CARCINOGENESIS

Lelly Yuniarti
 What is Cancer ?

Tumors: loss of cell cycle control

 de-differentiation and proliferation

benign: encapsulated by connective tissue

rarely life-threatening

malignant: invasive growth cell

shedding  metastasis cancer life-
threatening
 Molecular basis of cancer
Causes of cancer: - carcinogens, radiation, viruses, random
- hereditary vs. spontaneous tumors
- multi step process

Genes and gene products involved in cancer:

• activation of proto-oncogenes to oncogenes (gain-of-function)
• inactivation of tumor suppressor genes (loss-of-function)
• altered activity of modulator genes
 Cause of cancer?
 Multiple genetic abnormalities
 Internal factors : defect genetic/inherited
 External factors :
 X RAYS
 Environment : Viral infections : HPV, EBV,
HBV, HIV
 Carcinogenic compounds :
 food, working area,
 lifestyle: smoking , alcohol, HIV, HBV, HPV, EBV
Molecular Basis of multistep
 Retroviruses
 Retroviruses  family of RNA viruses that cause cancer
in variety of animals and humans.
 The Retrovirus : 3 main genes gag, pol & env 
required for virus replication, not play role in cell
transformation.
 a retrovirus can transform cells from normal to cancer if
they include a specific gene that is capable of inducing
cell transformation this gene is known as “Oncogene”.

Retrovirus

Cancerous
Retrovirus Oncogene
 Retrovirus oncogene
 Two main types of oncogenes:
 Viral oncogene: gene from the retrovirus itself
 Non-Viral oncogene (Cellular oncogene):
genes derived from the genes of the host cell
that are in an inactive form usually.
Occasionally if the gene incorporates with the
viral genome will form a highly oncogenic
virus.
 Proto-oncogenes: are the form of cellular genes that
inactive normally but can incorporate with the viral
genome to produce a highly oncogenic virus.
 EBV-NPC in Indonesia

 Central Java map

Late stage

 Most are Javanese, the 1st among men

 Hot Spot areas  way of life? Salted fish?
 Special tobacco ingredients?
 EBV-associated tumors
in man

Lymphoid tissues Epithelial tissues

Burkitt´s lymphoma, endemic 98% Oral hairy leukoplakia 100%

Burkitt´s lymphoma, sporadic 25% Gastric adenocarcinoma 5-10%

AIDS-immunoblastic lymphoma 60% Nasopharyngeal carcinoma,

-in CNS 100% undifferentiated 100%

Post-transplant lymphoma 100% Salivary gland carcinomas <100%

Hodgkin´s lymphoma 50% Muscle tissue

T-cell lymphomas 10-30%
--lethal midline granuloma >90% Leiomyosarcoma in
immunosuppressed 100%?
 Epstein-Barr virus
Latent Membrane Protein 1 (LMP 1 )
- Cell aggregation - Cycline D2
- CD 23 - EGFR
- CD 18, 21,39,40,44 - tumorigenicity
TRAF-1
TRAF-3 + - LFA 1
- LFA 3
- CAM (Ca2+ pr
kinase G1
TRADD/TANK - ICAM 1 - vimentin?

- - MHC Class II

p65/p50
?
+
IkB -epithelial cell
differentiation EBNA 2
Bcl-2
A20 PU1 Jk
+/- LMP1 LRS
cellular genes blocks apoptosis

N C W Y F HQ M E K B G D A
 EBV – latent membrane protein (LMP1)
as an oncoprotein

- Transform rodent cells, loss of contact inhibition

Anchorage-independent growth

- Morphological transformation of human keratinocytes

tumour in nude mice
- Inhibit human epithelial cells differentiation

- Induction of bcl-2 to protect infected B cells from apoptosis

- A20 in epithelial cells

-Essential for B-lymphocyte growth transformation

 LMP 1

NF-kB
EGFR NF-kB MMP9
(invasiveness
Cox-2 IV collagenase)

Prostaglandin E2 IL-8
(angiogenic factor)

VEGF
(angiogenesis)
 So what …….. ( from results??)
Clinical application ??

