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Molecular Basis of

Oncogenesis
Felina R. Masadao-Adefuin, MD

Fellow, Philippine College of Physicians


Fellow, Philippine Society of Medical Oncology
Certified Fellow, Molecular Oncology Society of the Philippines
Fellow, Philippine Society of Oncologists
Initiation Promotion Progression
Hallmarks of cancer
Oncogenes
Cell Cycle and Cell Cycle Regulation
Apoptosis
Metastasis
Angiogenesis
OVERVIEW OF
CARCINOGENESIS
HALLMARKS OF
CANCER
(Weinberg &
RADIATION
RADIATION Hanahan, 2000)

CHEMICAL
CHEMICAL
S
S ACCUMULATIO
ACCUMULATIO
1. SELF-SUFFICIENCY
N
N OF
OF GENETIC
GENETIC IN GROWTH SIGNALS
&
& EPIGENETIC
EPIGENETIC
ONCOGENES CHANGES
CHANGES
2. INSENSITIVITY TO
ANTI-GROWTH SIGNALS
INFECTIOU
INFECTIOU INACTIVATED
S
S TUMOUR SUPPRESSOR 3. EVASION OF APOPTOSIS
AGENTS
AGENTS GENES

4. LIMITLESS REPLICATIVE
HEREDITY
HEREDITY POTENTIAL

additional mutations
5. SUSTAINED ANGIOGENESIS

GENETIC
GENETIC 6. TISSUE INVASION
INSTABILIT
INSTABILIT AND METASTASIS
Y
Y

??? INFLAMMATION
7 Hallmark?
th

Francesco Colotta, et.al. (May 2009)


Link between inflammation and cancer
Inflammation contributes to proliferation and
survival of malignant cells, angiogenesis,
metastasis, subversion of adaptive
immunity, reduced response to hormones
and chemotherapy
Inflammation induces genetic instability via
inflammatory mediators, leading to
accumulation of random genetic alterations
in cancer cells
SCHEME OF
CARCINOGENESIS
Carcinogenesis
Normal cells

INITIATION

Initiated cells
CARCINOGEN

PROMOTION

Tumour cells
Inactive metabolite
Clones

PROGRESSIO Gross Tumour


N
Sequential Relationship Between
Initiation and Promotion
X
NO TUMOURS

X
TUMOURS

X
TUMOUR
S
X
NO TUMOURS

NO TUMOURS

X Initiator
Promoter
Knudsons Two-Hit Hypothesis
2nd
mutation
Uncontrolled proliferation;
Cancer formation

1st
mutation

Controlled growth;
No cancer
Cancer Mutations
Sporadic : majority of cancers; acquired
during ones lifetime; incidence increases
with age; due to environmental causes;
not passed on to offspring
Hereditary : less than 10% of cancers;
germline mutation (present in reproductive
cells) can be passed on to next
generation(s); cancers usually occur at
younger ages
ONCOGENES
Definitions
Proto-oncogenes : genes that
encode proteins that regulate normal
cell proliferation or apoptosis

Oncogenes : arise from mutant


proto-oncogenes; more active than
normal or active at inappropriate
times and stimulate unregulated cell
proliferation
General Classes of Cancer Genes
Gatekeeper genes : genes that directly affect cell growth
Oncogenes : positive Activated by :
Point mutation
Genes that induce or continue uncontrolled cell proliferation, e.g. ras, raf, jun,
fos, myc DNA amplification
Chromosomal rearrangement
Tumor suppressor genes : negative
Inactivated by :
Genes that normally restrain cell growth, e.g. p53, Rb, BRCA1, BRCA2
Point
Both alleles must be inactivated to completely lose the mutation
function (Knudsons two-
hit hypothesis) Large deletions LOH (hallmark
for
presence of a TSG at a particular
Caretaker genes : genes that do not directly affect locus)cell growth,
Gene of
but affect ability of cell to maintain the integrity silencing
the genome
DNA repair genes : Genes that encode proteins that repair any
damage/mutation to the cell
Defective DNA repair -> increased rate of failure to repair mutations ->
accumulation of mutations -> genomic instability
Mechanisms of Converting
Protooncogenes to Oncogenes
Excess normal
protein
Abnormal
Excess normal
(hyperactive)
protein Translocated
protein
chromosome

