch, Aaron Z.; Gibney, Patrick A.; Botstein, David; Koshland, Douglas E.

    2013-01-01

    Tolerance to desiccation in cultures of Saccharomyces cerevisiae is inducible; only one in a million cells from an exponential culture survive desiccation compared with one in five cells in stationary phase. Here we exploit the desiccation sensitivity of exponentially dividing cells to understand the stresses imposed by desiccation and their stress response pathways. We found that induction of desiccation tolerance is cell autonomous and that there is an inverse correlation between desiccation tolerance and growth rate in glucose-, ammonia-, or phosphate-limited continuous cultures. A transient heat shock induces a 5000–fold increase in desiccation tolerance, whereas hyper-ionic, -reductive, -oxidative, or -osmotic stress induced much less. Furthermore, we provide evidence that the Sch9p-regulated branch of the TOR and Ras-cAMP pathway inhibits desiccation tolerance by inhibiting the stress response transcription factors Gis1p, Msn2p, and Msn4p and by activating Sfp1p, a ribosome biogenesis transcription factor. Among 41 mutants defective in ribosome biogenesis, a subset defective in 60S showed a dramatic increase in desiccation tolerance independent of growth rate. We suggest that reduction of a specific intermediate in 60S biogenesis, resulting from conditions such as heat shock and nutrient deprivation, increases desiccation tolerance. PMID:23171550

  17. Sur8/Shoc2 promotes cell motility and metastasis through activation of Ras-PI3K signaling

    PubMed Central

    Kaduwal, Saluja; Jeong, Woo-Jeong; Park, Jong-Chan; Lee, Kug Hwa; Lee, Young-Mi; Jeon, Soung-Hoo; Lim, Yong-Beom; Min, Do Sik; Choi, Kang-Yell

    2015-01-01

    Sur8 (also known as Shoc2) is a Ras-Raf scaffold protein that modulates signaling through extracellular signal-regulated kinase (ERK) pathway. Although Sur8 has been shown to be a scaffold protein of the Ras-ERK pathway, its interaction with other signaling pathways and its involvement in tumor malignancy has not been reported. We identified that Sur8 interacts with the p110α subunit of phosphatidylinositol 3-kinase (PI3K), as well as with Ras and Raf, and these interactions are increased in an epidermal growth factor (EGF)- and oncogenic Ras-dependent manner. Sur8 regulates cell migration and invasion via activation of Rac and matrix metalloproteinases (MMPs). Interestingly, using inhibitors of MEK and PI3K we found Sur8 mediates these cellular behaviors predominantly through PI3K pathway. We further found that human metastatic melanoma tissues had higher Sur8 content followed by activations of Akt, ERK, and Rac. Lentivirus-mediated Sur8-knockdown attenuated metastatic potential of highly invasive B16-F10 melanoma cells indicating the role of Sur8 in melanoma metastasis. This is the first report to identify the role of scaffold protein Sur8 in regulating cell motility, invasion, and metastasis through activation of both ERK and PI3K pathways. PMID:26384305

  18. The novel plant homeodomain protein rhinoceros antagonizes Ras signaling in the Drosophila eye.

    PubMed Central

    Voas, Matthew G; Rebay, Ilaria

    2003-01-01

    The sequential specification of cell fates in the Drosophila eye requires repeated activation of the epidermal growth factor receptor (EGFR)/Ras/MAP kinase (MAPK) pathway. Equally important are the multiple layers of inhibitory regulation that prevent excessive or inappropriate signaling. Here we describe the molecular and genetic analysis of a previously uncharacterized gene, rhinoceros (rno), that we propose functions to restrict EGFR signaling in the eye. Loss of rno results in the overproduction of photoreceptors, cone cells, and pigment cells and a corresponding reduction in programmed cell death, all phenotypes characteristic of hyperactivated EGFR signaling. Genetic interactions between rno and multiple EGFR pathway components support this hypothesis. rno encodes a novel but evolutionarily conserved nuclear protein with a PHD zinc-finger domain, a motif commonly found in chromatin-remodeling factors. Future analyses of rno will help to elucidate the regulatory strategies that modulate EGFR signaling in the fly eye. PMID:14704181

  19. EGFR/Ras/MAPK signaling mediates adult midgut epithelial homeostasis and regeneration in Drosophila

    PubMed Central

    Jiang, Huaqi; Grenley, Marc O.; Bravo, Maria-Jose; Blumhagen, Rachel Z.; Edgar, Bruce A.

    2010-01-01

    Many tissues in higher animals undergo dynamic homeostatic growth, wherein damaged or aged cells are replaced by the progeny of resident stem cells. To maintain homeostasis, stem cells must respond to tissue needs. Here we show that in response to damage or stress in the intestinal (midgut) epithelium of adult Drosophila, multiple EGFR ligands and rhomboids (intramembrane proteases that activate some EGFR ligands) are induced, leading to the activation of EGFR signaling in intestinal stem cells (ISCs). Activation of EGFR signaling promotes ISC division and midgut epithelium regeneration, thus maintaining tissue homeostasis. ISCs defective in EGFR signaling cannot grow or divide, are poorly maintained, and cannot support midgut epithelium regeneration following enteric infection by the bacterium, Pseudomonas entomophila. Furthermore, ISC proliferation induced by Jak/Stat signaling is dependent upon EGFR signaling. Thus the EGFR/Ras/MAPK signaling pathway plays central, essential roles in ISC maintenance and the feedback system that mediates intestinal homeostasis. PMID:21167805

  20. CaM interaction and Ser181 phosphorylation as new K-Ras signaling modulators

    PubMed Central

    Alvarez-Moya, Blanca; Barceló, Carles; Tebar, Francesc; Jaumot, Montserrat

    2011-01-01

    The small G-protein Ras was the first oncogene to be identified and has a very important contribution to human cancer development (20–23% prevalence). K-RasB, one of the members of the Ras family, is the one that is most mutated and plays a prominent role in pancreatic, colon and lung cancer development. Ras proteins are membrane bound GTPases that cycle between inactive, GDP-bound and active, GTP-bound, states. Most of the research into K-RasB activity regulation has focused on the analysis of how GTP-exchange factors (GEFs) and GTPase activating proteins (GAPs) are regulated by external and internal signals. In contrast, oncogenic K-RasB has a very low GTPase activity and furthermore is not deactivated by GAPs. Consequently, the consensus was that activity of oncogenic K-RasB was not modulated. In this extra view we recapitulate some recent data showing that calmodulin binding to K-RasB inhibits phosphorylation of K-RasB at Ser181, near to the membrane anchoring domain, modulating signaling of both non-oncogenic and oncogenic K-RasB. This may be relevant to normal cell physiology, but also opens new therapeutic perspectives for the inhibition of oncogenic K-RasB signaling in tumors. PMID:21776410

  1. Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities

    PubMed Central

    Hubbard, Paul A.; Moody, Colleen L.; Murali, Ramachandran

    2014-01-01

    GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC. Although these observations suggest that targeting one of these nodes might lead to more effective treatment options for patients with pancreatic and other cancers, the complex regulatory mechanisms and the number of sequence-conserved isoforms of these proteins have been viewed as significant barriers in drug development. Emerging insights into the allosteric regulatory mechanisms of these proteins suggest novel opportunities for development of selective allosteric inhibitors with fragment-based drug discovery (FBDD) helping make significant inroads. The fact that allosteric inhibitors of Ras and AKT are currently in pre-clinical development lends support to this approach. In this article, we will focus on the recent advances and merits of developing allosteric drugs targeting these two inter-related signaling pathways. PMID:25566081

  2. Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities.

    PubMed

    Hubbard, Paul A; Moody, Colleen L; Murali, Ramachandran

    2014-01-01

    GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC. Although these observations suggest that targeting one of these nodes might lead to more effective treatment options for patients with pancreatic and other cancers, the complex regulatory mechanisms and the number of sequence-conserved isoforms of these proteins have been viewed as significant barriers in drug development. Emerging insights into the allosteric regulatory mechanisms of these proteins suggest novel opportunities for development of selective allosteric inhibitors with fragment-based drug discovery (FBDD) helping make significant inroads. The fact that allosteric inhibitors of Ras and AKT are currently in pre-clinical development lends support to this approach. In this article, we will focus on the recent advances and merits of developing allosteric drugs targeting these two inter-related signaling pathways. PMID:25566081

  3. H-ras Inhibits the Hippo Pathway by Promoting Mst1/Mst2 Heterodimerization.

    PubMed

    Rawat, Sonali J; Araiza-Olivera, Daniela; Arias-Romero, Luis E; Villamar-Cruz, Olga; Prudnikova, Tatiana Y; Roder, Heinrich; Chernoff, Jonathan

    2016-06-20

    The protein kinases Mst1 and Mst2 have tumor suppressor activity, but their mode of regulation is not well established. Mst1 and Mst2 are broadly expressed and may have certain overlapping functions in mammals, as deletions of both Mst1 and Mst2 together are required for tumorigenesis in mouse models [1-3]. These kinases act via a three-component signaling cascade comprising Mst1 and Mst2, the protein kinases Lats1 and Lats2, and the transcriptional coactivators Yap and Taz [4-6]. Mst1 and Mst2 contain C-terminal SARAH domains that mediate their homodimerization as well as heterodimerization with other SARAH domain-containing proteins, which may regulate Mst1/Mst2 activity. Here we show that, in addition to forming homodimers, Mst1 and Mst2 heterodimerize in cells, this interaction is mediated by their SARAH domains and is favored over homodimers, and these heterodimers have much-reduced protein kinase activity compared to Mst1 or Mst2 homodimers. Mst1/Mst2 heterodimerization is strongly promoted by oncogenic H-ras, and this effect requires activation of the Erk pathway. Cells lacking Mst1, in which Mst1/Mst2 heterodimers are not possible, are resistant to H-ras-mediated transformation and maintain active hippo pathway signaling compared to wild-type cells or cells lacking both Mst1 and Mst2. Our results suggest that H-ras, via an Erk-dependent mechanism, downregulates Mst1/Mst2 activity by inducing the formation of inactive Mst1/Mst2 heterodimers. PMID:27238285

  4. Orchestration of Morphogenesis in Filamentous Fungi: Conserved Roles for Ras Signaling Networks

    PubMed Central

    Fortwendel, Jarrod R.

    2015-01-01

    Filamentous fungi undergo complex developmental programs including conidial germination, polarized morphogenesis, and differentiation of sexual and asexual structures. For many fungi, the coordinated completion of development is required for pathogenicity, as specialized morphological structures must be produced by the invading fungus. Ras proteins are highly conserved GTPase signal transducers and function as major regulators of growth and development in eukaryotes. Filamentous fungi typically express two Ras homologues, comprising distinct groups of Ras1-like and Ras2-like proteins based on sequence homology. Recent evidence suggests shared roles for both Ras1 and Ras2 homologues, but also supports the existence of unique functions in the areas of stress response and virulence. This review focuses on the roles played by both Ras protein groups during growth, development, and pathogenicity of a diverse array of filamentous fungi. PMID:26257821

  5. Tyrosine 763 of the murine granulocyte colony-stimulating factor receptor mediates Ras-dependent activation of the JNK/SAPK mitogen-activated protein kinase pathway.

    PubMed Central

    Rausch, O; Marshall, C J

    1997-01-01

    The receptor for granulocyte colony-stimulating factor (G-CSF) can mediate differentiation and proliferation of hemopoietic cells. A proliferative signal is associated with activation of the ERK mitogen-activated protein kinase (MAPK) pathway. To determine whether other MAPK pathways are activated by G-CSF signalling, we have investigated activation of JNK/SAPK in cells proliferating in response to G-CSF. Here we show that G-CSF and interleukin-3 activate JNK/SAPK in two hemopoietic cell lines. The region of the G-CSF receptor required for G-CSF-induced JNK/SAPK activation is located within the C-terminal 68 amino acids of the cytoplasmic domain, which contains Tyr 763. Mutation of Tyr 763 to Phe completely blocks JNK/SAPK activation. However, the C-terminal 68 amino acids are not required for ERK2 activation. We show that activation of JNK/SAPK, like that of ERK2, is dependent on Ras but that higher levels of Ras-GTP are associated with activation of JNK/SAPK than with activation of ERK2. Two separate functional regions of the G-CSF receptor contribute to activation of Ras. The Y763F mutation reduces G-CSF-induced Ras activation from 30 to 35% Ras-GTP to 10 to 13% Ras-GTP. Low levels of Ras activation (10 to 13% Ras-GTP), which are sufficient for ERK2 activation, require only the 100 membrane-proximal amino acids. High levels of Ras-GTP provided by expression of oncogenic Ras are not sufficient to activate JNK/SAPK. An additional signal, also mediated by Tyr 763, is required for activation of JNK/SAPK. PMID:9032244

  6. E-Ras improves the efficiency of reprogramming by facilitating cell cycle progression through JNK-Sp1 pathway.

    PubMed

    Kwon, Yoo-Wook; Jang, Seulgi; Paek, Jae-Seung; Lee, Jae-Woong; Cho, Hyun-Jai; Yang, Han-Mo; Kim, Hyo-Soo

    2015-11-01

    We have previously shown that pluripotent stem cells can be induced from adult somatic cells which were exposed to protein extracts isolated from mouse embryonic stem cells (mESC). Interestingly, generation of induced pluripotent stem (iPS) cells depended on the background of ES cell lines; possible by extracts from C57, but not from E14. Proteomic analysis of two different mES cell lines (C57 and E14) shows that embryonic Ras (E-Ras) is expressed differently in two mES cell lines; high level of E-Ras only in C57 mESC whose extracts allows iPS cells production from somatic cells. Here, we show that E-Ras augments the efficiency in reprogramming of fibroblast by promoting cell proliferation. We found that over-expression of E-Ras in fibroblast increased cell proliferation which was caused by specific up-regulation of cyclins D and E, not A or B, leading to the accelerated G1 to S phase transition. To figure out the common transcription factor of cyclins D and E, we used TRANSFAC database and selected SP1 as a candidate which was confirmed as enhancer of cyclins D and E by luciferase promoter assay using mutants. As downstream signaling pathways, E-Ras activated only c-Jun N-terminal kinases (JNK) but not ERK or p38. Inhibition of JNK prevented E-Ras-mediated induction of pSP1, cyclins D, E, and cell proliferation. Finally, E-Ras transduction to fibroblast enhanced the efficiency of iPS cell generation by 4 factors (Oct4/Klf4/Sox2/C-myc), which was prevented by JNK inhibitor. In conclusion, E-Ras stimulates JNK, enhances binding of Sp1 on the promoter of cyclins D and E, leading to cell proliferation. E-Ras/JNK axis is a critical mechanism to generate iPS cells by transduction of 4 factors or by treatment of mESC protein extracts. PMID:26413787

  7. Mutations in the phosphatidylinositol-3-kinase pathway predict for antitumor activity of the inhibitor PX-866 while oncogenic Ras is a dominant predictor for resistance

    PubMed Central

    Ihle, NathanT.; Lemos, Robert; Wipf, Peter; Yacoub, Adly; Mitchell, Clint; Siwak, Doris; Mills, Gordon B.; Dent, Paul; Kirkpatrick, D Lynn.; Powis, Garth

    2008-01-01

    The novel phosphatidylinositol-3-kinase (PI-3-kinase) inhibitor PX-866 was tested against 13 experimental human tumor xenografts derived from cell lines of various tissue origins. Mutant PI-3-kinase (PIK3CA) and loss of PTEN activity were sufficient but not necessary as predictors of sensitivity to the antitumor activity of the PI-3-K inhibitor PX-866 in the presence of wild type Ras, while mutant oncogenic Ras was a dominant determinant of resistance, even in tumors with coexisting mutations in PIK3CA. The level of activation of PI-3-kinase signaling measured by tumor phospho-Ser473-Akt was insufficient to predict in vivo antitumor response to PX-866. Reverse phase protein array (RPPA) revealed that the Ras dependent down stream targets c-Myc and cyclin B were elevated in cell lines resistant to PX-866 in vivo. Studies using an H-Ras construct to constitutively and preferentially activate the three best defined downstream targets of Ras, namely Raf, RalGDS, and PI-3-kinase, showed that mutant Ras mediates resistance through its ability to utilize multiple pathways for tumorigenesis. The identification of Ras and downstream signaling pathways driving resistance to PI-3-kinase inhibition may serve as an important guide for patient selection as inhibitors enter clinical trials, and for the development of rational combinations with other molecularly targeted agents. PMID:19117997

  8. Opposing activities of the Ras and Hippo pathways converge on regulation of YAP protein turnover.

    PubMed

    Hong, Xin; Nguyen, Hung Thanh; Chen, Qingfeng; Zhang, Rui; Hagman, Zandra; Voorhoeve, P Mathijs; Cohen, Stephen M

    2014-11-01

    Cancer genomes accumulate numerous genetic and epigenetic modifications. Yet, human cellular transformation can be accomplished by a few genetically defined elements. These elements activate key pathways required to support replicative immortality and anchorage independent growth, a predictor of tumorigenesis in vivo. Here, we provide evidence that the Hippo tumor suppressor pathway is a key barrier to Ras-mediated cellular transformation. The Hippo pathway targets YAP1 for degradation via the βTrCP-SCF ubiquitin ligase complex. In contrast, the Ras pathway acts oppositely, to promote YAP1 stability through downregulation of the ubiquitin ligase complex substrate recognition factors SOCS5/6. Depletion of SOCS5/6 or upregulation of YAP1 can bypass the requirement for oncogenic Ras in anchorage independent growth in vitro and tumor formation in vivo. Through the YAP1 target, Amphiregulin, Ras activates the endogenous EGFR pathway, which is required for transformation. Thus, the oncogenic activity of Ras(V12) depends on its ability to counteract Hippo pathway activity, creating a positive feedback loop, which depends on stabilization of YAP1. PMID:25180228

  9. Opposing activities of the Ras and Hippo pathways converge on regulation of YAP protein turnover

    PubMed Central

    Hong, Xin; Nguyen, Hung Thanh; Chen, Qingfeng; Zhang, Rui; Hagman, Zandra; Voorhoeve, P Mathijs; Cohen, Stephen M

    2014-01-01

    Cancer genomes accumulate numerous genetic and epigenetic modifications. Yet, human cellular transformation can be accomplished by a few genetically defined elements. These elements activate key pathways required to support replicative immortality and anchorage independent growth, a predictor of tumorigenesis in vivo. Here, we provide evidence that the Hippo tumor suppressor pathway is a key barrier to Ras-mediated cellular transformation. The Hippo pathway targets YAP1 for degradation via the βTrCP-SCF ubiquitin ligase complex. In contrast, the Ras pathway acts oppositely, to promote YAP1 stability through downregulation of the ubiquitin ligase complex substrate recognition factors SOCS5/6. Depletion of SOCS5/6 or upregulation of YAP1 can bypass the requirement for oncogenic Ras in anchorage independent growth in vitro and tumor formation in vivo. Through the YAP1 target, Amphiregulin, Ras activates the endogenous EGFR pathway, which is required for transformation. Thus, the oncogenic activity of RasV12 depends on its ability to counteract Hippo pathway activity, creating a positive feedback loop, which depends on stabilization of YAP1. PMID:25180228

  10. First step of glycosylphosphatidylinositol (GPI) biosynthesis cross-talks with ergosterol biosynthesis and Ras signaling in Candida albicans.

    PubMed

    Yadav, Bhawna; Bhatnagar, Shilpi; Ahmad, Mohammad Faiz; Jain, Priyanka; Pratyusha, Vavilala A; Kumar, Pravin; Komath, Sneha Sudha

    2014-02-01

    Candida albicans is a leading cause of fungal infections worldwide. It has several glycosylphosphatidylinositol (GPI)-anchored virulence factors. Inhibiting GPI biosynthesis attenuates its virulence. Building on our previous work, we explore the interaction of GPI biosynthesis in C. albicans with ergosterol biosynthesis and hyphal morphogenesis. This study is also the first report of transcriptional co-regulation existing between two subunits of the multisubunit enzyme complex, GPI-N-acetylglucosaminyltransferase (GPI-GnT), involved in the first step of GPI anchor biosynthesis in eukaryotes. Using mutational analysis, we show that the accessory subunits, GPI2 and GPI19, of GPI-GnT exhibit opposite effects on ergosterol biosynthesis and Ras signaling (which determines hyphal morphogenesis). This is because the two subunits negatively regulate one another; GPI19 mutants show up-regulation of GPI2, whereas GPI2 mutants show up-regulation of GPI19. Two different models were examined as follows. First, the two GPI-GnT subunits independently interact with ergosterol biosynthesis and Ras signaling. Second, the two subunits mutually regulate one another and thereby regulate sterol levels and Ras signaling. Analysis of double mutants of these subunits indicates that GPI19 controls ergosterol biosynthesis through ERG11 levels, whereas GPI2 determines the filamentation by cross-talk with Ras1 signaling. Taken together, this suggests that the first step of GPI biosynthesis talks to and regulates two very important pathways in C. albicans. This could have implications for designing new antifungal strategies. PMID:24356967

  11. The E3 ubiquitin ligase Trim7 mediates c-Jun/AP-1 activation by Ras signalling

    PubMed Central

    Chakraborty, Atanu; Diefenbacher, Markus E.; Mylona, Anastasia; Kassel, Olivier; Behrens, Axel

    2015-01-01

    The c-Jun/AP-1 transcription factor controls key cellular behaviours, including proliferation and apoptosis, in response to JNK and Ras/MAPK signalling. While the JNK pathway has been well characterised, the mechanism of activation by Ras was elusive. Here we identify the uncharacterised ubiquitin ligase Trim7 as a critical component of AP-1 activation via Ras. We found that MSK1 directly phosphorylates Trim7 in response to direct activation by the Ras–Raf–MEK–ERK pathway, and this modification stimulates Trim7 E3 ubiquitin ligase activity. Trim7 mediates Lys63-linked ubiquitination of the AP-1 coactivator RACO-1, leading to RACO-1 protein stabilisation. Consequently, Trim7 depletion reduces RACO-1 levels and AP-1-dependent gene expression. Moreover, transgenic overexpression of Trim7 increases lung tumour burden in a Ras-driven cancer model, and knockdown of Trim7 in established xenografts reduces tumour growth. Thus, phosphorylation-ubiquitination crosstalk between MSK1, Trim7 and RACO-1 completes the long sought-after mechanism linking growth factor signalling and AP-1 activation. PMID:25851810

  12. Functional Cross-talk between Ras and Rho Pathways

    PubMed Central

    Jaiswal, Mamta; Dvorsky, Radovan; Amin, Ehsan; Risse, Sarah L.; Fansa, Eyad K.; Zhang, Si-Cai; Taha, Mohamed S.; Gauhar, Aziz R.; Nakhaei-Rad, Saeideh; Kordes, Claus; Koessmeier, Katja T.; Cirstea, Ion C.; Olayioye, Monilola A.; Häussinger, Dieter; Ahmadian, Mohammad R.

    2014-01-01

    The three deleted in liver cancer genes (DLC1–3) encode Rho-specific GTPase-activating proteins (RhoGAPs). Their expression is frequently silenced in a variety of cancers. The RhoGAP activity, which is required for full DLC-dependent tumor suppressor activity, can be inhibited by the Src homology 3 (SH3) domain of a Ras-specific GAP (p120RasGAP). Here, we comprehensively investigated the molecular mechanism underlying cross-talk between two distinct regulators of small GTP-binding proteins using structural and biochemical methods. We demonstrate that only the SH3 domain of p120 selectively inhibits the RhoGAP activity of all three DLC isoforms as compared with a large set of other representative SH3 or RhoGAP proteins. Structural and mutational analyses provide new insights into a putative interaction mode of the p120 SH3 domain with the DLC1 RhoGAP domain that is atypical and does not follow the classical PXXP-directed interaction. Hence, p120 associates with the DLC1 RhoGAP domain by targeting the catalytic arginine finger and thus by competitively and very potently inhibiting RhoGAP activity. The novel findings of this study shed light on the molecular mechanisms underlying the DLC inhibitory effects of p120 and suggest a functional cross-talk between Ras and Rho proteins at the level of regulatory proteins. PMID:24443565

  13. Doxycycline enhances the Ras-MAPK signaling and proliferation of mouse thymic epithelial cells.

    PubMed

    Chen, Xun; Xia, Sheng; Li, Rong; Liu, Hui; Huang, Ying; Qian, Xiaoping; Xiao, Xueyuan; Xu, Xun; Lin, Xin; Tian, Yuxiang; Zong, Yangyong; He, Dacheng; Chen, Weifeng; Zhang, Yu; Shao, Qixiang

    2009-06-01

    Depletion of T-cell-dependent immunity is a major consideration for patients suffering from infections of human immunodeficiency virus (HIV), those undergoing organ transplantation, and those receiving anti-cancer chemotherapy and/or radiotherapy. In general, T-cell regeneration occurs in the thymus through thymopoiesis. We have found that doxycycline (Dox), a tetracycline derivative, enhances the proliferation of mouse thymic epithelial cells, which are unique in their capacity to support positive selection and are essential throughout the development of thymocytes. Cell cycle analysis indicates that the increased cell proliferation is due to a shortened G(0)/G(1) phase. To reveal the underlying mechanisms, we examined the expression of an array of molecules that regulate the cell cycle. The results show that in mouse thymic medullary-type epithelial cell line 1 (MTEC1) Dox leads to elevated levels of H-Ras, phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), cyclin E, cyclin dependent kinase 4/2 (CDK4/CDK2), E2F3, and c-myc. These data, and the observation that the proliferation-enhancing effect is largely abolished following treatment with an ERK inhibitor support an active role of the Ras-ERK/mitogen-activated protein kinase (MAPK) signaling pathway. In conclusion, the present study reveals a new activity of an old family of antibiotics. The in vivo effect of Dox on immune reconstitution warrants further exploration. PMID:19330805

  14. A p53-inducible microRNA-34a downregulates Ras signaling by targeting IMPDH

    SciTech Connect

    Kim, Hwa-Ryeon; Roe, Jae-Seok; Lee, Ji-Eun; Hwang, In-Young; Cho, Eun-Jung; Youn, Hong-Duk

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer p53 downregulates IMPDH. Black-Right-Pointing-Pointer p53-dependent miR-34a transactivation inhibits IMPDH transcription. Black-Right-Pointing-Pointer miR-34a-mediated inhibition of IMPDH downregulates GTP-dependent Ras signal. -- Abstract: p53 is a well-known transcription factor that controls cell cycle arrest and cell death in response to a wide range of stresses. Moreover, p53 regulates glucose metabolism and its mutation results in the metabolic switch to the Warburg effect found in cancer cells. Nucleotide biosynthesis is also critical for cell proliferation and the cell division cycle. Nonetheless, little is known about whether p53 regulates nucleotide biosynthesis. Here we demonstrated that p53-inducible microRNA-34a (miR-34a) repressed inosine 5 Prime -monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme of de novo GTP biosynthesis. Treatment with anti-miR-34a inhibitor relieved the expression of IMPDH upon DNA damage. Ultimately, miR-34a-mediated inhibition of IMPDH resulted in repressed activation of the GTP-dependent Ras signaling pathway. In summary, we suggest that p53 has a novel function in regulating purine biosynthesis, aided by miR-34a-dependent IMPDH repression.