 Potential to be developed and further explored

 Better diagnostic approach
 Non invasive for follow up
 Field epidemiology for early screening/ high risk
 Molecular epidemiology  The profil EBV- NPC
 to develop vaccine, immunotherapy (LMP1,
LMP2, EBNA’s)
 Serology markers.
 EBV cycle in vivo Infected resting B
cells
IgA? Latent reservoir of EBV
Virus replication in Corresponds
oropharyngeal epithelium To neuronal
Axon of
neurotropic
Herpesvirus

Infection of
circulating B cells

Ag?

T cell mediated control of

proliferation

CTL

Proliferation of EBV
activated blasts
Eva Szabo &
Gail L. Shaw

http://www.moffitt.usf.edu/pubs/ccj/v4n2/article1.html
http://www2.scitech.sussex.ac.uk/undergrad/coursenotes/ehh/lec4/4.html
 http://www.belleonline.com/n2v91.html

Epigenetic Mechanisms of Chemical Carcinogenesis

James E. Klaunig, Lisa M. Kamendulis, and Yong Xu

Division of Toxicology, Department of Pharmacology and Toxicology,
Indiana University School of Medicine
 Different Steps of Carcinogenesis

Initiation: Mutation in one or more cellular genes controlling

key regulatory pathways of the cell (irreversible)—must be a
heritable DNA alteration.

Promotion: selective growth enhancement induced in the

initiated cell and its progeny by the continuous exposure to a
promoting agent.

Progression: results from continuing evolution of unstable

chromosomes; further mutations from genetic instability during
promotion—results in further degrees of independence,
invasiveness, metastasis, etc.
Initiation
• Initiation is the induction of a mutation in a critical gene
involved in the control of cell proliferation.
• As with mutational events, initiation requires one or
more rounds of cell division for the “fixation” of the
process.
• The metabolism of initiating agents to non-reactive
forms and the high efficiency of DNA repair of the
tissue can alter the process of initiation.
• Initiation is irreversible although the initiated cell may
eventually die during the development of the neoplasm.
 Types of mutations
Chemical carcinogens can cause:
1. Point mutations- the replacement of a single nucleotide base
with another nucleotide.
2. Frameshift mutations- addition or deletion of a nucleotide such
that the protein sequence from that point onward is altered.
3. Chromosomal aberrations- any change in the normal structure
or number of chromosomes
4. Aneuploidy- chromosome number is not a multiple of the
normal haploid (23)
5. Polyploidy- more than twice the haploid number of
chromosomes
 Mechanisms of DNA Repair
The persistence of chemically-induced DNA adducts is
predominantly the result of failure of DNA repair, due to
either:
• carcinogen-induced mutational inactivation of DNA
repair enzymes.
• failure of the DNA repair mechanisms to recognize
carcinogen-induced mutation.
 Targets of Initiation
Chemical carcinogens initiate cells via:
1. Mutational activation of oncogenic (proliferative)
pathways (e.g. growth factor receptors and
downstream signaling proteins, proteins involved in
cell cycle checkpoints.
2. Mutational inactivation of apoptotic (cell death)
pathways (e.g. growth inhibitory receptors, proteins
involved in apoptosis, tumor suppressors).
3. Mutational inactivation of DNA repair mechanisms
(e.g. BER, NER, etc).
4. Mutational inactivation of antioxidant response (e.g.
SOD).
 Tumor suppressor p53 signaling
mitogen mitogen

unstressed cell MAP kinase

PKB/Akt AP1/Ets binds
phosphorylates Mdm2 Mdm2 promoter

p53 binds Mdm2

nuclear export of p53

p53 gets ubiquinated

and degraded by proteosome

cell survival
 p53 is a an important tumor suppressor (transcriptional factor) that
controls cell cycle, apoptosis, DNA repair mechanisms.
 Mdm2 is a negative regulator of p53 that functions both as an E3
ubiquitin ligase and an inhibitor of p53 transcriptional activation.
 DNA damage, cell damage increased E2F
(uncontrolled cell cycle) p53—tumor suppressor:
from mutated Rb
ATM kinase activated

Mutated in most cancers.

