Cancer Genetics-lec 3, (akramism@tums.ac.ir)-ABAN 1389

Objectives
‫ﺩﺍﻧﺸﮕﺎﻩ ﻋﻠﻮﻡ ﭘﺰﺷﻜﻲ ﺗﻬﺮﺍﻥ‬
• Name the TS genes, with some examples
• Name the MMR genes, with some examples
‫ﮔﺮﻭﻩ ﮊﻧﺘﻴﻚ ﭘﺰﺷﻜﻲ‬
Tumor Suppressor and Mismatch repair genes
•
Understand the mechanism of tumor suppressor inactivation in cancer
formation (two-hit hypothesis)
•
For MSc students-ZUMS
Aban 1389
Common examples of tumor suppressor genes, their function in normal
cells, the effects of their loss
•
Seyed Mohammad Akrami [MD, PhD]
akramism@tums.ac.ir
role of cellular maintenance genes in cancer prevention and the
consequences of their inactivation
Tumor Suppressor Genes
~ 350 genes; >1% of the total; mutated
in multiple cancers, are probable cancer genes
Of these, ~ 90% are somatically mutated in cancers,
20% have germline mutations; predispose to cancer
10% exhibit both somatic and germline mutations.
Mutations in cancers; 2 functional categories:
(1) mutations that are required for tumorigenesis;
(2) mutations that merely occur during tumorigenesis
and do not contribute to the process.
referred to as drivers and passengers, respectively.
Cells require a balance of tumor suppressor
and oncogene activity
~20 genes
Definition; a gene that protects a cell from one step on the path to cancer. When this
gene is mutated to cause a loss or reduction in its function, the cell can progress to
cancer, usually in combination with other genetic changes.
Encode proteins that act to suppress cell growth, respond to
signals outside the cell, maintain DNA, and repair damage.
•
Control cell growth; normally suppress cell growth and proliferation. Inhibitor of cell
division, help for apoptosis
•
Cancer is caused by inactivating mutations in both alleles
•
Responsible for most inherited cancer syndromes
Too many tumor suppressor activities can lead to
cell growth arrest or death
Slow down
Obey
Stay put
Repair carefully
Tumor
Suppressors
Oncogenes
Oncogenes
Cancer Genetics-lec 3,
(akramism@tums.ac.ir)-ABAN 1389-ZUMS
Tumor
Suppressors
1
Too much oncogene activity can lead to
increased/uncontrolled cell division
Grow!
Grow!
Grow!
Grow!
Tumor
Suppressors
Loss
Tumor supressor gene
Gain
Oncogene
The development of solid tumors is associated with the acquisition of
complex genetic alterations that modify normal cell growth and
survival.
Oncogenes
Ways of gene status changing
-- Activating point mutations
Oncogenes
Genomic Alterations in Oncology
Many of these changes involve gains and/or losses of parts of the
genome;: Amplification of an oncogene or deletion of a tumor
suppressor gene; considered as important mechanisms for
tumorigenesis.
INSTABILITY OF GENOME
as a fundamental feature
of a cancer cell
-- Translocation under strong promoter
-- Amplification
-- Overexpression
-- Inactivating point mutations
Tumor
suppressors
-- Promotor methylation
-- Gross chromosomal deletions
-- Underexpression
http://www.genetics.psu.edu/Faculty/photos/research/
The two products of the CDKN2A gene.
This gene (also known as MTS and INK4A) encodes two completely unrelated
proteins. CDKN2A, or p16INK4A, is transcribed from exons 1, 2 and 3, and
ARF, or p19ARF, from exons 1, 2 and 3 - but with a different reading frame
of exons 2 and 3. The two gene products are active in the RB1 and p53 arms
of cell cycle control, respectively.
Cancer Genetics-lec 3,
(akramism@tums.ac.ir)-ABAN 1389-ZUMS
2
Discovery of Tumor Suppressors
•
fusion of malignant murine tumor cells with nonmalignant cells
often created nontumorigenic cells
•
These nontumorigenic cells could revert to tumorigenicity after a
number of cell divisions in culture; the revertants routinely lost
specific chromosomes
•
Single chromosome transfer found that specific DNA sequences
were sufficient to suppress the tumor phenotype
Tumor Suppressor Gene (TSG)
•
Block tumor development by regulating
cell growth
•
Mutated form – loss of function, leading to
uncontrolled cell division or defective
apoptosis
•
have a recessive effect (at the cellular
level); both alleles must be inactivated to
change cellular phenotype from normal to
malignant
Loss of both alleles; Two
Two--hit origin of cancer
might be heterogzyous for a mutation (hit 1) and then over lifetime, acquire a second
mutation in other allele (hit 2). Now, missing 2 copies!