  15. Simvastatin induces a central hypotensive effect via Ras-mediated signalling to cause eNOS up-regulation

    PubMed Central

    Cheng, Wen-Han; Ho, Wen-Yu; Chang, Chien-Feng; Lu, Pei-Jung; Cheng, Pei-Wen; Yeh, Tung-Chen; Hong, Ling-Zong; Sun, Gwo-Ching; Hsiao, Michael; Tseng, Ching-Jiunn

    2013-01-01

    BACKGROUND AND PURPOSE Clinical studies indicate that statins have a BP-lowering effect in hypercholesterolemic individuals with hypertension. Specifically, statins modulate BP through the up-regulation of endothelial NOS (eNOS) activation in the brain. However, the signalling mechanisms through which statins enhance eNOS activation remain unclear. Therefore, we examined the possible signalling pathways involved in statin-mediated BP regulation in the nucleus tractus solitarii (NTS). EXPERIMENTAL APPROACH To investigate the involvement of Ras and other signalling pathways in simvastatin-induced effects on BP, BP and renal sympathetic nerve activity (RSNA) were determined in spontaneously hypertensive rats (SHRs) before and after i.c.v. administration of simvastatin in the absence and presence of a Ras-specific inhibitor (farnesyl thiosalicylic acid, FTS), a geranylgeranyltransferase inhibitor (GGTI-2133), a PI3K inhibitor (LY294002) or a MAPK-ERK kinase (MEK) inhibitor (PD98059). KEY RESULTS FTS significantly attenuated the decrease in BP and increased NO evoked by simvastatin and reversed the decrease in basal RSNA induced by simvastatin. Immunoblotting and pharmacological studies showed that inhibition of Ras activity by FTS significantly abolished simvastatin-induced phosphorylation of ERK1/2, ribosomal protein S6 kinase (RSK), Akt and decreased eNOS phosphorylation. Likewise, administration of Akt and ERK1/2 signalling inhibitors, LY294002 and PD98059, attenuated the reduction in BP evoked by simvastatin. Furthermore, i.c.v. simvastatin decreased Rac1 activation and the number of ROS-positive cells in the NTS. CONCLUSIONS AND IMPLICATIONS Simvastatin modulates central BP control in the NTS of SHRs by increasing Ras-mediated activation of the PI3K-Akt and ERK1/2-RSK signalling pathways, which then up-regulates eNOS activation. PMID:23889671

  16. RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3.

    PubMed

    Cailliau, Katia; Lescuyer, Arlette; Burnol, Anne-Françoise; Cuesta-Marbán, Álvaro; Widmann, Christian; Browaeys-Poly, Edith

    2015-08-01

    Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling. PMID:26109071

  17. The Fourth International Symposium on Genetic Disorders of the Ras/MAPK Pathway

    PubMed Central

    Stevenson, David A.; Schill, Lisa; Schoyer, Lisa; Andresen, Brage S.; Bakker, Annette; Bayrak-Toydemir, Pinar; Burkitt-Wright, Emma; Chatfield, Kathryn; Elefteriou, Florent; Elgersma, Ype; Fisher, Michael J.; Franz, David; Gelb, Bruce D.; Goriely, Anne; Gripp, Karen W.; Hardan, Antonio Y.; Keppler-Noreuil, Kim M.; Kerr, Bronwyn; Korf, Bruce; Leoni, Chiara; McCormick, Frank; Plotkin, Scott R.; Rauen, Katherine A.; Reilly, Karlyne; Roberts, Amy; Sandler, Abby; Siegel, Dawn; Walsh, Karin; Widemann, Brigitte C.

    2016-01-01

    The RASopathies are a group of disorders due to variations of genes associated with the Ras/MAPK pathway. Some of the RASopathies include neurofibromatosis type 1 (NF1), Noonan syndrome, Noonan syndrome with multiple lentigines, cardiofaciocutaneous (CFC) syndrome, Costello syndrome, Legius syndrome, and capillary malformation–arteriovenous malformation (CM-AVM) syndrome. In combination, the RASopathies are a frequent group of genetic disorders. This report summarizes the proceedings of the 4th International Symposium on Genetic Disorders of the Ras/MAPK pathway and highlights gaps in the field. PMID:27155140

  18. The Fourth International Symposium on Genetic Disorders of the Ras/MAPK pathway.

    PubMed

    Stevenson, David A; Schill, Lisa; Schoyer, Lisa; Andresen, Brage S; Bakker, Annette; Bayrak-Toydemir, Pinar; Burkitt-Wright, Emma; Chatfield, Kathryn; Elefteriou, Florent; Elgersma, Ype; Fisher, Michael J; Franz, David; Gelb, Bruce D; Goriely, Anne; Gripp, Karen W; Hardan, Antonio Y; Keppler-Noreuil, Kim M; Kerr, Bronwyn; Korf, Bruce; Leoni, Chiara; McCormick, Frank; Plotkin, Scott R; Rauen, Katherine A; Reilly, Karlyne; Roberts, Amy; Sandler, Abby; Siegel, Dawn; Walsh, Karin; Widemann, Brigitte C

    2016-08-01

    The RASopathies are a group of disorders due to variations of genes associated with the Ras/MAPK pathway. Some of the RASopathies include neurofibromatosis type 1 (NF1), Noonan syndrome, Noonan syndrome with multiple lentigines, cardiofaciocutaneous (CFC) syndrome, Costello syndrome, Legius syndrome, and capillary malformation-arteriovenous malformation (CM-AVM) syndrome. In combination, the RASopathies are a frequent group of genetic disorders. This report summarizes the proceedings of the 4th International Symposium on Genetic Disorders of the Ras/MAPK pathway and highlights gaps in the field. © 2016 Wiley Periodicals, Inc. PMID:27155140

  19. Hexa (ethylene glycol) derivative of benzothiazole aniline promotes dendritic spine formation through the RasGRF1-Ras dependent pathway.

    PubMed

    Lee, Nathanael J; Song, Jung Min; Cho, Hyun-Ji; Sung, You Me; Lee, Taehee; Chung, Andrew; Hong, Sung-Ha; Cifelli, Jessica L; Rubinshtein, Mark; Habib, Lila K; Capule, Christina C; Turner, R Scott; Pak, Daniel T S; Yang, Jerry; Hoe, Hyang-Sook

    2016-02-01

    Our recent study demonstrated that an amyloid-β binding molecule, BTA-EG4, increases dendritic spine number via Ras-mediated signaling. To potentially optimize the potency of the BTA compounds, we synthesized and evaluated an amyloid-β binding analog of BTA-EG4 with increased solubility in aqueous solution, BTA-EG6. We initially examined the effects of BTA-EG6 on dendritic spine formation and found that BTA-EG6-treated primary hippocampal neurons had significantly increased dendritic spine number compared to control treatment. In addition, BTA-EG6 significantly increased the surface level of AMPA receptors. Upon investigation into the molecular mechanism by which BTA-EG6 promotes dendritic spine formation, we found that BTA-EG6 may exert its effects on spinogenesis via RasGRF1-ERK signaling, with potential involvement of other spinogenesis-related proteins such as Cdc42 and CDK5. Taken together, our data suggest that BTA-EG6 boosts spine and synapse number, which may have a beneficial effect of enhancing neuronal and synaptic function in the normal healthy brain. PMID:26675527

  20. Interplay Between HGF/SF-Met-Ras Signaling, Tumor Metabolism and Blood Flow as a Potential Target for Breast Cancer Therapy.

    PubMed

    Natan, Sari; Tsarfaty, Galia; Horev, Judith; Haklai, Roni; Kloog, Yoel; Tsarfaty, Ilan

    2014-01-01

    High glucose uptake and increase blood flow is a characteristic of most metastatic tumors. Activation of Ras signaling increases glycolytic flux into lactate, de novo nucleic acid synthesis and uncoupling of ATP synthase from the proton gradient. Met tyrosine kinase receptor signaling upon activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF), increases glycolysis, oxidative phosporylation, oxygen consumption, and tumor blood volume. Ras is a key factor in Met signaling. Using the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS), we investigated interplay between HGF/SF-Met-Ras signaling, metabolism, and tumor blood-flow regulation. In vitro, HGF/SF-activated Met increased Ras activity, Erk phosphorylation, cell motility and glucose uptake, but did not affect ATP. FTS inhibited basal and HGF/SF-induced signaling and cell motility, while further increasing glucose uptake and inhibiting ATP production. In vivo, HGF/SF rapidly increased tumor blood volume. FTS did not affect basal blood-flow but abolished the HGF/SF effect. Our results further demonstrate the complex interplay between growth-factor-receptor signaling and cellular and tumor metabolism, as reflected in blood flow. Inhibition of Ras signaling does not affect glucose consumption or basal tumor blood flow but dramatically decreases ATP synthesis and the HGF/SF induced increase in tumor blood volume. These findings demonstrate that the HGF/SF-Met-Ras pathway critically influences tumor-cell metabolism and tumor blood-flow regulation. This pathway could potentially be used to individualize tumor therapy based on functional molecular imaging, and for combined signaling/anti-metabolic targeted therapy. PMID:25593982

  1. A lin-45 raf enhancer screen identifies eor-1, eor-2 and unusual alleles of Ras pathway genes in Caenorhabditis elegans.

    PubMed Central

    Rocheleau, Christian E; Howard, Robyn M; Goldman, Alissa P; Volk, Mandy L; Girard, Laura J; Sundaram, Meera V

    2002-01-01

    In Caenorhabditis elegans, the Ras/Raf/MEK/ERK signal transduction pathway controls multiple processes including excretory system development, P12 fate specification, and vulval cell fate specification. To identify positive regulators of Ras signaling, we conducted a genetic screen for mutations that enhance the excretory system and egg-laying defects of hypomorphic lin-45 raf mutants. This screen identified unusual alleles of several known Ras pathway genes, including a mutation removing the second SH3 domain of the sem-5/Grb2 adaptor, a temperature-sensitive mutation in the helical hairpin of let-341/Sos, a gain-of-function mutation affecting a potential phosphorylation site of the lin-1 Ets domain transcription factor, a dominant-negative allele of ksr-1, and hypomorphic alleles of sur-6/PP2A-B, sur-2/Mediator, and lin-25. In addition, this screen identified multiple alleles of two newly identified genes, eor-1 and eor-2, that play a relatively weak role in vulval fate specification but positively regulate Ras signaling during excretory system development and P12 fate specification. The spectrum of identified mutations argues strongly for the specificity of the enhancer screen and for a close involvement of eor-1 and eor-2 in Ras signaling. PMID:12019228

  2. Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution

    PubMed Central

    Coyle, Scott M; Lim, Wendell A

    2016-01-01

    The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras’s ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease. DOI: http://dx.doi.org/10.7554/eLife.12435.001 PMID:26765565

  3. Nitric oxide induces thioredoxin-1 nuclear translocation: Possible association with the p21Ras survival pathway

    SciTech Connect

    Arai, Roberto J.; Yodoi, J.; Debbas, V.; Laurindo, Francisco R.; Stern, A.; Monteiro, Hugo P. . E-mail: hpmonte@uol.com.br

    2006-10-06

    One of the major redox-regulating molecules with thiol reducing activity is thioredoxin-1 (TRX-1). TRX-1 is a multifunctional protein that exists in the extracellular millieu, cytoplasm, and nucleus, and has a distinct role in each environment. It is well known that TRX-1 promptly migrates to the nuclear compartment in cells exposed to oxidants. However, the intracellular location of TRX-1 in cells exposed to nitrosothiols has not been investigated. Here, we demonstrated that the exposure of HeLa cells to increasing concentrations of the nitrosothiol S-nitroso-N-acetylpenicillamine (SNAP) promoted TRX-1 nuclear accumulation. The SNAP-induced TRX-1 translocation to the nucleus was inhibited by FPTIII, a selective inhibitor of p21Ras. Furthermore, TRX-1 migration was attenuated in cells stably transfected with NO insensitive p21Ras (p21{sup RasC118S}). Downstream to p21Ras, the MAP Kinases ERK1/2 were activated by SNAP under conditions that promote TRX-1 nuclear translocation. Inhibition of MEK prevented SNAP-stimulated ERK1/2 activation and TRX-1 nuclear migration. In addition, cells treated with p21Ras or MEK inhibitor showed increased susceptibility to cell death induced by SNAP. In conclusion, our observations suggest that the nuclear translocation of TRX-1 is induced by SNAP involving p21Ras survival pathway.

  4. RAS oncogenes: weaving a tumorigenic web

    PubMed Central

    Pylayeva-Gupta, Yuliya; Grabocka, Elda; Bar-Sagi, Dafna

    2013-01-01

    RAS proteins are essential components of signalling pathways that emanate from cell surface receptors. Oncogenic activation of these proteins owing to missense mutations is frequently detected in several types of cancer. A wealth of biochemical and genetic studies indicates that RAS proteins control a complex molecular circuitry that consists of a wide array of interconnecting pathways. In this Review, we describe how RAS oncogenes exploit their extensive signalling reach to affect multiple cellular processes that drive tumorigenesis. PMID:21993244

  5. Effects of mutant human Ki-ras{sup G12C} gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

    SciTech Connect

    Dance-Barnes, Stephanie T.; Kock, Nancy D.; Floyd, Heather S.; Moore, Joseph E.; Mosley, Libyadda J.; D'Agostino, Ralph B.; Pettenati, Mark J.; Miller, Mark Steven

    2008-08-15

    Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-ras{sup G12C} allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 {mu}g/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-ras{sup G12C} allele in the lung, and resulted in the development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 {mu}g/ml of DOX resulted in dose-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 {mu}g/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 {mu}g/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-ras{sup G12C} expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models.

  6. Ras in Cancer and Developmental Diseases

    PubMed Central

    Fernández-Medarde, Alberto; Santos, Eugenio

    2011-01-01

    Somatic, gain-of-function mutations in ras genes were the first specific genetic alterations identified in human cancer about 3 decades ago. Studies during the last quarter century have characterized the Ras proteins as essential components of signaling networks controlling cellular proliferation, differentiation, or survival. The oncogenic mutations of the H-ras, N-ras, or K-ras genes frequently found in human tumors are known to throw off balance the normal outcome of those signaling pathways, thus leading to tumor development. Oncogenic mutations in a number of other upstream or downstream components of Ras signaling pathways (including membrane RTKs or cytosolic kinases) have been detected more recently in association with a variety of cancers. Interestingly, the oncogenic Ras mutations and the mutations in other components of Ras/MAPK signaling pathways appear to be mutually exclusive events in most tumors, indicating that deregulation of Ras-dependent signaling is the essential requirement for tumorigenesis. In contrast to sporadic tumors, separate studies have identified germline mutations in Ras and various other components of Ras signaling pathways that occur in specific association with a number of different familial, developmental syndromes frequently sharing common phenotypic cardiofaciocutaneous features. Finally, even without being a causative force, defective Ras signaling has been cited as a contributing factor to many other human illnesses, including diabetes and immunological and inflammatory disorders. We aim this review at summarizing and updating current knowledge on the contribution of Ras mutations and altered Ras signaling to development of various tumoral and nontumoral pathologies. PMID:21779504

  7. Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells.

    PubMed Central

    Migliaccio, A; Di Domenico, M; Castoria, G; de Falco, A; Bontempo, P; Nola, E; Auricchio, F

    1996-01-01

    The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol-dependent growth, estradiol treatment of human mammary cancer MCF-7 cells triggers rapid and transient activation of the mitogen-activated (MAP) kinases, erk-1 and erk-2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras-GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP-kinase pathway in MCF-7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild-type 67 kDa estradiol receptor cDNA that Cos cells become estradiol-responsive in terms of erk-2 activity. This finding, together with the inhibition by the pure anti-estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF-7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c-src show that the estradiol receptor activates c-src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c-src is an initial and integral part of the signaling events mediated by the estradiol receptor. Images PMID:8635462

  8. Role of Ras signaling in the induction of snail by transforming growth factor-beta.

    PubMed

    Horiguchi, Kana; Shirakihara, Takuya; Nakano, Ayako; Imamura, Takeshi; Miyazono, Kohei; Saitoh, Masao

    2009-01-01

    The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during the progression of epithelial tumors. EMT can be induced by transforming growth factor (TGF)-beta in some tumor cells. Here, we demonstrate the molecular mechanism whereby Snail, a key regulator of EMT, is induced by TGF-beta in tumor cells. Snail induction by TGF-beta was highly dependent on cooperation with active Ras signals, and silencing of Ras abolished Snail induction by TGF-beta in pancreatic cancer Panc-1 cells. Transfection of constitutively active Ras into HeLa cells led to induction of Snail by TGF-beta, while representative direct targets of TGF-beta, including Smad7 and PAI-1, were not affected by Ras signaling. Using mitogen-activated protein kinase inhibitors or Smad3 or Smad2 mutants, we found that phosphorylation at the linker region of Smad2/3 was not required for the induction of Snail by TGF-beta. Taken together, these findings indicate that Ras and TGF-beta-Smad signaling selectively cooperate in the induction of Snail, which occurs in a Smad-dependent manner, but independently of phosphorylation at the linker region of R-Smads by Ras signaling. PMID:19010789

  9. Potential roles of the RGMa-FAK-Ras pathway in hippocampal mossy fiber sprouting in the pentylenetetrazole kindling model

    PubMed Central

    SONG, MING-YU; TIAN, FA-FA; WANG, YU-ZHONG; HUANG, XIA; GUO, JIA-LING; DING, DONG-XUE

    2015-01-01

    Mossy fiber sprouting (MFS) is a unique feature of chronic epilepsy. However, the molecular signals underlying MFS are still unclear. The repulsive guidance molecule A (RGMa) appears to contribute to axon growth and axonal guidance, and may exert its biological effects by dephosphorylating focal adhesion kinase (FAK) at Tyr397, then regulating the activation of Ras. The objective of this study was to explore the expression patterns of RGMa, FAK (Tyr397) and Ras in epileptogenesis, and their correlation with MFS. The epileptic models were established by intraperitoneal pentylenetetrazole (PTZ) injection of Sprague-Dawley rats. At 3 days and at 1, 2, 4 and 6 weeks after the first PTZ injection, Timm staining was scored at different time points in the CA3 region of the hippocampus and dentate gyrus. The protein levels of RGMa, FAK (Tyr397) and Ras were analyzed at different time points in the CA3 region of the hippocampus using immunofluorescence, immunohistochemistry and western blot analysis. Compared with the control (saline-injected) group, the expression of RGMa in the CA3 area was significantly downregulated (P<0.05) from 3 days and still maintained the low expression at 6 weeks in the PTZ group. The expression of FAK (Tyr397) and Ras was upregulated (P<0.05) in the PTZ groups. The Timm score in the CA3 region was significantly higher than that in the control group at different time points and reached a peak at 4 weeks. In the CA3 region, no obvious distinction was observed at the different time points in the control group. To the best of our knowledge, these are the first results to indicate that the RGMa-FAK-Ras pathway may be involved in MFS and the development of temporal lobe epilepsy. PMID:25420768

  10. Low proliferation and high apoptosis of osteoblastic cells on hydrophobic surface are associated with defective Ras signaling

    SciTech Connect

    Chang, Eun-Ju; Kim, Hong-Hee; Huh, Jung-Eun; Kim, In-Ae; Seung Ko, Jea; Chung, Chong-Pyoung; Kim, Hyun-Man . E-mail: hyunmkim@plaza.snu.ac.kr

    2005-02-01

    The hydrophobic (HPB) nature of most polymeric biomaterials has been a major obstacle in using those materials in vivo due to low compatibility with cells. However, there is little knowledge of the molecular detail to explain how surface hydrophobicity affects cell responses. In this study, we compared the proliferation and apoptosis of human osteoblastic MG63 cells adhered to hydrophilic (HPL) and hydrophobic surfaces. On the hydrophobic surface, less formation of focal contacts and actin stress fibers, a delay in cell cycle progression, and an increase in apoptosis were observed. By using fibroblast growth factor 1 (FGF1) as a model growth factor, we also investigated intracellular signaling pathways on hydrophilic and hydrophobic surfaces. The activation of Ras, Akt, and ERK by FGF1 was impaired in MG63 cells on the hydrophobic surface. The overexpression of constitutively active form of Ras and Akt rescued those cells from apoptosis and recovered cell cycle progression. Furthermore, their overexpression also restored the actin cytoskeletal organization on the hydrophobic surface. Finally, the proliferative, antiapoptotic, and cytoskeletal effects of constitutively active Ras in MG63 cells on the hydrophobic surface were blocked by wortmannin and PD98059 that inhibit Akt and ERK activation, respectively. Therefore, our results suggest that the activation of Ras and its downstream molecules Akt and ERK to an appropriate level is one of crucial elements in the determination of osteoblast cell responses. The Ras pathway may represent a cell biological target that should be considered for successful surface modification of biomaterials to induce adequate cell responses in the bone tissue.

  11. The Fibroblast Growth Factor signaling pathway

    PubMed Central

    Ornitz, David M; Itoh, Nobuyuki

    2015-01-01

    The signaling component of the mammalian Fibroblast Growth Factor (FGF) family is comprised of eighteen secreted proteins that interact with four signaling tyrosine kinase FGF receptors (FGFRs). Interaction of FGF ligands with their signaling receptors is regulated by protein or proteoglycan cofactors and by extracellular binding proteins. Activated FGFRs phosphorylate specific tyrosine residues that mediate interaction with cytosolic adaptor proteins and the RAS-MAPK, PI3K-AKT, PLCγ, and STAT intracellular signaling pathways. Four structurally related intracellular non-signaling FGFs interact with and regulate the family of voltage gated sodium channels. Members of the FGF family function in the earliest stages of embryonic development and during organogenesis to maintain progenitor cells and mediate their growth, differentiation, survival, and patterning. FGFs also have roles in adult tissues where they mediate metabolic functions, tissue repair, and regeneration, often by reactivating developmental signaling pathways. Consistent with the presence of FGFs in almost all tissues and organs, aberrant activity of the pathway is associated with developmental defects that disrupt organogenesis, impair the response to injury, and result in metabolic disorders, and cancer. © 2015 Wiley Periodicals, Inc. PMID:25772309

  12. Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Inhibitors: Rationale and Importance to Inhibiting These Pathways in Human Health

    PubMed Central

    Chappell, William H.; Steelman, Linda S.; Long, Jacquelyn M.; Kempf, Ruth C.; Abrams, Stephen L.; Franklin, Richard A.; Bäsecke, Jörg; Stivala, Franca; Donia, Marco; Fagone, Paolo; Malaponte, Graziella; Mazzarino, Maria C.; Nicoletti, Ferdinando; Libra, Massimo; Maksimovic-Ivanic, Danijela; Mijatovic, Sanja; Montalto, Giuseppe; Cervello, Melchiorre; Laidler, Piotr; Milella, Michele; Tafuri, Agostino; Bonati, Antonio; Evangelisti, Camilla; Cocco, Lucio; Martelli, Alberto M.; McCubrey, James A.