phosphorylates phosphorylates Mdm2
p53 so it can't bind Mdm2 prevents ubiquitination of p53

increased
Carcinogens often
inc re as e d p53 p14ARF
sequesters
mutationally inactivate p53 as
(te trame ric TF) Mdm2 well as proteins that control
binds tandem sequence of
increased Apaf p53 function (e.g. Mdm2, p14)
(apoptosis)
Fas ligand PuPuPuC A/t T/a G PyPyPy increased p21 (G1
arrest)

increased IGF-BP3
increased Fas receptor increased Bax

IGF-1
FADD/DISC complex Bax dimer
depolarizes mitochondrial
membrane
IGF-1 receptor
activates caspase 8 + 10

cyt c released into cytosol

PKB/Akt activation

cytc, Apaf-1, caspase 9

form apoptosome cell survival

activates executioner caspases 3,6,7

apo pto s is
Ras oncogene: involved in control of cell cycle progression and apoptosis
norepinephrine growth factor
serotonin, etc (PDGF, IGF, EGF, NGF)

binds G-protein coupled binds receptor tyrosine kinase

receptor and dimerizes to autophosphorylate
PLC cytosolic Tyr on receptor

IP3 DAG recruits Grb2/Sos to phospho-Tyr PI-4,5-P2

PI3-Kinase
Ca2+ Ras-GDP Ras -GTP binds Ras (active)
PKC
PIP3

Raf Ral-GDS binds Akt/PKB and is

activated by
CaMK MEK phosphorylation by PDK
Rac
MAP
ERK Kinas e pathway PAK
P-bad P-p21, P-p27 P-Mdm2 inhibits
p90 MEK (inactive) (inactive) (sequesters p53) TSC1/2
c-Myc (mTor active)
CREB MMK4

cyclin D, E2F1-3 Fos JNK

CDK4 Jun
apo pto s is s uppre s s e d activates
decreases p21, p15 AP1 protein
synthesis
increased cyclin D

c e ll c yc le pro g re s s ion
 CYP/PHS O
CYP/PHS EH

HO HO
O OH OH
benzo[a]pyrene (+) benzo[a]pyrene (+) benzo[a]pyrene
7,8-oxide (-) benzo[a]pyrene
7,8-dihydrodiol-9,10-epoxide
7,8-dihydrodiol
ULTIMATE CARCINOGEN
CYP/PHS GST/GSH

DNA
Phase II and
excretion
O N
GS HN N
O OH N DNA
inactive (excreted) HN
HO

HO
OH

OH BaP-N2-dG DNA adduct

OH
inactive

Phase II
 Benzopyrene Leads to Mutations in K-Ras and p53 in the Genomic
Loci Found to be Mutated in Smoking-Induced Lung Cancers

• K-Ras and p53 are the two oncogenes most frequently mutated in
smoking-related lung cancers
• If not corrected by the cell’s DNA repair mechanism, this guanine “adduct” is
misread as a thymine by the DNA polymerase that copies chromosomes
during replication
• Ultimately, the original G—C base pair may be replaced by a T—A base pair,
a mutation called a traversion
• Cells treated with Benzopyrene show the same spectrum of G—T
transversions as found in the K-RAS and p53 of smokers.
• These mutational “hot spots” map well to the guanine binding sites of BaP
epoxide
Promotion
 Promotion
 Epigenetic event—change in gene expression
without change in DNA.
 Mitogenic (Not mutagenic) Stimulates proliferation.
Causes both mutated and normal cells to
proliferate.
 Enhances the effect of the genotoxic initiating agent
by establishing clones of initiated cells.
 Long delay possible between administration of
initiating agent and promoting agent.
 Promotion is reversible.
 Promoters

1. Reactive Oxygen Species (ROS) and

redox active xenobiotics and metals
2. Phorbol esters (e.g. TPA)
3. Polycyclic aromatic compounds (e.g.
Dioxin)
4. Peroxisome Proliferators (oxidized fats)
5. Endocrine Disruptors (estradiol, DES)
 Structures of Representative Promoters

TPA and other phorbol esters

activate protein kinase C,
which leads to signal
transduction pathways that
increase DNA replication, cell
division

TCDD (dioxin) activates aryl

hydrocarbon receptor (AhR) and
induces the expression of
cytochrome P450increases
oxidative stresscan oxidatively
activate oncogenic pathways (e.g.
RAS)
 Endocrine Receptors and Carcinogenesis

Endocrine disruptors are involved in breast, ovarian, colon,

prostate cancers.

