DOCSLIB.ORG

High-Intensity Raf Signals Convert Mitotic Cell Cycling Into Cellular Growth'

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
High-Intensity Raf Signals Convert Mitotic Cell Cycling Into Cellular Growth' (CANCERRESEARCH58. 1636-1640. April 15. 19981 Advances in Brief High-intensity Raf Signals Convert Mitotic Cell Cycling into Cellular Growth' Eugen Kerkhoff and Ulf R. Rapp2 lnstitutfürmedizinischeStrahlenkundeund Zellforschung(MSZ). Universityof Wurzburg,97078 Wiirzburg, Germany Abstract oncogernc c-Raf-1-estrogen receptor fusion protein (c-Raf-1-BxB ERTM). Activation of the exogenous kinase induces high and sustained The selectionof NIH 3T3 cellsexpressinga hydroxytamoxifen-induci c-Raf signals and inhibits DNA synthesis and mitosis. Despite the ble c-Raf-1-estrogen receptor fusion protein (c-Raf-1-BxB-ER@) in the inhibition of cell cycle progression, cell growth remains unaffected, absence or presence of the inducer results in dramatic differences in the expression levels of the fusion protein. Hydroxytamoxifen-mediated con leading to cells with a DNA content of G1 cells and a cell size of stitutive activation of the Rafsignal favors the selection ofcells expressing G2-M-phase cells. Therefore, high-intensity Raf signals uncouple the low levels of c-R.af-1-BxB-ER@. Cells selected in the absence of hy regulation of cell growth from DNA replication and mitosis. droxytamoxifen express up to 20 tImes higher levels of the inducible Raf kinase. Activation ofthe oncogenic Rafkinase in cells expressing low levels Materials and Methods leads to a weak activation of the Raf/Mek/Erk cascade and the induction of S phase In confluentcells.The activationof cellsexpressinghigh levels Expression Vector. The pBabe-puro-BxB-ER@ vector was constructed by ofthe kinaseleadsto a strongpersIstentsignalandinhibitsDNAsynthesis releasing sequences encoding the c-Raf-l-BxB-ER@ protein from the BJ4- andmitOsisinproliferatingcells.TheinhibitionofDNAsynthesisandcell BxB-ER@ vector (7) by EcoRI digestion (2.2-kb fragment) and inserting them dIvision is presumably due to the elevated expression ofthe cyclin-depend into the unique EcoRI recognition site of the pBabe-puro vector (13). @@ ent kinaseinhibitorp21@', cellsexposedto ionizingradiation. Cell Culture and Transfections. NIH 313 and N-BxB-ER'@ cells were Despite the inhibition of DNA synthesis and mitOSIS, the constltutive grown in DMEM (Life Technologies, Inc.) supplemented with glucose (Life activity of the Raf signaling pathway is still able to initiate cell growth. Technologies, Inc.), L-glutamine (Life Technologies, Inc.), 10% FCS (Hy Activationofthe high-intensityRafsignalin arrestedserum-starvedcells clone), 100 units/mi penicillin (Life Technologies,Inc.), and 100 @&g/mi induces cell growth up to a size corresponding to that of M-phase cells in streptomycin (Life Technologies, Inc.). The cells were incubated at 37°C,5% the absenceof DNAsynthesis.High-intensityRafsignalsin proliferating CO2.and90%humidity.FortheinductionofcellswithOHT,asolutionof 1 cells consistently lead to an accumulation of cells with the size of M-phase mM OHT in ethanol was added to the medium to a final concentration of 200 cellsandthe DNAcontentof G1cellsor G2-M-phasecells.Therefore,the flM (5000 X dilution). For puromycin selection, the cells were grown in the activation of Raf kinase is sufficient to drive cell growth, even in the presence of 6 @tg/mipuromycin (Sigma). The cells were transfected with a presence of high levels of the cydlin-dependent kinase inhibitor p21@1. high-efficiency liposome transfection method, using LipofectAMiNE reagent supplied by Life Technologies, Inc. (7). Introduction Immunoblot and Antibodies. Immunobbot experiments were performed as described