Objectives for this lecture
• Understand the mechanism of tumour suppressor
inactivation in cancer formation (the two-hit hypothesis)
• Gain familiarity with common examples of oncogenes and
tumour suppressor genes
– their function in normal cells
– the effects of aberrations in cancer
• Recognise the role of cellular maintenance genes in cancer
prevention and grasp the consequences of their inactivation
• Understand the concepts of microsatellite instability (MIN)
and chromosomal instability (CIN)
• Understand the potential use of molecular genetic analysis
in cancer diagnosis and treatment selection
Retinoblastoma
autosomal dominant inheritance
•
Paediatric tumour of the retina
1/20,000 births – two forms of the
disease – familial and sporadic
•
Due to mutations in the
Retinoblastoma (Rb) tumour
suppressor gene
Knudson’s Two Hit Hypothesis
Inherited
One normal allele
+
One mutant allele
(1st “hit”)
Two mutant alleles
(2nd “hit”)
Tumorigenesis
Sporadic
Two normal alleles
One normal allele
+
One mutant allele
(1st “hit”)
Two mutant alleles
(2nd “hit”)
Tumorigenesis
Genomic instability
An abnormal cell state associated with an increased rate
of heritable genomic alterations including
mutations
chromosomal rearrangements
deletions
inversions
Genomic instability
Mutator hypothesis:
The proposal that genomic instability promotes
tumorigenesis by increasing the rate at which mutations in
oncogenes and tumour suppressor genes arise during the
multistep development of cancer
Genomic instability
Microsatellite instability (MIN)
also known as Replication Error phenotype
associated with errors in the DNA mismatch repair system that may
lead to an elevated DNA mutation rate
genome-wide alterations in repetitive DNA sequences
found in tumours of patients with hereditary non-polyposis cancer
syndrome and 15% of sporadic (non-inherited) colorectal cancers
also found in other types of sporadic cancer
Genomic instability
Microsatellite instability (MIN)
caused by defects in nucleotide mismatch repair machinery
HNPCC patients have germline mutations in MMR genes
hMSH2, hMLH1, hPMS1, hPMS2
Tumours with MIN have somatic mutations in MMR genes
Mismatch repair process
G
G
parental strand
newly replicated strand
G
G
Mismatch recognized by
hMSH2/GTBP complex
G
G
hMLH1 & hPMS2 join repair
complex
G
C
Excision of mismatch and
resynthesis of correct base
Genomic instability
Microsatellite instability (MIN)
general increase in mutation rate
specifically related to frameshift mutations in genes with repetitive sequences
TGFb receptor II has polyA tract mutated in 90% of MIN+ colorectal ca
BAX gene involved in apoptosis mutated in 50% of MIN+ colorectal ca
Mismatch repair
and methylation tolerance
Paradox: DNA damaging environment potentiates growth
advantage of repair deficiency
Methylating agents produce mutations at guanine residues
O6-methylguanine-DNA methyltransferase (MGMT) reverses these
animals deficient in MGMT hypersensitive to mutagenic and toxic
effects of methylating agents
MGMT-deficient cells surviving exposure often evolve tolerance
due to defects in MMR pathway
Mismatch repair
and methylation tolerance
Paradox: DNA damaging environment potentiates growth
advantage of repair deficiency
MGMT-/MGMT-, MLH1+/MLH1+ mice are hypersensitive to toxic effects
of MNU exposure
MGMT-/MGMT-, MLH1-/MLH1- mice are as resistant to toxic effects
of MNU exposure as wild-type mice, but develop numerous tumours
Explanation:
methylating carcinogens produce mutations in growthpromoting genes AND result in general methylation and (MMR)
gene silencing
Chromosomal Instability
and bulky adduct-forming
carcinogens
Bulky adduct-forming carcinogens
UV radiation, free oxygen radicals, many chemicals
DNA damage repaired by Nucleotide Excision Repair (NER)
involves removal and resynthesis of large DNA fragments
promotes chromosomal rearrangments, may distort spindle formation
and chromosomal segregation; activates mitotic checkpoint (MCP)
Explanation:
MCP deficiency may give growth advantage to cells exposed
to BAF carcinogens
Genomic instability
Chromosomal instability (CIN)
chromosomal rearrangements, losses and gains
measured as abnormal number of chromosomes, shift in nuclear DNA content
aneuploidy associated with defect in chromosomal segregation
Genomic instability
Ways to acquire
genomic instability
Type
Biological process
Genes in pathway
Associated disorder
Mutations:
MIN Mismatch repair (MMR) MSH2, PMS1, PMS2, MLH1 HNPCC
Nucleotide excision repair (NER) XPA-XPG, CSA, CSB Xeroderma
pigmentosum
Deletion
DNA damage signalling
ATM, BRCA1, p53
& Translocation
Double-strand break repair
Blm, Wrn
DNA cross-link repair
FANCA-FANCG
Ataxia telangiectasia
Bloom’s & Werner’s
syndromes
Fanconi’s anaemia
Ways to acquire
genomic instability
Type
Chromosomal instability
Biological process
Sister chromatid cohesion
Genes in pathway Associated disorder
PTTG
Pituitary tumours
and condensation