1. ERβ/ERα (estrogen receptors) ratio is decreased in cancers

(ligands include estradiol); ERs are transcription factors.

2. ERβ inhibits ERα

a.ERα-ERα dimerization (homodimer) leads to mitogenic
activation.
b.ERβ-ERα dimerization (heterodimer) leads to an inactivation.

3. Androgen Receptor (prostate) (AR) can also homodimerize

with AR leading to mitogenic activation; AR can heterodimerize
with ERβ to cause growth arrest (prostate also dependent on
estrogenic signals).
 Estrogen Receptor Interactions

e s tro g e n

E R b e ta
c y to s o l

E R a lp h a

m it o g e n ic n o p r o life r a tio n

n u c le u s
 Examples of Endocrine Disruptors

Other examples include dioxin, polychlorinated biphenyls

(PCBs), DDT, bisphenol A (BPA) and atrazine.
Progression
 Mechanisms of Progression
Progression is an irreversible process and leads to metastasis.
Progression requires:
1. Further mutations from genetic instability (chromosomal
instability) during promotion.
2. Recruitment of inflammatory immune cells to the tumor.
3. The tumor cell acquiring “wound-healing” characteristics
(secretion of chemo-attractants to attract inflammatory immune
cells, angiogenesis factors, proteases, etc).
Examples of progressor agents: inflammation, asbestos fibers,
benzene, benzoyl peroxide, other peroxides, oxidative stress,
inflammation
 Chronic Toxicant Exposure
Decreased ATP, increased Ca2+,
increased oxidative stress

Cellular Necrosis
Intracellular contents
(e.g. ATP, dsDNA)

Activation of Resident
Macrophages
Cytokines, chemokines,
Eicosanoids (TNFa, IL1b, PGE2)

Recruitment and Activation

TGFb
of More Macrophages Epithelial-to-mesenchymal
transition (EMT)
TGFb, IGF1,
Growth factors
PDGF, TNFa
(e.g. TGFb, IGF1,
Leakier basement
PDGF, ROS)
membrane
Fibroblast proliferation,
VEGF
Cell proliferation differentiation TNFa, Infiltration of more
ROS
immune cells into damaged
Growth factors
(e.g. TGFb, IGF1, tissues
PDGF, ROS) Excessive formation of
Genetic instability angiogenesis hardened extracellular
Mutations matrix (ECM) Tissue Cells
Cell proliferation And Macrophage
Cellular transformation Cellular Necrosis
Growth factors fibrosis Tissue
(e.g. TGFb, IGF1, dysfunction,
PDGF, ROS) tissue damage,
Malignant Cytokines, chemokines, degeneration,
progression of cancer Eicosanoids (TNFa, IL1b, PGE2) organ failure
TGFb cells Recruitment and Activation
proteases
Epithelial-to-mesenchymal of More Macrophages
Cancer cells
transition (EMT) breakdown Proteases,
extravagate with
TGFb
of ECM (invasion) macrophages and metastasis
EMT and breakdown of ECM
blood supply into
circulation
 Inflammation and Cancer
• Inflammation acts at all stages of tumorigenesis
• It may contribute to tumor initiation through mutations,
genomic instability
• Inflammation activates tissue repair responses, induces
proliferation of premalignant cells, and enhances their
survival
• Inflammation also stimulates angiogenesis, causes
localized immunosuppression, and promotes the
formation hospitable microenvironment in which
premalignant cells can survive, expand, and accumulate
additional mutations
• Inflammation also promotes metastatic spread.
Clinical relevance: There is mounting evidence
that cellular senescence acts as a "cancer brake"
because it takes many divisions to accumulate all
the changes needed to become a cancer cell. In
addition to the accumulation of several mutations
in oncogenes and tumor suppressor genes,
almost all cancer cells are immortal and, thus,
have overcome the normal cellular signals that
prevent continued division. Young normal cells
can divide many times, but these cells are not
cancer cells since they have not accumulated all
the other changes needed to make a cell
malignant. In most instances a cell becomes
senescent before it can become a cancer cell.
Therefore, aging and cancer are two ends of the
same spectrum. The key issue is to find out how
to make our cancer cells mortal and our healthy
cells immortal, or at least longer-lasting. Inhibition
of telomerase in cancer cells may be a viable
target for anti-cancer therapeutics while
expression of telomerase in normal cells may
have important biopharmaceutical and medical
applications. In summary, telomerase is both an
important target for cancer and for the treatment
of age-related disease.