previously (7). The following antibodies were used: (a) HL7, The Ras/Raf signal transduction cascade is an important mediator mouseestrogenreceptor-sensitiverabbitpolycbonalantibody(a gift from A. in the regulation of cell proliferation and differentiation. Experiments Sewing and H. Land); (b) C-19, cyclin A-sensitive affmity-purified rabbit in the rat pheochromocytoma cell line PC12 led to the discovery that polyclonal antibody (Santa Cruz); (c) C-19, p2l@@'-scnsifive affmity-purified the intensity and duration of the signal determine proliferation or rabbit pobyclonal antibody (Santa Cruz); (d) M-2, cdk2-sensitive affinity neuronal differentiation and cell cycle arrest (1). The extended activity purified rabbit pobycbonal antibody (Santa Cruz); (e) C-22, cdk4-sensitive affinity-purified rabbit polycbonal antibody (Santa Cruz); (f) phospho Eat of the RasfMeklErk cascade in this respect favors cellular differenti sensitive affinity-purified rabbit polyclonab antibody (New England Biolabs); ation. Constitutive activation of the Ras/Raf cascade in immortalized and (g) C-l6, Erkl-sensitive affinity-purified rabbit polycbonalantibody (Santa fibroblast cell lines can induce either cell cycle progression or cell Cruz). cycle arrest in the G@or G2-M phases (2—9).Ithas long been proposed Immunocomplex Kinase Assay. The kinase assays were performed as that high activity of the Ras signal is responsible for the growth arrest, described previously (7), with the following specifications. Cells were lysed because the expression of an activated Ras oncoprotein is only toler with a buffer containing 50 nmi HEPES (pH 7.5), 150 mMNaG, 1 mMEDTA, ated at very low levels in the absence of a second cooperating 1 mMEGTA, 0.1% (v/v) Tween 20, 10% (v/v) glycerol, 2 @&g/mileupeptin,2 oncogene (2, 3). Experiments using dominant negative mutants of p53 lLg/mlaprotinin, 1 m@iphenylmethylsulfonybfluoride, 10mMsodium fluoride, protein have led to the conclusion that p53-related activity is an and 0.2 mM sodium orthovanadate. For immunodetection, the following anti important mediator of Ras-induced cell cycle arrest (10). The p21'@'@ bodies were used: (a) M-2, cdk2-sensitive affinity-purified rabbit polycbonal antibody (Santa Cruz); and (b) C-22, cdk4-sensitive affinity-purified rabbit cdk3 inhibitor is induced by p53 or p73, a recently discovered p53 polycbonalantibody (Santa Cruz). As substrates for the kinase reaction, 1 g@g homobogue (11, 12). Fibroblast cells isolated from p2l@'1-deficient of histone Hi (Boebringer) or 6 g.@gofGST-Rb (an 769-921; a kind gift from mice cannot be arrested by high-intensity Raf signals, indicating that R. Lilischkis) were used for each reaction. the cdk inhibitor is essential for Ras/Raf-mediated cell cycle arrest (9). Flow CytOmetry Analysis. The cells were fixed in ethanol, stained with We have established cell lines that express high levels of an inducible propidium iodide (Sigma), and analyzed with a FACSCalibur flow cytometer (BectonDickinson;Ref.7). Received 2/25/98; accepted 3/2/98. Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage Results charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. High expression of constitutively active forms of the Ran or Raf I Supported by the Wilhelm Sander-Stiftung and the SFB 456. 