ONCOGENE AND SIGNAL TRANSDUCTION Chanitra Thuwajit Ph.D., M.D. Department of Immunology Faculty of Medicine Siriraj Hospital Mahidol University Thai Society of Clinical Oncology : Basic Sciences, Dec 22, 2018 Finding of human oncogenes ■ DNA extracted from a human bladder carcinoma induced transformation of recipient mouse cells in culture ■ This transformation of mouse cells resulted from integration and expression of an oncogene derived from the human tumor https://www.ncbi.nlm.nih.gov/books/NBK9840/ Pagliarini R et al, EMBO Rep 2015 What is an oncogene? ■ An oncogene is a mutated form of a normal cellular gene – called a proto-oncogene that contributes to the development of a cancer ■ Proto-oncogenes typically regulate cell growth and cell differentiation ■ Alterations of proto-oncogenes that cause their conversion to oncogenes cause many of the perturbations in cell growth and differentiation that are commonly seen in cancer cells ■ It is estimated that fully 1% of the ~21,000 genes in the human genome are proto-oncogenes Functions of oncoproteins in cells Gene functions Examples Ligands Epidermal Growth Factor (EGF) Receptors EGF Receptor (EGFR, HER1), HER2/NEU Intracellular signaling RAS, SRC, RAF, ABL, BCR/ABL cascade proteins Transcription factors AP-1, Jun and Fos heterodimer, MYC DNA repairing proteins Ataxia telangiectasia mutated (ATM) http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/O/Oncogenes.html HOW ARE ONCOGENES ACTIVATED ?? Representative human oncogenes and activation mechanism https://www.ncbi.nlm.nih.gov/books/NBK9840/ Functional gain mutation Hyperactive Overexpressed Overexpressed Overexpressed Fusion to protein in normal protein normal protein normal protein actively normal transcribed amounts gene produces hyperactive fusion protein Activation mutation by point mutation ■ RAS - usually activated by single nucleotide substitutions RAS oncogene ■ RAS protein involves in kinase signaling pathways that control the transcription of genes, which then regulate cell growth and differentiation ■ Encodes GTPase protein : GDP-bound (off) state and GTP-bound (on) state ■ RAS protein must bind to a particular molecule (GTP) in the cell to be activated and break up the GTP molecule to be inactivated ■ Alterations in the RAS gene can change the RAS protein so that it is no longer able to break up and release the GTP ■ Mutant RAS has been identified in cancers of many different origins, including: pancreas (90%), colon (50%), lung (30%), thyroid (50%), bladder (6%), ovarian (15%), breast, skin, liver, kidney, and some leukemias http://www.cancerquest.org/ras-oncogene.html Types of RAS oncoproteins HRAS : Harvey rat sarcoma viral Ch.11p15.5 oncogene homolog (HRAS) NRAS : neuroblastoma RAS viral Ch.1p13.2 (v-ras) oncogene homolog (NRAS) KRAS : Kirsten rat sarcoma viral Ch.12p12.1 oncogene homolog (KRAS) KRAS4A and KRAS4B RAS structure • RAS proteins share a high level of sequence homology in the G domain and diverse in the HVR (hypervariable region), especially CAAX motif • CAAX motif regulates a diverse set of effector proteins Ryan MB and Corcoran RB. Nature Rev Clin Oncol 2018 RAS and RAS effector pathways GTP-RAS binds to numerous effectors to trigger various signaling cascades and in turn modulate different cellular processes ranging from cell growth, survival, cell migration, differentiation, and death Raialingam K et al. BBA 2007 A comprehensive survey of RAS mutation in cancer • KRAS is the most commonly mutated oncogene in cancer • Mutations in residues G12, G13, and Q61 alter RAS interaction with regulatory protein, leading to constitutive activation Prior IA et al. Cancer Res 2012 Incidence of RAS isoform mutations in cancer Prior IA et al. Cancer Res 2012 RAS isoform specific codon mutation bias Prior IA et al. Cancer Res 2012 https://www.youtube.com/watch?v=RqdlfZP26BI Gene amplification ■ MYC - typically activated by complex genomic rearrangements MYC oncogene ■ The MYC protein acts as a transcription factor and it controls the expression of several genes ■ The MYC family of oncogenes may become activated by gene rearrangement or amplification ■ Mutations in the MYC gene have been found in many different cancers, including Burkitt's lymphoma, B-cell leukemia, and lung cancer ■ The amount of MYC protein present in the cell is important because the activity of MYC is balanced by another protein that opposes MYC activity http://www.cancerquest.org/myc-oncogene.html Mechanism of MYC oncoprotein MAX dsDNA MYC Bind with MYC-associated factor X (MAX), a member of the basic helix-loop-helix leucine zipper family of transcription factors Max binds to itself and to other transcription factors through its leucine zipper to form homo- and hetero-dimers, respectively