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Carcinogénesis
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Dr. Alejandro Claude
Instituto de Bioquímica
UACH
Carcinogenesis

a). Multistep carcinogenesis

i). Stages in the evolution of colon cancer
ii). Growth characteristics of cells in culture
e
d
b). Genes responsible for tumorigenic cell growth
u
la
i). Proto-oncogenes and oncogenes
C
.
transforming viruses
A
r
functions of proto-oncogenes
o
es
oncogene activation
f
P ro
ii). Tumor suppressor genes
Knudson’s two-hit hypothesis
loss of heterozygosity
functions of tumor suppressor genes
retinoblastoma
p53
 Multistep carcinogenesis
Stages in the evolution of colon cancer

Chromosome 5q gene loss or mutation

Normal
colon cell

Increased
d e
cell growth
Ras gene mutationla u
Adenoma
I . C
A
e s or
Adenoma
II
Chromosome 18 loss or mutation
DCC tumor suppressor gene

ro f
Adenoma
P III

Chromosome 17 loss or mutation Carcinoma

p53 tumor suppressor gene

Other chromosome losses Metastasis

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 Growth characteristics of cells in culture
(parallels what happens in vivo)

• Normal primary cells: survive only a relatively short

time in culture (days to weeks)
• Immortalized (established) cells: immortalized, but not transformed
(they retain many of their in vivo characteristics)
d e
• anchorage dependence (prefer to adhere to culture plate)
a
• serum dependence (require growth factors)
l u
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• contact inhibited (stop growing when cells are confluent)
• Transformed cells: will form tumors (tumorigenic) when injected
r A
into a host, but will not necessarily kill the host
es o
• less dependent on substratum
f
• less dependent on growth factors
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• not contact inhibited
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• Metastatic cells: fully transformed cells that have the ability to
migrate and invade tissues; will establish new colonies
and kill the host

At least 2-3 steps (which are caused by mutations)

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What are the genes responsible for tumorigenic cell growth?

Normal
Proto-oncogenes +
d e
Cell growth and proliferation
Tumor suppressor genes
- la u
. C
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Cancer es o
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Mutated or “activated”
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oncogenes
++
Malignant transformation
Loss or mutation of
Tumor suppressor genes
Oncogenes are usually dominant (gain of function)

• cellular proto-oncogenes that have been mutated (and “activated”)

• cellular proto-oncogenes that have been captured by retroviruses
e
and have been mutated in the process (and “activated”)
d
la u
• virus-specific genes that behave like cellular proto-oncogenes
that have been mutated to oncogenes (i.e., “activated”)
. C
r A
Tumor suppressor genes
e s o
are usually recessive (loss of function)

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• loss of a cellular gene or chromosome region by deletion
• loss of gene function by an inactivating point mutation
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 Transforming viruses

Viral class Viral genome Oncogenes

adenovirus ds DNA E1A & E1B

papovavirus ds DNA e
T antigens
d
SV40 (monkey)
la u
Polyoma (human) C
A .
retrovirus

e s or
ss RNA mutated cellular
proto-oncogenes

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 Activities of viral oncogenes

Oncogene activity
Virus Immortalizing Transforming

adenovirus (human) E1A d e E1B

papovavirus
la u
SV40 (monkey) large T antigen
C small T antigen
Polyoma (human) .
large T antigen
A middle T antigen

o r
retrovirus
f es
Avian myelocytomatosis myc
P ro
Harvey murine sarcoma ras
 Retrovirus oncogenes derived from normal cellular genes

Retrovirus Viral oncogene Cellular proto-oncogene

Rous sarcoma virus v-src c-src (src)

Simian sarcoma v-sis
d ec-sis (sis)
Harvey murine sarcoma
Kirsten murine sarcoma
v-H-ras
v-K-ras la u c-H-ras (H-ras)
c-K-ras (K-ras)
FBJ murine osteosarcoma v-fos . C c-fos (fos)
Avian myelocytomatosis r A
v-myc c-myc (myc)
Abelson leukemia virus
es o v-abl c-abl (abl)
Avian erythroblastosis f v-erbB c-erbB (erbB)