2 To whom requests for reprints should be addressed, at Institut für medizinische oncoproteins has been shown to block cell proliferation (2, 3, 8, 9). To StrahlenkundeundZellforsehung.UniversityofWtirzburg,VersbacherStrasse5,97078 analyze the effects of high-intensity Raf signals on the cell cycle, it is Wurzburg, Germany. Phone: 49-931-201-5140; Fax: 49-931-201-3835. therefore essential to establish conditional cell lines expressing an 3 The abbreviations used are: cdk, cycin-dependent kinase; OHT, 4-hydroxytamox ifen; as. amino acid. inducible form of the oncoprotein. Fusion of the oncogernc c-Raf-l 1636 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1998 American Association for Cancer Research. @@@@@— — — Raf SIGNALS AND CELL GROWTH BxB deletion mutant to the hormone-binding domain of the estrogen N-BxB-ER@cells receptor (c-Raf-1-BxB-ER@) provides a 4 OHT-inducible kinase (7). On treatment with the inducer, the fusion protein is constitutively active. We transfected NIH 3T3 cells with a eukaryotic expression vector driving the constitutive expression of the c@Raf@l@BxB@ERTM kinase and a puromycin resistance protein (pBabe-puro-BxB-ER@). F)*d.1+@' fi4el+CIfI 0.1+0111 Selection for puromycin-resistant colonies in the presence or absence O@O30 224 30 2 24 0 10 30 2 24 of OHT resultsin dramaticdifferences in the expression levels of the oncogenic Raf kinase. Clonal cell lines selected while the kinase was —@----@----- @@@@@@@1@— —cyclinA active (N-BxB-ER@-FA clones) express around 20-fold less of the c-Raf-1-BxB-ER@ protein than clones selected in the absence of 01ff (N@BxB@ERTM@Clclones),in whichthe kinaseis inactive(Fig. 1). This is in agreement with the finding that high levels of activity of the oncogenic Raf kinase areinhibitoryfor cell proliferation.The fact @ that all four isolated FA clones express comparable low levels of the .@——- —Ii =pbosphoErkl,2 inducible c-Raf-1 kinase suggests that there is a sharp tolerance threshold up to which proliferation is possible. The addition of 01ff to all clones expressing high levels of the :::__@__@_@_ —ii: .-Erkl,2 inducible Raf kinase causes a cell cycle arrest in the G1 and G2-M phases (Fig. 4B; data not shown). Within 24 h, the cells go out of S Fig. 2. Activation of the c-Rrf-l-BxB-ER@ protein in N@BxB@ERTM@Cl@lcellsby OHT leads to the induction of a strong and persistent Raf signal as measured by Erk phospho phase and accumulate with a 2N and 4N DNA content. All additional rylation and a rapid induction of overexpression of the cdk inhibitor [email protected] analyses were performed with the cell clones N-BxB-ER@-Cl-l
Load more

Recommended publications
  • Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling
    G C A T T A C G G C A T genes Review Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling Aoife A. Nolan 1, Nourhan K. Aboud 1, Walter Kolch 1,2,* and David Matallanas 1,* 1 Systems Biology Ireland, School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland; [email protected] (A.A.N.); [email protected] (N.K.A.) 2 Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland * Correspondence: [email protected] (W.K.); [email protected] (D.M.) Abstract: Oncogenic RAS (Rat sarcoma) mutations drive more than half of human cancers, and RAS inhibition is the holy grail of oncology. Thirty years of relentless efforts and harsh disappointments have taught us about the intricacies of oncogenic RAS signalling that allow us to now get a pharma- cological grip on this elusive protein. The inhibition of effector pathways, such as the RAF-MEK-ERK pathway, has largely proven disappointing. Thus far, most of these efforts were aimed at blocking the activation of ERK. Here, we discuss RAF-dependent pathways that are regulated through RAF functions independent of catalytic activity and their potential role as targets to block oncogenic RAS signalling. We focus on the now well documented roles of RAF kinase-independent functions in apoptosis, cell cycle progression and cell migration. Keywords: RAF kinase-independent; RAS; MST2; ASK; PLK; RHO-α; apoptosis; cell cycle; cancer therapy Citation: Nolan, A.A.; Aboud, N.K.; Kolch, W.; Matallanas, D. Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling.