    2011-01-01

    The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Integral components of these pathways, Ras, B-Raf, PI3K, and PTEN are also activated/inactivated by mutations. These pathways have profound effects on proliferative, apoptotic and differentiation pathways. Dysregulation of these pathways can contribute to chemotherapeutic drug resistance, proliferation of cancer initiating cells (CICs) and premature aging. This review will evaluate more recently described potential uses of MEK, PI3K, Akt and mTOR inhibitors in the proliferation of malignant cells, suppression of CICs, cellular senescence and prevention of aging. Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways play key roles in the regulation of normal and malignant cell growth. Inhibitors targeting these pathways have many potential uses from suppression of cancer, proliferative diseases as well as aging. PMID:21411864

  13. Therapeutic Strategies for Targeting Ras Proteins

    PubMed Central

    Gysin, Stephan; Salt, Megan; Young, Amy; McCormick, Frank

    2011-01-01

    Ras genes are frequently activated in cancer. Attempts to develop drugs that target mutant Ras proteins have, so far, been unsuccessful. Tumors bearing these mutations, therefore, remain among the most difficult to treat. Most efforts to block activated Ras have focused on pathways downstream. Drugs that inhibit Raf kinase have shown clinical benefit in the treatment of malignant melanoma. However, these drugs have failed to show clinical benefit in Ras mutant tumors. It remains unclear to what extent Ras depends on Raf kinase for transforming activity, even though Raf proteins bind directly to Ras and are certainly major effectors of Ras action in normal cells and in development. Furthermore, Raf kinase inhibitors can lead to paradoxical activation of the MAPK pathway. MEK inhibitors block the Ras-MAPK pathway, but often activate the PI3’-kinase, and have shown little clinical benefit as single agents. This activation is mediated by EGF-R and other receptor tyrosine kinases through relief of a negative feedback loop from ERK. Drug combinations that target multiple points within the Ras signaling network are likely to be necessary to achieve substantial clinical benefit. Other effectors may also contribute to Ras signaling and provide a source of targets. In addition, unbiased screens for genes necessary for Ras transformation have revealed new potential targets and have added to our understanding of Ras cancer biology. PMID:21779505

  14. A non-cell-autonomous role for Ras signaling in C. elegans neuroblast delamination

    PubMed Central

    Parry, Jean M.; Sundaram, Meera V.

    2014-01-01

    Receptor tyrosine kinase (RTK) signaling through Ras influences many aspects of normal cell behavior, including epithelial-to-mesenchymal transition, and aberrant signaling promotes both tumorigenesis and metastasis. Although many such effects are cell-autonomous, here we show a non-cell-autonomous role for RTK-Ras signaling in the delamination of a neuroblast from an epithelial organ. The C. elegans renal-like excretory organ is initially composed of three unicellular epithelial tubes, namely the canal, duct and G1 pore cells; however, the G1 cell later delaminates from the excretory system to become a neuroblast and is replaced by the G2 cell. G1 delamination and G2 intercalation involve cytoskeletal remodeling, interconversion of autocellular and intercellular junctions and migration over a luminal extracellular matrix, followed by G1 junction loss. LET-23/EGFR and SOS-1, an exchange factor for Ras, are required for G1 junction loss but not for initial cytoskeletal or junction remodeling. Surprisingly, expression of activated LET-60/Ras in the neighboring duct cell, but not in the G1 or G2 cells, is sufficient to rescue sos-1 delamination defects, revealing that Ras acts non-cell-autonomously to permit G1 delamination. We suggest that, similarly, oncogenic mutations in cells within a tumor might help create a microenvironment that is permissive for other cells to detach and ultimately metastasize. PMID:25371363

  15. Expression profiling of medulloblastoma: PDGFRA and the RAS/MAPK pathway as therapeutic targets for metastatic disease.

    PubMed

    MacDonald, T J; Brown, K M; LaFleur, B; Peterson, K; Lawlor, C; Chen, Y; Packer, R J; Cogen, P; Stephan, D A

    2001-10-01

    Little is known about the genetic regulation of medulloblastoma dissemination, but metastatic medulloblastoma is highly associated with poor outcome. We obtained expression profiles of 23 primary medulloblastomas clinically designated as either metastatic (M+) or non-metastatic (M0) and identified 85 genes whose expression differed significantly between classes. Using a class prediction algorithm based on these genes and a leave-one-out approach, we assigned sample class to these tumors (M+ or M0) with 72% accuracy and to four additional independent tumors with 100% accuracy. We also assigned the metastatic medulloblastoma cell line Daoy to the metastatic class. Notably, platelet-derived growth factor receptor alpha (PDGFRA) and members of the downstream RAS/mitogen-activated protein kinase (MAPK) signal transduction pathway are upregulated in M+ tumors. Immunohistochemical validation on an independent set of tumors shows significant overexpression of PDGFRA in M+ tumors compared to M0 tumors. Using in vitro assays, we show that platelet-derived growth factor alpha (PDGFA) enhances medulloblastoma migration and increases downstream MAP2K1 (MEK1), MAP2K2 (MEK2), MAPK1 (p42 MAPK) and MAPK3 (p44 MAPK) phosphorylation in a dose-dependent manner. Neutralizing antibodies to PDGFRA blocks MAP2K1, MAP2K2 and MAPK1/3 phosphorylation, whereas U0126, a highly specific inhibitor of MAP2K1 and MAP2K2, also blocks MAPK1/3. Both inhibit migration and prevent PDGFA-stimulated migration. These results provide the first insight into the genetic regulation of medulloblastoma metastasis and are the first to suggest a role for PDGFRA and the RAS/MAPK signaling pathway in medulloblastoma metastasis. Inhibitors of PDGFRA and RAS proteins should therefore be considered for investigation as possible novel therapeutic strategies against medulloblastoma. PMID:11544480

  16. Genetic biomarkers of drug response for small-molecule therapeutics targeting the RTK/Ras/PI3K, p53 or Rb pathway in glioblastoma.

    PubMed

    Venkatesan, Subramanian; Lamfers, Martine Lm; Dirven, Clemens Mf; Leenstra, Sieger

    2016-04-01

    Glioblastoma is the most deadly and frequently occurring primary malignant tumor of the central nervous system. Genomic studies have shown that mutated oncogenes and tumor suppressor genes in glioblastoma mainly occur in three pathways: the RTK/Ras/PI3K signaling, the p53 and the Rb pathways. In this review, we summarize the modulatory effects of genetic aberrations in these three pathways to drugs targeting these specific pathways. We also provide an overview of the preclinical efforts made to identify genetic biomarkers of response and resistance. Knowledge of biomarkers will finally promote patient stratification in clinical trials, a prerequisite for trial design in the era of precision medicine. PMID:26986934

  17. Relapsed neuroblastomas show frequent RAS-MAPK pathway mutations | Office of Cancer Genomics

    Cancer.gov

    The majority of patients with neuroblastoma have tumors that initially respond to chemotherapy, but a large proportion will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole-genome sequencing of 23 paired diagnostic and relapse neuroblastomas showed clonal evolution from the diagnostic tumor, with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK pathway.

  18. Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling.

    PubMed Central

    Denhardt, D T

    1996-01-01

    The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras-->Raf-->MEK-->ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein alpha and beta gamma subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families

  19. RAS diseases in children

    PubMed Central

    Niemeyer, Charlotte M.

    2014-01-01

    RAS genes encode a family of 21 kDa proteins that are an essential hub for a number of survival, proliferation, differentiation and senescence pathways. Signaling of the RAS-GTPases through the RAF-MEK-ERK pathway, the first identified mitogen-associated protein kinase (MAPK) cascade is essential in development. A group of genetic syndromes, named “RASopathies”, had been identified which are caused by heterozygosity for germline mutations in genes that encode protein components of the RAS/MAPK pathway. Several of these clinically overlapping disorders, including Noonan syndrome, Noonan-like CBL syndrome, Costello syndrome, cardio-facio-cutaneous (CFC) syndrome, neurofibromatosis type I, and Legius syndrome, predispose to cancer and abnormal myelopoiesis in infancy. This review focuses on juvenile myelomonocytic leukemia (JMML), a malignancy of early childhood characterized by initiating germline and/or somatic mutations in five genes of the RAS/MAPK pathway: PTPN11, CBL, NF-1, KRAS and NRAS. Natural courses of these five subtypes differ, although hematopoietic stem cell transplantation remains the only curative therapy option for most children with JMML. With whole-exome sequencing studies revealing few secondary lesions it will be crucial to better understand the RAS/MAPK signaling network with its crosstalks and feed-back loops to carefully design early clinical trials with novel pharmacological agents in this still puzzling leukemia. PMID:25420281

  20. Alphavirus production is inhibited in neurofibromin 1-deficient cells through activated RAS signalling

    SciTech Connect

    Kolokoltsova, Olga A. Domina, Aaron M. Kolokoltsov, Andrey A. Davey, Robert A. | Weaver, Scott C. || Watowich, Stanley J. ||

    2008-07-20

    Virus-host interactions essential for alphavirus pathogenesis are poorly understood. To address this shortcoming, we coupled retrovirus insertional mutagenesis and a cell survival selection strategy to generate clonal cell lines broadly resistant to Sindbis virus (SINV) and other alphaviruses. Resistant cells had significantly impaired SINV production relative to wild-type (WT) cells, although virus binding and fusion events were similar in both sets of cells. Analysis of the retroviral integration sites identified the neurofibromin 1 (NF1) gene as disrupted in alphavirus-resistant cell lines. Subsequent analysis indicated that expression of NF1 was significantly reduced in alphavirus-resistant cells. Importantly, independent down-regulation of NF1 expression in WT HEK 293 cells decreased virus production and increased cell viability during SINV infection, relative to infected WT cells. Additionally, we observed hyperactive RAS signalling in the resistant HEK 293 cells, which was anticipated because NF1 is a negative regulator of RAS. Expression of constitutively active RAS (HRAS-G12V) in a WT HEK 293 cell line resulted in a marked delay in virus production, compared with infected cells transfected with parental plasmid or dominant-negative RAS (HRAS-S17N). This work highlights novel host cell determinants required for alphavirus pathogenesis and suggests that RAS signalling may play an important role in neuronal susceptibility to SINV infection.

  1. A Flt3 and Ras-dependent Pathway Primes B Cell Development by Inducing A State of IL7-responsiveness

    PubMed Central

    Li, Lin-Xi; Goetz, Christine A.; Katerndahl, Casey D.S.; Sakaguchi, Nobuo; Farrar, Michael A.

    2009-01-01

    Ras plays an important role in B cell development. However, the stage at which Ras governs B cell development remains unclear. Moreover, the upstream receptors and downstream effectors of Ras that govern B cell differentiation remain undefined. Using mice that express a dominant negative form of Ras, we demonstrate that Ras-mediated signaling plays a critical role in the development of common lymphoid progenitors (CLP). This developmental block parallels that found in flt3−/− mice, suggesting that Flt3 is an important upstream activator of Ras in early B cell progenitors. Ras inhibition impaired proliferation of CLP and pre-pro-B cells but not pro-B cells. Rather, Ras promotes STAT5-dependent pro-B cell differentiation by enhancing IL7Rα levels and suppressing socs2 and socs3 expression. Our results suggest a model in which Flt3/Ras-dependent signals play a critical role in B cell development by priming early B cell progenitors for subsequent STAT5-dependent B cell differentiation. PMID:20065110

  2. Nore1a drives Ras to flick the P53 senescence switch

    PubMed Central

    Donninger, Howard; Clark, Geoffrey J.

    2016-01-01

    ABSTRACT RAS-induced senescence is a protective mechanism to avoid unrestricted cell growth due to aberrant mitogenic signals; however, the exact mechanism by which RAS induces senescence is not known. We recently identified a novel pathway linking RAS to p53 via NORE1A and HIPK2 that mechanistically explains how Ras induces senescence. PMID:27314075

  3. RasGRP1 and RasGRP3 Are Required for Efficient Generation of Early Thymic Progenitors.

    PubMed

    Golec, Dominic P; Henao Caviedes, Laura M; Baldwin, Troy A

    2016-09-01

    T cell development is dependent on the migration of progenitor cells from the bone marrow to the thymus. Upon reaching the thymus, progenitors undergo a complex developmental program that requires inputs from various highly conserved signaling pathways including the Notch and Wnt pathways. To date, Ras signaling has not been implicated in the very earliest stages of T cell differentiation, but members of a family of Ras activators called RasGRPs have been shown to be involved at multiple stages of T cell development. We examined early T cell development in mice lacking RasGRP1, RasGRP3, and RasGRPs 1 and 3. We report that RasGRP1- and RasGRP3-deficient thymi show significantly reduced numbers of early thymic progenitors (ETPs) relative to wild type thymi. Furthermore, RasGRP1/3 double-deficient thymi show significant reductions in ETP numbers compared with either RasGRP1 or RasGRP3 single-deficient thymi, suggesting that both RasGRP1 and RasGRP3 regulate the generation of ETPs. In addition, competitive bone marrow chimera experiments reveal that RasGRP1/3 double-deficient progenitors intrinsically generate ETPs less efficiently than wild type progenitors. Finally, RasGRP1/3-deficient progenitors show impaired migration toward the CCR9 ligand, CCL25, suggesting that RasGRP1 and RasGRP3 may regulate progenitor entry into the thymus through a CCR9-dependent mechanism. These data demonstrate that, in addition to Notch and Wnt, the highly conserved Ras pathway is critical for the earliest stages of T cell development and further highlight the importance of Ras signaling during thymocyte maturation. PMID:27465532

  4. Probing a 3,4'-bis-guanidinium diaryl derivative as an allosteric inhibitor of the Ras pathway.

    PubMed

    Diez-Cecilia, Elena; Carson, Robert; Kelly, Brendan; van Schaeybroeck, Sandra; Murray, James T; Rozas, Isabel

    2015-10-01

    Mutations in the Ras-pathway occur in 40-45% of colorectal cancer patients and these are refractory to treatment with anti-EGFR-targeted therapies. With this in mind, we have studied novel guanidinium-based compounds with demonstrated ability to inhibit protein kinases. We have performed docking studies with several proteins involved in the Ras-pathway and evaluated 3,4'-bis-guanidinium derivatives as inhibitors of B-Raf. Compound 3, the most potent in this series, demonstrated strong cytotoxicity in (WT)B-Raf colorectal cancer cells and also cells with (V600E)B-Raf mutations. Cell death was induced by apoptosis, detected by cleavage of PARP. Compound 3 also potently inhibited ERK1/2 signalling, inhibited EGFR activation, as well as Src, STAT3 and AKT phosphorylation. Mechanistically, compound 3 did not inhibit ATP binding to B-Raf, but direct assay of B-Raf activity was inhibited in vitro. Summarizing, we have identified a novel B-Raf type-III inhibitor that exhibits potent cellular cytotoxicity. PMID:26318998

  5. RAS and Hedgehog--partners in crime.

    PubMed

    Lauth, Matthias

    2011-01-01

    Both RAS and Hedgehog (HH) pathway activation can be found in approximately one third of all cancers. In many cases, this activation occurs in the same tumor types, suggesting a positive impact of a simultaneous activation of RAS and HH on tumor development. This review aims to summarize the current knowledge about the molecular and functional crosstalk of RAS and HH signaling in the development of hyperproliferative disease. PMID:21622175

  6. Regulation of Ras Exchange Factors and Cellular Localization of Ras Activation by Lipid Messengers in T Cells

    PubMed Central

    Jun, Jesse E.; Rubio, Ignacio; Roose, Jeroen P.

    2013-01-01

    The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs) catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and Son of Sevenless (SOS)-family GEFs. Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood. One large group of biomolecules critically involved in the control of RasGEFs functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR) stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells. PMID:24027568

  7. Retroactive Signaling in Short Signaling Pathways

    PubMed Central

    Sepulchre, Jacques-Alexandre; Merajver, Sofía D.; Ventura, Alejandra C.

    2012-01-01

    In biochemical signaling pathways without explicit feedback connections, the core signal transduction is usually described as a one-way communication, going from upstream to downstream in a feedforward chain or network of covalent modification cycles. In this paper we explore the possibility of a new type of signaling called retroactive signaling, offered by the recently demonstrated property of retroactivity in signaling cascades. The possibility of retroactive signaling is analysed in the simplest case of the stationary states of a bicyclic cascade of signaling cycles. In this case, we work out the conditions for which variables of the upstream cycle are affected by a change of the total amount of protein in the downstream cycle, or by a variation of the phosphatase deactivating the same protein. Particularly, we predict the characteristic ranges of the downstream protein, or of the downstream phosphatase, for which a retroactive effect can be observed on the upstream cycle variables. Next, we extend the possibility of retroactive signaling in short but nonlinear signaling pathways involving a few covalent modification cycles. PMID:22848403

  8. Signaling on the endocytic pathway.

    PubMed

    McPherson, P S; Kay, B K; Hussain, N K

    2001-06-01

    Ligand binding to receptor tyrosine kinases and G-protein-coupled receptors initiates signal transduction events and induces receptor endocytosis via clathrin-coated pits and vesicles. While receptor-mediated endocytosis has been traditionally considered an effective mechanism to attenuate ligand-activated responses, more recent studies demonstrate that signaling continues on the endocytic pathway. In fact, certain signaling events, such as the activation of the extracellular signal-regulated kinases, appear to require endocytosis. Protein components of signal transduction cascades can assemble at clathrin coated pits and remain associated with endocytic vesicles following their dynamin-dependent release from the plasma membrane. Thus, endocytic vesicles can function as a signaling compartment distinct from the plasma membrane. These observations demonstrate that endocytosis plays an important role in the activation and propagation of signaling pathways. PMID:11389765

  9. Ras and TGF-β signaling enhance cancer progression by promoting the ΔNp63 transcriptional program.

    PubMed

    Vasilaki, Eleftheria; Morikawa, Masato; Koinuma, Daizo; Mizutani, Anna; Hirano, Yudai; Ehata, Shogo; Sundqvist, Anders; Kawasaki, Natsumi; Cedervall, Jessica; Olsson, Anna-Karin; Aburatani, Hiroyuki; Moustakas, Aristidis; Miyazono, Kohei; Heldin, Carl-Henrik

    2016-01-01

    The p53 family of transcription factors includes p63, which is a master regulator of gene expression in epithelial cells. Determining whether p63 is tumor-suppressive or tumorigenic is complicated by isoform-specific and cellular context-dependent protein associations, as well as antagonism from mutant p53. ΔNp63 is an amino-terminal-truncated isoform, that is, the predominant isoform expressed in cancer cells of epithelial origin. In HaCaT keratinocytes, which have mutant p53 and ΔNp63, we found that mutant p53 antagonized ΔNp63 transcriptional activity but that activation of Ras or transforming growth factor-β (TGF-β) signaling pathways reduced the abundance of mutant p53 and strengthened target gene binding and activity of ΔNp63. Among the products of ΔNp63-induced genes was dual-specificity phosphatase 6 (DUSP6), which promoted the degradation of mutant p53, likely by dephosphorylating p53. Knocking down all forms of p63 or DUSP6 and DUSP7 (DUSP6/7) inhibited the basal or TGF-β-induced or epidermal growth factor (which activates Ras)-induced migration and invasion in cultures of p53-mutant breast cancer and squamous skin cancer cells. Alternatively, overexpressing ΔNp63 in the breast cancer cells increased their capacity to colonize various tissues upon intracardiac injection in mice, and this was inhibited by knocking down DUSP6/7 in these ΔNp63-overexpressing cells. High abundance of ΔNp63 in various tumors correlated with poor prognosis in patients, and this correlation was stronger in patients whose tumors also had a mutation in the gene encoding p53. Thus, oncogenic Ras and TGF-β signaling stimulate cancer progression through activation of the ΔNp63 transcriptional program. PMID:27555661

  10. Signaling Pathways in Melanogenesis.

    PubMed

    D'Mello, Stacey A N; Finlay, Graeme J; Baguley, Bruce C; Askarian-Amiri, Marjan E

    2016-01-01

    Melanocytes are melanin-producing cells found in skin, hair follicles, eyes, inner ear, bones, heart and brain of humans. They arise from pluripotent neural crest cells and differentiate in response to a complex network of interacting regulatory pathways. Melanins are pigment molecules that are endogenously synthesized by melanocytes. The light absorption of melanin in skin and hair leads to photoreceptor shielding, thermoregulation, photoprotection, camouflage and display coloring. Melanins are also powerful cation chelators and may act as free radical sinks. Melanin formation is a product of complex biochemical events that starts from amino acid tyrosine and its metabolite, dopa. The types and amounts of melanin produced by melanocytes are determined genetically and are influenced by a variety of extrinsic and intrinsic factors such as hormonal changes, inflammation, age and exposure to UV light. These stimuli affect the different pathways in melanogenesis. In this review we will discuss the regulatory mechanisms involved in melanogenesis and explain how intrinsic and extrinsic factors regulate melanin production. We will also explain the regulatory roles of different proteins involved in melanogenesis. PMID:27428965

  11. Signaling Pathways in Melanogenesis

    PubMed Central

    D’Mello, Stacey A. N.; Finlay, Graeme J.; Baguley, Bruce C.; Askarian-Amiri, Marjan E.

    2016-01-01

    Melanocytes are melanin-producing cells found in skin, hair follicles, eyes, inner ear, bones, heart and brain of humans. They arise from pluripotent neural crest cells and differentiate in response to a complex network of interacting regulatory pathways. Melanins are pigment molecules that are endogenously synthesized by melanocytes. The light absorption of melanin in skin and hair leads to photoreceptor shielding, thermoregulation, photoprotection, camouflage and display coloring. Melanins are also powerful cation chelators and may act as free radical sinks. Melanin formation is a product of complex biochemical events that starts from amino acid tyrosine and its metabolite, dopa. The types and amounts of melanin produced by melanocytes are determined genetically and are influenced by a variety of extrinsic and intrinsic factors such as hormonal changes, inflammation, age and exposure to UV light. These stimuli affect the different pathways in melanogenesis. In this review we will discuss the regulatory mechanisms involved in melanogenesis and explain how intrinsic and extrinsic factors regulate melanin production. We will also explain the regulatory roles of different proteins involved in melanogenesis. PMID:27428965

  12. DISC1 regulates astrogenesis in the embryonic brain via modulation of RAS/MEK/ERK signaling through RASSF7.

    PubMed

    Wang, Shukun; Liang, Qingli; Qiao, Huimin; Li, Hong; Shen, Tianjin; Ji, Fen; Jiao, Jianwei

    2016-08-01

    Disrupted in schizophrenia 1 (DISC1) is known as a high susceptibility gene for schizophrenia. Recent studies have indicated that schizophrenia might be caused by glia defects and dysfunction. However, there is no direct evidence of a link between the schizophrenia gene DISC1 and gliogenesis defects. Thus, an investigation into the involvement of DISC1 (a ubiquitously expressed brain protein) in astrogenesis during the late stage of mouse embryonic brain development is warranted. Here, we show that suppression of DISC1 expression represses astrogenesis in vitro and in vivo, and that DISC1 overexpression substantially enhances the process. Furthermore, mouse and human DISC1 overexpression rescued the astrogenesis defects caused by DISC1 knockdown. Mechanistically, DISC1 activates the RAS/MEK/ERK signaling pathway via direct association with RASSF7. Also, the pERK complex undergoes nuclear translocation and influences the expression of genes related to astrogenesis. In summary, our results demonstrate that DISC1 regulates astrogenesis by modulating RAS/MEK/ERK signaling via RASSF7 and provide a framework for understanding how DISC1 dysfunction might lead to neuropsychiatric diseases. PMID:27287808

  13. Past, Present, and Future of Targeting Ras for Cancer Therapies.

    PubMed

    Tan, Zhi; Zhang, Shuxing

    2016-01-01

    For decades, mutant Ras (mut-Ras) proteins have been identified as drivers of multiple cancers including pancreatic, lung, and colon cancers. However, targeting this oncogene has been challenging and no Ras inhibitors are on the market to date. Lately several candidates targeting the downstream pathways of Ras signaling, including PI3K and Raf, were approved for cancer treatment. However, they do not present promising therapeutic effects on patients harboring Ras mutations. Recently, a variety of compounds have been reported to impair the activity of Ras, and these exciting discoveries reignite the hope for development of novel drugs targeting mut-Ras. In this article, we will review the progress made in this field and the current state-of-the-art technologies to develop Ras inhibitors. Also we will discuss the future direction of targeting Ras. PMID:26423695

  14. Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

    PubMed Central

    Kauko, Otto; Laajala, Teemu Daniel; Jumppanen, Mikael; Hintsanen, Petteri; Suni, Veronika; Haapaniemi, Pekka; Corthals, Garry; Aittokallio, Tero; Westermarck, Jukka; Imanishi, Susumu Y.

    2015-01-01

    Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification. To allow data normalization in situations where depletion of RAS or PP2A inhibitor CIP2A causes a large uni-directional change in the phosphopeptide abundance, we developed a novel normalization strategy, named pairwise normalization. This normalization is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of the pairwise normalization was verified by various independent methods. Additionally, we demonstrate how the selected normalization method influences the downstream analyses and interpretation of pathway activities. Consequently, bioinformatics analysis of RAS and CIP2A regulated phosphoproteomes revealed a significant overlap in their functional pathways. This is most likely biologically meaningful as we observed a synergistic survival effect between CIP2A and RAS expression as well as KRAS activating mutations in TCGA pan-cancer data set, and synergistic relationship between CIP2A and KRAS depletion in colony growth assays. PMID:26278961

  15. Signaling Pathways in Osteoclast Differentiation.

    PubMed

    Kim, Jung Ha; Kim, Nacksung

    2016-01-01

    Osteoclasts are multinucleated cells of hematopoietic origin that are responsible for the degradation of old bone matrix. Osteoclast differentiation and activity are controlled by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-κB ligand (RANKL). M-CSF and RANKL bind to their respective receptors c-Fms and RANK to stimulate osteoclast differentiation through regulation of delicate signaling systems. Here, we summarize the critical or essential signaling pathways for osteoclast differentiation including M-CSF-c-Fms signaling, RANKL-RANK signaling, and costimulatory signaling for RANK. PMID:26865996

  16. Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras farnesylation

    SciTech Connect

    Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen

    2012-04-15

    Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2 activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular

  17. Phosphorylation of ASPP2 by RAS/MAPK Pathway Is Critical for Its Full Pro-Apoptotic Function

    PubMed Central

    Slee, Elizabeth; Lu, Xin

    2013-01-01

    We reported recently that apoptosis-stimulating protein of p53 (ASPP) 2, an activator of p53, co-operates with oncogenic RAS to enhance the transcription and apoptotic function of p53. However, the detailed mechanism remains unknown. Here we show that ASPP2 is a novel substrate of mitogen-activated protein kinase (MAPK). Phosphorylation of ASPP2 by MAPK is required for RAS-induced increased binding to p53 and increased transactivation of pro-apoptotic genes. In contrast, an ASPP2 phosphorylation mutant exhibits reduced p53 binding and fails to enhance transactivation and apoptosis. Thus phosphorylation of ASPP2 by RAS/MAPK pathway provides a novel link between RAS and p53 in regulating apoptosis. PMID:24312625

  18. Bisphosphonates and statins inhibit expression and secretion of MIP-1α via suppression of Ras/MEK/ERK/AML-1A and Ras/PI3K/Akt/AML-1A pathways.