P ro
• viral oncogenes are ~80-99% homologous to cellular proto-oncogenes
• viral oncogenes in general are copies of cellular mRNA and lack introns
 Gene organization of a retrovirus
gag pol env
5’ 3’

Gene organization of a transforming retrovirus

gag pol env onc
5’ 3’d e
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.
gag = group specific antigen C
A
pol = reverse transcriptase
r
env = envelopees o
f
onc = oncogene
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Gene organization of a cellular proto-oncogene

5’ 3’ mRNA
 Functions of cellular proto-oncogenes

1. Secreted Growth Factors

e
2. Growth Factor Receptors
d
la u
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r A 4. Nuclear
es o Proteins:
ro f Transcription
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3. Cytoplasmic Factors
Signal Transduction
Proteins
Cell Growth
Genes
 Functions of selected proto-oncogenes

Proto-oncogene Biochemical property

1. Secreted growth factors

c-sis Platelet derived growth factor
d e
2. Growth factor receptors
la u
c-erbB C
Epidermal growth factor receptor
.
r
3. Signal transduction proteins
A
c-abl
es o Protein kinase
c-src
ro f Protein kinase
H-ras P Small G-protein
K-ras Small G-protein

4. Nuclear proteins
c-myc Transcription factor
c-fos Transcription factor
 Oncogenes in human tumors

Mechanisms of activation of proto-oncogenes

• point mutations e
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• chromosomal rearrangements or translocations
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• gene amplifications C la
A .
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f es
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 Identification of oncogene mutations in human tumors

• most human tumors contain mutated or “activated” proto-oncogenes

• demonstrated by isolating the mutated genes from human tumors

Isolate DNA

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Transfer fragments of
Tumor cells DNA into la u
normal cells
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s o
Isolate transformed
e clones of cells
o f
(by virtue
r of their growth advantage)
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Isolate new DNA gained
by transformed cells

10-20% of spontaneous human tumors have DNA that will

transform cells in culture; most are due to ras gene mutations
 Ras family proteins
• the c-ras family contains three genes: H-ras, K-ras, and N-ras
• the Ras proteins encoded by these genes are small G-proteins
• the proteins transmit growth signals from cell surface receptors
• the Ras proteins are activated by binding GTP
• the proteins are inactivated by GTP to GDP hydrolysis

d e
• mutations in the c-ras genes inactivate the Ras GTPase
la
• mutated Ras proteins are constitutively active
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• constitutively active Ras proteins result in uncontrolled cell growth
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 Amino acid substitutions in Ras family proteins

amino acid position

Ras gene 12 59 61 Tumor

c-ras (H, K, N) Gly Ala Gln normal cells

d e
H-ras Gly Ala Leu
la ulung carcinoma
Val Ala Gln C bladder carcinoma
K-ras Cys Ala .
Gln
A lung carcinoma
Arg Ala
o r Gln lung carcinoma

N-ras
Val
f
Gly esAla
Ala
Gln
Lys
colon carcinoma
neuroblastoma
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Gly Ala Arg lung carcinoma

Murine sarcoma virus

H-ras Arg Thr Gln Harvey strain

K-ras Ser Thr Gln Kirsten strain
 Chromosomal rearrangements or translocations

Neoplasm Translocation Proto-oncogene

Burkitt lymphoma t(8;14) 80% of cases c-myc1

t(8;22) 15% of cases
t(2;8) 5% of cases
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Chronic myelogenous la
t(9;22) 90-95% of cases
u bcr-abl2
leukemia . C
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Acute lymphocytic
leukemia es o
t(9;22) 10-15% of cases bcr-abl2

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1c-myc
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is translocated to the IgG locus, which results in its activated expression
2bcr-abl fusion protein is produced, which results in a constitutively active abl kinase
 c-myc is translocated to the IgG locus,
which results in its activated expression

c-myc IgG
c-myc is activated by
IgG enhancer
u de
the IgG enhancer in
C la
lymphocytes
A .
o r
es
bcr-abl fusion protein is produced,
f
ro
which results in a constitutively active abl kinase
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bcr

bcr-abl
abl
 Gene amplification

Oncogene Amplification Source of tumor

c-myc ~20-fold leukemia and lung carcinoma

N-myc 5-1,000-fold e
neuroblastoma
d
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retinoblastoma