Abnormal
(hyperactive)
GENE
GENE protein
AMPLIFICATIO
AMPLIFICATIO CHROMOSOMAL
CHROMOSOMAL
POINT
POINT N
N TRANSLOCATION
TRANSLOCATION
MUTATION
MUTATION
DNA

LOCAL
LOCAL DNA
DNA proto-oncogene INSERTIONAL
INSERTIONAL
REARRANGEMENT
REARRANGEMENT MUTAGENESIS
MUTAGENESIS
S
S

Insertion or
Viral DNA
Insertion Deletion Transposition

Excess normal
protein
Abnormal
(hyperactive)
protein
Functions of Cellular Proto-oncogenes

1. Secreted Growth Factors

2. Growth Factor Receptors

4. Nuclear
Proteins:
Transcription
3. Cytoplasmic Factors
Signal Transduction
Proteins
Cell Growth
Genes
Oncogenes : Growth Factors
sis : PDGF B chain
transduced by retrovirus
autocrine loop in some glioblastomas
int-2 : FGF-related growth factor
activated by integration of retrovirus
Oncogenes : Growth Factor
Receptors
ErbB/B2 : EGF receptor family
truncated erbB is transduced by retrovirus
overexpression is due to increased transcription or gene
amplification (CAs of breast, ovary, stomach)
Src : membrane-associated nonreceptor PTK
transduced by retrovirus
deleted regulatory domain so constitutively active
mas : angiotensin receptor
transduced by retrovirus
RET : involved in thyroid CA
Oncogenes : Signal Transduction
Proteins
abl : nonreceptor PTK; detects DNA
damage
translocation-fusion w/ BCR in CML
ras family : monomeric GTP-binding
proteins
aa substitutions reduce GTPase activity or
increase rate of exchange of GDP for GTP so it
remains active
raf : MAPKKK Ser/Thr kinase
Mutated/rearranged in stomach cancers
Oncogenes : Transcription Factors

myc : early response genes/sequence


specific DNA-binding transcription factor
overexpression due to gene amplification or
translocation to Ig heavy chain locus in
lymphomas
fos/jun : early response
genes/transcription factor; combine to
form AP-1
erb A : thyroxine (T3) receptor
transcription factor
Other Oncogenes
bcl-2 : anti-apoptotic factor
overexpression is due to translocation to
Ig heavy chain locus/enhancer in
follicular lymphoma
bcl-1 : also called PRAD-1; codes for
cyclin D1; involved in BRCA and H&N
cancers
mdm2 : p53 antagonist; involved in
sarcomas
Common Oncogenes Altered in
Human Cancers
ONCOGENE FXN ALTERATION NEOPLASM
IN CANCER
AKT1 Serine/threonine Amplification Gastric CA
kinase
AKT2 Serine/threonine Amplification Ovarian, breast,
kinase pancreas cancer
BRAF Serine/threonine Point mutation Melanoma, lung,
kinase colorectal cancer

CTNNB1 Signal transduction Point mutation Colon, prostate,


melanoma, skin, etc
FOS Transcription factor Overexpression Osteosarcomas
ERBB2 Receptor tyrosine Point mutation, Breast, ovary,
kinase amplification stomach,
neuroblastoma
JUN Transcription factor Overexpression Lung cancer

MET Receptor tyrosine Point mutation, Osteocarcinoma,


kinase rearrangement kidney, glioma
MYB Transcription factor Amplification AML, CML, colon
cancer, melanoma
Common Oncogenes Altered in
Human Cancers
ONCOGENE FXN ALTERATION NEOPLASM
IN CANCER
C-MYC Transcription factor Amplification Breast, colon,
gastric, lung
L-MYC Transcription factor Amplification Lung carcinoma,
bladder
N-MYC Transcription factor Amplification Neuroblastoma, lung
cancer
HRAS GTPase Point mutation Colon, lung, pancreas