http://claim.springer.de/EncRef/CancerResearch/samples/0001.htm
 Application of Proteomics in Cancer Research

Characteristics of cancer cells

• general changes: - loss of division limits (immortality)
- uncontrolled proliferation

• genetic changes: - point mutations …

- chromosomal changes

• structural changes: - less organized cytoskeleton

- increased membrane fluidity

• biochemical changes: - altered protein expression

- altered protein modification
Cancer Arise by multistage Proses
How cell cancer arise
How cell cancer arise
Tumorigenesis
Step of tumorigenesis
Cancer Tissue consist of
Heterogeneous cell
Multistep Change In Tissue
Architecture
Hallmark of Cancer
 The genome is an organism’s complete set of DNA.
 a bacteria contains about 600,000 DNA base
pairs
 human and mouse genomes have some 3 billion.
 human genome has 24 distinct chromosomes.
 Each chromosome contains many genes.
 Gene
 basic physical and functional units of heredity.
 specific sequences of DNA bases that encode
instructions on how to make proteins.
 Proteins
 Make up the cellular structure
 large, complex molecules made up of smaller
subunits called amino acids.
Protooncogenes
Protooncogenes
Protooncogenes become Oncogene
Oncogene
 Proto-Oncogene Oncogene
 The proto-oncogene become oncogene by:
1. Mutation:
 Example: mutation in Ras gene Continuous
activation of Ras by (constitutively in the GTP-bound
conformation ) Unregulated cell proliferation
Cell transformation.
 Proto-Oncogene Oncogene
2. Abnormal Activity:
Example: Removal of the Regulatory domain in the Raf gene
and replaced by gag gene Raf kinase domain
consciously active Cell transformation

Raf Proto-oncogene
Regulatory Domain Protein Kinase Domain

Raf oncogene
gag Protein Kinase Domain
 Proto-Oncogene Oncogene
3. Gene translocation:
Example: c-myc gene is
translocated from
chromosome 8 to the IgH
on the chromosome 14
resulting in abnormal c-
myc expression Cell
transformation
Proto-Oncogene Oncogene
4. Amplification:
Example: Amplification of n-myc
neuroblastoma. Amplification of erbB-2
Breast & ovarian carcinomas
How does a Proto-oncogene become an
Oncogene?
Proto-Oncogene Oncogene

1.Mutation 2. Abnormal 3.Gene Translocation 4. Amplification

Activity

Abnormal Activity
 Functions of oncogene
1. Growth Factor (example, Epithelium growth
factor EGF , and platelet derived growth factor
PDGF)
2. Growth Factor Receptor (Example; PDGFR)
3. Signal transudation (example; Ras, Raf, &
MEK)
4. Transcription Factor (example; Jun, Fos, Elk-1
& myc)
 Oncogenes
 Oncogene causes cancer by affecting:
1. Cell Proliferation: (example; Ras, Raf, EGF)
2. Cell differentiation (example, PML/RAR that
inhibits the differentiation of promyelocyte to
granulocyte which will maintain the cell in its
active proliferate state)
3. Cell Survival (example; Pl-3/AKT which will
activate BCL-2 inhibit Apoptosis &
maintain cell survival.
Tumor suppressor genes
Tumor suppressor genes
Protooncogenes and Tumor
suppressor genes
 Tumour Suppressor Genes
 Tumour Suppressor genes: are genes that act to
inhibit cell proliferation and tumour
development.
If Tumor Suppresor Gene was

Mutated OR Inactivated

It will lead to cell transformation

 Hereditary
Mutation

Non-
hereditary
Mutation
 Function of Tumour Suppressor gene
1. Antagonize the action of oncogene. (ex.PTEN which
converts PIPIII to PIPII because PIPIII will activate Pl-
3/AKT which will activate BCL-2 that will inhibit apoptosis
and induce cell transformation)