Retinoblastoma (Rb)
Familial retinoblastoma
AD inheritance
Hereditary form (1/3rd of tumors)
a) Bilateral or multifocal
b) Predisposition inherited as an AD
trait
•
Pediatric tumor of the retina
1/20,000 births, two forms: familial
and sporadic
c) Mutations inherited from a carrier
parent in 25% of the cases
d) A new mutation occurring very
early in embryogenesis in
75% of cases
e) Overall estimates of the penetrance
http://www.djo.harvard.edu/meei/OA
•
of the trait: 85–95%
Due to mutations in the Rb
tumor suppressor gene
Retinoblastoma
Retinoblastoma
 The 2 hit hypothesis was originally devised to explain how retinoblastoma could
be both sporadic and familial. Families would be heterozygous for the mutation.
Sporadic patient need 2 mutations in both alleles.
•
Inheritance of retinoblastoma is autosomal dominant because it only requires one
mutation in heterozygous carriers to see a phenotype. This is thought to occur in
the eye because of the rapid rate of proliferation of a large number of primordial
retinoblasts.
•
Infants with the heritable form of retinoblastoma have a 400-fold increased risk of
developing osteogenic sarcomas, fibrosarcomas and melanomas in early adult
life. The risk is much higher if the child has received radiotherapy.
Cancer Genetics-lec 3,
(akramism@tums.ac.ir)-ABAN 1389-ZUMS
3
Rb normal function•
13q14, 27 exons, 928 aa, 106KDa
•
> 100 different mutations reported to date;
gatekeeper
Deletions of normal cellular genes leading to
predispositions for cancer...
a) Missense
b) Nonsense
c) Splice-site
d) Small and large deletions
•
Regulates cell cycle - growth inhibitor –
binds to and inhibits transcription
factors
•
Indirectly controls the expression of a
number of genes
•
Rb is regulated via phosphorylation
during progression in cell cycle
• individuals with
deletion in this region
are at increased risk for
cancer of the retinas
• the loss of a gene in
this region leaves only
one functional
copy…its loss leads to
cancer
when it is unphosphorylated, it arrests cell cycle,
phosphorylation releases arrest
Initial deletion (first hit) increases likelihood for cancer with
loss of other allele...
Rb protein is important in cell cycle control:
Cyclic inactivation of Rb protein will lead to
progression to S phase: increase cell
proliferation….
Rb gene involved in cell
cycle regulation
Frees transcription factor
which can now activate
genes required for DNA
synthesis
Cells must pass
“checkpoint” before
entering S phase….
P53
53,,
17p
17
p13
13..1, 11
11exons,
exons, 393
393aa,
aa, 53
53KDa
KDa
Li-Fraumeni Syndrome (LFS)
rare “cancer families” with
Loss of Rb protein has same effect as
inactivation…frees EF2 and
stimulates proliferation...
constitutional mutations in TP53 are found in families with the LiFraumeni syndrome (AD).
Affected family members suffer multiple primary tumors, typically
including soft tissue sarcomas, osteosarcomas, tumors of the breast, brain
and adrenal cortex, and leukemia
history of many different forms
of cancer, affected at early age,
AD inheritance
Most of the LFS families carry a germline mutation in the P53
gene
Cancer Genetics-lec 3,
(akramism@tums.ac.ir)-ABAN 1389-ZUMS
A typical pedigree
Malignancies typical of Li-Fraumeni
syndrome;
(I2); bilateral breast cancer
diagnosed at 40
(II1) a brain tumor at 35
(II3) soft tissue sarcoma at 19 and
breast cancer at 33
(II5) breast cancer at 32
(III3) osteosarcoma at 8
(III4) leukemia at age 2
(III5) soft tissue sarcoma at 3.