Loss/Gain of chromosomes
Spindle checkpoint
BUB1
Colorectal cancers
Altered Ploidy
Centrosome cytokinesis
Aurora A
Colorectal cancers
Cell death following
p53, Bcl-2
Breast cancers
prolonged mitotic arrest
TP53 is inactivated in many
forms of human cancer
•
one of the most commonly deleted mutated genes in human cancer
•
Complete loss of functional P53 occurs in over 50% of all human tumours
•
Loss of function is generally due to point mutation for one allele and loss of
the other
•
Majority of mutations occur in central region of coding sequence
TP53 function
•
Encodes p53 – 393 a.a. nuclear phosphoprotein
•
DNA-binding protein: role in transcriptional
regulation
•
Controls cell’s decision to replicate DNA at
G1/S checkpoint
•
Causes cells with DNA damage to arrest at G1
•
Exerts control over cell’s decision to undergo
apoptosis
P53 binding DNA
BRCA1
•
Accounts for 1/2 of the autosomal dominant familial breast cancers
•
Confers high risk for ovarian cancer as well
•
May also predispose to prostate and colon cancer
•
Encodes 1863 a.a. nuclear protein
•
Most identified mutations result in a truncated protein
BRCA2
•
Accounts for 1/3 of the autosomal dominant familial breast cancers
•
Confers high risk for ovarian cancer as well (but not as high as BRCA1)
•
Confers high risk for male breast cancer (10-20% of all cases have BRCA2
mutations)
•
May also predispose to malignant melanoma, prostate, pancreatic, gall bladder,
bile duct and stomach cancer
•
Encodes 3418 a.a. nuclear protein
Potential roles of BRCA1
& BRCA2 in DNA repair
•
BRCA1 and BRCA2 function in the
same multiprotein complex
•
May help maintain genomic integrity
by promoting repair of DNA double
strand breaks that result from
damage
•
Evidence also suggests the
complex may play a role in
transcriptional regulation
BRCA1 & BRCA2 mutations
and cancer predisposition
What is the lifetime risk for
developing breast cancer for
women carrying mutations in
BRCA1 and BRCA2?
Originally thought to be 80%,
however, when risk was estimated
from pop.studies, 45-60%
High penetrance families have
additional genetic and/or
environmental factors present
– many women in the family
are affected
BRCA1 & BRCA2 mutations
in sporadic breast cancer
•
Initially, very few sporadic tumours were found to have detectable BRCA1 or
BRCA2 mutations
•
It now appears that promoter hypermethylation may represent an important
mechanism for BRCA1 inactivation – leads to closed chromatin conformation
Telomerase represents a novel
proto-oncogene
•
Full length telomeres are
approximately 15 kb long
•
In germline cells,
telomerase, a reverse
transcriptase, adds a
hexameric DNA repeat to the
end to maintain full telomere
length after DNA replication
Telomerase represents a novel
proto-oncogene
•
As cells differentiate during fetal development, telomerase function declines
and the telomeres shorten - with each successive round of DNA replication,
the telomere shortens by about 35 bases
•
Ultimately, as telomeres shorten, chromosome ends become damaged and
the cells stop dividing-may be the cause of normal cellular senescence
•
In transformed cells and many tumors, telomerase activity reappears,
enhancing the ability of tumor cells to divide without limit
– Telomerase activity detected in more than 30 cancer types
– Telomerase activity detected in over 80% of cancer samples
Fusion Genes
in Solid Cancers
Gene A
Promoter
Breakpoints
Gene B
Promoter
Fusion Gene A/B
Promoter
Fusion protein
Domain from A
Domain from B
Oncogenes activated by
chromosome translocations
• Breakpoint can occur within introns of two genes:
chimeric protein with novel properties:
Chronic Myelogenous Leukaemia
• Alternately, translocation may place protooncogene downstream of a strong constitutive
promoter from another gene – proto-oncogene is
now expressed at inappropriate time/place –
Burkitt Lymphoma
Chronic Myelogenous
Leukemia (CML)
•
Proto-oncogene ABL (tyrosine
kinase) moves from 9q to the
“breakpoint cluster region (BCR)
on 22q
•
Chimeric protein has increased
tyrosine kinase activity but altered
structure and function
•
Requires secondary mutation to
move into crisis phase
•
Effective drug therapy developed
to target novel protein
Burkitt Lymphoma
•
B-cell tumour
•
C-MYC proto-oncogene
(transcription factor) translocated
from 8q24 to 14q32, distal to the Ig
heavy chain locus
•
Ig enhancers or activating
sequences act on C-MYC –
allowing unregulated expression
and uncontrolled cell growth
http://tooldoc.wncc.edu/Infections/lymphoma.JPG
• Solid tumour of B-lymphocytes
• Predominantly affecting young children
in Africa
• one of the fastest growing malignancies
in humans
• manifested most often as a large jaw
lesion expanding rapidly over a period of
a few weeks to invade the orbit
• Visceral involvement, usually an
abdominal mass
• Treatment of the jaw and eye areas is by
radiotherapy,while visceral involvement
requires systemic chemotherapy.