KRAS GTPase Point mutation Melanoma, colorectal


cancer, AML
NRAS GTPase Point mutation Various carcinomas,
melanoma
REL Transcription factor Rearrangement, Lymphomas
amplification
WNT1 Growth factor Amplification Retinoblastoma
CELL CYCLE AND CELL
CYCLE REGULATION
Cell 100:5770, 2000
The Hallmarks of Cancer Review
Douglas Hanahan* and Robert A. Weinberg
Cell Cycle

9
9
h
h

1
1
0
0
h
h

4
4
h
h
1
1
h
h
Rao & Johnson :
cell fusion experiments
G1-phase cells + S-phase cells = DNA
REPLICATION IN HYBRID CELL
G2-phase cells + S-phase cells = NO DNA
REPLICATION
G1-phase cells + M-phase cells =
PREMATURE CHROMOSOMAL COMPACTION
G2-phase cells + M-phase cells =
PREMATURE CHROMOSOMAL COMPACTION
S-phase cells + M-phase cells =
CHROMATIN COMPACTION
Cell Cycle Regulation : Importance

to ensure the orderly progression of


events so that nuclear cycle is
coordinated with cell growth and physical
separation
to make sure that replication must occur
once per cell cycle and precede
chromosome segregation
to ensure that segregation must be
complete before cytokinesis (cell division)
Cyclin-CDK complex

CDK : CYCLIN-DEPENDENT PROTEIN KINASE


(CATALYTIC UNIT)

CYCLIN : REGULATORY UNIT


cdc25
A
cdc25
B
cdc25 Cyclin B,
C +
CDK1
Cyclin A
+ G0
CDK1
M
cdc25
G2 B
Cyclin D1,
D2,
D3
+
cdc25 CDK 4, 6
B G1
Cyclin A S
+
CDK2

cdc25
A
Cyclin E1, E2
+
CDK 2
Role of Rb/E2F in cell cycle

M G1

G2 E2F
Rb/p107/
p130

S
SWI/
SNF METHYLASE
S

HDAC
s

Rb/p107/ p13 p10 RB


p130 0 7

E2F
E2F E2F E2F E2F E2F E2F
6 5 4 1 2 3
TRANSCRIPTIONAL WEAK TRANSCRIPTIONAL STRONG TRANSCRIPTIONAL
REPRESSOR ACTIVATORS ACTIVATORS
Role of Rb/E2F in cell cycle

M G1 Cyclin
Cyclin
D1,2,3
D1,2,3
++
CDK4,6
CDK4,6
G2 E2F
P Rb/p107/
p130
P P Cyclin
Cyclin
P E E
E2F
S ++
P CDK2
CDK2
P

Cyclin A,
DNApolDHFR, E; CDK2, c-myc, c-
TK, TS E2F1 jun, c-fos
Cyclin B,
+
CDK1
Cyclin A
+ G0
CDK1
M

G2
Cyclin D1,
D2,
D3
+
CDK 4, 6
G1
Cyclin A S
+
CDK2
p15ink4b
ink4b

p16ink4
ink4

p21waf1/cip
waf1/cip p18ink4c
ink4c

1
1 p19ink4d
ink4d

p27kip1
kip1 Cyclin E1, E2 p21waf1/cip1
waf1/cip1
+
p57kip2
kip2
CDK 2
p27kip1
kip1

p130 p57kip2
kip2

p107
CELL CYCLE CHECKPOINTS
G1S
Are we ready to replicate our DNA?
Is the DNA repair machinery in place to
fix any mutations that are detected?
Are the DNA replicating enzymes
available? Is there an adequate supply
of nucleotides? Is there sufficient
energy?
DNA
DAMAGE
Ionizing radiation, ds DNA breaks UV, replication errors