    [Show full text]
  • RAF Protein-Serine/Threonine Kinases: Structure and Regulation
    Biochemical and Biophysical Research Communications 399 (2010) 313–317 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc Mini Review RAF protein-serine/threonine kinases: Structure and regulation Robert Roskoski Jr. * Blue Ridge Institute for Medical Research, 3754 Brevard Road, Suite 116, Box 19, Horse Shoe, NC 28742, USA article info abstract Article history: A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the Received 12 July 2010 RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large vari- Available online 30 July 2010 ety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transforma- tion to the cancerous state. RAS mutations occur in 15–30% of all human cancers, and B-RAF mutations Keywords: occur in 30–60% of melanomas, 30–50% of thyroid cancers, and 5–20% of colorectal cancers. Activation 14-3-3 of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phos- ERK phorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The for- GDC-0879 mation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are MEK Melanoma effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu PLX4032 activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells PLX4720 containing wild-type B-RAF and wild-type or activated mutant RAS.
    [Show full text]
  • Mutation-Specific RAS Oncogenicity Explains NRAS Codon 61 Selection in Melanoma
    Published OnlineFirst September 24, 2014; DOI: 10.1158/2159-8290.CD-14-0729 reseArch Article Mutation-Specific RAS Oncogenicity Explains NRAS Codon 61 Selection in Melanoma Christin E. Burd1,2, Wenjin Liu3,4, Minh V. Huynh5, Meriam A. Waqas1, James E. Gillahan1,2, Kelly S. Clark3,4, Kailing Fu3,4, Brit L. Martin1, William R. Jeck3,4,. George P Souroullas3,4, David B. Darr3,4, Daniel C. Zedek6, Michael J. Miley7, Bruce C. Baguley8, Sharon L. Campbell4,5, and Norman E. Sharpless3,4 Abstrt Ac NRAS mutation at codons 12, 13, or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an NrasQ61R knock-in allele to similarly designed KrasG12D and NrasG12D alleles. With concomitant p16INK4a inactivation, KrasG12D or NrasQ61R expression efficiently promoted melanoma in vivo, whereas NrasG12D did not. In addition, NrasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of NrasQ61R and NrasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, NrasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity, and increased stability when compared with NrasG12D. This work identifies a faithful model of human NRAS-mutant melanoma, and suggests that the increased melanomagenecity of NrasQ61R over NrasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways. SIGNIFICANCE: This work explains the curious predominance in human melanoma of mutations of codon 61 of NRAS over other oncogenic NRAS mutations.
    [Show full text]
  • Interaction Between the Protein Kinase B-Raf and the A-Subunit of the IIS Proteasome Regulator1
    (CANCER RESEARCH 58. 2986-2990, July 15, I998| Advances in Brief Interaction between the Protein Kinase B-Raf and the a-Subunit of the IIS Proteasome Regulator1 Andreas Kalmes,2 Garsten Hagemann,2 Christoph K. Weber, Ludmilla Wixler, Tillman Schuster, and Ulf R. Kapp* InstituífürMedizinische Strahlenkunde und Zettforschung (MSZ). University of Würzburg,97078 Würzburg,Germany ¡C.H.. C. K. W.. L. W.. T. S.. U. K. R.], and Department of Surgery, University of Washington, Seattle. Washington 9SI95 ¡A.KJ Abstract using the yeast two-hybrid system. In addition to the already known B-Raf-binding proteins (MEK and several 14-3-3 isoforms), we iso Protein kinases of the Raf family act as signal-transducing elements lated PA28a, the subunit of the 1IS proteasome regulator (11, 12), as downstream of activated cell surface receptors and are involved in the a novel B-Raf-binding partner. regulation of proliferation, differentiation, and cell survival. Whereas the role of c-Raf-1 as a mitogen-activated protein/extracellular signal-regu The 11S regulator is one of two known activators of the 20S lated kinase activator within the mitogenic cascade is well established, less proteasome (13). Both of these activators seem to possess different is known about the mammalian Raf isoforins A-Raf and B-Raf. Here we functions in vivo. The PA700 activator complex (19S regulator) binds report that B-Raf binds to PA28a, one of two subunits of the 1IS regulator to the 20S proteasome core complex to form the active 26S protea of proteasomes. PA28<* was isolated as a B-Raf-binding protein in a yeast some, which mediates the ATP- and ubiquitination-dependent degra two-hybrid screen of a PC 12 cDNA library.