    PubMed

    Tsubaki, Masanobu; Takeda, Tomoya; Sakamoto, Kotaro; Shimaoka, Hirotaka; Fujita, Arisa; Itoh, Tatsuki; Imano, Motohiro; Mashimo, Kenji; Fujiwara, Daiichiro; Sakaguchi, Katsuhiko; Satou, Takao; Nishida, Shozo

    2015-01-01

    Osteolytic bone disease in multiple myeloma (MM) is associated with upregulated osteoclast activity. Macrophage inflammatory protein-1α (MIP-1α) is crucially involved in the development of osteolytic bone lesions in MM. We previously reported that minodronate inhibited lipopolysaccharide-induced MIP-1α secretion in mouse myeloma cells. However, it remains unknown whether bisphosphonates and statins inhibit MIP-1α secretion by human MM cells. In present study, we investigated whether bisphosphonates and statins had any inhibitory effect on MIP-1α secretion by human myeloma cells and the mechanism underlying this effect. In this study, we found that bisphosphonates and statins inhibited MIP-1α mRNA and MIP-1α secretion and suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation by inhibiting Ras prenylation. Moreover, bisphosphonates and statins suppressed the expression of acute myeloid leukemia-1A (AML-1A) mRNA, a MIP-1α transcription factor. These results indicate that bisphosphonates and statins suppress the Ras/mitogen-activated protein kinase kinase/ERK/AML-1A and Ras/phosphatidylinositol-3 kinase/Akt/AML-1A pathways, thereby inhibiting MIP-1α secretion by MM cells. Therefore, use of MIP-1α expression inhibitors such as bisphosphonates and statins may provide a new therapeutic approach to inhibiting tumour progression and bone destruction in MM patients. PMID:25628928

  19. RasGRP3 regulates the migration of glioma cells via interaction with Arp3

    PubMed Central

    Lee, Hae Kyung; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Poisson, Laila M.; Blumberg, Peter M.; Brodie, Chaya

    2015-01-01

    Glioblastoma (GBM), the most aggressive primary brain tumors, are highly infiltrative. Although GBM express high Ras activity and Ras proteins have been implicated in gliomagenesis, Ras-activating mutations are not frequent in these tumors. RasGRP3, an important signaling protein responsive to diacylglycerol (DAG), increases Ras activation. Here, we examined the expression and functions of RasGRP3 in GBM and glioma cells. RasGRP3 expression was upregulated in GBM specimens and glioma stem cells compared with normal brains and neural stem cells, respectively. RasGRP3 activated Ras and Rap1 in glioma cells and increased cell migration and invasion partially via Ras activation. Using pull-down assay and mass spectroscopy we identified the actin-related protein, Arp3, as a novel interacting protein of RasGRP3. The interaction of RasGRP3 and Arp3 was validated by immunofluorescence staining and co-immunoprecipitation, and PMA, which activates RasGRP3 and induces its translocation to the peri-nuclear region, increased the association of Arp3 and RasGRP3. Arp3 was upregulated in GBM, regulated cell spreading and migration and its silencing partially decreased these effects of RasGRP3 in glioma cells. In summary, RasGRP3 acts as an important integrating signaling protein of the DAG and Ras signaling pathways and actin polymerization and represents an important therapeutic target in GBM. PMID:25682201

  20. Cancer stem cell signaling pathways.

    PubMed

    Matsui, William H

    2016-09-01

    Tissue development and homeostasis are governed by the actions of stem cells. Multipotent cells are capable of self-renewal during the course of one's lifetime. The accurate and appropriate regulation of stem cell functions is absolutely critical for normal biological activity. Several key developmental or signaling pathways have been shown to play essential roles in this regulatory capacity. Specifically, the Janus-activated kinase/signal transducer and activator of transcription, Hedgehog, Wnt, Notch, phosphatidylinositol 3-kinase/phosphatase and tensin homolog, and nuclear factor-κB signaling pathways have all been shown experimentally to mediate various stem cell properties, such as self-renewal, cell fate decisions, survival, proliferation, and differentiation. Unsurprisingly, many of these crucial signaling pathways are dysregulated in cancer. Growing evidence suggests that overactive or abnormal signaling within and among these pathways may contribute to the survival of cancer stem cells (CSCs). CSCs are a relatively rare population of cancer cells capable of self-renewal, differentiation, and generation of serially transplantable heterogeneous tumors of several types of cancer. PMID:27611937

  1. Signaling pathways in diabetic nephropathy.

    PubMed

    Kawanami, Daiji; Matoba, Keiichiro; Utsunomiya, Kazunori

    2016-10-01

    Diabetic nephropathy (DN) is a major cause of end-stage renal disease (ESRD), however, specific treatment for DN has not yet been elucidated. Therefore, it is critically important to understand the molecular mechanism underlying DN to develop cause-related therapeutic strategy. To date, various factors such as hemodynamic changes and metabolic pathways have been shown to be involved in the pathogenesis of DN. Excessive glucose influx activates cellular signaling pathways, including the diacylglycerol (DAG)-protein kinase C (PKC) pathway, advanced glycation end-products (AGE), polyol pathway, hexosamine pathway and oxidative stress. These factors interact with one another, thereby facilitating inflammatory processes, leading to the development of glomerulosclerosis under diabetic conditions. In addition to metabolic pathways, Rho-kinase, an effector of small-GTPase binding protein Rho, has been implicated as an important factor in the pathogenesis of DN. A number of studies have demonstrated that Rho-kinase plays key roles in the development of DN by inducing endothelial dysfunction, mesangial excessive extracellular matrix (ECM) production, podocyte abnormality, and tubulointerstitial fibrosis. In this review article, we describe our current understanding of the signaling pathways in DN. PMID:27094540

  2. Signaling pathways mediating alcohol effects.

    PubMed

    Ron, Dorit; Messing, Robert O

    2013-01-01

    Ethanol's effects on intracellular signaling pathways contribute to acute effects of ethanol as well as to neuroadaptive responses to repeated ethanol exposure. In this chapter we review recent discoveries that demonstrate how ethanol alters signaling pathways involving several receptor tyrosine kinases and intracellular tyrosine and serine-threonine kinases, with consequences for regulation of cell surface receptor function, gene expression, protein translation, neuronal excitability and animal behavior. We also describe recent work that demonstrates a key role for ethanol in regulating the function of scaffolding proteins that organize signaling complexes into functional units. Finally, we review recent exciting studies demonstrating ethanol modulation of DNA and histone modification and the expression of microRNAs, indicating epigenetic mechanisms by which ethanol regulates neuronal gene expression and addictive behaviors. PMID:21877259

  3. Carcinogen-specific mutations in preferred Ras-Raf pathway oncogenes directed by strand bias.

    PubMed

    Keller, Ross R; Gestl, Shelley A; Lu, Amy Q; Hoke, Alicia; Feith, David J; Gunther, Edward J

    2016-08-01

    Carcinogen exposures inscribe mutation patterns on cancer genomes and sometimes bias the acquisition of driver mutations toward preferred oncogenes, potentially dictating sensitivity to targeted agents. Whether and how carcinogen-specific mutation patterns direct activation of preferred oncogenes remains poorly understood. Here, mouse models of breast cancer were exploited to uncover a mechanistic link between strand-biased mutagenesis and oncogene preference. When chemical carcinogens were employed during Wnt1-initiated mammary tumorigenesis, exposure to either 7,12-dimethylbenz(a)anthracene (DMBA) or N-ethyl-N-nitrosourea (ENU) dramatically accelerated tumor onset. Mammary tumors that followed DMBA exposure nearly always activated the Ras pathway via somatic Hras(CAA61CTA) mutations. Surprisingly, mammary tumors that followed ENU exposure typically lacked Hras mutations, and instead activated the Ras pathway downstream via Braf(GTG636GAG) mutations. Hras(CAA61CTA) mutations involve an A-to-T change on the sense strand, whereas Braf(GTG636GAG) mutations involve an inverse T-to-A change, suggesting that strand-biased mutagenesis may determine oncogene preference. To examine this possibility further, we turned to an alternative Wnt-driven tumor model in which carcinogen exposures augment a latent mammary tumor predisposition in Apc(min) mice. DMBA and ENU each accelerated mammary tumor onset in Apc(min) mice by introducing somatic, "second-hit" Apc mutations. Consistent with our strand bias model, DMBA and ENU generated strikingly distinct Apc mutation patterns, including stringently strand-inverse mutation signatures at A:T sites. Crucially, these contrasting signatures precisely match those proposed to confer bias toward Hras(CAA61CTA) versus Braf(GTG636GAG) mutations in the original tumor sets. Our findings highlight a novel mechanism whereby exposure history acts through strand-biased mutagenesis to specify activation of preferred oncogenes. PMID:27207659

  4. The Pore-Forming α-Toxin from Clostridium septicum Activates the MAPK Pathway in a Ras-c-Raf-Dependent and Independent Manner

    PubMed Central

    Chakravorty, Anjana; Awad, Milena M.; Cheung, Jackie K.; Hiscox, Thomas J.; Lyras, Dena; Rood, Julian I.

    2015-01-01

    Clostridium septicum is the causative agent of atraumatic gas gangrene, with α-toxin, an extracellular pore-forming toxin, essential for disease. How C. septicum modulates the host’s innate immune response is poorly defined, although α-toxin-intoxicated muscle cells undergo cellular oncosis, characterised by mitochondrial dysfunction and release of reactive oxygen species. Nonetheless, the signalling events that occur prior to the initiation of oncosis are poorly characterised. Our aims were to characterise the ability of α-toxin to activate the host mitogen activated protein kinase (MAPK) signalling pathway both in vitro and in vivo. Treatment of Vero cells with purified α-toxin activated the extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 arms of the MAPK pathway and stimulated the release of TNF-α in a dose-dependent manner. Studies using inhibitors of all three MAPK components suggested that activation of ERK occurred in a Ras-c-Raf dependent manner, whereas activation of JNK and p38 occurred by a Ras-independent mechanism. Toxin-mediated activation was dependent on efficient receptor binding and pore formation and on an influx of extracellular calcium ions. In the mouse myonecrosis model we showed that the MAPK pathway was activated in tissues of infected mice, implying that it has an important role in the disease process. PMID:25675415

  5. The pore-forming α-toxin from clostridium septicum activates the MAPK pathway in a Ras-c-Raf-dependent and independent manner.

    PubMed

    Chakravorty, Anjana; Awad, Milena M; Cheung, Jackie K; Hiscox, Thomas J; Lyras, Dena; Rood, Julian I

    2015-02-01

    Clostridium septicum is the causative agent of atraumatic gas gangrene, with α-toxin, an extracellular pore-forming toxin, essential for disease. How C. septicum modulates the host's innate immune response is poorly defined, although α-toxin-intoxicated muscle cells undergo cellular oncosis, characterised by mitochondrial dysfunction and release of reactive oxygen species. Nonetheless, the signalling events that occur prior to the initiation of oncosis are poorly characterised. Our aims were to characterise the ability of α-toxin to activate the host mitogen activated protein kinase (MAPK) signalling pathway both in vitro and in vivo. Treatment of Vero cells with purified α-toxin activated the extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 arms of the MAPK pathway and stimulated the release of TNF-α in a dose-dependent manner. Studies using inhibitors of all three MAPK components suggested that activation of ERK occurred in a Ras-c-Raf dependent manner, whereas activation of JNK and p38 occurred by a Ras-independent mechanism. Toxin-mediated activation was dependent on efficient receptor binding and pore formation and on an influx of extracellular calcium ions. In the mouse myonecrosis model we showed that the MAPK pathway was activated in tissues of infected mice, implying that it has an important role in the disease process. PMID:25675415

  6. The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells

    PubMed Central

    Melillo, Rosa Marina; Castellone, Maria Domenica; Guarino, Valentina; De Falco, Valentina; Cirafici, Anna Maria; Salvatore, Giuliana; Caiazzo, Fiorina; Basolo, Fulvio; Giannini, Riccardo; Kruhoffer, Mogens; Orntoft, Torben; Fusco, Alfredo; Santoro, Massimo

    2005-01-01

    In papillary thyroid carcinomas (PTCs), rearrangements of the RET receptor (RET/PTC) and activating mutations in the BRAF or RAS oncogenes are mutually exclusive. Here we show that the 3 proteins function along a linear oncogenic signaling cascade in which RET/PTC induces RAS-dependent BRAF activation and RAS- and BRAF-dependent ERK activation. Adoptive activation of the RET/PTC-RAS-BRAF axis induced cell proliferation and Matrigel invasion of thyroid follicular cells. Gene expression profiling revealed that the 3 oncogenes activate a common transcriptional program in thyroid cells that includes upregulation of the CXCL1 and CXCL10 chemokines, which in turn stimulate proliferation and invasion. Thus, motile and mitogenic properties are intrinsic to transformed thyroid cells and are governed by an epistatic oncogenic signaling cascade. PMID:15761501

  7. Ras history

    PubMed Central

    2010-01-01

    Although the roots of Ras sprouted from the rich history of retrovirus research, it was the discovery of mutationally activated RAS genes in human cancer in 1982 that stimulated an intensive research effort to understand Ras protein structure, biochemistry and biology. While the ultimate goal has been developing anti-Ras drugs for cancer treatment, discoveries from Ras have laid the foundation for three broad areas of science. First, they focused studies on the origins of cancer to the molecular level, with the subsequent discovery of genes mutated in cancer that now number in the thousands. Second, elucidation of the biochemical mechanisms by which Ras facilitates signal transduction established many of our fundamental concepts of how a normal cell orchestrates responses to extracellular cues. Third, Ras proteins are also founding members of a large superfamily of small GTPases that regulate all key cellular processes and established the versatile role of small GTP-binding proteins in biology. We highlight some of the key findings of the last 28 years. PMID:21686117

  8. Oog1, an oocyte-specific protein, interacts with Ras and Ras-signaling proteins during early embryogenesis

    SciTech Connect

    Tsukamoto, Satoshi; Ihara, Ryo; Aizawa, Akira; Kishida, Shosei; Kikuchi, Akira; Imai, Hiroshi; Minami, Naojiro . E-mail: naojiro@kais.kyoto-u.ac.jp

    2006-05-19

    We previously identified an oocyte-specific gene, Oogenesin 1 (Oog1), that encodes 326 amino acids containing a leucine zipper structure and a leucine-rich repeat. In the present study, to identify the interacting proteins of Oog1, we performed a yeast two-hybrid screening using a GV-oocyte cDNA library and found that Ral guanine nucleotide dissociation stimulator (RalGDS) is the binding partner of Oog1. Coimmunoprecipitation assay confirmed the interaction between Oog1 and RalGDS proteins. Colocalization experiments provide the evidence that the nuclear localization of RalGDS depends on the expression of Oog1. Interestingly, RalGDS protein localized in the nucleus rather than the cytoplasm between late 1-cell and early 2-cell stages, the time when Oog1 localizes in the nucleus. We also examined the interaction between Oog1 and Ras by GST pull-down assay and revealed that Oog1 interacts with Ras in a GTP-dependent manner. These findings suggest a role of Oog1 as a Ras-binding protein.

  9. Epigenomic Regulation of Smad1 Signaling During Cellular Senescence Induced by Ras Activation.

    PubMed

    Kaneda, Atsushi; Nonaka, Aya; Fujita, Takanori; Yamanaka, Ryota; Fujimoto, Mai; Miyazono, Kohei; Aburatani, Hiroyuki

    2016-01-01

    Epigenomic modification plays important roles in regulating gene expression during development, differentiation, and cellular senescence. When oncogenes are activated, cells fall into stable growth arrest to block cellular proliferation, which is called oncogene-induced senescence. We recently identified through genome-wide analyses that Bmp2-Smad1 signal and its regulation by harmonized epigenomic alteration play an important role in Ras-induced senescence of mouse embryonic fibroblasts. We describe in this chapter the methods for analyses of epigenomic alteration and Smad1 targets on genome-wide scale. PMID:26520136

  10. Para-phenylenediamine-induces apoptosis via a pathway dependent on PTK-Ras-Raf-JNK activation but independent of the PI3K/Akt pathway in NRK-52E cells.

    PubMed

    Kasi, Reena A P; Moi, Chye Soi; Kien, Yip Wai; Yian, Koh Rhun; Chin, Ng Wei; Yen, Ng Khuen; Ponnudurai, Gnanajothy; Fong, Seow Heng

    2015-03-01

    para‑Phenylenediamine (p‑PD) is a potential carcinogen, and widely used in marketed hair dye formulations. In the present study, the role of the protein tyrosine kinase (PTK)/Ras/Raf/c‑Jun N‑terminal kinase (JNK) and phosphoinositide 3‑kinase (PI3k)/protein kinase B (Akt) pathways on the growth of NRK‑52E cells was investigated. The results demonstrated that p‑PD reduced cell viability in a dose‑dependent manner. The cell death due to apoptosis was confirmed by cell cycle analysis and an Annexin‑V‑fluorescein isothiocyanate binding assay. Subsequent to staining with 2',7'‑dichlorofluorescin diacetate, the treated cells demonstrated a significant increase in reactive oxygen species (ROS) generation compared with the controls. The effects of p‑PD on the signalling pathways were analysed by western blotting. p‑PD‑treated cells exhibited an upregulated phospho‑stress‑activated protein kinase/JNK protein expression level and downregulated Ras and Raf protein expression levels; however, Akt, Bcl‑2, Bcl‑XL and Bad protein expression levels were not significantly altered compared with the control. In conclusion, p‑PD induced apoptosis by a PTK/Ras/Raf/JNK‑dependent pathway and was independent of the PI3K/Akt pathway in NRK‑52E cells. PMID:25411820

  11. NF1 regulation of RAS/ERK signaling is required for appropriate granule neuron progenitor expansion and migration in cerebellar development.

    PubMed

    Sanchez-Ortiz, Efrain; Cho, Woosung; Nazarenko, Inga; Mo, Wei; Chen, Jian; Parada, Luis F

    2014-11-01

    Cerebellar development is regulated by a coordinated spatiotemporal interplay between granule neuron progenitors (GNPs), Purkinje neurons, and glia. Abnormal development can trigger motor deficits, and more recent data indicate important roles in aspects of memory, behavior, and autism spectrum disorders (ASDs). Germline mutation in the NF1 tumor suppressor gene underlies Neurofibromatosis type 1, a complex disease that enhances susceptibility to certain cancers and neurological disorders, including intellectual deficits and ASD. The NF1 gene encodes for neurofibromin, a RAS GTPase-activating protein, and thus negatively regulates the RAS signaling pathway. Here, using mouse models to direct conditional NF1 ablation in either embryonic cerebellar progenitors or neonatal GNPs, we show that neurofibromin is required for appropriate development of cerebellar folia layering and structure. Remarkably, neonatal administration of inhibitors of the ERK pathway reversed the morphological defects. Thus, our findings establish a critical cell-autonomous role for the NF1-RAS-ERK pathway in the appropriate regulation of cerebellar development and provide a basis for using neonatal ERK inhibitor-based therapies to treat NF1-induced cerebellar disorders. PMID:25367036

  12. Uncoupling of EGFR-RAS signaling and nuclear localization of YBX1 in colorectal cancer.

    PubMed

    Roßner, F; Gieseler, C; Morkel, M; Royer, H-D; Rivera, M; Bläker, H; Dietel, M; Schäfer, R; Sers, C

    2016-01-01

    The transcription factor YBX1 can act as a mediator of signals transmitted via the EGFR-RAS-MAPK axis. YBX1 expression has been associated with tumor progression and prognosis in multiple types of cancer. Immunohistochemical studies have revealed dependency between YBX1 expression and individual EGFR family members. We analyzed YBX1 and EGFR family proteins in a colorectal cancer (CRC) cohort and provide functional analyses of YBX1 in the context of EGFR-RAS-MAPK signaling. Immunohistochemistry for YBX1 and EGFR family receptors with two antibodies for YBX1 and EGFR were performed and related to clinicopathological data. We employed Caco2 cells expressing an inducible KRASV12 gene to determine effects on localization and levels of YBX1. Mouse xenografts of Caco2-KRASV12 cells were used to determine YBX1 dynamics in a tissue context. The two different antibodies against YBX1 showed discordant immunohistochemical stainings in cell culture and clinical specimens. Expression of YBX1 and EGFR family members were not correlated in CRC. Analysis of Caco2 xenografts displayed again heterogeneity of YBX1 staining with both antibodies. Our results suggest that YBX1 is controlled via complex regulatory mechanisms involving tumor stroma interaction and signal transduction processes. Our study highlights that YBX1 antibodies have different specificities, advocating their use in a combined manner. PMID:26779809

  13. Activated K-Ras, But Not H-Ras or N-Ras, Regulates Brain Neural Stem Cell Proliferation in a Raf/Rb-Dependent Manner

    PubMed Central

    Bender, R. Hugh F.; Haigis, Kevin M.; Gutmann, David H.

    2016-01-01

    Neural stem cells (NSCs) give rise to all the major cell types in the brain, including neurons, oligodendrocytes, and astrocytes. However, the intracellular signaling pathways that govern brain NSC proliferation and differentiation have been incompletely characterized to date. Since some neurodevelopmental brain disorders (Costello syndrome and Noonan syndrome) are caused by germline activating mutations in the RAS genes, Ras small GTPases are likely critical regulators of brain NSC function. In the mammalian brain, Ras exists as three distinct molecules (H-Ras, K-Ras, and N-Ras), each with different subcellular localizations, downstream signaling effectors, and biological effects. Leveraging a novel series of conditional-activated Ras molecule-expressing genetically engineered mouse strains, we demonstrate that activated K-Ras, but not H-Ras or N-Ras, expression increases brain NSC growth in a Raf-dependent, but Mek-independent, manner. Moreover, we show that activated K-Ras regulation of brain NSC proliferation requires Raf binding and suppression of retinoblastoma (Rb) function. Collectively, these observations establish tissue-specific differences in activated Ras molecule regulation of brain cell growth that operate through a noncanonical mechanism. PMID:25788415

  14. Pharmacology of intracellular signalling pathways

    PubMed Central

    Nahorski, Stefan R

    2006-01-01

    This article provides a brief and somewhat personalized review of the dramatic developments that have occurred over the last 45 years in our understanding of intracellular signalling pathways associated with G-protein-coupled receptor activation. Signalling via cyclic AMP, the phosphoinositides and Ca2+ is emphasized and these systems have already been revealed as new pharmacological targets. The therapeutic benefits of most of such targets are, however, yet to be realized, but it is certain that the discipline of pharmacology needs to widen its boundaries to meet these challenges in the future. PMID:16402119

  15. Signaling Pathways in Cartilage Repair

    PubMed Central

    Mariani, Erminia; Pulsatelli, Lia; Facchini, Andrea

    2014-01-01

    In adult healthy cartilage, chondrocytes are in a quiescent phase characterized by a fine balance between anabolic and catabolic activities. In ageing, degenerative joint diseases and traumatic injuries of cartilage, a loss of homeostatic conditions and an up-regulation of catabolic pathways occur. Since cartilage differentiation and maintenance of homeostasis are finely tuned by a complex network of signaling molecules and biophysical factors, shedding light on these mechanisms appears to be extremely relevant for both the identification of pathogenic key factors, as specific therapeutic targets, and the development of biological approaches for cartilage regeneration. This review will focus on the main signaling pathways that can activate cellular and molecular processes, regulating the functional behavior of cartilage in both physiological and pathological conditions. These networks may be relevant in the crosstalk among joint compartments and increased knowledge in this field may lead to the development of more effective strategies for inducing cartilage repair. PMID:24837833

  16. Alterations in K-ras, APC and p53-multiple genetic pathway in colorectal cancer among Indians.

    PubMed

    Malhotra, Pooja; Anwar, Mumtaz; Nanda, Neha; Kochhar, Rakesh; Wig, Jai Dev; Vaiphei, Kim; Mahmood, Safrun

    2013-06-01

    The incidence of colorectal cancer (CRC) is increasing rapidly in Asian countries during the past few decades, but no comprehensive analysis has been done to find out the exact cause of this disease. In this study, we investigated the frequencies of mutations and expression pattern of K-ras, APC (adenomatosis polyposis coli) and p53 in tumor, adjoining and distant normal mucosa and to correlate these alterations with patients clinicopathological parameters as well as with the survival. Polymerase chain reaction (PCR)-restriction digestion was used to detect mutations in K-ras and PCR-SSCP (Single Strand Conformation Polymorphism) followed by DNA sequencing was used to detect mutations in APC and p53 genes. Immunohistochemistry was used to detect the expression pattern of K-ras, APC and p53 proteins. The frequencies of mutations of K-ras, APC and p53 in 30 tumor tissues samples were 26.7 %, 46.7 % and 20 %, respectively. Only 3.3 % of tumors contained mutations in all the three genes. The most common combination of mutation was APC and p53 whereas mutation in both p53 and K-ras were extremely rare. There was no association between the mutations and expression pattern of K-ras, APC and p53 (p>0.05). In Indians, the frequency of alterations of K-ras and APC is similar as in Westerns, whereas the frequency of p53 mutation is slightly lower. The lack of multiple mutations in tumor specimens suggests that these genetic alterations might have independent influences on CRC development and there could be multiple alternative genetic pathways to CRC in our present study cohort. PMID:23526092