4
Tumor Suppressor Genes involved in
Hereditary Cancer (I)
Tumor Suppressor Genes involved in
Hereditary Cancer (II)
APC
Familial Adenomatous Polyposis
P53
Li Fraumeni syndrome
VHL
Von Hippel-Lindau Syndrome
P16/CDK4
Hereditary Melanoma syndromes
WT1
Wilms tumor syndromes
PTCH
Nevoid Basal Cell carcinoma syndrome
RB1
Hereditary Retinoblastoma
MEN1
Multiple Endocrine Neoplasia 1
NF1
Neurofibromatosis 1
BRCA1
Breast Ovarian Cancer Syndrome
NF2
Neurofibromatosis 2
BRCA2
Breast Ovarian Cancer Syndrome
Tumor Suppressor Genes
The TwoTwo-Hit Hypothesis
The two-hit hypothesis
•
Tumorigenesis requires loss of function of both copies of tumor
suppressor
•
At clinical level – dominant inheritance (looks like only a single
mutation is required)
Knudson, 1971
First Hit
Second Hit
•
At cellular level – two mutations required for tumor to develop
Mutation in one allele
Mutation in the other
allele
•
First hit may be inherited or occur somatically
•
Second hit occurs somatically
•
One mechanism – loss of heterozygosity - LOH: individual is
heterozygous in normal tissues at a specific marker but tumor cells
contain only 1 of the 2 alleles
CANCER
Tumor suppressor genes
Have been
Loss of suppressor-gene
function
theoretically predicted
by Alfred Knudson
in 1971
(Two-hit hypothesis)
Calvert, P. M. et. al. -Ann Intern Med 2002;137:603-612
Cancer Genetics-lec 3,
(akramism@tums.ac.ir)-ABAN 1389-ZUMS
5
Tumor Suppressor Genes
Gene
Locus
Tumor type
RB1
WT1
TP53
NF1
APC
NF2
VHL
CDKN2A
CDK4
BRCA1
BRCA2
13q14
11p13
17p13
17q11
5q21
22q12
3p25
9p21
12q13
17q21
13q12
Retinoblastoma
Wilms tumor
Li-Fraumeni syndrome
Neurofibromatosis 1
Familial adenomatous polyposis
Neurofibromatosis 2
von-Hippel-Lindau syndrome
Hereditary
melanoma
HPC1
PTEN
1q24
10q23
Hereditary prostate cancer
Cowden disease
Hereditary breast cancer
Deletion or inactivation
mutation
Tumor suppressor genes can have … functions
(phenotypes)
TSGs;
"recessive" in their action.
because of a "loss of
function" mutation,
such as a deletion or
inactivating point mutation
•
•
•
haploinsufficiency,
gain of function
dominant negative
a reevaluation of the ‘two-hit’ model of tumor
suppressor inactivation.
Haploinsufficiency
• is not an absolute property all the time; partial or complete.
• can vary depending on tissue type, other epistatic interactions, and
environmental factors
the phenotypic penetrance of these mutations can vary depending on;
•
•
•
•
•
Nature of the mutation itself,
The genetic background,
The Tissue type,
Environmental factors
Other variables.
Incorporating these new findings into existing models of the clonal
evolution will be a challenge for the future.
LOH
(loss of
heterozygosity)
Loss of one allele of
polymorphic markers arranged
on the same chromosome
(usually by PCR)
DETECTION of LOH
Informative microsatellite
(polymorphic in this particular normal sample)
TWO alleles in normal tissue
versus ONE allele in tumor tissue
Cancer Genetics-lec 3,
(akramism@tums.ac.ir)-ABAN 1389-ZUMS
6
LOH appears on a certain stage of tumor
development
Mismatch Repair genes (MMR)
http://www.bentham.org/cmm1-1/miatra/Miatra-fig3-pg159.jpg
Importance
DNA-damage Response Genes
• Repair DNA-damage
“DNA Mismatch Repair”; 2508 papers in Nov. 2010
386 review
36 OMIM
Examples;
The role of DNA damage repair in aging of adult stem cells.
Microsatellite analysis and hMLH1/hMSH2 expression detection in young patients with colorectal cancer: value in
screening HNPCC.
Multiple functions for the N-terminal region of Msh6.
Constitutive deficiency in DNA mismatch repair: is it time for Lynch III?
