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Home , Signal transduction

AND SIGNAL

Chanitra Thuwajit Ph.D., M.D. Department of Faculty of Medicine Siriraj Hospital Mahidol University

Thai Society of Clinical Oncology : Basic Sciences, Dec 22, 2018 Finding of human

■ 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 ■ Proto-oncogenes typically regulate growth and cell differentiation ■ Alterations of proto-oncogenes that cause their conversion to oncogenes cause many of the perturbations in 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 (EGF)

Receptors EGF (EGFR, HER1), HER2/NEU

Intracellular signaling RAS, SRC, RAF, ABL, BCR/ABL cascade

Transcription factors AP-1, Jun and Fos heterodimer,

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 in normal protein normal protein normal protein actively normal transcribed amounts gene produces hyperactive fusion protein Activation mutation by ■ RAS - usually activated by single nucleotide substitutions RAS oncogene

■ RAS protein involves in signaling pathways that control the 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 of many different origins, including: pancreas (90%), colon (50%), lung (30%), (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, , 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 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 ■ 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 as well as

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 kinase, and the BCR-ABL transcript is also translated into a , "always on" or continuously activated, which results in unregulated ■ 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/ responsive elements (HREs)

- is a specific region of DNA where -hormone complex attaches - HREs are located in the 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 ( fingers) 2. -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 and through second messengers

- an increasing number of enzymes (amount and activity) and other molecules in the events that proceed from the initial

- 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

RTKs : growth factors,

Cancer cells

KiAutophos - Kinase phorylation

Growth factors (GFs): (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, ,

G-protein

cAMP dependent ()

Campbell, Biochemistry, Ed 3 GPCR-phosphatidylinositol

Phosphatidyl diphosphate (PIP2) Diacylglycerol (DAG) Inositol PKC triphosphate (IP3)

• in ER is released to • binds to or may help to activate PKC Phosphopr otein Abnormal 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 cancer - colon cancer - non-small cell lung cancer (NSCLC) - head neck squamous cell carcinoma (HNSCC) EGF signaling pathway in cancer

Alterations of EGFR or HER signaling pathways cause the inappropriate cell growth that leads to proliferation, migration, and survival of cancer cells

http://www.gene.com/gene/products/information/oncology/tarceva/egfr_factsheet.html Inhibitors for EGFR signaling pathway Colon cancer, HNSCC, NSCLC

RTK signaling

Nyati MK, Nat Rev Cancer 2006, 6, 876-885 Inhibitor for EGF signaling pathway

Cetuximab (EGFR inhibitor) Erbitux® • is a chimeric (mouse/human) monoclonal Ab • FDA approved for treatment of KRAS wild type colon cancer, since Cetuximab had little or no effect in colorectal tumors harboring a KRAS mutation (The first genetic test to guide treatment of cancer) • 75% of patients with metastatic colon cancer have an EGFR-over-expressing tumor (normal form)

Gefitinib (EGFR-specific tyrosine kinase inhibitor) Iressa® • inhibit autophosphorylation of the receptors • used for NSCLC which shows increased activity of the EGFR by EGFR mutation Inhibitors for HER2 signaling pathway

In patients with HER2 positive

In patients with transtuzumab resistance

http://am.asco.org/her2-pathway-breast-cancer Transtuzumab (Herceptin®): mechanism of action

It is the only approved Immune cells HER2 therapy designed to targeting cancer bind to HER2+ cancer cells bound by cells and flag them for Herceptin destruction by the

Dimerized HER2 Rps signal cancer cells to the drug It blocks down-stream HER2 signaling leading to inhibit proliferation of cells Inhibit receptor dimerization Induce apoptosis Hynes NE, Lane HA, Nature Rev Cancer 2005, 341-64 BCR-ABL- targeted therapy  Philadelphia Chromosome, the result of a reciprocal translocation between chromosomes 9 and 22; breakpoint cluster region (BCR) of chromosome 22 is fused with part of the Abelson (ABL) gene on chromosome 9; resulting BCR-ABL which codes for a mutant tyrosine kinase