ATM ATR

PS395 ATR
S15
mdm2 P
S20
p5
3 T68 P P
Chk
Chk Chk
Chk
2
2 1
1

p5
3

Cyclin
Cyclin
E E
GADD p2
++
mdm2 45 CDK2
1 CDK2

Cyclin
Cyclin
PCNA
D D
+ + G1
CDK4/6
CDK4/6 ARREST
DNA
DAMAGE
Ionizing radiation, ds DNA breaks UV, replication errors

ATM ATR
ATR

T68 P
Chk
Chk
2
2
BASC : BRCA1-associated genome surveillance
complex
NBS 1 : Nimejen Breakage Syndrome 1
BRCA 1 : breast cancer susceptibility gene 1
P SMC1 : structural maintenance of chromosome
protein 1
NBS1/BRCA
P
S123
1/SMC1
cdc25A
cdc25A

Cyclin E/A
Cyclin E/A
++
CDK2
CDK2

S-PHASE DELAY
G2M
Have all of the chromosomes been fully
duplicated?
Were any segments of DNA copied more
than once?
Do we have the right number of
chromosomes and the right amount of
DNA?
DNA
DAMAGE
Ionizing radiation, ds DNA breaks UV, replication errors

ATM ATR
ATR

P
p5
P P S216
P
3 T68
Chk
Chk Chk
Chk cdc25C
cdc25C
2
2 1
1

S216
P
p5
3 cdc25C
cdc25C

14-3-
3

p2
1

Cyclin
Cyclin
B1B1
++
GADD cdc2
cdc2
45

G2
ARREST
DNA
DAMAGE
Ionizing radiation, ds DNA breaks UV, replication errors

ATM ATR

P ATR

mdm2 P
p5
P P P
3
Chk
Chk Chk
Chk cdc25C
cdc25C
2
2 1
1

P
p5
3 cdc25C
cdc25C
NBS1/BRC
P
14-3-
A1/SMC1 3
cdc25A
cdc25A

GADD
P
p2
mdm2 45 1

Cyclin Cyclin
Cyclin
Cyclin Cyclin E/A
E E Cyclin E/A B1B1
++ ++
++
CDK2
CDK2 cdc2
cdc2
CDK2
CDK2

G1 G2
S-PHASE DELAY
ARREST ARREST
APOPTOSIS
Cell 100:5770, 2000
The Hallmarks of Cancer Review
Douglas Hanahan* and Robert A.
Weinberg
Definition
derived from greek word
meaning dropping off or
falling off of petals from the
trees
programmed cell death
Morphological Events

chromatin
condensation
DNA fragmentation
nuclear breakdown
membrane
"blebbing"
cell fragmentation
(apoptotic bodies)
phagocytosis
Apoptosis vs Necrosis
Internal / external
signals Ischemia/toxins/radn
Energy dependent
Single cells Non-energy dependent
The cell shrinks and is Groups of cells
engulfed by The cells swell and
macrophages or release their content
neighbouring cells into the surroundings
Normally, no and the circulation
inflammatory reaction Marked inflammatory
reaction
bcl-2 Family

PORE MEMBRANE
REGULATION FORMATION ANCHOR
Anti-apoptotic
BH BH BH BH
bcl-2, bcl-XL, bcl-w 4 3 1 2
TM

Pro-apoptotic
BH BH BH
Bax, Bak, Bok 3 1 2
TM

BH3-only Pro-apoptotic
BH
Bik, Blk, Hrk, Bnip3 3
TM

Bid, Bad, Bim, Puma, Noxa, Bmf BH


3
Caspases
(cysteine aspartate proteases)
ICE subfamily ->
inflammation procaspase-9
Caspase -13, -5, -4,
-1
procaspase-8

CED-3 subfamily ->


apoptosis
procaspase-3
Caspase -7, -3, -6
Caspase -8, -10, -2, caspase recruitment domai
-9 death effector domain
Activation of Caspases