    [Show full text]
  • A-Raf and Raf-1 Work Together to Influence Transient ERK Phosphorylation and Gl/S Cell Cycle Progression
    Oncogene (2005) 24, 5207–5217 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc A-Raf and Raf-1 work together to influence transient ERK phosphorylation and Gl/S cell cycle progression Kathryn Mercer1, Susan Giblett1, Anthony Oakden2, Jane Brown2, Richard Marais3 and Catrin Pritchard*,1 1Department of Biochemistry, University of Leicester, Adrian Building, University Road, Leicester LEI 7RH, UK; 2Division of Biomedical Services, University of Leicester, University Road, Leicester LEI 7RH, UK; 3Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK The Raf/MEK/ERK (extracellular regulated kinase) Kerkhoff and Rapp, 1997, 1998; Woods et al., 1997; signal transduction pathway controls the ability of cells Marshall, 1999; Squires et al., 2002). This function to respond to proliferative, apoptotic, migratory and is mediated by the ability of ERKs to translocate to the differentiation signals. We have investigated the combined nucleus where they are able to phosphorylate transcrip- contribution of A-Raf and Raf-1 isotypes to signalling tion factors, particularly the AP-1 complex that through this pathway by generating mice with knockout comprises various heterodimers of c-fos (c-fos, FosB, mutations of both A-raf and raf-1 genes. Double knockout Fra-1, Fra-2) and c-jun (c-Jun, JunB, JunD) family (DKO) mice have a more severe phenotype than single null members (Whitmarsh and Davis, 1996; Balmanno mutations of either gene, dying in embryogenesis at E10.5. and Cook, 1999; Cook et al., 1999; Shaulian and The DKO embryos show no changes in apoptosis, but Karin, 2002).
    [Show full text]
  • Inhibition of Mtorc1 Leads to MAPK Pathway Activation Through a PI3K-Dependent Feedback Loop in Human Cancer
    Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer Arkaitz Carracedo, … , Jose Baselga, Pier Paolo Pandolfi J Clin Invest. 2008;118(9):3065-3074. https://doi.org/10.1172/JCI34739. Research Article Numerous studies have established a causal link between aberrant mammalian target of rapamycin (mTOR) activation and tumorigenesis, indicating that mTOR inhibition may have therapeutic potential. In this study, we show that rapamycin and its analogs activate the MAPK pathway in human cancer, in what represents a novel mTORC1-MAPK feedback loop. We found that tumor samples from patients with biopsy-accessible solid tumors of advanced disease treated with RAD001, a rapamycin derivative, showed an administration schedule–dependent increase in activation of the MAPK pathway. RAD001 treatment also led to MAPK activation in a mouse model of prostate cancer. We further show that rapamycin-induced MAPK activation occurs in both normal cells and cancer cells lines and that this feedback loop depends on an S6K-PI3K-Ras pathway. Significantly, pharmacological inhibition of the MAPK pathway enhanced the antitumoral effect of mTORC1 inhibition by rapamycin in cancer cells in vitro and in a xenograft mouse model. Taken together, our findings identify MAPK activation as a consequence of mTORC1 inhibition and underscore the potential of a combined therapeutic approach with mTORC1 and MAPK inhibitors, currently employed as single agents in the clinic, for the treatment of human cancers. Find the latest version: https://jci.me/34739/pdf Related Commentary, page 3003 Research article Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer Arkaitz Carracedo,1,2,3 Li Ma,2,3,4 Julie Teruya-Feldstein,3 Federico Rojo,5,6 Leonardo Salmena,1,2,3 Andrea Alimonti,1,2,3 Ainara Egia,1,2,3 Atsuo T.