L-myc 10-20-fold . C
small-cell lung cancer
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c-abl ~5-fold
es o chronic myoloid leukemia

c-myb ro f
5-10-fold acute myeloid leukemia
P colon carcinoma

c-erbB ~30-fold epidermoid carcinoma

K-ras 4-20-fold colon carcinoma

30-60-fold adrenocortical carcinoma
 Tumor suppressor genes

Disorders in which gene is affected

Gene (locus) Function Familial Sporadic

DCC (18q) cell surface unknown colorectal

interactions d e cancer

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WT1 (11p) transcription C
Wilm’s tumor
. lung cancer

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Rb1 (13q)
es
transcription
o retinoblastoma small-cell lung

ro f carcinoma

p53 (17p)
P
transcription Li-Fraumeni breast, colon,
syndrome & lung cancer
 Sporadic and familial (Mendelian) forms of cancer
Knudson’s two-hit hypothesis

Normal
Sporadic tumor
suppressor
gene
d e Somatic
la u mutation
C in one allele
A .
o r Somatic

f es mutation
in other allele
P ro
Single tumors,
unilateral,
later-onset

• two mutations (two hits) are required for loss of tumor suppressor function
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 Sporadic and familial (Mendelian) forms of cancer
Knudson’s two-hit hypothesis
Tumor suppressor gene
containing a germline
Familial
mutation in one allele -
heterozygous for the
mutation
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mutation
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Multiple tumors,
bilateral,
early-onset

• two mutations (two hits) are required for loss of tumor suppressor function
• the first “hit” is inherited and the second “hit” is somatic
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 Cell-cycle dependent phosphorylation of Rb
Phosphorylation of Rb Hyperphosphorylated Rb
allows cells to transit p p p p
the restriction point
Rb Rb
and enter S phase p p p p
Restriction point
Sd e
p
la u
phase
Rb . C
p A
o G1
rphase
f es G2
G0
P ro phase
Quiescent cells M p p
p
phase Rb
p p
Hypophosphorylated Rb Rb
p p p
Rb
p p
 Function of the Rb protein
Growth suppression Relief of growth suppression
p p
p
E2F Rb E2F Rb
p
p p
e
G1 phase phosphorylation
d
• E2F is a transcription factor releases E2F
la u p
that mediates growth-dependent
activation of genes required to . C
A
E2F E1A Rb
make the transition into and
through S phase
e s or p
ro f Adenovirus E1A oncoprotein binding
• Rb binds and inactivates E2F
P releases E2F
under conditions of growth p
suppression
E2F Rb
• There are several ways to
alleviate growth suppression p
resulting in controlled or Gene mutation affecting binding pocket
uncontrolled cell growth releases E2F
Li-Fraumeni syndrome - caused by mutations in the p53 gene

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Breast cancer Sarcoma Other malignant neoplasms

There are multiple neoplasms in Li-Fraumeni families that are inherited

in an autosomal dominant fashion
 p53 is the “guardian of the genome”

• germline p53 mutations are found in Li-Fraumeni syndrome

• p53 is frequently found mutated in human tumors
• the p53 protein functions as a transcription factor that
d
regulates cell-cycle and DNA repair genes
e
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• UV irradiation causes cell-cycle arrest in G1 that is dependent
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on p53; cells that contain a mutated p53 cannot arrest
and go into S phase and replicate damaged DNA
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• p53 loss-of-function mutations result in the replication of
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cells with damaged DNA and to the further accumulation
f
of other mutations affecting oncogenes and tumor
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suppressor genes, and to an increased
P
likelihood of cancer
 The role of p53 in the cell cycle

apoptosis (cell death)

DNA synthesis
UV irradiation leads
p53 S d e
to cell cycle arrest phasela u
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G0 G1 r A
s
phase
e o
Quiescent cells o f G2
Pr phase
Growth and
M preparation for
phase
cell division

Mitosis
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