  17. Signalling pathways in endometrial cancer.

    PubMed

    Markowska, Anna; Pawałowska, Monika; Lubin, Jolanta; Markowska, Janina

    2014-01-01

    Carcinogenesis is a multistage process, during which the activity of signalling pathways responsible for cell cycle regulation and division is disrupted which leads to inhibition of apoptosis and enhanced proliferation. Improper activation of Wnt/β-catenin and PI3K. Akt pathways play essential role in endometrial cancers (EC), mainly type I. Mutations in APC, axin or CTNBB1 may lead to β-catenin overactivation leading to excessive gene expression. PTEN inactivation, mutations in the PIK3CA or Akt result in increased transmission in the PI3K/Akt pathway, apoptosis inhibition, intensive cell division, mTOR excitation. In non-endometrioid cancers, key mutations include suppressor gene TP53 responsible for repairing damaged DNA or apoptosis initiation. Irregularities in gene P16, encoding a protein forming the p16-cyclinD/CDK-pRb have also been described. Understanding the complex relations between specific proteins taking part in signal transduction of the abovementioned pathways is key to research on drugs used in targeted therapy. PMID:25520571

  18. Modulation of Ras signaling alters the toxicity of hydroquinone, a benzene metabolite and component of cigarette smoke

    PubMed Central

    2014-01-01

    Background Benzene is an established human leukemogen, with a ubiquitous environmental presence leading to significant population exposure. In a genome-wide functional screen in the yeast Saccharomyces cerevisiae, inactivation of IRA2, a yeast ortholog of the human tumor suppressor gene NF1 (Neurofibromin), enhanced sensitivity to hydroquinone, an important benzene metabolite. Increased Ras signaling is implicated as a causal factor in the increased pre-disposition to leukemia of individuals with mutations in NF1. Methods Growth inhibition of yeast by hydroquinone was assessed in mutant strains exhibiting varying levels of Ras activity. Subsequently, effects of hydroquinone on both genotoxicity (measured by micronucleus formation) and proliferation of WT and Nf1 null murine hematopoietic precursors were assessed. Results Here we show that the Ras status of both yeast and mammalian cells modulates hydroquinone toxicity, indicating potential synergy between Ras signaling and benzene toxicity. Specifically, enhanced Ras signaling increases both hydroquinone-mediated growth inhibition in yeast and genotoxicity in mammalian hematopoetic precursors as measured by an in vitro erythroid micronucleus assay. Hydroquinone also increases proliferation of CFU-GM progenitor cells in mice with Nf1 null bone marrow relative to WT, the same cell type associated with benzene-associated leukemia. Conclusions Together our findings show that hydroquinone toxicity is modulated by Ras signaling. Individuals with abnormal Ras signaling could be more vulnerable to developing myeloid diseases after exposure to benzene. We note that hydroquinone is used cosmetically as a skin-bleaching agent, including by individuals with cafe-au-lait spots (which may be present in individuals with neurofibromatosis who have a mutation in NF1), which could be unadvisable given our findings. PMID:24386979

  19. Regulation of the Notch target gene Hes-1 by TGF{alpha} induced Ras/MAPK signaling in human neuroblastoma cells

    SciTech Connect

    Stockhausen, Marie-Therese; Sjoelund, Jonas; Axelson, Hakan . E-mail: hakan.axelson@molmed.mas.lu.se

    2005-10-15

    Ras and Notch signaling have recently been shown to cooperate in the maintenance of neoplastic transformation. Here, we show that TGF{alpha}, a known activator of Ras signaling, can drive cell proliferation and at the same time induce the expression of the Notch target Hes-1 in the neuroblastoma cell line SK-N-BE(2)c. The up-regulation of Hes-1 occurred both at the transcriptional and protein levels and by use of EGFR and MEK inhibitors we could show that the Hes-1 response was dependent on activation of the MAP kinase ERK. Blocking Notch activation by {gamma}-secretase inhibition did not profoundly affect the Hes-1 levels, neither in untreated nor in TGF{alpha} treated cells. The up-regulation of Hes-1 was associated with down-regulation of its pro-neuronal target gene Hash-1. Taken together, these results show that TGF{alpha} is a potent mitogen of neuroblastoma cells and suggest a connection between activation of ERK and Hes-1, thus providing a link between the Ras and Notch signaling pathways.

  20. Decreased bone mineralization in Children with Noonan Syndrome: Another Consequence of Dysregulated RAS MAPKinase Pathway?

    PubMed Central

    Choudhry, Kiran S.; Grover, Monica; Tran, Alyssa; O'Brian Smith, E.; Ellis, Kenneth J.; Lee, Brendan H.

    2012-01-01

    Introduction Noonan syndrome (NS) is a disorder of RAS- mitogen activated protein kinase (MAPK) pathway with clinical features of skeletal dysplasia. This pathway is essential for regulation of cell differentiation and growth including bone homeostasis. Currently, limited information exists regarding bone mineralization in NS. Material and Methods Using dual-energy X-ray absorptiometry (DXA), bone mineralization was evaluated in 12 subjects (mean age 8.7 years) with clinical features of NS. All subjects underwent genetic testing which showed mutations in PTPN11 gene (N=9) and SOS1 gene (N=1). In a subgroup of subjects with low bone mass, indices of calcium-phosphate metabolism and bone turnover were obtained. Results 50% of subjects had low bone mass as measured by DXA. Z-scores for bone mineral content (BMC) were calculated based on age, gender, height, and ethnicity. Mean BMC z-score was marginally decreased at -0.89 {95% CI -2.01 to 0.23; p=0.1}. Mean total body bone mineral density (BMD) z-score was significantly reduced at -1.87 {95% CI -2.73 to -1.0; p= 0.001}. Mean height percentile was close to -2 SD for this cohort, thus total body BMD z-scores were recalculated, adjusting for height age. Adjusted mean total body BMD z-score was less reduced but still significant at -0.82 {95% CI -1.39 to -0.25; p= 0.009}. Biochemical evaluation for bone turnover was unremarkable except serum IGF- I and IGF-BP3 levels which were low-normal for age. Discussion Children with Noonan syndrome have a significantly lower total body BMD compared to age, gender, ethnicity and height matched controls. In addition, total BMC appears to trend lower in children with Noonan syndrome compared to controls. We conclude that the metabolic bone disease present resulted from a subtle variation in the interplay of osteoclast and osteoblast activity, without clear abnormalities being defined in the metabolism of either. Clinical significance of this finding needs to be validated by larger

  1. RAS Interaction with PI3K

    PubMed Central

    Castellano, Esther; Downward, Julian

    2011-01-01

    RAS proteins are small GTPases known for their involvement in oncogenesis: around 25% of human tumors present mutations in a member of this family. RAS operates in a complex signaling network with multiple activators and effectors, which allows them to regulate many cellular functions such as cell proliferation, differentiation, apoptosis, and senescence. Phosphatidylinositol 3-kinase (PI3K) is one of the main effector pathways of RAS, regulating cell growth, cell cycle entry, cell survival, cytoskeleton reorganization, and metabolism. However, it is the involvement of this pathway in human tumors that has attracted most attention. PI3K has proven to be necessary for RAS-induced transformation in vitro, and more importantly, mice with mutations in the PI3K catalytic subunit p110α that block its ability to interact with RAS are highly resistant to endogenous oncogenic KRAS-induced lung tumorigenesis and HRAS-induced skin carcinogenesis. These animals also have a delayed development of the lymphatic vasculature. Many PI3K inhibitors have been developed that are now in clinical trials. However, it is a complex pathway with many feedback loops, and interactions with other pathways make the results of its inhibition hard to predict. Combined therapy with another RAS-regulated pathway such as RAF/MEK/ERK may be the most effective way to treat cancer, at least in animal models mimicking the human disease. In this review, we will summarize current knowledge about how RAS regulates one of its best-known effectors, PI3K. PMID:21779497

  2. Reduction of metastasis, cell invasion, and adhesion in mouse osteosarcoma by YM529/ONO-5920-induced blockade of the Ras/MEK/ERK and Ras/PI3K/Akt pathway

    SciTech Connect

    Tsubaki, Masanobu; Satou, Takao; Itoh, Tatsuki; Imano, Motohiro; Ogaki, Mitsuhiko; Yanae, Masashi; Nishida, Shozo

    2012-03-15

    Osteosarcoma is one of the most common primary malignant bone tumors in children and adolescents. Some patients continue to have a poor prognosis, because of the metastatic disease. YM529/ONO-5920 is a nitrogen-containing bisphosphonate that has been used for the treatment of osteoporosis. YM529/ONO-5920 has recently been reported to induce apoptosis in various tumors including osteosarcoma. However, the mode of metastasis suppression in osteosarcoma by YM529/ONO-5920 is unclear. In the present study, we investigated whether YM529/ONO-5920 inhibited tumor cell migration, invasion, adhesion, or metastasis in the LM8 mouse osteosarcoma cell line. We found that YM529/ONO-5920 significantly inhibited metastasis, cell migration, invasion, and adhesion at concentrations that did not have antiproliferative effects on LM8 cells. YM529/ONO-5920 also inhibited the mRNA expression and protein activities of matrix metalloproteinases (MMPs). In addition, YM529/ONO-5920 suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and the serine/threonine protein kinase B (Akt) by the inhibition of Ras prenylation. Moreover, U0126, a mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, also inhibited LM8 cell migration, invasion, adhesion, and metastasis, as well as the mRNA expression and protein activities of MMP-1, MMP-2, MMP-9, and MT1-MMP. The results indicated that YM529/ONO-5920 suppressed the Ras/MEK/ERK and Ras/PI3K/Akt pathways, thereby inhibiting LM8 cell migration, invasion, adhesion, and metastasis. These findings suggest that YM529/ONO-5920 has potential clinical applications for the treatment of tumor cell metastasis in osteosarcoma. -- Highlights: ► We investigated whether YM529/ONO-5920 inhibited tumor metastasis in osteosarcoma. ► YM529/ONO-5920 inhibited metastasis, cell migration, invasion, and adhesion. ► YM529/ONO-5920 suppressed Ras signalings. ► YM529/ONO-5920

  3. Genomic classification of the RAS network identifies a personalized treatment strategy for lung cancer

    PubMed Central

    El-Chaar, Nader N.; Piccolo, Stephen R.; Boucher, Kenneth M.; Cohen, Adam L.; Chang, Jeffrey T.; Moos, Philip J.; Bild, Andrea H.

    2014-01-01

    Better approaches are needed to evaluate a single patient's drug response at the genomic level. Targeted therapy for signaling pathways in cancer has met limited success in part due to the exceedingly interwoven nature of the pathways. In particular, the highly complex RAS network has been challenging to target. Effectively targeting the pathway requires development of techniques that measure global network activity to account for pathway complexity. For this purpose, we used a gene-expression-based biomarker for RAS network activity in non-small cell lung cancer (NSCLC) cells, and screened for drugs whose efficacy were significantly highly correlated to RAS network activity. Results identified EGFR and MEK co-inhibition as the most effective treatment for RAS-active NSCLC amongst a panel of over 360 compounds and fractions. RAS activity was identified in both RAS-mutant and wild-type lines, indicating broad characterization of RAS signaling inclusive of multiple mechanisms of RAS activity, and not solely based on mutation status. Mechanistic studies demonstrated that co-inhibition of EGFR and MEK induced apoptosis and blocked both EGFR-RAS-RAF-MEK-ERK and EGFR-PI3K-AKT-RPS6 nodes simultaneously in RAS-active, but not RAS-inactive NSCLC. These results provide a comprehensive strategy to personalize treatment of NSCLC based on RAS network dysregulation and provide proof-of-concept of a genomic approach to classify and target complex signaling networks. PMID:24908424

  4. Effects of RAF inhibitors on PI3K/AKT signalling depend on mutational status of the RAS/RAF signalling axis.

    PubMed

    Fritsche-Guenther, Raphaela; Witzel, Franziska; Kempa, Stefan; Brummer, Tilman; Sers, Christine; Blüthgen, Nils

    2016-02-16

    Targeted therapies within the RAS/RAF/MEK/ERK signalling axis become increasingly popular, yet cross-talk and feedbacks in the signalling network lead to unexpected effects. Here we look systematically into how inhibiting RAF and MEK with clinically relevant inhibitors result in changes in PI3K/AKT activation. We measure the signalling response using a bead-based ELISA, and use a panel of three cell lines, and isogenic cell lines that express mutant forms of the oncogenes KRAS and BRAF to interrogate the effects of the MEK and RAF inhibitors on signalling. We find that treatment with the RAF inhibitors have opposing effects on AKT phosphorylation depending on the mutational status of two important oncogenes, KRAS and BRAF. If these two genes are in wildtype configuration, RAF inhibitors reduce AKT phosphorylation. In contrast, if BRAF or KRAS are mutant, RAF inhibitors will leave AKT phosphorylation unaffected or lead to an increase of AKT phosphorylation. Down-regulation of phospho-AKT by RAF inhibitors also extends to downstream transcription factors, and correlates with apoptosis induction. Our results show that oncogenes rewire signalling such that targeted therapies can have opposing effects on parallel pathways, which depend on the mutational status of the cell. PMID:26799289

  5. Effects of RAF inhibitors on PI3K/AKT signalling depend on mutational status of the RAS/RAF signalling axis

    PubMed Central

    Fritsche-Guenther, Raphaela; Witzel, Franziska; Kempa, Stefan; Brummer, Tilman; Sers, Christine; Blüthgen, Nils

    2016-01-01

    Targeted therapies within the RAS/RAF/MEK/ERK signalling axis become increasingly popular, yet cross-talk and feedbacks in the signalling network lead to unexpected effects. Here we look systematically into how inhibiting RAF and MEK with clinically relevant inhibitors result in changes in PI3K/AKT activation. We measure the signalling response using a bead-based ELISA, and use a panel of three cell lines, and isogenic cell lines that express mutant forms of the oncogenes KRAS and BRAF to interrogate the effects of the MEK and RAF inhibitors on signalling. We find that treatment with the RAF inhibitors have opposing effects on AKT phosphorylation depending on the mutational status of two important oncogenes, KRAS and BRAF. If these two genes are in wildtype configuration, RAF inhibitors reduce AKT phosphorylation. In contrast, if BRAF or KRAS are mutant, RAF inhibitors will leave AKT phosphorylation unaffected or lead to an increase of AKT phosphorylation. Down-regulation of phospho-AKT by RAF inhibitors also extends to downstream transcription factors, and correlates with apoptosis induction. Our results show that oncogenes rewire signalling such that targeted therapies can have opposing effects on parallel pathways, which depend on the mutational status of the cell. PMID:26799289

  6. Pharmacological modulation of oncogenic Ras by natural products and their derivatives: Renewed hope in the discovery of novel anti-Ras drugs.

    PubMed

    Quah, Shun Ying; Tan, Michelle Siying; Teh, Yuan Han; Stanslas, Johnson

    2016-06-01

    Oncogenic rat sarcoma (Ras) is linked to the most fatal cancers such as those of the pancreas, colon, and lung. Decades of research to discover an efficacious drug that can block oncogenic Ras signaling have yielded disappointing results; thus, Ras was considered "undruggable" until recently. Inhibitors that directly target Ras by binding to previously undiscovered pockets have been recently identified. Some of these molecules are either isolated from natural products or derived from natural compounds. In this review, we described the potential of these compounds and other inhibitors of Ras signaling in drugging Ras. We highlighted the modes of action of these compounds in suppressing signaling pathways activated by oncogenic Ras, such as mitogen-activated protein kinase (MAPK) signaling and the phosphoinositide-3-kinase (PI3K) pathways. The anti-Ras strategy of these compounds can be categorized into four main types: inhibition of Ras-effector interaction, interference of Ras membrane association, prevention of Ras-guanosine triphosphate (GTP) formation, and downregulation of Ras proteins. Another promising strategy that must be validated experimentally is enhancement of the intrinsic Ras-guanosine triphosphatase (GTPase) activity by small chemical entities. Among the inhibitors of Ras signaling that were reported thus far, salirasib and TLN-4601 have been tested for their clinical efficacy. Although both compounds passed phase I trials, they failed in their respective phase II trials. Therefore, new compounds of natural origin with relevant clinical activity against Ras-driven malignancies are urgently needed. Apart from salirasib and TLN-4601, some other compounds with a proven inhibitory effect on Ras signaling include derivatives of salirasib, sulindac, polyamine, andrographolide, lipstatin, levoglucosenone, rasfonin, and quercetin. PMID:27016467

  7. FGFR1 signaling in hypertrophic chondrocytes is attenuated by the Ras-GAP neurofibromin during endochondral bone formation

    PubMed Central

    Karolak, Matthew R.; Yang, Xiangli; Elefteriou, Florent

    2015-01-01

    Aberrant fibroblast growth factor receptor 3 (FGFR3) signaling disrupts chondrocyte proliferation and growth plate size and architecture, leading to various chondrodysplasias or bone overgrowth. These observations suggest that the duration, intensity and cellular context of FGFR signaling during growth plate chondrocyte maturation require tight, regulated control for proper bone elongation. However, the machinery fine-tuning FGFR signaling in chondrocytes is incompletely defined. We report here that neurofibromin, a Ras-GAP encoded by Nf1, has an overlapping expression pattern with FGFR1 and FGFR3 in prehypertrophic chondrocytes, and with FGFR1 in hypertrophic chondrocytes during endochondral ossification. Based on previous evidence that neurofibromin inhibits Ras-ERK signaling in chondrocytes and phenotypic analogies between mice with constitutive FGFR1 activation and Nf1 deficiency in Col2a1-positive chondrocytes, we asked whether neurofibromin is required to control FGFR1-Ras-ERK signaling in maturing chondrocytes in vivo. Genetic Nf1 ablation in Fgfr1-deficient chondrocytes reactivated Ras-ERK1/2 signaling in hypertrophic chondrocytes and reversed the expansion of the hypertrophic zone observed in mice lacking Fgfr1 in Col2a1-positive chondrocytes. Histomorphometric and gene expression analyses suggested that neurofibromin, by inhibiting Rankl expression, attenuates pro-osteoclastogenic FGFR1 signaling in hypertrophic chondrocytes. We also provide evidence suggesting that neurofibromin in prehypertrophic chondrocytes, downstream of FGFRs and via an indirect mechanism, is required for normal extension and organization of proliferative columns. Collectively, this study indicates that FGFR signaling provides an important input into the Ras-Raf-MEK-ERK1/2 signaling axis in chondrocytes, and that this input is differentially regulated during chondrocyte maturation by a complex intracellular machinery, of which neurofibromin is a critical component. PMID:25616962

  8. FGFR1 signaling in hypertrophic chondrocytes is attenuated by the Ras-GAP neurofibromin during endochondral bone formation.

    PubMed

    Karolak, Matthew R; Yang, Xiangli; Elefteriou, Florent

    2015-05-01

    Aberrant fibroblast growth factor receptor 3 (FGFR3) signaling disrupts chondrocyte proliferation and growth plate size and architecture, leading to various chondrodysplasias or bone overgrowth. These observations suggest that the duration, intensity and cellular context of FGFR signaling during growth plate chondrocyte maturation require tight, regulated control for proper bone elongation. However, the machinery fine-tuning FGFR signaling in chondrocytes is incompletely defined. We report here that neurofibromin, a Ras-GAP encoded by Nf1, has an overlapping expression pattern with FGFR1 and FGFR3 in prehypertrophic chondrocytes, and with FGFR1 in hypertrophic chondrocytes during endochondral ossification. Based on previous evidence that neurofibromin inhibits Ras-ERK signaling in chondrocytes and phenotypic analogies between mice with constitutive FGFR1 activation and Nf1 deficiency in Col2a1-positive chondrocytes, we asked whether neurofibromin is required to control FGFR1-Ras-ERK signaling in maturing chondrocytes in vivo. Genetic Nf1 ablation in Fgfr1-deficient chondrocytes reactivated Ras-ERK1/2 signaling in hypertrophic chondrocytes and reversed the expansion of the hypertrophic zone observed in mice lacking Fgfr1 in Col2a1-positive chondrocytes. Histomorphometric and gene expression analyses suggested that neurofibromin, by inhibiting Rankl expression, attenuates pro-osteoclastogenic FGFR1 signaling in hypertrophic chondrocytes. We also provide evidence suggesting that neurofibromin in prehypertrophic chondrocytes, downstream of FGFRs and via an indirect mechanism, is required for normal extension and organization of proliferative columns. Collectively, this study indicates that FGFR signaling provides an important input into the Ras-Raf-MEK-ERK1/2 signaling axis in chondrocytes, and that this input is differentially regulated during chondrocyte maturation by a complex intracellular machinery, of which neurofibromin is a critical component. PMID:25616962

  9. A Model for Direction Sensing in Dictyostelium discoideum: Ras Activity and Symmetry Breaking Driven by a Gβγ-Mediated, Gα2-Ric8 -- Dependent Signal Transduction Network.

    PubMed

    Cheng, Yougan; Othmer, Hans

    2016-05-01

    Chemotaxis is a dynamic cellular process, comprised of direction sensing, polarization and locomotion, that leads to the directed movement of eukaryotic cells along extracellular gradients. As a primary step in the response of an individual cell to a spatial stimulus, direction sensing has attracted numerous theoretical treatments aimed at explaining experimental observations in a variety of cell types. Here we propose a new model of direction sensing based on experiments using Dictyostelium discoideum (Dicty). The model is built around a reaction-diffusion-translocation system that involves three main component processes: a signal detection step based on G-protein-coupled receptors (GPCR) for cyclic AMP (cAMP), a transduction step based on a heterotrimetic G protein Gα2βγ, and an activation step of a monomeric G-protein Ras. The model can predict the experimentally-observed response of cells treated with latrunculin A, which removes feedback from downstream processes, under a variety of stimulus protocols. We show that [Formula: see text] cycling modulated by Ric8, a nonreceptor guanine exchange factor for [Formula: see text] in Dicty, drives multiple phases of Ras activation and leads to direction sensing and signal amplification in cAMP gradients. The model predicts that both [Formula: see text] and Gβγ are essential for direction sensing, in that membrane-localized [Formula: see text], the activated GTP-bearing form of [Formula: see text], leads to asymmetrical recruitment of RasGEF and Ric8, while globally-diffusing Gβγ mediates their activation. We show that the predicted response at the level of Ras activation encodes sufficient 'memory' to eliminate the 'back-of-the wave' problem, and the effects of diffusion and cell shape on direction sensing are also investigated. In contrast with existing LEGI models of chemotaxis, the results do not require a disparity between the diffusion coefficients of the Ras activator GEF and the Ras inhibitor GAP. Since

  10. A Model for Direction Sensing in Dictyostelium discoideum: Ras Activity and Symmetry Breaking Driven by a Gβγ-Mediated, Gα2-Ric8 -- Dependent Signal Transduction Network

    PubMed Central

    Cheng, Yougan; Othmer, Hans

    2016-01-01

    Chemotaxis is a dynamic cellular process, comprised of direction sensing, polarization and locomotion, that leads to the directed movement of eukaryotic cells along extracellular gradients. As a primary step in the response of an individual cell to a spatial stimulus, direction sensing has attracted numerous theoretical treatments aimed at explaining experimental observations in a variety of cell types. Here we propose a new model of direction sensing based on experiments using Dictyostelium discoideum (Dicty). The model is built around a reaction-diffusion-translocation system that involves three main component processes: a signal detection step based on G-protein-coupled receptors (GPCR) for cyclic AMP (cAMP), a transduction step based on a heterotrimetic G protein Gα2βγ, and an activation step of a monomeric G-protein Ras. The model can predict the experimentally-observed response of cells treated with latrunculin A, which removes feedback from downstream processes, under a variety of stimulus protocols. We show that Gα2βγ cycling modulated by Ric8, a nonreceptor guanine exchange factor for Gα2 in Dicty, drives multiple phases of Ras activation and leads to direction sensing and signal amplification in cAMP gradients. The model predicts that both Gα2 and Gβγ are essential for direction sensing, in that membrane-localized Gα2*, the activated GTP-bearing form of Gα2, leads to asymmetrical recruitment of RasGEF and Ric8, while globally-diffusing Gβγ mediates their activation. We show that the predicted response at the level of Ras activation encodes sufficient ‘memory’ to eliminate the ‘back-of-the wave’ problem, and the effects of diffusion and cell shape on direction sensing are also investigated. In contrast with existing LEGI models of chemotaxis, the results do not require a disparity between the diffusion coefficients of the Ras activator GEF and the Ras inhibitor GAP. Since the signal pathways we study are highly conserved between Dicty

  11. Human iPS Cell-Derived Neurons Uncover the Impact of Increased Ras Signaling in Costello Syndrome

    PubMed Central

    Rooney, Gemma E.; Goodwin, Alice F.; Depeille, Philippe; Sharir, Amnon; Schofield, Claude M.; Yeh, Erika; Roose, Jeroen P.; Klein, Ophir D.; Rauen, Katherine A.; Weiss, Lauren A.