    [Show full text]
  • Cholera Toxin, a Typical Protein Kinase a Activator, Induces G1 Phase
    MOLECULAR MEDICINE REPORTS 9: 1773-1779, 2014 Cholera toxin, a typical protein kinase A activator, induces G1 phase growth arrest in human bladder transitional cell carcinoma cells via inhibiting the c‑Raf/MEK/ERK signaling pathway XIAOKE ZHENG1*, YANQIU OU2*, MINFENG SHU3, YOUQIONG WANG3, YUXI ZHOU3, XINGWEN SU3, WENBO ZHU3, WEI YIN4, SHIFENG LI3, PENGXIN QIU3, GUANGMEI YAN3, JINGXIA ZHANG3, JUN HU5 and DONG XU3 1Department of Pathology, The First Affiliated Hospital, Sun Yat‑Sen University, Guangzhou, Guangdong 510089; 2Department of Cardiovascular Epidemiology, Guangdong General Hospital, Guangzhou, Guangdong 510080; 3Department of Pharmacology, Zhongshan School of Medicine; 4Department of Biochemistry, Zhongshan Medical College; 5Department of Microbiology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong 510089, P.R. China Received September 10, 2013; Accepted February 19, 2014 DOI: 10.3892/mmr.2014.2054 Abstract. The biotoxin cholera toxin has been demonstrated cycle regulation. The results revealed that cholera toxin signifi- to have anti-tumor activity in numerous types of cancer, cantly induced G1 arrest and downregulated the expression of including glioma. However, the role of cholera toxin in the cyclin D1 and cyclin-dependent kinase 4/6 in the TCC cell tumorigenesis of transitional cell carcinoma (TCC), the most lines, and this was rescued by KT5720. Furthermore, it was common malignant tumor of the bladder, remains to be eluci- demonstrated that cholera toxin downregulated the activation dated. To address this, in the present study, two TCC cell lines, of the c-Raf/Mek/Erk cascade, an important mediator of tumor T24 and UM-UC-3, were treated with cholera toxin [protein cell proliferation, via the PKA-dependent c-Raf phosphoryla- kinase A (PKA) activator] and KT5720 (PKA inhibitor).
    [Show full text]
  • Discovery of a Novel Raf Kinase Inhibitor
    Endocrine-Related Cancer (2001) 8 219–225 Discovery of a novel Raf kinase inhibitor J F Lyons, S Wilhelm1, B Hibner 2 and G Bollag Onyx Pharmaceuticals, 3031 Research Drive, Richmond, California 94806, USA 1Bayer Corporation, West Haven, Connecticut, Connecticut 06516, USA 2Chiron Corporation, 4560 Horton Street, Emeryville, California 94608-2916, USA (Requests for offprints should be addressed to J F Lyons; Email: [email protected]) Abstract We discuss the biology of Ras signal transduction and the epidemiology of ras mutations in association with disease as a background for the development of a Raf kinase inhibitor, BAY 43-9006. Knowledge of Ras effector pathways has permitted genetic validation of numerous targets involved in the Ras signaling cascade. A key Ras effector pathway involves the kinase cascade RAF/MEK/ ERK (MEK: MAP/ERK kinase; ERK: extracellular signal related kinase). Indeed, we present studies of cell lines stably expressing mutant MEK constructs, which point to Raf kinase as a target for therapeutics with selective anti-tumor activity. Finally, a small molecule drug discovery program based on inhibition of Raf kinase activity is outlined and the initial pre-clinical development process of the Raf kinase inhibitor BAY 43-9006 is discussed. Endocrine-Related Cancer (2001) 8 219–225 Ras epidemiology communication). In particular, patients with the more severe Duke’s C stage cancer had a 50% increased risk of relapse In human tumors, mutant ras oncogenes are frequently or death associated with the valine mutation. Therefore, of associated with disease progression (Rodenhuis 1992, every 1 000 000 patients who contract colorectal cancer, Sanchez-Cespedes et al.