DNA damage-response genes
involved in hereditary cancer
• Cancer is caused by inactivating mutations
in both the alleles (considered as a subset
of Tumor Suppressor Genes)
• Loss of their function lead to accumulation
of mutations in other critical genes
Dominantly Inherited Cancer Syndromes
Syndrome
Associated gene
hMLH1
Hereditary Nonpolyposis Colon Cancer
Hereditary NonPolyposis Colorectal Cancer
(HNPCC)
hMSH2, hMSH6
Hereditary Nonpolyposis Colon Cancer
Breast and Ovarian Cancer
BRCA1, BRCA2
hPMS1, hPMS2
Hereditary Nonpolyposis Colon Cancer
Familial Adenomatous Polyposis (FAP)
APC
Adenomatous polyposis coli
Li Fraumeni
P53
ATM
Ataxia Telangiectasia
Familial Retinoblastoma
RB1
XP A,C,D,F
Xeroderma Pigmentosum
BLM
Bloom Syndrome
MutYH
Cancer Genetics-lec 3,
(akramism@tums.ac.ir)-ABAN 1389-ZUMS
MLH1, MSH2, MSH6,
PMS1, PMS2
…and many other
7
Recessively Inherited Cancer Syndromes
Mismatch Repair (MMR)
• Accounts for 99% of all repairs
• Mismatch from replication
Syndrome
Associated gene
Ataxia Telangiectasia
ATM
Bloom Syndrome
BLM
Xeroderma Pigmentosum
XPB, XPD, XPA
Fanconi Anemia
FACC, FACA
Colorectal polyposis
MYH
How does system recognize progeny strand
rather than parent strand as one with mismatch?
•
•
•
•
•
•
•
•
Because of methylation
DNA methylase (coded for by dam [DNA adenine methylase] locus)
methylates 5'-GATC-3' sequence in DNA at A residue
Mismatch from replication recognized by mutL and mutS gene
products
mutH gene product nicks DNA strand (progeny strand) on either side
of mismatch
DNA helicase II from mutU gene (also called uvrD gene)
unwinds DNA duplex and releases nicked region
Gap filled in by DNA Pol I and ligase
• behind replication fork
• Two ways to correct mistakes made during replication:
1) 3'>5' exonuclease - proofreading
2) Mismatch repair
• mutL
• mutS
• mutH
• and mutU (same UvrD) gene products involved (mut for mutator
because if gene is mutated, cell has increased levels of spontaneous
mutations)
Replication error (RER)
and mismatch
DNA mismatch repair is a system for recognising and repairing
erroneous insertion, deletion and mis-incorporation of bases that can arise
during DNA replication and recombination, as-well as repairing some
forms of DNA damage. The fact that the damage detection and repair
systems are as complex as the replication machinery itself highlights the
importance evolution has attached to DNA fidelity.
From Wikipedia
DNA polymerases copy the DNA
DNA polymerases make mistakes!
Sometimes polymerases put in the wrong nucleotide
A
GC
AC
AT
GC
GC
AT
G
C
G
C
A A T G
T T A C
A
G
A A T G
T T A C
Mutation?
A
G
Cancer Genetics-lec 3,
(akramism@tums.ac.ir)-ABAN 1389-ZUMS
Base substitution
8
Mismatch Repair
•The proof-reading repair system used to correct polymerase errors
Hereditary
Hereditary Non
NonPolyposis
PolyposisColorectal
Colorectal Cancer
Cancer(HNPCC)
(HNPCC)defects
defects in
in
mismatch
mismatch repair
repair(MMR)
(MMR) genes
genes
~~5%
of
all
colorectal
cancer
5% of all colorectal cancer
Hallmarks:
Hallmarks: family
familyhistory
history of
ofcolorectal
colorectal cancer
cancer
early
earlyonset
onset
autosomal
autosomaldominant
dominant
microsatellite
microsatellite instability
instability
AATCAGCAGCATCATA
AC
C
GC
CC
GG
AC
T T T T A TCAGCAGCA C T A
AC
TA
TTAG
GTCCGTTCG
GTAAGTTAG
GTCCGTTCG
GTAAGAT
C
AAA
ATTAGG
TCC
GTTCGG
TAA
GTTAGG
TCG
AT
G C
T G
AT
CG
DNA repair
During DNA replication, some errors may be
produced in which the proofreading activity
of DNA polymerase immediately corrects.
Overall proofreading activities reduce the rate
of replication errors to about 1 per 1012bp.
good but not perfect!
Xeroderma Pigmentosum (XP)
• XP is a rare inherited disease of humans
• predisposes the patient to:
– pigmented lesions on areas of the skin exposed to the sun
– an elevated incidence of skin cancer
Ultraviolet light causes cancer
Xeroderma Pigmentosum
UV
TAC GAC TAC G
AC G A C T A C G A C
T G C T G A T G C T G A AA T G C T G A T G C
Cells from which tissues become tumors?
Cancer Genetics-lec 3,
(akramism@tums.ac.ir)-ABAN 1389-ZUMS
9
...thank you for your attention,
and ... let's remain optimistic
let's look forward to
a safe landing
akramism@tums.ac.ir
Cancer Genetics-lec 3,
(akramism@tums.ac.ir)-ABAN 1389-ZUMS
10