    2016-01-01

    Increasing evidence implicates abnormal Ras signaling as a major contributor in neurodevelopmental disorders, yet how such signaling causes cortical pathogenesis is unknown. We examined the consequences of aberrant Ras signaling in the developing mouse brain and uncovered several critical phenotypes, including increased production of cortical neurons and morphological deficits. To determine whether these phenotypes are recapitulated in humans, we generated induced pluripotent stem (iPS) cell lines from patients with Costello syndrome (CS), a developmental disorder caused by abnormal Ras signaling and characterized by neurodevelopmental abnormalities, such as cognitive impairment and autism. Directed differentiation toward a neuroectodermal fate revealed an extended progenitor phase and subsequent increased production of cortical neurons. Morphological analysis of mature neurons revealed significantly altered neurite length and soma size in CS patients. This study demonstrates the synergy between mouse and human models and validates the use of iPS cells as a platform to study the underlying cellular pathologies resulting from signaling deficits. SIGNIFICANCE STATEMENT Increasing evidence implicates Ras signaling dysfunction as a major contributor in psychiatric and neurodevelopmental disorders, such as cognitive impairment and autism, but the underlying cortical cellular pathogenesis remains unclear. This study is the first to reveal human neuronal pathogenesis resulting from abnormal Ras signaling and provides insights into how these phenotypic abnormalities likely contribute to neurodevelopmental disorders. We also demonstrate the synergy between mouse and human models, thereby validating the use of iPS cells as a platform to study underlying cellular pathologies resulting from signaling deficits. Recapitulating human cellular pathologies in vitro facilitates the future high throughput screening of potential therapeutic agents that may reverse phenotypic and

  12. The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Toward a Therapeutic Approach

    PubMed Central

    Korf, Bruce; Ahmadian, Reza; Allanson, Judith; Aoki, Yoko; Bakker, Annette; Wright, Emma Burkitt; Denger, Brian; Elgersma, Ype; Gelb, Bruce D.; Gripp, Karen W.; Kerr, Bronwyn; Kontaridis, Maria; Lazaro, Conxi; Linardic, Corinne; Lozano, Reymundo; MacRae, Calum A.; Messiaen, Ludwine; Mulero-Navarro, Sonia; Neel, Benjamin; Plotkin, Scott; Rauen, Katherine A.; Roberts, Amy; Silva, Alcino J.; Sittampalam, Sitta G.; Zhang, Chao; Schoyer, Lisa

    2015-01-01

    “The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Towards a Therapeutic Approach” was held at the Renaissance Orlando at SeaWorld Hotel (August 2–4, 2013). Seventy-one physicians and scientists attended the meeting, and parallel meetings were held by patient advocacy groups (CFC International, Costello Syndrome Family Network, NF Network and Noonan Syndrome Foundation). Parent and patient advocates opened the meeting with a panel discussion to set the stage regarding their hopes and expectations for therapeutic advances. In keeping with the theme on therapeutic development, the sessions followed a progression from description of the phenotype and definition of therapeutic endpoints, to definition of genomic changes, to identification of therapeutic targets in the RAS/MAPK pathway, to preclinical drug development and testing, to clinical trials. These proceedings will review the major points of discussion. PMID:25900621

  13. The third international meeting on genetic disorders in the RAS/MAPK pathway: towards a therapeutic approach.

    PubMed

    Korf, Bruce; Ahmadian, Reza; Allanson, Judith; Aoki, Yoko; Bakker, Annette; Wright, Emma Burkitt; Denger, Brian; Elgersma, Ype; Gelb, Bruce D; Gripp, Karen W; Kerr, Bronwyn; Kontaridis, Maria; Lazaro, Conxi; Linardic, Corinne; Lozano, Reymundo; MacRae, Calum A; Messiaen, Ludwine; Mulero-Navarro, Sonia; Neel, Benjamin; Plotkin, Scott; Rauen, Katherine A; Roberts, Amy; Silva, Alcino J; Sittampalam, Sitta G; Zhang, Chao; Schoyer, Lisa

    2015-08-01

    "The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Towards a Therapeutic Approach" was held at the Renaissance Orlando at SeaWorld Hotel (August 2-4, 2013). Seventy-one physicians and scientists attended the meeting, and parallel meetings were held by patient advocacy groups (CFC International, Costello Syndrome Family Network, NF Network and Noonan Syndrome Foundation). Parent and patient advocates opened the meeting with a panel discussion to set the stage regarding their hopes and expectations for therapeutic advances. In keeping with the theme on therapeutic development, the sessions followed a progression from description of the phenotype and definition of therapeutic endpoints, to definition of genomic changes, to identification of therapeutic targets in the RAS/MAPK pathway, to preclinical drug development and testing, to clinical trials. These proceedings will review the major points of discussion. PMID:25900621

  14. Biochemical similarity of Schizosaccharomyces pombe ras1 protein with RAS2 protein of Saccharomyces cervisiae.

    PubMed

    Onozawa, T; Danjoh, I; Fujiyama, A

    1995-07-01

    Schizosaccharomyces pombe contains single ras oncogene homologue, ras1, that functions in the signal transduction pathway conducting the cell's mating processes. To understand the biochemical basis of yeast ras proteins, we have purified the ras1 protein and compared the major biochemical constants with those of RAS2 protein from Saccharomyces cerevisiae and mammalian ras proteins. The purified ras1 protein showed a remarkably high Kd value for GDP binding (178 nM) and for binding with ATP. In contrast, the Kd value for GTP binding and the rate of GTPase activity were 64 nM and 77 x 10(-6) s-1 at 37 degrees C, respectively; both were higher than normal p21ras protein, but at the same level as the RAS2 protein. We directly measured rate of GTP binding and GDP binding which were 3.9 x 10(-3) s-1 and 1.8 x 10(-3) s-1 at 30 degrees C, respectively. On the other hand, exchange rates between bound and free nucleotides remained almost constant throughout the tested combination of GTP and GDP, and were several-fold lower than the binding rate. These results suggest that the release of the guanine nucleotide is the rate-limiting step in the ras-GTP/GDP cycle. As a whole, the biochemical properties of the ras1 protein are close to those of the RAS2 protein, although these two proteins function differently in the signal transduction pathway in the cells. PMID:7483844

  15. Superoxide Inhibits Guanine Nucleotide Exchange Factor (GEF) Action on Ras, but not on Rho, through Desensitization of Ras to GEF

    PubMed Central

    2015-01-01

    Ras and Rho GTPases are molecular switches for various vital cellular signaling pathways. Overactivation of these GTPases often causes development of cancer. Guanine nucleotide exchange factors (GEFs) and oxidants function to upregulate these GTPases through facilitation of guanine nucleotide exchange (GNE) of these GTPases. However, the effect of oxidants on GEF functions, or vice versa, has not been known. We show that, via targeting Ras Cys51, an oxidant inhibits the catalytic action of Cdc25—the catalytic domain of RasGEFs—on Ras. However, the enhancement of Ras GNE by an oxidant continues regardless of the presence of Cdc25. Limiting RasGEF action by an oxidant may function to prevent the pathophysiological overactivation of Ras in the presence of both RasGEFs and oxidants. The continuous exposure of Ras to nitric oxide and its derivatives can form S-nitrosated Ras (Ras-SNO). This study also shows that an oxidant not only inhibits the catalytic action of Cdc25 on Ras-SNO but also fails to enhance Ras-SNO GNE. This lack of enhancement then populates the biologically inactive Ras-SNO in cells, which may function to prevent the continued redox signaling of the Ras pathophysiological response. Finally, this study also demonstrates that, unlike the case with RasGEFs, an oxidant does not inhibit the catalytic action of RhoGEF—Vav or Dbs—on Rho GTPases such as Rac1, RhoA, RhoC, and Cdc42. This result explains the results of the previous study in which, despite the presence of an oxidant, the catalytic action of Dbs in cells continued to enhance RhoC GNE. PMID:24422478

  16. Ras regulates kinesin 13 family members to control cell migration pathways in transformed human bronchial epithelial cells

    PubMed Central

    Zaganjor, Elma; Osborne, Jihan K.; Weil, Lauren M.; Diaz-Martinez, Laura A.; Gonzales, Joshua X.; Singel, Stina M.; Larsen, Jill E.; Girard, Luc; Minna, John D.; Cobb, Melanie H.

    2014-01-01

    We show that expression of the microtubule depolymerizing kinesin KIF2C is induced by transformation of immortalized human bronchial epithelial cells by expression of K-RasG12V and knockdown of p53. Further investigation demonstrates that this is due to the K-Ras/ERK1/2 MAPK pathway, as loss of p53 had little effect on KIF2C expression. In addition to KIF2C, we also found that the related kinesin KIF2A is modestly upregulated in this model system; both proteins are expressed more highly in many lung cancer cell lines compared to normal tissue. As a consequence of their depolymerizing activity, these kinesins increase dynamic instability of microtubules. Depletion of either of these kinesins impairs the ability of cells transformed with mutant K-Ras to migrate and invade matrigel. However, depletion of these kinesins does not reverse the epithelial-mesenchymal transition caused by mutant K-Ras. Our studies indicate that increased expression of microtubule destabilizing factors can occur during oncogenesis to support enhanced migration and invasion of tumor cells. PMID:24240690

  17. EGFR/Ras Signaling Controls Drosophila Intestinal Stem Cell Proliferation via Capicua-Regulated Genes

    PubMed Central

    Jin, Yinhua; Ha, Nati; Forés, Marta; Xiang, Jinyi; Gläßer, Christine; Maldera, Julieta; Jiménez, Gerardo; Edgar, Bruce A.

    2015-01-01

    Epithelial renewal in the Drosophila intestine is orchestrated by Intestinal Stem Cells (ISCs). Following damage or stress the intestinal epithelium produces ligands that activate the epidermal growth factor receptor (EGFR) in ISCs. This promotes their growth and division and, thereby, epithelial regeneration. Here we demonstrate that the HMG-box transcriptional repressor, Capicua (Cic), mediates these functions of EGFR signaling. Depleting Cic in ISCs activated them for division, whereas overexpressed Cic inhibited ISC proliferation and midgut regeneration. Epistasis tests showed that Cic acted as an essential downstream effector of EGFR/Ras signaling, and immunofluorescence showed that Cic’s nuclear localization was regulated by EGFR signaling. ISC-specific mRNA expression profiling and DNA binding mapping using DamID indicated that Cic represses cell proliferation via direct targets including string (Cdc25), Cyclin E, and the ETS domain transcription factors Ets21C and Pointed (pnt). pnt was required for ISC over-proliferation following Cic depletion, and ectopic pnt restored ISC proliferation even in the presence of overexpressed dominant-active Cic. These studies identify Cic, Pnt, and Ets21C as critical downstream effectors of EGFR signaling in Drosophila ISCs. PMID:26683696

  18. The tumor suppressor p53 inhibits Net, an effector of Ras/extracellular signal-regulated kinase signaling.

    PubMed

    Nakade, Koji; Zheng, Hong; Ganguli, Gitali; Buchwalter, Gilles; Gross, Christian; Wasylyk, Bohdan

    2004-02-01

    The tumor suppressor function of p53 is linked to its ability to repress gene expression, but the mechanisms of specific gene repression are poorly understood. We report that wild-type p53 inhibits an effector of the Ras oncogene/mitogen-activated protein (MAP) kinase pathway, the transcription factor Net. Tumor-associated mutant p53s are less efficient inhibitors. p53 inhibits by preventing phosphorylation of Net by MAP kinases. Loss of p53 in vivo leads to increased Net phosphorylation in response to wound healing and UV irradiation of skin. Our results show that p53 can repress specific gene expression by inhibiting Net, a factor implicated in cell cycle entry. PMID:14729959

  19. Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence*

    PubMed Central

    Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L.; Embrey, Kevin J.; Golovanov, Alexander P.

    2016-01-01

    The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly 15N-labeled Ras as well as [13C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. PMID:26565026

  20. Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

    PubMed

    Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L; Embrey, Kevin J; Golovanov, Alexander P

    2016-01-22

    The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. PMID:26565026

  1. Gankyrin plays an essential role in Ras-induced tumorigenesis through regulation of the RhoA/ROCK pathway in mammalian cells

    PubMed Central

    Man, Jiang-Hong; Liang, Bing; Gu, Yue-Xi; Zhou, Tao; Li, Ai-Ling; Li, Tao; Jin, Bao-Feng; Bai, Bing; Zhang, Hai-Ying; Zhang, Wei-Na; Li, Wei-Hua; Gong, Wei-Li; Li, Hui-Yan; Zhang, Xue-Min

    2010-01-01

    Activating mutations in Ras proteins are present in about 30% of human cancers. Despite tremendous progress in the study of Ras oncogenes, many aspects of the molecular mechanisms underlying Ras-induced tumorigenesis remain unknown. Through proteomics analysis, we previously found that the protein Gankyrin, a known oncoprotein in hepatocellular carcinoma, was upregulated during Ras-mediated transformation, although the functional consequences of this were not clear. Here we present evidence that Gankyrin plays an essential role in Ras-initiated tumorigenesis in mouse and human cells. We found that the increased Gankyrin present following Ras activation increased the interaction between the RhoA GTPase and its GDP dissociation inhibitor RhoGDI, which resulted in inhibition of the RhoA effector kinase Rho-associated coiled coil–containing protein kinase (ROCK). Importantly, Gankyrin-mediated ROCK inhibition led to prolonged Akt activation, a critical step in activated Ras–induced transformation and tumorigenesis. In addition, we found that Gankyrin is highly expressed in human lung cancers that have Ras mutations and that increased Gankyrin expression is required for the constitutive activation of Akt and tumorigenesis in these lung cancers. Our findings suggest that Gankyrin is a key regulator of Ras-mediated activation of Akt through inhibition of the downstream RhoA/ROCK pathway and thus plays an essential role in Ras-induced tumorigenesis. PMID:20628200

  2. Differential involvement of Ras-GRF1 and Ras-GRF2 in L-DOPA-induced dyskinesia

    PubMed Central

    Bido, Simone; Solari, Nicola; Indrigo, Marzia; D’Antoni, Angela; Brambilla, Riccardo; Morari, Michele; Fasano, Stefania

    2015-01-01

    Objective Recent findings have shown that pharmacogenetic manipulations of the Ras-ERK pathway provide a therapeutic means to tackle l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID). First, we investigated whether a prolonged l-DOPA treatment differentially affected ERK signaling in medium spiny neurons of the direct pathway (dMSNs) and in cholinergic aspiny interneurons (ChIs) and assessed the role of Ras-GRF1 in both subpopulations. Second, using viral-assisted technology, we probed Ras-GRF1 and Ras-GRF2 as potential targets in this pathway. We investigated how selective blockade of striatal Ras-GRF1 or Ras-GRF2 expression impacted on LID (induction, maintenance, and reversion) and its neurochemical correlates. Methods We used both Ras-GRF1 knockout mice and lentiviral vectors (LVs) delivering short-hairpin RNA sequences (shRNAs) to obtain striatum-specific gene knockdown of Ras-GRF1 and Ras-GRF2. The consequences of these genetic manipulations were evaluated in the 6-hydroxydopamine mouse model of Parkinson’s disease. Escalating doses of l-DOPA were administered and then behavioral analysis with immunohistochemical assays and in vivo microdialysis were performed. Results Ras-GRF1 was found essential in controlling ERK signaling in dMSNs, but its ablation did not prevent ERK activation in ChIs. Moreover, striatal injection of LV-shRNA/Ras-GRF1 attenuated dyskinesia development and ERK-dependent signaling, whereas LV-shRNA/Ras-GRF2 was without effect, ruling out the involvement of Ras-GRF2 in LID expression. Accordingly, Ras-GRF1 but not Ras-GRF2 striatal gene-knockdown reduced l-DOPA-induced GABA and glutamate release in the substantia nigra pars reticulata, a neurochemical correlate of dyskinesia. Finally, inactivation of Ras-GRF1 provided a prolonged anti-dyskinetic effect for up to 7 weeks and significantly attenuated symptoms in animals with established LID. Interpretation Our results suggest that Ras-GRF1 is a promising target for LID

  3. MK2 Regulates Ras Oncogenesis through Stimulating ROS Production

    PubMed Central

    Kobayashi, Yusuke; Qi, Xiaomei

    2012-01-01

    Ras signals through both mitogenic and stress pathways and studies of Ras regulatory effects of stress pathways hold great promise to control Ras-dependent malignancies. Our previous work showed Ras activation of a stress kinase (MAPK-activated protein kinase 2 [MK2]), and here, we examine regulatory effects of MK2 on Ras oncogenesis. MK2 knockout was shown to increase Ras transformation in mouse embryonic fibroblasts (MEFs) in vitro and to enhance the resultant tumor growth in mice, indicating a tumor suppressor activity. In Ras-dependent and -independent human colon cancer, however, MK2-forced expression increases and MK2 depletion decreases the malignant growth, suggesting its oncogenic activity. The oncogenic activity of MK2 couples with its activation by both stress and mitogenic signals through extracellular signal–regulated kinase and p38α pathways, whereas its tumor-suppressing effect links to its stimulation only by stress downstream of p38α. Of interest, MK2 was shown to decrease intracellular levels of reactive oxygen species (ROS) in MEFs but increase its production in human colon cancer cells, and experiments with antioxidants revealed that ROS is required for Ras oncogenesis in both systems. These results indicate that MK2 can increase or decrease Ras oncogenesis dependent of its ROS regulatory activities. PMID:23264852

  4. The renewed battle against RAS-mutant cancers.

    PubMed

    Zhang, Fuquan; Cheong, Jit Kong

    2016-05-01

    The RAS genes encode for members of a large superfamily of guanosine-5'-triphosphate (GTP)-binding proteins that control diverse intracellular signaling pathways to promote cell proliferation. Somatic mutations in the RAS oncogenes are the most common activating lesions found in human cancers. These mutations invariably result in the gain-of-function of RAS by impairing GTP hydrolysis and are frequently associated with poor responses to standard cancer therapies. In this review, we summarize key findings of past and present landmark studies that have deepened our understanding of the RAS biology in the context of oncogenesis. We also discuss how emerging areas of research could further bolster a renewed global effort to target the largely undruggable oncogenic RAS and/or its activated downstream effector signaling cascades to achieve better treatment outcomes for RAS-mutant cancer patients. PMID:26892781

  5. DA-Raf-Mediated Suppression of the Ras--ERK Pathway Is Essential for TGF-β1-Induced Epithelial-Mesenchymal Transition in Alveolar Epithelial Type 2 Cells.

    PubMed

    Watanabe-Takano, Haruko; Takano, Kazunori; Hatano, Masahiko; Tokuhisa, Takeshi; Endo, Takeshi

    2015-01-01

    Myofibroblasts play critical roles in the development of idiopathic pulmonary fibrosis by depositing components of extracellular matrix. One source of lung myofibroblasts is thought to be alveolar epithelial type 2 cells that undergo epithelial-mesenchymal transition (EMT). Rat RLE-6TN alveolar epithelial type 2 cells treated with transforming growth factor-β1 (TGF-β1) are converted into myofibroblasts through EMT. TGF-β induces both canonical Smad signaling and non-canonical signaling, including the Ras-induced ERK pathway (Raf-MEK-ERK). However, the signaling mechanisms regulating TGF-β1-induced EMT are not fully understood. Here, we show that the Ras-ERK pathway negatively regulates TGF-β1-induced EMT in RLE-6TN cells and that DA-Raf1 (DA-Raf), a splicing isoform of A-Raf and a dominant-negative antagonist of the Ras-ERK pathway, plays an essential role in EMT. Stimulation of the cells with fibroblast growth factor 2 (FGF2), which activated the ERK pathway, prominently suppressed TGF-β1-induced EMT. An inhibitor of MEK, but not an inhibitor of phosphatidylinositol 3-kinase, rescued the TGF-β1-treated cells from the suppression of EMT by FGF2. Overexpression of a constitutively active mutant of a component of the Ras-ERK pathway, i.e., H-Ras, B-Raf, or MEK1, interfered with EMT. Knockdown of DA-Raf expression with siRNAs facilitated the activity of MEK and ERK, which were only weakly and transiently activated by TGF-β1. Although DA-Raf knockdown abrogated TGF-β1-induced EMT, the abrogation of EMT was reversed by the addition of the MEK inhibitor. Furthermore, DA-Raf knockdown impaired the TGF-β1-induced nuclear translocation of Smad2, which mediates the transcription required for EMT. These results imply that intrinsic DA-Raf exerts essential functions for EMT by antagonizing the TGF-β1-induced Ras-ERK pathway in RLE-6TN cells. PMID:25996975

  6. Qingfei Xiaoyan Wan, a traditional Chinese medicine formula, ameliorates Pseudomonas aeruginosa–induced acute lung inflammation by regulation of PI3K/AKT and Ras/MAPK pathways

    PubMed Central

    Hou, Yuanyuan; Nie, Yan; Cheng, Binfeng; Tao, Jin; Ma, Xiaoyao; Jiang, Min; Gao, Jie; Bai, Gang

    2016-01-01

    Gram-negative pathogen–induced nosocomial infections and resistance are a most serious menace to global public health. Qingfei Xiaoyan Wan (QF), a traditional Chinese medicine (TCM) formula, has been used clinically in China for the treatment of upper respiratory tract infections, acute or chronic bronchitis and pulmonary infection. In this study, the effects of QF on Pseudomonas aeruginosa–induced acute pneumonia in mice were evaluated. The mechanisms by which four typical anti-inflammatory ingredients from QF, arctigenin (ATG), cholic acid (CLA), chlorogenic acid (CGA) and sinapic acid (SPA), regulate anti-inflammatory signaling pathways and related targets were investigated using molecular biology and molecular docking techniques. The results showed that pretreatment with QF significantly inhibits the release of cytokines (TNF-α and IL-6) and chemokines (IL-8 and RANTES), reduces leukocytes recruitment into inflamed tissues and ameliorates pulmonary edema and necrosis. In addition, ATG was identified as the primary anti-inflammatory agent with action on the PI3K/AKT and Ras/MAPK pathways. CLA and CGA enhanced the actions of ATG and exhibited synergistic NF-κB inactivation effects possibly via the Ras/MAPK signaling pathway. Moreover, CLA is speculated to target FGFR and MEK firstly. Overall, QF regulated the PI3K/AKT and Ras/MAPK pathways to inhibit pathogenic bacterial infections effectively. PMID:27175332

  7. Qingfei Xiaoyan Wan, a traditional Chinese medicine formula, ameliorates Pseudomonas aeruginosa-induced acute lung inflammation by regulation of PI3K/AKT and Ras/MAPK pathways.

    PubMed

    Hou, Yuanyuan; Nie, Yan; Cheng, Binfeng; Tao, Jin; Ma, Xiaoyao; Jiang, Min; Gao, Jie; Bai, Gang

    2016-05-01

    Gram-negative pathogen-induced nosocomial infections and resistance are a most serious menace to global public health. Qingfei Xiaoyan Wan (QF), a traditional Chinese medicine (TCM) formula, has been used clinically in China for the treatment of upper respiratory tract infections, acute or chronic bronchitis and pulmonary infection. In this study, the effects of QF on Pseudomonas aeruginosa-induced acute pneumonia in mice were evaluated. The mechanisms by which four typical anti-inflammatory ingredients from QF, arctigenin (ATG), cholic acid (CLA), chlorogenic acid (CGA) and sinapic acid (SPA), regulate anti-inflammatory signaling pathways and related targets were investigated using molecular biology and molecular docking techniques. The results showed that pretreatment with QF significantly inhibits the release of cytokines (TNF-α and IL-6) and chemokines (IL-8 and RANTES), reduces leukocytes recruitment into inflamed tissues and ameliorates pulmonary edema and necrosis. In addition, ATG was identified as the primary anti-inflammatory agent with action on the PI3K/AKT and Ras/MAPK pathways. CLA and CGA enhanced the actions of ATG and exhibited synergistic NF-κB inactivation effects possibly via the Ras/MAPK signaling pathway. Moreover, CLA is speculated to target FGFR and MEK firstly. Overall, QF regulated the PI3K/AKT and Ras/MAPK pathways to inhibit pathogenic bacterial infections effectively. PMID:27175332

  8. The combinatorial activation of the PI3K and Ras/MAPK pathways is sufficient for aggressive tumor formation, while individual pathway activation supports cell persistence

    PubMed Central

    Thompson, Keyata N.; Whipple, Rebecca A.; Yoon, Jennifer R.; Lipsky, Michael; Charpentier, Monica S.; Boggs, Amanda E.; Chakrabarti, Kristi R.; Bhandary, Lekhana; Hessler, Lindsay K.; Martin, Stuart S.; Vitolo, Michele I.