    [Show full text]
  • Inhibition of Galectin-1 Sensitizes HRAS-Driven Tumor Growth to Rapamycin Treatment JAMES V
    ANTICANCER RESEARCH 36 : 5053-5062 (2016) doi:10.21873/anticanres.11074 Inhibition of Galectin-1 Sensitizes HRAS-driven Tumor Growth to Rapamycin Treatment JAMES V. MICHAEL 1, JEREMY G.T. WURTZEL 1 and LAWRENCE E. GOLDFINGER 1,2 1Department of Anatomy and Cell Biology, The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, U.S.A.; 2Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA, U.S.A. Abstract. The goal of this study was to develop The rat sarcoma (RAS) genes most prominently associated combinatorial application of two drugs currently either in with cancer, Harvey RAS ( HRAS ), neuroblastoma RAS active use as anticancer agents (rapamycin) or in clinical (NRAS ) and Kirsten RAS ( KRAS ), are ubiquitously expressed trials (OTX008) as a novel strategy to inhibit Harvey RAS and have overlapping, yet non-redundant functions (1). RAS (HRAS)-driven tumor progression. HRAS anchored to the proteins cycle between an active GTP-bound and an inactive plasma membrane shuttles from the lipid ordered (L o) GDP-bound state. Active RAS stimulates mitogenic and domain to the lipid ordered/lipid disordered border upon survival signal transduction by coupling to effectors activation, and retention of HRAS at these sites requires including rapidly accelerated fibrosarcoma (RAF) kinase for galectin-1. We recently showed that genetically enforced L o propagation of the mitogen-activated protein kinase (MAPK) sequestration of HRAS inhibited mitogen-activated protein pathway [RAS/RAF/MAPK kinase (MEK)/extracellular kinase (MAPK) signaling, but not phoshatidylinositol 3- regulated kinase (ERK)], and phosphatidylinositol 3-kinase kinase (PI3K) activation. Here we show that inhibition of (PI3K) pathways (2).
    [Show full text]
  • Structural and Mechanistic Insights Into RAF Kinase Regulation by the KSR/CNK/HYP Complex
    Structural and mechanistic insights into RAF kinase regulation by the KSR/CNK/HYP complex by Thanashan Rajakulendran A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Thanashan Rajakulendran, 2011 Structural and mechanistic insights into RAF kinase regulation by the KSR/CNK/HYP complex Thanashan Rajakulendran Doctor of Philosophy Department of Molecular Genetics University of Toronto 2011 Abstract The RAS/RAF/MEK/ERK pathway is the prototypical cellular signal transduction cascade and has been the focus of intense scrutiny over the last two decades. As a mitogenic pathway, its activation is a potent driver of cellular growth and survival, and its deregulation underlies many cancers. While RAS family GTPases have long been recognized as prolific human oncogenes, a landmark study in 2002 also established the RAF family kinase as a bona fide oncogene (Davies et al., 2002). Indeed, aberrant RAS-RAF signaling underlies nearly one-third of all human cancers (Wellbrock et al., 2004). Notably, mutations in RAF are found with astounding frequency in certain cancers (e.g. 70% of malignant melanomas) (Dhomen and Marais, 2007). These findings have identified intercepting aberrant RAF function as an ideal therapeutic target. RAF is a Ser/Thr protein kinase and its activity is strictly regulated by a core complex of at least three proteins, namely, KSR, CNK and HYP (Claperon and Therrien, 2007). The mechanism by which the KSR/CNK/HYP complex regulates RAF function remains enigmatic. In particular, the function of KSR in regulating RAF activity is highly controversial.