PROCASPASE

Proteolytic cleavage

CASPAS
E
Targets of Caspases
INACTIVATION OF FAK ->
DISRUPTION OF CELL ADHESION
DETACHMENT OF APOPTOTIC CELL
Protein kinases
IN INNER FROM
LININGNEIGHBOURING
OF NUCLEAR CELLS
ENVELOPE; CLEAVAGE
Lamins CLEAVAGE OFOF
DISASSEMBLY INTERM FILAMENTS,
NUCLEAR LAMINA &
ACTIN, GELSOLINOF
SHRINKAGE NUCLEUS
CHANGES IN
Proteins required for cell structure
CELL SHAPE
CASPASE-ACTIVATED
Endonuclease (CAD) DNAase ATTACKS DNA,
SEVERING IT INTO
Enzymes involved in DNA repairFRAGMENTS
DEATH SIGNALS
*EXTRINSIC FAS FasL (CD95L)
* TRAIL-R
TNF-R DISC
TNF
DR4, DR5 (TRAIL DEATH RECEPTORS
DD
DDDD
CELL MEMBRANE
FADD

CASPASE 10
CASPASE 8
INITIATOR
CASPASES

CASPASE 3
CASPASE 7
CASPASE 6

EXECUTIONER CASPASES

NUCLEUS
APOPTOSIS
INTERNAL STIMULI :
DNA DAMAGE 1. Cytochrome c
*INTRINSIC* ONCOGENE-INDUCED
release
PROLIFERATION
LOSS OF ATTACHMENT TO 2.
ECMBax translocation
CHEMOTHERAPY, RADIATION
THERAPY
to mitochondria
CELL MEMBRANE 3.*INTRINSIC*
Bax/Bak
PRO-APOPTOTIC oligomerization
PROTEINS :
Bak,Bax, Bad, Bid, Bik, MITOCHONDRIA
Bim, Puma, NOXA, BmftBID
CASPASE 8
CYTOCHROME C
INITIATOR
CASPASE
CASPASE 9

APAF-1
CASPASE 3
APOPTOSOME
CASPASE 7
CASPASE 6

EXECUTIONER CASPASES

NUCLEUS
APOPTOSIS
PRO-APOPTOTIC PROTEINS
REGULATION FAS
TRAIL ANTI-APOPTOTIC PROTEINS
TNF
DDDD
TRAF2
CELL MEMBRANE Ras
FADD
NFKB P13K
MITOCHONDRIA
PTEN Akt
CASPASE 10
Smac/Diabl
o IAP
FLIP CASPASE 8

CASPASE 9

APAF-1
CASPASE 3
CASPASE 7
CASPASE 6
CASPASE 2
EFFECTOR CASPASES
RAIDD
ICH1-CED3

NUCLEUS PIDD

p53 APOPTOSIS
Mdm2
Hypoxia, DNA damage (UV, gamma, chemotherapy),
ribonucleotide depletion, telomere shortening

P53 INDUCTION
Cell 100:5770, 2000
The Hallmarks of Cancer Review
Douglas Hanahan* and Robert A.
Weinberg
Guardian of the Genome