    2015-01-01

    A high proportion of human tumors maintain activation of both the PI3K and Ras/MAPK pathways. In basal-like breast cancer (BBC), PTEN expression is decreased/lost in over 50% of cases, leading to aberrant activation of the PI3K pathway. Additionally, BBC cell lines and tumor models have been shown to exhibit an oncogenic Ras-like gene transcriptional signature, indicating activation of the Ras/MAPK pathway. To directly test how the PI3K and Ras/MAPK pathways contribute to tumorigenesis, we deleted PTEN and activated KRas within non-tumorigenic MCF-10A breast cells. Neither individual mutation was sufficient to promote tumorigenesis, but the combination promoted robust tumor growth in mice. However, in vivo bioluminescence reveals that each mutation has the ability to promote a persistent phenotype. Inherent in the concept of tumor cell dormancy, a stage in which residual disease is present but remains asymptomatic, viable cells with each individual mutation can persist in vivo during a period of latency. The persistent cells were excised from the mice and showed increased levels of the cell cycle arrest proteins p21 and p27 compared to the aggressively growing PTEN−/−KRAS(G12V) cells. Additionally, when these persistent cells were placed into growth-promoting conditions, they were able to re-enter the cell cycle and proliferate. These results highlight the potential for either PTEN loss or KRAS activation to promote cell survival in vivo, and the unique ability of the combined mutations to yield rapid tumor growth. This could have important implications in determining recurrence risk and disease progression in tumor subtypes where these mutations are common. PMID:26497685

  9. Role of H-Ras/ERK signaling in carbon nanotube-induced neoplastic-like transformation of human mesothelial cells

    PubMed Central

    Lohcharoenkal, Warangkana; Wang, Liying; Stueckle, Todd A.; Park, Jino; Tse, William; Dinu, Cerasela-Zoica; Rojanasakul, Yon

    2014-01-01

    Rapid development and deployment of engineered nanomaterials such as carbon nanotubes (CNTs) in various commercial and biomedical applications have raised concerns about their potential adverse health effects, especially their long-term effects which have not been well addressed. We demonstrated here that prolonged exposure of human mesothelial cells to single-walled CNT (SWCNT) induced neoplastic-like transformation as indicated by anchorage-independent cell growth and increased cell invasiveness. Such transformation was associated with an up-regulation of H-Ras and activation of ERK1/2. Downregulation of H-Ras by siRNA or inactivation of ERK by chemical inhibitor effectively inhibited the aggressive phenotype of SWCNT-exposed cells. Integrin alpha V and cortactin, but not epithelial-mesenchymal transition (EMT) transcriptional regulators, were up-regulated in the SWCNT-exposed cells, suggesting their role in the aggressive phenotype. Cortactin expression was shown to be controlled by the H-Ras/ERK signaling. Thus, our results indicate a novel role of H-Ras/ERK signaling and cortactin in the aggressive transformation of human mesothelial cells by SWCNT. PMID:24971065

  10. Genetic and Proteomic Evidence for Roles of Drosophila SUMO in Cell Cycle Control, Ras Signaling, and Early Pattern Formation

    PubMed Central

    Nie, Minghua; Xie, Yongming; Loo, Joseph A.; Courey, Albert J.

    2009-01-01

    SUMO is a protein modifier that is vital for multicellular development. Here we present the first system-wide analysis, combining multiple approaches, to correlate the sumoylated proteome (SUMO-ome) in a multicellular organism with the developmental roles of SUMO. Using mass-spectrometry-based protein identification, we found over 140 largely novel SUMO conjugates in the early Drosophila embryo. Enriched functional groups include proteins involved in Ras signaling, cell cycle, and pattern formation. In support of the functional significance of these findings, sumo germline clone embryos exhibited phenotypes indicative of defects in these same three processes. Our cell culture and immunolocalization studies further substantiate roles for SUMO in Ras signaling and cell cycle regulation. For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation. We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody. Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites. These studies show that SUMO coordinates multiple regulatory processes during oogenesis and early embryogenesis. In addition, our database of sumoylated proteins provides a valuable resource for those studying the roles of SUMO in development. PMID:19529778

  11. The Rho-GTPase Rnd1 Suppresses Mammary Tumorigenesis and EMT by Restraining Ras-MAPK signaling

    PubMed Central

    Okada, Tomoyo; Sinha, Surajit; Esposito, Ilaria; Schiavon, Gaia; López-Lago, Miguel A.; Su, Wenjing; Pratilas, Christine A.; Abele, Cristina; Hernandez, Jonathan M.; Ohara, Masahiro; Okada, Morihito; Viale, Agnes; Heguy, Adriana; Socci, Nicholas D.; Sapino, Anna; Seshan, Venkatraman E.; Long, Stephen; Inghirami, Giorgio; Rosen, Neal; Giancotti, Filippo G.

    2015-01-01

    SUMMARY We identified the Rho-GTPase Rnd1 as a candidate metastasis suppressor through bioinformatics analysis and showed that its depletion disrupt epithelial adhesion and polarity, induced Epithelial-to-Mesenchymal Transition (EMT), and cooperated with deregulated expression of c-Myc or loss of p53 to cause neoplastic conversion. Mechanistic studies revealed that Rnd1 suppresses Ras signalling by activating the GAP domain of Plexin B1, which inhibits Rap1. Rap1 inhibition in turn led to derepression of p120-RasGAP, which was able to inhibit Ras. Inactivation of Rnd1 in mammary epithelial cells induced highly undifferentiated and invasive tumors in mice. Conversely, Rnd1 expression inhibited spontaneous and experimental lung colonization in mouse models of metastasis. Genomic studies indicated that gene deletion in combination with epigenetic silencing or, more rarely, point mutation inactivates RND1 in human breast cancer. These results reveal a previously unappreciated mechanism through which Rnd1 restrains activation of Ras-MAPK signaling and breast tumor initiation and progression. PMID:25531777

  12. Signalling pathways: jack of all cascades.

    PubMed

    Cahill, M A; Janknecht, R; Nordheim, A

    1996-01-01

    The transcription factors that bind the c-fos promoter element SRE are targeted by multiple, independent signalling cascades; the identities of these signalling pathways and their modes of activation are being elucidated. PMID:8805215

  13. Ras and autophagy in cancer development and therapy

    PubMed Central

    Schmukler, Eran; Kloog, Yoel; Pinkas-Kramarski, Ronit

    2014-01-01

    Autophagy, a process of self-degradation and turnover of cellular components, plays a complex role in cancer. Evidence exists to show that autophagy may support tumor growth and cell survival, whereas it can also contribute to tumor suppression and have anti-survival characteristics in different cellular systems. Numerous studies have described the effects of various oncogenes and tumor suppressors on autophagy. The small GTPase Ras is an oncogene involved in the regulation of various cell-signaling pathways, and is mutated in 33% of human cancers. In the present review, we discuss the interplay between Ras and autophagy in relation to oncogenesis. It appears that Ras can upregulate or downregulate autophagy through several signaling pathways. In turn, autophagy can affect the tumorigenicity driven by Ras, resulting in either tumor progression or repression, depending on the cellular context. Furthermore, Ras inhibitors were shown to induce autophagy in several cancer cell lines. PMID:24583697

  14. Visualizing and Quantitating the Spatiotemporal Regulation of Ras/ERK Signaling by Dual-Specificity Mitogen-Activated Protein Phosphatases (MKPs).

    PubMed

    Caunt, Christopher J; Kidger, Andrew M; Keyse, Stephen M

    2016-01-01

    The spatiotemporal regulation of the Ras/ERK pathway is critical in determining the physiological and pathophysiological outcome of signaling. Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (DUSPs or MKPs) are key regulators of pathway activity and may also localize ERK to distinct subcellular locations. Here we present methods largely based on the use of high content microscopy to both visualize and quantitate the subcellular distribution of activated (p-ERK) and total ERK in populations of mouse embryonic fibroblasts derived from mice lacking DUSP5, a nuclear ERK-specific MKP. Such methods in combination with rescue experiments using adenoviral vectors encoding wild-type and mutant forms of DUSP5 have allowed us to visualize specific defects in ERK regulation in these cells thus confirming the role of this phosphatase as both a nuclear regulator of ERK activity and localization. PMID:27514808

  15. The hypervariable region of K-Ras4B is responsible for its specific interactions with Calmodulin

    PubMed Central

    Abraham, Sherwin J.; Nolet, Ryan P.; Calvert, Richard J.; Anderson, Lucy M.; Gaponenko, Vadim

    2009-01-01

    K-Ras4B belongs to the family of p21 Ras GTPases, which play an important role in cell proliferation, survival and motility. The p21 Ras proteins such as K-Ras4B, K-Ras4A, H-Ras, and N-Ras, share 85% sequence homology and activate very similar signaling pathways. Only the C-terminal hypervariable regions differ significantly. A growing body of literature demonstrates that each Ras isoform possesses unique functions in normal physiological processes as well as in pathogenesis. One of the central questions in the field of Ras biology is how these very similar proteins achieve such remarkable specificity in protein-protein interactions that regulate signal transduction pathways. Here we explore specific binding of K-Ras4B to calmodulin. Using NMR techniques and isothermal titration calorimetry we demonstrate that the hypervariable region of K-Ras contributes in a major way to the interaction with calmodulin while the catalytic domain of K-Ras4B provides a way to control the interaction by nucleotide binding. The hypervariable region of K-Ras4B binds specifically to the C-terminal domain of Ca2+-loaded calmodulin with micromolar affinity, while the GTP-γ-S loaded catalytic domain of K-Ras4B may interact with the N-terminal domain of calmodulin. PMID:19583261

  16. Leptin signalling pathways in hypothalamic neurons.

    PubMed

    Kwon, Obin; Kim, Ki Woo; Kim, Min-Seon

    2016-04-01

    Leptin is the most critical hormone in the homeostatic regulation of energy balance among those so far discovered. Leptin primarily acts on the neurons of the mediobasal part of hypothalamus to regulate food intake, thermogenesis, and the blood glucose level. In the hypothalamic neurons, leptin binding to the long form leptin receptors on the plasma membrane initiates multiple signaling cascades. The signaling pathways known to mediate the actions of leptin include JAK-STAT signaling, PI3K-Akt-FoxO1 signaling, SHP2-ERK signaling, AMPK signaling, and mTOR-S6K signaling. Recent evidence suggests that leptin signaling in hypothalamic neurons is also linked to primary cilia function. On the other hand, signaling molecules/pathways mitigating leptin actions in hypothalamic neurons have been extensively investigated in an effort to treat leptin resistance observed in obesity. These include SOCS3, tyrosine phosphatase PTP1B, and inflammatory signaling pathways such as IKK-NFκB and JNK signaling, and ER stress-mitochondrial signaling. In this review, we discuss leptin signaling pathways in the hypothalamus, with a particular focus on the most recently discovered pathways. PMID:26786898

  17. Novel Ras pathway inhibitor induces apoptosis and growth inhibition of K-ras-mutated cancer cells in vitro and in vivo.

    PubMed

    Jasinski, Piotr; Zwolak, Pawel; Terai, Kaoru; Dudek, Arkadiusz Z

    2008-11-01

    MT477 is a novel quinoline with potential activity in Ras-mutated cancers. In this study, MT477 preferentially inhibited the proliferation of K-ras-mutated human pulmonary (A549) and pancreatic (MiaPaCa-2) adenocarcinoma cell lines, compared with a non-Ras-mutated human lung squamous carcinoma cell line (H226) and normal human lung fibroblasts. MT477 treatment induced apoptosis in A549 cells and was associated with caspase-3 activation. MT477 also induced sub-G1 cell-cycle arrest in A549 cells. Although we found that MT477 partially inhibited protein kinase C (PKC), it inhibited Ras directly followed in time by inhibition of 2 Ras downstream molecules, Erk1/2 and Ral. MT477 also caused a reorganization of the actin cytoskeleton and formation of filopodias in A549 cells; this event may lead to decreased migration and invasion of tumor cells. In a xenograft mouse model, A549 tumor growth was inhibited significantly by MT477 at a dose of 1 mg/kg (P < 0.05 vs vehicle control). Taken together, these results support the conclusion that MT477 acts as a direct Ras inhibitor. This quinoline, therefore, could potentially be active in Ras-mutated cancers and could be developed extensively as an anticancer molecule with this in mind. PMID:19010291

  18. K-RAS(V12) Induces Autocrine Production of EGFR Ligands and Mediates Radioresistance Through EGFR-Dependent Akt Signaling and Activation of DNA-PKcs

    SciTech Connect

    Minjgee, Minjmaa; Toulany, Mahmoud; Kehlbach, Rainer; Giehl, Klaudia; Rodemann, H. Peter

    2011-12-01

    Purpose: It is known that postirradiation survival of tumor cells presenting mutated K-RAS is mediated through autocrine activation of epidermal growth factor receptor (EGFR). In this study the molecular mechanism of radioresistance of cells overexpressing mutated K-RAS(V12) was investigated. Methods and Materials: Head-and-neck cancer cells (FaDu) presenting wild-type K-RAS were transfected with empty vector or vector expressing mutated K-RAS(V12). The effect of K-RAS(V12) on autocrine production of EGFR ligands, activation of EGFR downstream pathways, DNA damage repair, and postirradiation survival was analyzed. Results: Conditioned medium collected from K-RAS(V12)-transfected cells enhanced activation of the phosphatidylinositol-3-kinase-Akt pathway and increased postirradiation survival of wild-type K-RAS parental cells when compared with controls. These effects were reversed by amphiregulin (AREG)-neutralizing antibody. In addition, secretion of the EGFR ligands AREG and transforming growth factor {alpha} was significantly increased upon overexpression of K-RAS(V12). Expression of mutated K-RAS(V12) resulted in an increase in radiation-induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation at S2056. This increase was accompanied by increased repair of DNA double-strand breaks. Abrogation of DNA-PKcs phosphorylation by serum depletion or AREG-neutralizing antibody underscored the role of autocrine production of EGFR ligands, namely, AREG, in regulating DNA-PKcs activation in K-RAS mutated cells. Conclusions: These data indicate that radioresistance of K-RAS mutated tumor cells is at least in part due to constitutive production of EGFR ligands, which mediate enhanced repair of DNA double-strand breaks through the EGFR-phosphatidylinositol-3-kinase-Akt cascade.

  19. YES oncogenic activity is specified by its SH4 domain and regulates RAS/MAPK signaling in colon carcinoma cells

    PubMed Central

    Dubois, Fanny; Leroy, Cédric; Simon, Valérie; Benistant, Christine; Roche, Serge

    2015-01-01

    Members of the SRC family of tyrosine kinases (SFK) display important functions in human cancer, but their specific role in tumorigenesis remains unclear. We previously demonstrated that YES regulates a unique oncogenic signaling important for colorectal cancer (CRC) progression that is not shared with SRC. Here, we addressed the underlying mechanism involved in this process. We show that YES oncogenic signaling relies on palmitoylation of its SH4 domain that controls YES localization in cholesterol-enriched membrane micro-domains. Specifically, deletion of the palmitoylation site compromised YES transforming activity, while addition of a palmitoylation site in the SH4 domain of SRC was sufficient for SRC to restore the transforming properties of cells in which YES had been silenced. Subsequently, SILAC phosphoproteomic analysis revealed that micro-domain-associated cell adhesive components and receptor tyrosine kinases are major YES substrates. YES also phosphorylates upstream regulators of RAS/MAPK signaling, including EGFR, SHC and SHP2, which were not targeted by SRC due to the absence of palmitoylation. Accordingly, EGFR-induced MAPK activity was attenuated by YES down-regulation, while increased RAS activity significantly restored cell transformation that was lost upon YES silencing. Collectively, these results uncover a critical role for the SH4 domain in the specification of SFK oncogenic activity and a selective role for YES in the induction of RAS/MAPK signaling in CRC cells. PMID:26269757

  20. Unbiased RNAi screen for hepcidin regulators links hepcidin suppression to proliferative Ras/RAF and nutrient-dependent mTOR signaling.

    PubMed

    Mleczko-Sanecka, Katarzyna; Roche, Franziska; da Silva, Ana Rita; Call, Debora; D'Alessio, Flavia; Ragab, Anan; Lapinski, Philip E; Ummanni, Ramesh; Korf, Ulrike; Oakes, Christopher; Damm, Georg; D'Alessandro, Lorenza A; Klingmüller, Ursula; King, Philip D; Boutros, Michael; Hentze, Matthias W; Muckenthaler, Martina U

    2014-03-01

    The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6-triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism. PMID:24385536

  1. Angiotensin II activates different calcium signaling pathways in adipocytes.

    PubMed

    Dolgacheva, Lyudmila P; Turovskaya, Maria V; Dynnik, Vladimir V; Zinchenko, Valery P; Goncharov, Nikolay V; Davletov, Bazbek; Turovsky, Egor A

    2016-03-01

    Angiotensin II (Ang II) is an important mammalian neurohormone involved in reninangiotensin system. Ang II is produced both constitutively and locally by RAS systems, including white fat adipocytes. The influence of Ang II on adipocytes is complex, affecting different systems of signal transduction from early Са(2+) responses to cell proliferation and differentiation, triglyceride accumulation, expression of adipokine-encoding genes and adipokine secretion. It is known that white fat adipocytes express all RAS components and Ang II receptors (АТ1 and АТ2). The current work was carried out with the primary white adipocytes culture, and Са(2+) signaling pathways activated by Ang II were investigated using fluorescent microscopy. Са(2+)-oscillations and transient responses of differentiated adipocytes to Ang II were registered in cells with both small and multiple lipid inclusions. Using inhibitory analysis and selective antagonists, we now show that Ang II initiates periodic Са(2+)-oscillations and transient responses by activating АТ1 and АТ2 receptors and involving branched signaling cascades: 1) Ang II → Gq → PLC → IP3 → IP3Rs → Ca(2+) 2) Gβγ → PI3Kγ → PKB 3) PKB → eNOS → NO → PKG 4) CD38 → cADPR → RyRs → Ca(2+) In these cascades, AT1 receptors play the leading role. The results of the present work open a perspective of using Ang II for correction of signal resistance of adipocytes often observed during obesity and type 2 diabetes. PMID:26850364

  2. Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS.

    PubMed

    Taylor-Harding, Barbie; Aspuria, Paul-Joseph; Agadjanian, Hasmik; Cheon, Dong-Joo; Mizuno, Takako; Greenberg, Danielle; Allen, Jenieke R; Spurka, Lindsay; Funari, Vincent; Spiteri, Elizabeth; Wang, Qiang; Orsulic, Sandra; Walsh, Christine; Karlan, Beth Y; Wiedemeyer, W Ruprecht

    2015-01-20

    High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2F target gene expression. We identify CCNE1 gain and RB1 loss as mechanisms of resistance to CDK4/6 inhibition, whereas receptor tyrosine kinase (RTK) and RAS signaling is associated with CDK2 inhibitor resistance. Mechanistically, we show that ETS factors are mediators of RTK/RAS signaling that cooperate with E2F in cell cycle progression. Consequently, CDK2 inhibition sensitizes cyclin E1-driven but not RAS-driven ovarian cancer cells to platinum-based chemotherapy. In summary, this study outlines a rational approach for incorporating CDKi into treatment regimens for HGSOC. PMID:25557169

  3. Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

    PubMed Central

    Taylor-Harding, Barbie; Aspuria, Paul-Joseph; Agadjanian, Hasmik; Cheon, Dong-Joo; Mizuno, Takako; Greenberg, Danielle; Allen, Jenieke R.; Spurka, Lindsay; Funari, Vincent; Spiteri, Elizabeth; Wang, Qiang; Orsulic, Sandra; Walsh, Christine; Karlan, Beth Y.; Wiedemeyer, W. Ruprecht

    2015-01-01

    High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2F target gene expression. We identify CCNE1 gain and RB1 loss as mechanisms of resistance to CDK4/6 inhibition, whereas receptor tyrosine kinase (RTK) and RAS signaling is associated with CDK2 inhibitor resistance. Mechanistically, we show that ETS factors are mediators of RTK/RAS signaling that cooperate with E2F in cell cycle progression. Consequently, CDK2 inhibition sensitizes cyclin E1-driven but not RAS-driven ovarian cancer cells to platinum-based chemotherapy. In summary, this study outlines a rational approach for incorporating CDKi into treatment regimens for HGSOC. PMID:25557169

  4. Cutaneous adverse effects of targeted therapies: Part II: Inhibitors of intracellular molecular signaling pathways.

    PubMed

    Macdonald, James B; Macdonald, Brooke; Golitz, Loren E; LoRusso, Patricia; Sekulic, Aleksandar

    2015-02-01

    The last decade has spawned an exciting new era of oncotherapy in dermatology, including the development of targeted therapies for metastatic melanoma and basal cell carcinoma. Along with skin cancer, deregulation of the PI3K-AKT-mTOR and RAS-RAF-MEK-ERK intracellular signaling pathways contributes to tumorigenesis of a multitude of other cancers, and inhibitors of these pathways are being actively studied. Similar to other classes of targeted therapies, cutaneous adverse effects are among the most frequent toxicities observed with mitogen-activated protein kinase pathway inhibitors, PI3K-AKT-mTOR inhibitors, hedgehog signaling pathway inhibitors, and immunotherapies. Given the rapid expansion of these families of targeted treatments, dermatologists will be essential in offering dermatologic supportive care measures to cancer patients being treated with these agents. Part II of this continuing medical education article reviews skin-related adverse sequelae, including the frequency of occurrence and the implications associated with on- and off-target cutaneous toxicities of inhibitors of the RAS-RAF-MEK-ERK pathway, PI3K-AKT-mTOR pathway, hedgehog signaling pathway, and immunotherapies. PMID:25592339

  5. Involvement of H- and N-Ras isoforms in transforming growth factor-{beta}1-induced proliferation and in collagen and fibronectin synthesis

    SciTech Connect

    Martinez-Salgado, Carlos . E-mail: carloms@usal.es; Fuentes-Calvo, Isabel; Garcia-Cenador, Begona; Santos, Eugenio; Lopez-Novoa, Jose M.

    2006-07-01

    Transforming growth factor {beta}1 (TGF-{beta}1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-{beta} and Ras signaling pathways are closely related: TGF-{beta}1 overcomes Ras mitogenic effects and Ras counteracts TGF-{beta} signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-{beta}1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras {sup -/-}/N-ras {sup -/-}) isoforms and from heterozygote mice (H-ras {sup +/-}/N-ras {sup +/-}). ECM synthesis is increased in basal conditions in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts, this increase being higher after stimulation with TGF-{beta}1. TGF-{beta}1-induced fibroblast proliferation is smaller in H-ras {sup -/-}/N-ras {sup -/-} than in H-ras {sup +/-}/N-ras {sup +/-} fibroblasts. Erk activation is decreased in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras.

  6. MicroRNA-132/212 family enhances arteriogenesis after hindlimb ischaemia through modulation of the Ras-MAPK pathway.

    PubMed

    Lei, Zhiyong; van Mil, Alain; Brandt, Maarten M; Grundmann, Sebastian; Hoefer, Imo; Smits, Michiel; El Azzouzi, Hamid; Fukao, Taro; Cheng, Caroline; Doevendans, Pieter A; Sluijter, Joost P G

    2015-08-01

    Arteriogenesis is a complicated process induced by increased local shear-and radial wall-stress, leading to an increase in arterial diameter. This process is enhanced by growth factors secreted by both inflammatory and endothelial cells in response to physical stress. Although therapeutic promotion of arteriogenesis is of great interest for ischaemic diseases, little is known about the modulation of the signalling cascades via microRNAs. We observed that miR-132/212 expression was significantly upregulated after occlusion of the femoral artery. miR-132/212 knockout (KO) mice display a slower perfusion recovery after hind-limb ischaemia compared to wildtype (WT) mice. Immunohistochemical analysis demonstrates a clear trend towards smaller collateral arteries in KO mice. Although Ex vivo aortic ring assays score similar number of branches in miR-132/212 KO mice compared to WT, it can be stimulated with exogenous miR-132, a dominant member of the miR-132/212 family. Moreover, in in vitro pericyte-endothelial co-culture cell assays, overexpression of miR-132 and mir-212 in endothelial cells results in enhanced vascularization, as shown by an increase in tubular structures and junctions. Our results suggested that miR-132/212 may exert their effects by enhancing the Ras-Mitogen-activated protein kinases MAPK signalling pathway through direct inhibition of Rasa1, and Spred1. The miR-132/212 cluster promotes arteriogenesis by modulating Ras-MAPK signalling via direct targeting of its inhibitors Rasa1 and Spred1. PMID:25945589

  7. [Hedgehog signaling pathway and human disorders].

    PubMed

    Fujii, Katsunori; Miyashita, Toshiyuki

    2009-07-01

    The hedgehog signaling pathway plays pivotal roles in embryonic development and cancer formation. This pathway in mammals consists of multiple molecules such as Sonic Hedgehog, PTCH, SMO, and GLI. Mutations of these components result in various human malformations or tumors, i.e., holoprosencephaly, Gorlin syndrome, Greig encephalopolysyndactyly, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, basal cell carcinomas, and medulloblastomas. Recently, small molecules that inhibit this signaling pathway were developed, and clinically applied to cancer therapy. Thus, understanding of these molecular relationships may facilitate the development of new therapies and treatments for diseases caused by hedgehog signaling disorders. PMID:19618878

  8. Metabolic Dependencies in RAS-Driven Cancers.

    PubMed

    Kimmelman, Alec C

    2015-04-15

    The ability to inhibit the RAS oncogene has been the holy grail of oncology because of the critical role of this gene in a multitude of tumor types. In addition, RAS-mutant tumors are among the most aggressive and refractory to treatment. Although directly targeting the RAS oncogene has proven challenging, an alternative approach for treating RAS-driven cancers is to inhibit critical downstream events that are required for tumor maintenance. Indeed, much focus has been put on inhibiting signaling cascades downstream of RAS. Recent studies have shown that oncogenic RAS promotes a metabolic reprogramming of tumor cells, shifting them toward an anabolic metabolism necessary to produce biomass to support unconstrained proliferation. These cancers also use a diverse set of fuel sources to meet their metabolic needs and have even developed a variety of mechanisms to act as metabolic scavengers to obtain necessary metabolic substrates from both extracellular and intracellular sources. Collectively, these adaptations can create "metabolic bottlenecks" whereby tumor cells rely on particular pathways or rate-limiting metabolites. In this regard, inhibiting individual or combinations of these metabolic pathways can attenuate growth in preclinical models. Because these dependencies are tumor selective and downstream of oncogenic RAS, there is the opportunity for therapeutic intervention. Although targeting tumor metabolism is still in the early days of translation to patients, our continued advances in understanding critical metabolic adaptations in RAS-driven cancers, as well as the ability to study this altered metabolism in relevant tumor models, will accelerate the development of new therapeutic approaches. Clin Cancer Res; 21(8); 1828-34. ©2015 AACR. See all articles in this CCR Focus section, "Targeting RAS-Driven Cancers." PMID:25878364

  9. R-Ras contributes to LTP and contextual discrimination.

    PubMed

    Darcy, M J; Jin, S-X; Feig, L A

    2014-09-26

    The ability to discriminate between closely related contexts is a specific form of hippocampal-dependent learning that may be impaired in certain neurodegenerative disorders such as Alzheimer's and Down Syndrome. However, signaling pathways regulating this form of learning are poorly understood. Previous studies have shown that the calcium-dependent exchange factor Ras-GRF1, an activator of Rac, Ras and R-Ras GTPases, is important for this form of learning and memory. Moreover, the ability to discriminate contexts was linked to the ability of Ras-GRF1 to promote high-frequency stimulation long-term potentiation (HFS-LTP) via the activation of p38 Map kinase. Here, we show that R-Ras is involved in this form of learning by using virally-delivered miRNAs targeting R-Ras into the CA1 region of the dorsal hippocampus and observing impaired contextual discrimination. Like the loss of GRF1, knockdown of R-Ras in the CA1 also impairs the induction of HFS-LTP and p38 Map kinase. Nevertheless, experiments indicate that this involvement of R-Ras in HFS-LTP that is required for contextual discrimination is independent of Ras-GRF1. Thus, R-Ras is a novel regulator of a form of hippocampal-dependent LTP as well as learning and memory that is affected in certain forms of neurodegenerative diseases. PMID:25043327

  10. Ras1-Mediated Modulation of Drosophila Homeotic Function in Cell and Segment Identity

    PubMed Central

    Boube, M.; Benassayag, C.; Seroude, L.; Cribbs, D. L.