 BCR-ABL is constitutively active; not require activation by the cellular messaging proteins to stimulate cellular replication; resulting to acceleration of cell division, an inhibition of DNA repair, overall genomic instability, and the fatal blast crisis characteristic of chronic myelogenous leukemia (CML)

Inhibitors for tyrosine kinase

 Imatinib (Gleevec®), the first in a class of drugs that specifically target and competively inhibit the ATP on BCR-ABL tyrosine kinase. This prevents the ABL domain from phosphorylating the tyrosine residue, and as a result preventing the proliferation of hematopoietic cells that express BCR-ABL

The drug inhibits proliferation and induces apoptosis in BCR-ABL positive cells and in fresh leukemic cells

GPCR-targeted therapy GPCR in cancers from cancer cells and chemokines from cells in tumor microenvironment

Innamorati G, Pharmaceuticals 2011, 4, 567-589 bind agonist

Rp internalization with

antagonist isotope and toxin

Specific inhibitor

Sustain stimulation

Acylation block binding to

a: antagonists; b: ; c: quenching agonist itself; d: sustained stimulation; e: acylated GPCR intracellular loop; f: specific inhibitors; g: Rp internalization

Innamorati G, Pharmaceuticals 2011, 4, 567-589 Target GPCR in cancer treatment

More than one third of marketed drugs targeted GPCRs leaving a hundreds of potential new opportunities for developing novel anticancer agents

Innamorati G, Pharmaceuticals 2011, 4, 567-589 Combination treatment of targeting signal transduction

Hynes NE, Lane HA. Nature Rev Cancer 2005 Combination treatment of targeting signal transduction

Target PI3K TKI TKI resistance

Fruman DA and Rommel C. Nature Rev Drug Discov 2014 Cancer signaling networks - the wide variety of intra- and intercellular signals affected in cancer - focusing on Ras-ERK and PI3K-Akt signaling

Sever R and Brugg JS. Cold Spring Harb Perspect Med 2015 Resistance to oncogenic targeted therapy Pagliarini R et al. EMBO Rep 2015 Pagliarini R et al. EMBO Rep 2015 Pagliarini R et al. EMBO Rep 2015 Oncogenic targeted therapy and immune response Concha-Benavente F and Ferris R. Curr Opin Immunol 2017 EGFR/HER2 mediated immunoescape

Concha-Benavente F and Ferris R. Curr Opin Immunol 2017

EGFR stimulation

Phosphatase SHP2

 HLA class I

 PD- RAS signaling and immune interplay

• Cancer cells with the presence of KRAS mutation can result in the immunosuppressive TME and reduce the no. of TILs but increased the expression of PD-L1 • In BRAF-mutant melanoma patients, combined BRAF inhibition and immune checkpoint inhibitors (ICIs) (i.e. ipilimumab : anti-CTLA4, nivolumab/pembrolizumab/ atezolizumab : anti-PD-L1) led to the increased CD8+ T cells infiltrated into TME • These suggest the potential of improve the immune response by targeting RAS-RAS effectors to be a promising therapeutic strategy

Ryan MR and Corcoran RB. Nature Rev Clin Oncol 2018 Take home message

• There is a dysregulation of cellular signal transduction (induced by the genetic and epigenetic changes) that drive cancer

• Pharmacologic and antibody-based inhibitors that target signaling proteins mutated in tumors or proteins downstream from these have had significant impact as cancer treatments

• Because of the heterogeneous population in cancer, a deeper understanding of the nature of resistance mechanisms and how different cellular signaling programs mediate resistant states are required

• Combination therapies that target these signal transduction molecules should increase the efficacy of targeted therapies Reference : 2015, page 1104-1144 THANK YOU FOR YOUR ATTENTION https://en.wikibooks.org/wiki/An_Introduction_to_Molecular_Biology/Genetic_Code