PTEN
PIPII PIPIII
PI-3
AKT

BCL-2

Inhibit apoptosis & induce

cell transformation
 Function of Tumour Suppressor gene

2. Transcription factors
 Repressor transcription factors: example;
WT1 is a repressor that appears to suppress
transcription factor ( Insulin like growth factor)
which will contribute in the development of
tumour.
 Activator transcription factors: example;
SMAD family that are activated by TGF-β,
leading to inhibition of cell proliferation.
 Function of Tumour Suppressor gene
3. Regulate cell cycle :
 Rb gene: that inhibits the cell cycle in the G1
phase decrease cell proliferation.
 INK-4 gene: that produces P16 that
inhibits cdk4/cyclin D action ( to
phosphorylate Rb gene to inactivate it’s
action)
 P53: that produces P21 that has the same
action of P16 in inhibiting the action of
cdk4/cyclin D
 Function of Tumour Suppressor gene

4. Induce apoptosis:
 P53 release will increase Bax form
holes in the mitochondria release
cytochrom c activate apoptosis
Hallmark of Cancer
 Sustained angiogenesis
Cancer cells can stimulate neo-angiogenesis, during
which new vessels sprout from previously existing
capillaries, or, in some cases, vasculogenesis, in which
endothelial cells are recruited from the bone marrow.

Angiogenesis (Neovascularization) is a necessary biologic

event in malignancy as it is required for

1. Perfusion that supplies needed nutrients and oxygen

2. Growth of neoplastic cells by newly formed endothelial
cells that secrete growth factors (e.g., insulin-like
growth factors & PDGF)
3. Access to the blood vessels and hence for metastasis
 Invasive & metastasizing potentials
 A cancer first must breach the underlying basement
membrane, then traverse the interstitial connective tissue,
and ultimately gain access to the circulation by penetrating
the vascular basement membrane.
 This cycle is repeated when tumor cell emboli extravasate
at a distant site.
 Invasion of Extracellular Matrix (ECM)

requires four steps:

 1. Detachment of tumor cells from each other
 2. Degradation of ECM
 3. Attachment to new ECM components
 4. Migration of tumor cells
 The first step  loosening of tumor cells.
E-cadherin molecules act as intercellular glues that
keep the cells together. E-cadherin function is lost in
almost all epithelial cancers by mutational inactivation
of E-cadherin genes.
 The second step  local degradation of the
basement membrane and interstitial connective
tissue.
 Tumor cells may either secrete proteolytic enzymes
themselves or induce stromal cells to elaborate
proteases. Multiple different families of proteases,
including matrix metalloproteinases (MMPs) have
been implicated in tumor cell invasion
 The third step  attachment of tumor cells to
another set of ECM proteins.
Normal epithelial cells have receptors (such as
integrins) for basement membrane laminin and
collagens that are polarized at their basal surface.

The matrix itself is modified in ways that promote

invasion and metastasis.
For example, cleavage of the basement membrane
proteins collagen IV and laminin by MMPs
generates new sites that bind to receptors on tumor
cells and stimulate migration.
 final step  propelling tumor cells through the
degraded basement membranes and zones of
matrix proteolysis. Migration is a complex
multistep process that eventually impinges on the
actin cytoskeleton. Such movement seems to be
augmented and directed by tumor cell-derived
cytokines, such as autocrine motility factors.
Stromal cells also produce paracrine effectors of
cell motility, such as hepatocyte growth
factor/scatter factor (HGF/SCF), which bind to
receptors on tumor cells.
 Vascular Dissemination and Homing of
Tumor Cells
 When in the circulation, tumor cells are vulnerable
to destruction by host immune cells. In the
bloodstream, some tumor cells form emboli by
aggregating and adhering to circulating
leukocytes & platelets.
 Extravasation of free tumor cells or tumor emboli
involves adhesion to the vascular endothelium,
followed by passing through the basement
membrane into the organ parenchyma by
mechanisms similar to those involved in invasion.
Genomic instability due to defects in
DNA repair
 Mutations are at the heart of carcinogenesis.
Although humans literally swim in environmental
agents that are mutagenic (e.g., chemicals,
radiation, sunlight), cancers are relatively rare
outcomes of these encounters
 Defective mismatch repair
 Defective recombination repair