    [Show full text]
  • CDK4/6 and MAPK—Crosstalk As Opportunity for Cancer Treatment
    pharmaceuticals Review CDK4/6 and MAPK—Crosstalk as Opportunity for Cancer Treatment Lisa Scheiblecker, Karoline Kollmann and Veronika Sexl * Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, 1210 Vienna, Austria; [email protected] (L.S.); [email protected] (K.K.) * Correspondence: [email protected] Received: 24 October 2020; Accepted: 22 November 2020; Published: 24 November 2020 Abstract: Despite the development of targeted therapies and novel inhibitors, cancer remains an undefeated disease. Resistance mechanisms arise quickly and alternative treatment options are urgently required, which may be partially met by drug combinations. Protein kinases as signaling switchboards are frequently deregulated in cancer and signify vulnerable nodes and potential therapeutic targets. We here focus on the cell cycle kinase CDK6 and on the MAPK pathway and on their interplay. We also provide an overview on clinical studies examining the effects of combinational treatments currently explored for several cancer types. Keywords: CDK6; MAPK; p38; combinational; palbociclib; inhibitors; cancer 1. Introduction Tumorigenesis requires that cells hijack regulatory networks to obtain their full oncogenic potential. The MAPK (mitogen-activated protein kinase) pathway is a tightly regulated signaling cascade frequently mutated or deregulated in cancer. Extra- and intracellular stimuli activate the MAPK cascade that regulates a broad variety of cellular programs including proliferation, differentiation, stress responses and apoptosis [1]. The best-studied MAPK pathways are the extracellular signal-regulated kinase (ERK) and stress-activated MAPK (c-Jun N-terminal kinase JNK and p38) pathways (Figure1). While the ERK pathway is predominantly activated by growth factors to drive survival and cell growth, activation of the JNK or p38 pathway is accomplished by environmental stressors and inflammatory cytokines and is associated with apoptosis and growth inhibition [1].
    [Show full text]
  • Is B-Raf a Good Therapeutic Target for Melanoma and Other Malignancies? Subbarao V
    Review Is B-Raf a Good Therapeutic Target for Melanoma and Other Malignancies? SubbaRao V. Madhunapantula1 and Gavin P. Robertson1,2,3,4,5 Departments of 1Pharmacology, 2Pathology, and 3Dermatology, The Pennsylvania State University College of Medicine; 4The Foreman Foundation for Melanoma Research; and 5Penn State Melanoma Therapeutics Program, Hershey, Pennsylvania Abstract normal cells and causes hyperactivity of the MAP kinase pathway The RAF family members, A-Raf, B-Raf, and C-Raf (or Raf-1), promoting tumor development (2, 3). Activation occurs as a result are intermediate molecules in the mitogen-activated protein of a conformational change in protein structure due to glutamic acid acting as a phosphomimetic between the Thr598 and Ser601 (MAP) kinase [Ras/Raf/MAP kinase/extracellular signal– B-Raf regulated kinase (Erk) kinase (MEK)/Erk] pathway, which phosphorylation sites (2, 4). Activating mutations are relays extracellular signals from the cell membrane to the acquired, somatic, post-zygotic events and are not inherited in families (5). Cell culture and/or animal based studies have shown nucleus via a cascade of phosphorylation events ultimately V600E promoting cancer development. This pathway is activated by the oncogenic potential conferred by B-Raf to developing mutation in f7% of all human cancers. B-Raf is one of the cancer cells (1, 2). Abnormally high activation of the MAP kinase pathway can proteins frequently mutated to an active form during tumor development. Therefore, B-Raf is an attractive cancer target inhibit cellular growth in a wide variety of normal and cancer cells but lack of clinical efficacy using agents targeting this protein by promoting cellular senescence (6).
    [Show full text]