    1997-01-01

    Mutations of the Drosophila homeotic proboscipedia gene (pb; the Hox-A2/B2 homologue) provoke dose-sensitive defects. These were used to search for dose-sensitive dominant modifiers of pb function. Two identified interacting genes were the proto-oncogene Ras1 and its functional antagonist Gap1, prominent intermediaries in known signal transduction pathways. Ras1(+) is a positive modifier of pb activity both in normal and ectopic cell contexts, while the Ras1-antagonist Gap1 has an opposite effect. A general role for Ras1 in homeotic function is likely, since Ras1(+) activity also modulates functions of the homeotic loci Sex combs reduced and Ultrabithorax. Our data suggest that the modulation occurs by a mechanism independent of transcriptional control of the homeotic loci themselves, or of the Ras1/Gap1 genes. Taken together our data support a role for Ras1-mediated cell signaling in the homeotic control of segmental differentiation. PMID:9178011

  11. Function and Regulation in MAPK Signaling Pathways

    PubMed Central

    Chen, Raymond E.; Thorner, Jeremy

    2007-01-01

    Signaling pathways that activate different mitogen-activated protein kinases (MAPKs) elicit many of the responses that are evoked in cells by changes in certain environmental conditions and upon exposure to a variety of hormonal and other stimuli. These pathways were first elucidated in the unicellular eukaryote Saccharomyces cerevisiae (budding yeast). Studies of MAPK pathways in this organism continue to be especially informative in revealing the molecular mechanisms by which MAPK cascades operate, propagate signals, modulate cellular processes, and are controlled by regulatory factors both internal to and external to the pathways. Here we highlight recent advances and new insights about MAPK-based signaling that have been made through studies in yeast, which provide lessons directly applicable to, and that enhance our understanding of, MAPK-mediated signaling in mammalian cells. PMID:17604854

  12. Modularized TGFbeta-Smad Signaling Pathway

    NASA Technical Reports Server (NTRS)

    Li, Yongfeng; Wang, M.; Carra, C.; Cucinotta, F. A.

    2011-01-01

    The Transforming Growth Factor beta (TGFbeta) signaling pathway is a prominent regulatory signaling pathway controlling various important cellular processes. It can be induced by several factors, including ionizing radiation. It is regulated by Smads in a negative feedback loop through promoting increases in the regulatory Smads in the cell nucleus, and subsequent expression of inhibitory Smad, Smad7 to form a ubiquitin ligase with Smurf targeting active TGF receptors for degradation. In this work, we proposed a mathematical model to study the radiation-induced Smad-regulated TGF signaling pathway. By modularization, we are able to analyze each module (subsystem) and recover the nonlinear dynamics of the entire network system. Meanwhile the excitability, a common feature observed in the biological systems, along the TGF signaling pathway is discussed by mathematical analysis and numerical simulation.

  13. Premetazoan origin of the Hippo signaling pathway

    PubMed Central

    Sebé-Pedrós, Arnau; Zheng, Yonggang; Ruiz-Trillo, Iñaki; Pan, Duojia

    2012-01-01

    Summary Non-aggregative multicellularity requires strict control of cell number. The Hippo signaling pathway coordinates cell proliferation and apoptosis and is a central regulator of organ size in animals. Recent studies have shown the presence of key members of the Hippo pathway in non-bilaterian animals, but failed to identify this pathway outside Metazoa. Through comparative analyses of recently sequenced holozoan genomes, we show that Hippo pathway components, such as the kinases Hippo and Warts, the co-activator Yorkie and the transcription factor Scalloped, were already present in the unicellular ancestors of animals. Remarkably, functional analysis of Hippo components of the amoeboid holozoan Capsaspora owczarzaki, performed in Drosophila, demonstrate that the growth-regulatory activity of the Hippo pathway is conserved in this unicellular lineage. Our findings show that the Hippo pathway evolved well before the origin of Metazoa and highlight the importance of Hippo signaling as a key developmental mechanism pre-dating the origin of Metazoa. PMID:22832104

  14. Fungal Communication Requires the MAK-2 Pathway Elements STE-20 and RAS-2, the NRC-1 Adapter STE-50 and the MAP Kinase Scaffold HAM-5

    PubMed Central

    Dettmann, Anne; Heilig, Yvonne; Valerius, Oliver; Ludwig, Sarah; Seiler, Stephan

    2014-01-01

    Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell–cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell–cell communication in fungi and higher eukaryotes. PMID:25411845

  15. NF2 loss promotes oncogenic RAS-induced thyroid cancers via YAP-dependent transactivation of RAS proteins and sensitizes them to MEK inhibition

    PubMed Central

    Garcia-Rendueles, Maria E.R.; Ricarte-Filho, Julio C.; Untch, Brian R.; Landa, Iňigo; Knauf, Jeffrey A.; Voza, Francesca; Smith, Vicki E.; Ganly, Ian; Taylor, Barry S.; Persaud, Yogindra; Oler, Gisele; Fang, Yuqiang; Jhanwar, Suresh C.; Viale, Agnes; Heguy, Adriana; Huberman, Kety H.; Giancotti, Filippo; Ghossein, Ronald; Fagin, James A.

    2015-01-01

    Ch22q LOH is preferentially associated with RAS mutations in papillary and in poorly differentiated thyroid cancer (PDTC). The 22q tumor suppressor NF2, encoding merlin, is implicated in this interaction because of its frequent loss of function in human thyroid cancer cell lines. Nf2 deletion or Hras mutation are insufficient for transformation, whereas their combined disruption leads to murine PDTC with increased MAPK signaling. Merlin loss induces RAS signaling in part through inactivation of Hippo, which activates a YAP-TEAD transcriptional program. We find that the three RAS genes are themselves YAP-TEAD1 transcriptional targets, providing a novel mechanism of promotion of RAS-induced tumorigenesis. Moreover, pharmacological disruption of YAP-TEAD with verteporfin blocks RAS transcription and signaling, and inhibits cell growth. The increased MAPK output generated by NF2 loss in RAS-mutant cancers may inform therapeutic strategies, as it generates greater dependency on the MAPK pathway for viability. PMID:26359368

  16. Nucleophosmin Mutants Promote Adhesion, Migration and Invasion of Human Leukemia THP-1 Cells through MMPs Up-regulation via Ras/ERK MAPK Signaling

    PubMed Central

    Xian, Jingrong; Shao, Huiyuan; Chen, Xianchun; Zhang, Shuaishuai; Quan, Jing; Zou, Qin; Jin, Hongjun; Zhang, Ling

    2016-01-01

    Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been defined as a unique subgroup in the new classification of myeloid neoplasm, and the AML patients with mutated NPM1 frequently present extramedullary infiltration, but how NPM1 mutants regulate this process remains elusive. In this study, we found that overexpression of type A NPM1 gene mutation (NPM1-mA) enhanced the adhesive, migratory and invasive potential in THP-1 AML cells lacking mutated NPM1. NPM1-mA had up-regulated expression and gelatinolytic matrix metalloprotease-2 (MMP-2)/MMP-9 activity, as assessed by real-time PCR, western blotting and gelatin zymography. Following immunoprecipitation analysis to identify the interaction of NPM1-mA with K-Ras, we focused on the effect of NPM1-mA overexpression on the Ras/Mitogen-activated protein kinase (MAPK) signaling axis and showed that NPM1-mA increased the MEK and ERK phosphorylation levels, as evaluated by western blotting. Notably, a specific inhibitor of the ERK/MAPK pathway (PD98059), but not p38/MAPK, JNK/MAPK or PI3-K/AKT inhibitors, markedly decreased the cell invasion numbers in a transwell assay. Further experiments demonstrated that blocking the ERK/MAPK pathway by PD98059 resulted in reduced MMP-2/9 protein levels and MMP-9 activity. Additionally, NPM1-mA overexpression had down-regulated gene expression and protein production of tissue inhibitor of MMP-2 (TIMP-2) in THP-1 cells. Furthermore, evaluation of gene expression data from The Cancer Genome Atlas (TCGA) dataset revealed that MMP-2 was overexpressed in AML patient samples with NPM1 mutated and high MMP-2 expression associated with leukemic skin infiltration. Taken together, our results reveal that NPM1 mutations contribute to the invasive potential of AML cells through MMPs up-regulation via Ras/ERK MAPK signaling pathway activation and offer novel insights into the potential role of NPM1 mutations in leukemogenesis. PMID:26884713

  17. A Novel Ras Effector Pathway Found to Play Significant Role in Tumor Suppression | Poster

    Cancer.gov

    By Nancy Parrish, Staff Writer; photo by Richard Frederickson, Staff Photographer Normal cells have mechanisms to prevent the development of cancer. Among these is a type of tumor suppressor mechanism known as oncogene-induced senescence, or OIS, which halts the uncontrolled growth of cells caused by mutations in oncogenes. The oncogene Ras plays a crucial role in inducing OIS through a specific cascade of proteins, as reported in a recent article in Molecular and Cellular Biology by Jacqueline Salotti, Ph.D., and colleagues in the Eukaryotic Transcriptional Regulation Section of the Mouse Cancer Genetics Program, Center for Cancer Research (CCR).

  18. A Novel Ras Effector Pathway Found to Play Significant Role in Tumor Suppression | Poster

    Cancer.gov

    By Nancy Parrish, Staff Writer; photo by Richard Frederickson, Staff Photographer Normal cells have mechanisms to prevent the development of cancer. Among these is a type of tumor suppressor mechanism known as oncogene-induced senescence, or OIS, which halts the uncontrolled growth of cells caused by mutations in oncogenes. The oncogene Ras plays a crucial role in inducing OIS through a specific cascade of proteins, as reported in a recent article in Molecular and Cellular Biology by Jacqueline Salotti, Ph.D., and colleagues in the Eukaryotic Transcriptional Regulation Section of the Mouse Cancer Genetics Program, Center for Cancer Research (CCR).

  19. Mutational analysis of PI3K/AKT and RAS/RAF pathway activation in malignant salivary gland tumours with a new mutation of PIK3CA.

    PubMed

    Shalmon, B; Drendel, M; Wolf, M; Hirshberg, A; Cohen, Y

    2016-06-01

    The phosphoinositide 3-kinase (PIK3)/v-akt murine thymoma (AKT) oncogene pathway and the RAS/RAF pathway are involved in regulating the signalling of multiple biological processes, including apoptosis, metabolism, cell proliferation, and cell growth. Mutations in the genes within these pathways are frequently found in several tumours. The aim of this study was to investigate the frequency of mutations in the PIK3CA, BRAF, and KRAS genes in cases of malignant salivary gland tumours. Mutational analysis of the PIK3CA, KRAS, and BRAF genes was performed by direct sequencing of material from 21 patients with malignant salivary gland tumours who underwent surgery between 1992 and 2001. No mutations were found in the KRAS exon 2, BRAF exon 15, or PIK3CA exon 9 genes. However, an unpublished mutation of the PIK3CA gene in exon 20 (W1051 stop mutation) was found in one case of adenocarcinoma NOS. The impact of this mutation on the biological behaviour of the tumour has yet to be explored, however the patient with adenocarcinoma NOS harbouring this mutation has survived for over 20 years following surgery despite a high stage at presentation. Further studies with more homogeneous patient cohorts are needed to address whether this mutation reflects a different clinical presentation and may benefit from targeted treatment strategies. PMID:26811072

  20. Optogenetic control of intracellular signaling pathways

    PubMed Central

    Zhang, Kai; Cui, Bianxiao

    2014-01-01

    Cells employ a plethora of signaling pathways to make their life-and-death decisions. Extensive genetic, biochemical, and physiological studies have led to the accumulation of knowledge about signaling components and their interactions within signaling networks. These conventional approaches, though useful, lack the ability to control the spatial and temporal aspects of signaling processes. The recently emerged optogenetic tools open up exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution, easy delivery, rapid reversibility, fewer off-target side effects, and the ability to dissect complex signaling networks. Here we review recent achievements in using light to control intracellular signaling pathways, and discuss future prospects for the field, including integration of new genetic approaches into optogenetics. PMID:25529484

  1. Analysis of corkscrew signaling in the Drosophila epidermal growth factor receptor pathway during myogenesis.

    PubMed Central

    Johnson Hamlet, M R; Perkins, L A

    2001-01-01

    The Drosophila nonreceptor protein tyrosine phosphatase, Corkscrew (Csw), functions positively in multiple receptor tyrosine kinase (RTK) pathways, including signaling by the epidermal growth factor receptor (EGFR). Detailed phenotypic analyses of csw mutations have revealed that Csw activity is required in many of the same developmental processes that require EGFR function. However, it is still unclear where in the signaling hierarchy Csw functions relative to other proteins whose activities are also required downstream of the receptor. To address this issue, genetic interaction experiments were performed to place csw gene activity relative to the EGFR, spitz (spi), rhomboid (rho), daughter of sevenless (DOS), kinase-suppressor of ras (ksr), ras1, D-raf, pointed (pnt), and moleskin. We followed the EGFR-dependent formation of VA2 muscle precursor cells as a sensitive assay for these genetic interaction studies. First, we established that Csw has a positive function during mesoderm development. Second, we found that tissue-specific expression of a gain-of-function csw construct rescues loss-of-function mutations in other positive signaling genes upstream of rolled (rl)/MAPK in the EGFR pathway. Third, we were able to infer levels of EGFR signaling in various mutant backgrounds during myogenesis. This work extends previous studies of Csw during Torso and Sevenless RTK signaling to include an in-depth analysis of the role of Csw in the EGFR signaling pathway. PMID:11729154

  2. Review: Ras GTPases and myosin: Qualitative conservation and quantitative diversification in signal and energy transduction.

    PubMed

    Mueller, Matthias P; Goody, Roger S

    2016-08-01

    Most GTPases and many ATPases belong to the P-loop class of proteins with significant structural and mechanistic similarities. Here we compare and contrast the basic properties of the Ras family GTPases and myosin, and conclude that there are fundamental similarities but also distinct differences related to their specific roles. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 422-430, 2016. PMID:27018658

  3. Advances in Targeting Signal Transduction Pathways

    PubMed Central

    McCubrey, James A.; Steelman, Linda S.; Chappell, William H.; Sun, Lin; Davis, Nicole M.; Abrams, Stephen L.; Franklin, Richard A.; Cocco, Lucio; Evangelisti, Camilla; Chiarini, Francesca; Martelli, Alberto M.; Libra, Massimo; Candido, Saverio; Ligresti, Giovanni; Malaponte, Grazia; Mazzarino, Maria C.; Fagone, Paolo; Donia, Marco; Nicoletti, Ferdinando; Polesel, Jerry; Talamini, Renato; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Dulińska-Litewka, Joanna; Laidler, Piotr; D'Assoro, Antonio B.; Drobot, Lyudmyla; Umezawa, Kazuo; Montalto, Giuseppe; Cervello, Melchiorre; Demidenko, Zoya N.

    2012-01-01

    Over the past few years, significant advances have occurred in both our understanding of the complexity of signal transduction pathways as well as the isolation of specific inhibitors which target key components in those pathways. Furthermore critical information is being accrued regarding how genetic mutations can affect the sensitivity of various types of patients to targeted therapy. Finally, genetic mechanisms responsible for the development of resistance after targeted therapy are being discovered which may allow the creation of alternative therapies to overcome resistance. This review will discuss some of the highlights over the past few years on the roles of key signaling pathways in various diseases, the targeting of signal transduction pathways and the genetic mechanisms governing sensitivity and resistance to targeted therapies. PMID:23455493

  4. Compartmentalized Ras Proteins Transform NIH 3T3 Cells with Different Efficiencies▿ †

    PubMed Central

    Cheng, Chiang-Min; Li, Huiling; Gasman, Stéphane; Huang, Jian; Schiff, Rachel; Chang, Eric C.

    2011-01-01

    Ras GTPases were long thought to function exclusively from the plasma membrane (PM). However, a current model suggests that Ras proteins can compartmentalize to regulate different functions, and an oncogenic H-Ras mutant that is restricted to the endomembrane can still transform cells. In this study, we demonstrated that cells transformed by endomembrane-restricted oncogenic H-Ras formed tumors in nude mice. To define downstream targets of endomembrane Ras pathways, we analyzed Cdc42, which concentrates in the endomembrane and has been shown to act downstream of Ras in Schizosaccharomyces pombe. Our data show that cell transformation induced by endomembrane-restricted oncogenic H-Ras was blocked when Cdc42 activity was inhibited. Moreover, H-Ras formed a complex with Cdc42 on the endomembrane, and this interaction was enhanced when H-Ras was GTP bound or when cells were stimulated by growth factors. H-Ras binding evidently induced Cdc42 activation by recruiting and/or activating Cdc42 exchange factors. In contrast, when constitutively active H-Ras was restricted to the PM by fusing to a PM localization signal from the Rit GTPase, the resulting protein did not detectably activate Cdc42 although it activated Raf-1 and efficiently induced hallmarks of Ras-induced senescence in human BJ foreskin fibroblasts. Surprisingly, PM-restricted oncogenic Ras when expressed alone could only weakly transform NIH 3T3 cells; however, when constitutively active Cdc42 was coexpressed, together they transformed cells much more efficiently than either one alone. These data suggest that efficient cell transformation requires Ras proteins to interact with Cdc42 on the endomembrane and that in order for a given Ras protein to fully transform cells, multiple compartment-specific Ras pathways need to work cooperatively. PMID:21189290

  5. The Hedgehog signalling pathway in bone formation

    PubMed Central

    Yang, Jing; Andre, Philipp; Ye, Ling; Yang, Ying-Zi

    2015-01-01

    The Hedgehog (Hh) signalling pathway plays many important roles in development, homeostasis and tumorigenesis. The critical function of Hh signalling in bone formation has been identified in the past two decades. Here, we review the evolutionarily conserved Hh signalling mechanisms with an emphasis on the functions of the Hh signalling pathway in bone development, homeostasis and diseases. In the early stages of embryonic limb development, Sonic Hedgehog (Shh) acts as a major morphogen in patterning the limb buds. Indian Hedgehog (Ihh) has an essential function in endochondral ossification and induces osteoblast differentiation in the perichondrium. Hh signalling is also involved intramembrane ossification. Interactions between Hh and Wnt signalling regulate cartilage development, endochondral bone formation and synovial joint formation. Hh also plays an important role in bone homeostasis, and reducing Hh signalling protects against age-related bone loss. Disruption of Hh signalling regulation leads to multiple bone diseases, such as progressive osseous heteroplasia. Therefore, understanding the signalling mechanisms and functions of Hh signalling in bone development, homeostasis and diseases will provide important insights into bone disease prevention, diagnoses and therapeutics. PMID:26023726

  6. Cell Type-Specific Activation of AKT and ERK Signaling Pathways by Small Negatively-Charged Magnetic Nanoparticles

    PubMed Central

    Rauch, Jens; Kolch, Walter; Mahmoudi, Morteza

    2012-01-01

    The interaction of nanoparticles (NPs) with living organisms has become a focus of public and scientific debate due to their potential wide applications in biomedicine, but also because of unwanted side effects. Here, we show that superparamagnetic iron oxide NPs (SPIONs) with different surface coatings can differentially affect signal transduction pathways. Using isogenic pairs of breast and colon derived cell lines we found that the stimulation of ERK and AKT signaling pathways by SPIONs is selectively dependent on the cell type and SPION type. In general, cells with Ras mutations respond better than their non-mutant counterparts. Small negatively charged SPIONs (snSPIONs) activated ERK to a similar extent as epidermal growth factor (EGF), and used the same upstream signaling components including activation of the EGF receptor. Importantly, snSPIONs stimulated the proliferation of Ras transformed breast epithelial cells as efficiently as EGF suggesting that NPs can mimic physiological growth factors. PMID:23162692

  7. Cell Type-Specific Activation of AKT and ERK Signaling Pathways by Small Negatively-Charged Magnetic Nanoparticles

    NASA Astrophysics Data System (ADS)

    Rauch, Jens; Kolch, Walter; Mahmoudi, Morteza

    2012-11-01

    The interaction of nanoparticles (NPs) with living organisms has become a focus of public and scientific debate due to their potential wide applications in biomedicine, but also because of unwanted side effects. Here, we show that superparamagnetic iron oxide NPs (SPIONs) with different surface coatings can differentially affect signal transduction pathways. Using isogenic pairs of breast and colon derived cell lines we found that the stimulation of ERK and AKT signaling pathways by SPIONs is selectively dependent on the cell type and SPION type. In general, cells with Ras mutations respond better than their non-mutant counterparts. Small negatively charged SPIONs (snSPIONs) activated ERK to a similar extent as epidermal growth factor (EGF), and used the same upstream signaling components including activation of the EGF receptor. Importantly, snSPIONs stimulated the proliferation of Ras transformed breast epithelial cells as efficiently as EGF suggesting that NPs can mimic physiological growth factors.

  8. GPCR signaling along the endocytic pathway

    PubMed Central

    Irannejad, Roshanak; von Zastrow, Mark

    2016-01-01

    Many G protein-coupled receptors (GPCRs) internalize after agonist-induced activation. While endocytosis has long been associated with homeostatic attenuation of cellular responsiveness, accumulating evidence from study of a wide range of eukaryotes reveals that the endocytic pathway also contributes to generating receptor-initiated signals themselves. Here we review recent progress in this area, discussing primarily but not exclusively GPCR signaling in mammalian cells. PMID:24680436

  9. Semaphorin-7a reverses the ERF-induced inhibition of EMT in Ras-dependent mouse mammary epithelial cells.

    PubMed

    Allegra, Maryline; Zaragkoulias, Andreas; Vorgia, Elena; Ioannou, Marina; Litos, Gabriele; Beug, Hartmut; Mavrothalassitis, George

    2012-10-01

    Epithelial-to-mesenchymal transition (EMT) is a key process in cancer progression and metastasis, requiring cooperation of the epidermal growth factor/Ras with the transforming growth factor-β (TGF-β) signaling pathway in a multistep process. The molecular mechanisms by which Ras signaling contributes to EMT, however, remain elusive to a large extent. We therefore examined the transcriptional repressor Ets2-repressor factor (ERF)-a bona fide Ras-extracellular signal-regulated kinase/mitogen-activated protein kinase effector-for its ability to interfere with TGF-β-induced EMT in mammary epithelial cells (EpH4) expressing oncogenic Ras (EpRas). ERF-overexpressing EpRas cells failed to undergo TGF-β-induced EMT, formed three-dimensional tubular structures in collagen gels, and retained expression of epithelial markers. Transcriptome analysis indicated that TGF-β signaling through Smads was mostly unaffected, and ERF suppressed the TGF-β-induced EMT via Semaphorin-7a repression. Forced expression of Semaphorin-7a in ERF-overexpressing EpRas cells reestablished their ability to undergo EMT. In contrast, inhibition of Semaphorin-7a in the parental EpRas cells inhibited their ability to undergo TGF-β-induced EMT. Our data suggest that oncogenic Ras may play an additional role in EMT via the ERF, regulating Semaphorin-7a and providing a new interconnection between the Ras- and the TGF-β-signaling pathways. PMID:22875994

  10. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding

    PubMed Central

    Ting, Pamela Y.; Johnson, Christian W.; Fang, Cong; Cao, Xiaoqing; Graeber, Thomas G.; Mattos, Carla; Colicelli, John

    2015-01-01

    RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr137. Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr137 phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr137 is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRASY137F and HRASY137E revealed conformation changes radiating from the mutated residue. Although consistent with Tyr137 participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr137 phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRASG12V with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr137 allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.—Ting, P. Y., Johnson, C. W., Fang, C., Cao, X., Graeber, T. G., Mattos, C., Colicelli, J. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding. PMID:25999467