In all cases, translocation of C-MYC is the cause
Fusion Genes
in Solid Cancers
CHOP
Myxoid liposarcoma
ERG
Myeloid leukaemia
TLS/FUS
FEV
FLI1
EWS
Ewing’s sarcoma
ETV1
E1AF
WT1
Desmoplastic small round cell tumour
ATF1
Clear cell sarcoma
TEC
Extraskeletal myxoid chondrosarcoma
Oncogene amplifications
in solid tumours
How do oncogenes amplify?
Intrachromosomal
tandem duplication during recombination, further unequal chromatid
exchange
double chromatid breaks at fragile site, subsequent telomere fusion,
breakage-fusion bridge cycles
Extrachromosomal
repair replication at fragile site
Oncogenes activated by
locus amplification
• Amplified sequences can be
seen in karyotypes as:
– double minute (DM)
chromosomes - very small
accessory chromosomes
– additional banding regions
called homogeneously staining
regions (HSR)
• Both contain 20-100s of copies
of a DNA region of several
hundred thousand bases-extra
copies of proto-oncogenes NMYC, HER2
DM
Oncogene amplifications
in solid tumours
N-MYC: originally identified as HSRs or DMs in 20% neuroblastoma
less frequent in
small cell lung cancer
retinoblastoma
malignant gliomas
peripheral neuroectodermal tumours
typically present as 50-100-fold amplification
co-amplification of DDX1 in 50% of
N- MYC+ retinoblastomas & neuroblastomas
Oncogene amplifications
in solid tumours
MDM2: amplified in neuroblastomas, sarcomas and gliomas
in neuroblastomas, only amplified in MYCN+ cases (never p53 mutant)
MDM2 protein complexes with p53
 overexpression causes p53 sequestration
sarcomas with MDM2 amplification plus p53 mutation: worse prognosis
HER2 is amplified in many
breast cancers
•
Encodes transmembrane receptor tyrosine
kinase, overexpression leads to homodimer
formation-> constitutively active expression
•
HER2 amplification is found in 20-25% of
breast cancers
•
leads to increased gene expression and an
increase in cell proliferation
•
amplification correlated with
– More likely lymph node metastasis
– Shortened time to relapse
– Reduced overall survival
Antibodies to HER2 may become
part of clinical treatment
•
Antibodies to erbB2
– are able to convert rapidly dividing breast
cancer cells into growth-arrested cells
– Remove the receptor from the cell
surface
– Attract natural killer cells to the cell,
targeting it for destruction
– Commercially available as Trastuzumab
(HerceptinTM) from Genentech and used
in conjunction with chemotherapy
Molecular Genetics
of Breast Cancer
Chromosomal
location
1p
1q
3p
6q
7q
8p
8q
9p
10q
11q
13q
16q
17p
17q
18q
20q
22q
Abnormality
% of tumours
deletion
45%
deletion/amplification 60%
deletion
40%
deletion
40%
deletion
0-80%
deletion
50%
amplification
15%
deletion
45%
deletion
rare
amplification
40%
deletion
50%
deletion
65%
deletion
50%
deletion/amplification 30-50%
deletion
40%
amplification
15%
deletion
40%
Oncogene
Suppressor gene
FHIT
MYC
PTEN
CCND1
HER2
BRCA2, RB1
ECDH
TP53
BRCA1
Molecular Detection
and Analysis of Cancer
•
Expression of a gene – its transcription from DNA to RNA
•
All genes are not expressed equally in every cell
•
Altered gene expression is part of the cancer transformation
process
•
Better monitoring of gene expression in tumour cells vs. normal
cells can:
– Provide better classification system
– Serve as predictors of outcome and response to treatment
options
Van’t Veer. L.J. et al. Nature,
415, 530-536 (2002)
patients
Expression
patterns of
different
tumours can
be compared
Red-upregulated
Green-downregulated
Identity of the genes is not important-- predictive profile is
Conclusions
• The three major classes of genes involved in cancer
development are
– Oncogenes
– Tumour suppressors
– Genes involved in cellular and genomic maintenance
Conclusions
• Oncogenes can be activated in several ways:
– Point mutations
• RAS
– Chromosomal translocation
• BCR/ABL - CML
• MYC/Ig - Burkitt’s Lymphoma
– Amplification
• HER2 – Breast, ovarian cancers
• Telomerase can serve as an oncogene by postponing cell
senescence
Conclusions
•
Molecular analysis is used to refine the classification of
various forms of cancer
molecular profiling
•
Patient prognosis can be predicted based on the profile of
their tumour
•
Response to various types of treatment can be predicted by
the profiles of the tumour
•
•
MIN+ colorectal cancers may have better response to
chemotherapy
HER2+ tumours are candidates for Herceptin therapy