MYC can heterodimerize with MAX to form heterodimers that can both bind DNA and transactivate The transcriptionally active MAX/MYC dimer promotes cell proliferation as well as apoptosis Fusion oncoprotein by chromosome rearrangement ■ The t(9;22) reciprocal translocation results in the creation of two separate fusions between the BCR and C-ABL genes ■ The BCR-ABL gene is created on the derivative of chromosome 22, the Philadelphia chromosome in CML Function of BCR-ABL oncoprotein ■ Reciprocal translocation, creating an elongated chromosome 9 (termed a derivative chromosome, or der 9), and a truncated chromosome 22 (the Philadelphia chromosome) ■ BCR ("breakpoint cluster region"), ABL stands for "Abelson", the name of a leukemia virus which carries a similar protein ■ The ABL gene expresses a membrane-associated protein, a tyrosine kinase, and the BCR-ABL transcript is also translated into a tyrosine kinase, "always on" or continuously activated, which results in unregulated cell division ■ Tyrosine kinase inhibitors (such as imatinib and sunitinib) http://www.pathophys.org/wp-content/uploads/2012/10/Cell-cycle-copy.jpg Two types of signaling pathways Intracellular Rp-mediated signaling Membrane Rp-mediated signaling Intracellular Rp-mediated signalling Estrogen receptor (ER) alpha DNA-bonding domain in complex with DNA http://pharmaceuticalintelligence.com/tag/signal-transduction-pathways/ Hormone responsive elements (HREs) - is a specific region of DNA where nuclear receptor-hormone complex attaches - HREs are located in the promoter region of the genes that after activation by the hormone-receptor complex, a certain set of genes are expressed Nuclear receptors 1. DNA-binding domain (zinc fingers) 2. ligand-binding domain Membrane receptors Three parts of membrane receptors 1. extracellular part: binds with mediators 2. transmembranous part: holds with membrane 3. cytoplasmic part: transduces external stimuli into the cell and activates second messengers Membrane receptor-mediated signalling http://pharmaceuticalintelligence.com/tag/signal-transduction-pathways/ Steps of membrane Rp-mediated signalling 1 2 3 4 Mechanism of Mb Rp-mediated signalling - involves a sequence of biochemical reactions inside cells, carried out by enzymes and through second messengers - an increasing number of enzymes (amount and activity) and other molecules in the events that proceed from the initial stimulus - results in a small stimulus eliciting a large response - take place in as little time as a millisecond or a few seconds http://en.wikipedia.org/wiki/Signal_transduction Membrane Rp-mediated signalling Catalytic receptors (Rp tyrosine kinase, RTKs) Receptors involving second messenger G-protein coupled Rp, GPCRs consists of 2 types o Rps couple to adenylate cyclase o Rps couple to phosphatidylinositol RTKs : growth factors, cytokines Cancer cells KiAutophos - Kinase phorylation Growth factors (GFs): epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) etc. http://www.biooncology.com/molecular-causes-of-cancer/proliferative-signaling RAS-targeted therapy Direct inhibition of RAS function Farnesyl transferase inhibitors Farnesylation of Cys residue Ki RAS-converting enz. (remove AAX) Isoprenylcysteine O-methyltransferase (methylation of carboxyl gr.) • RAS proteins are active and able to engage with downstream effectors when they are associated with mb • FTIs : effective in only HRAS-driven cancers : lung adenocarcinoma (1%), urothelial ca (5.5%), HNSCC (1%) Ryan MR and Corcoran RB. Nature Rev Clin Oncol 2018 Inhibitors of RAS-MAPK signaling pw Ki Ryan MR and Corcoran RB. Nature Rev Clin Oncol 2018 Approaches to inhibiting RAS functions 6 2 5 1 Ki 3 4 The overview of therapeutic approaches to targeting RAS Ryan MR and Corcoran RB. Nature Rev Clin Oncol 2018 GPCR-adenylate cyclase epinephrine, histamine, chemokines, prostaglandins G-protein cAMP dependent protein kinase (Protein kinase A) Campbell, Biochemistry, Ed 3 GPCR-phosphatidylinositol Phosphatidyl inositol diphosphate (PIP2) Diacylglycerol (DAG) Inositol PKC triphosphate (IP3) • Calcium in ER is released to cytosol • binds to calmodulin or may help to activate PKC Phosphopr otein Abnormal signal transduction pathways in cancer Medical applications of chemical mediators and their signal transduction pathways Abnormal signal transduction in cancer cell EGF, PDGF, HGF, FGF EGFR RAS BCR-ABL MYC TP53 Thuwajit C, Biochemistry Textbook (in Thai) 2015 Examples of approved oncogene-targeted therapy EGFR-targeted therapy EGF signaling pathway Epidermal growth factor receptor, EGFR (ERBB-1/ HER1) belongs to a family of receptors that compose of 3 additional proteins, ERBB-2 (NEU/HER2), ERBB-3 (HER3) and ERBB-4 (HER4) EGFRs are over-expressed in - breast