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

■ 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

Finding of human oncogenes

https://www.ncbi.nlm.nih.gov/books/NBK9840/

Pagliarini R et al, EMBO Rep 2015

■ 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

What is an oncogene?

Gene functions Examples

Ligands Epidermal Growth Factor (EGF)

Receptors EGF Receptor (EGFR, HER1), HER2/NEU

Intracellular signaling

cascade proteins

RAS, SRC, RAF, ABL, BCR/ABL

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

Functions of oncoproteins in cells

HOW ARE ONCOGENES ACTIVATED ??

Representative human oncogenes and activation mechanism

https://www.ncbi.nlm.nih.gov/books/NBK9840/

Functional gain mutation

Hyperactive protein in

normal amounts

Overexpressed normal protein

Overexpressed normal protein

Overexpressed normal protein

Fusion to actively

transcribed gene produces

hyperactive fusion protein

■ RAS - usually activated by single nucleotide substitutions

Activation mutation by point mutation

■ 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

RAS oncogene

Types of RAS oncoproteins

HRAS : Harvey rat sarcoma viral

oncogene homolog

(HRAS)

Ch.11p15.5

NRAS : neuroblastoma RAS viral

(v-ras) oncogene homolog

(NRAS)

Ch.1p13.2

KRAS : Kirsten rat sarcoma viral

oncogene homolog

(KRAS) KRAS4A and KRAS4B

Ch.12p12.1

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

Raialingam K et al. BBA 2007

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

A comprehensive survey of RAS mutation in cancer

Prior IA et al. Cancer Res 2012

• 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

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

■ MYC - typically activated by complex genomic rearrangements

Gene amplification

■ 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

MYC oncogene

Mechanism of MYC oncoprotein

MYC

MAX

dsDNA

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

■ 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

Fusion oncoprotein by chromosome rearrangement

■ 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)

Function of BCR-ABL oncoprotein

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

http://pharmaceuticalintelligence.com/tag/signal-transduction-pathways/

Estrogen receptor (ER) alpha DNA-bonding domain in

complex with DNA

Intracellular Rp-mediated signalling

- 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

Hormone responsive elements (HREs)

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 receptors

http://pharmaceuticalintelligence.com/tag/signal-transduction-pathways/

Membrane receptor-mediated signalling

1

2

3

4

Steps of membrane 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

Mechanism of Mb 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

Membrane Rp-mediated signalling

Ki

http://www.biooncology.com/molecular-causes-of-cancer/proliferative-signaling

Kinase

Growth factors (GFs): epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) etc.

Autophos-phorylation

Cancer cells

RTKs : growth factors, cytokines

RAS-targeted

therapy

Ki

Direct inhibition of RAS function

Ryan MR and Corcoran RB. Nature Rev Clin Oncol 2018

• 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%)

Farnesylation of Cys residue

RAS-converting enz. (remove AAX)

Isoprenylcysteine O-methyltransferase (methylation of carboxyl gr.)

Farnesyl transferase inhibitors

Ki

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

The overview of therapeutic approaches to targeting RAS

1

2

3

4

5

6

GPCR-adenylate cyclase epinephrine, histamine, chemokines, prostaglandins

cAMP dependent protein kinase

(Protein kinase A)

Campbell, Biochemistry, Ed 3

G-protein

GPCR-phosphatidylinositol

Phosphatidyl inositol diphosphate (PIP2) Diacylglycerol

(DAG)

PKC Inositol triphosphate (IP3)

• Calcium in ER is released to cytosol • binds to calmodulin or may help to activate PKC

Phosphoprotein

Abnormal signal transduction

pathways in cancer

Medical applications of chemical

mediators and their signal

transduction pathways

Abnormal signal transduction in cancer cell

MYC TP53

BCR-ABL

EGF, PDGF, HGF, FGF

Thuwajit C, Biochemistry Textbook (in Thai)

RAS

EGFR

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

Nyati MK, Nat Rev Cancer 2006, 6, 876-885

RTK signaling

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

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)

Inhibitors for HER2 signaling pathway

http://am.asco.org/her2-pathway-breast-cancer

In patients with HER2 positive

In patients with transtuzumab resistance

Transtuzumab (Herceptin®): mechanism of action

Immune cells

targeting cancer

cells bound by

Herceptin

Dimerized HER2

Rps signal cancer

cells to the drug

It is the only approved

HER2 therapy designed to

bind to HER2+ cancer

cells and flag them for

destruction by the

immune system

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)

Imatinib (Gleevec®), the first in a class of drugs that specifically target and competively inhibit the ATP binding site 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

Inhibitors for tyrosine kinase

GPCR-targeted

therapy

GPCR in cancers

Innamorati G, Pharmaceuticals 2011, 4, 567-589

Hormones from cancer cells and chemokines from cells in tumor microenvironment

Innamorati G, Pharmaceuticals 2011, 4, 567-589

a: antagonists; b: antibodies; c: quenching agonist itself; d: sustained stimulation; e: acylated GPCR intracellular loop;

f: specific inhibitors; g: Rp internalization

antagonist

antibody bind agonist

Rp internalization with isotope and toxin

Specific inhibitor

Sustain stimulation

Acylation block binding to G protein

Target GPCR in cancer treatment

Innamorati G, Pharmaceuticals 2011, 4, 567-589

More than one third of marketed drugs targeted GPCRs leaving a hundreds of potential

new opportunities for developing novel anticancer agents

Combination treatment of targeting signal transduction

Hynes NE, Lane HA. Nature Rev Cancer 2005

Fruman DA and Rommel C. Nature Rev Drug Discov 2014

Combination treatment of targeting signal transduction

Target PI3K

TKI resistance TKI

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

EGFR stimulation

Phosphatase SHP2

HLA class I

PD-L1

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

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

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