p5
3
1. DNA damage checkpoint p5 2. Oncogene checkpoint
P mdm2
3

ATM ATR E2F myc EIA


ATR

T68 P P
Chk Chk
Chk
2
2
Chk
1
1 p14ARF

Transcriptional activation of p53-responsive


genes
p21Cip1/Waf1, NOXA, PUMA, etc
ANGIOGENESIS
Vasculogenesis : formation of vascular
structures from circulating or tissue-
resident endothelial stem
cells(angioblasts), which proliferate into
de novo endothelial cells
this form particularly relates to the embryonal
development of the vascular system
Arteriogenesis : formation of medium-
sized blood vessels possessing tunica
media plus adventitia
Angiogenesis : formation of new
blood vessels from pre-existing vessels
(formation of thin-walled endothelium-
lined structures with /without muscular
smooth muscle wall and pericytes
(fibrocytes); relates to repair
mechanism and in spread of cancer
Sprouting angiogenesis
Intusussceptive or splitting angiogenesis
Angiogenic switch : phase in
tumour development when
proangiogenic factors > anti-
angiogenic factors
Pro-angiogenic : VEGF, bFGF, TGF, EGF,
TNF, Angiogenin, IL-8, Angiopoietin
Anti-angiogenic : Angiostatin,
Endostatin, Vasostatin, IFN, Prolactin, IL-
12, Tumstatin
Tumor blood vessels
Abnormal architecture
tortuous, dilated, uneven diameter, excessive branching,
shunting
Vessel walls have numerous openings, widened
interendothelial jxns, absent or discontinuous basement
membrane
Lack perivascular cells (pericytes, smooth muscle cells)
which regulate flow in response to tissue metabolic needs
Vascular lining = ECs (highly proliferative) + tumor cells

Variable blood flow : with areas of hypoxemia and


acidosis (variants resistant to hypoxemia-induced
apoptosis)
ENDOTHELIAL CELL-SPECIFIC LIGAND/RECEPTOR COMPLEXES

PIGF VEGF-A VEGF-B VEGF-C Ang-1 Ang-2 Ephrins ECM bFGF PDGF

VEGFR1 VEGFR2 VEGFR3 Tie-2 EPHB4 v3 FGFR PDGF


ENDOTHELIAL CELLS LYMPHATICS BV STABILIZATION ARTERY-VEIN ARTERY-VEIN RECRUITMENT OF
& REMODELING DIFFERENTIATION,DIFFERENTIATION, SMOOTH MUSCLE
VESSEL VESSEL CELLS AND
REMODELING REMODELING PERICYTES

DOWNSTREAM PATHWAYS
Ras/MAPK
PI3K/AKT
Rho/Rac/cdc42
NFB

ENDOTHELIAL CELL PROLIFERATION, MIGRATION, SURVIVAL


Steps in Angiogenesis
Stimulation of endothelial cells by
growth factors
Degradation of extracellular matrix by
proteases degradation of basement
membrane escape of endothelial
cells
Proliferation of endothelial cells and
migration into the tumour
Formation of new capillary tubes
CEP contributes
Vascular mimicry : tumor
newly
cells as part of vessel
differentiated EC
wall

Dilated, leaky Leaky vessels;


Tumor vessel High intratumoral Region of hypoxia
pressure HIF

VEGF

VEGF
VEGFR2 VEGFR2
Destabilizatio Tie 2
Ang
n Tie 2 v3
2 v3 v ECM
Tie 2

New sprout
Follows VEGF gradient to tumour

HOST BLOOD VESSEL


VEGFR2

Circulating endothelial precursors (CEP)


CD133

VEGFR1
Hematopoietic cell-derived leukocytes (HSC)

c-kit
NORMAL BLOOD TUMOR BLOOD
VESSEL VESSEL
Tortuous vessels
Hierarchical branching
Haphazard blood flow
Even blood distribution
High IP
Low IP
Hypoxemia
Normoxic

Acidosis
Physiologic pH
Loss of EC jxn
Tight jxns between EC
complexes
Well-formed BM
Absent or few
Pericyte coverage
pericytes
Normal permeability Increased permeability
METASTASIS
Major Features of Tissue Invasion

Cell adhesion to basement


membrane
Local proteolysis of the membrane
Movement of cell thru the rent in the
membrane and the ECM

Rate-limiting step : ability for tumour


cells to survive and expand in the
novel microenvironment of the
metastatic site
basement membrane

Normal epithelial cells lamina propria


Cytokeratin

Tumor-associated fibroblast

Adherens
junction
E-cadherin

New lymph vessel


Tumor cell

N-cadherin Tumor-associated macrophage


TGF-
TGF-
receptor MMP
Cytokines
GFs
Snail

HGF
Twist
New integrin
expression C-Met

N-cadherin VEGF-A New blood vessel

HOST STROMAL CELLS

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