Objectives for this lecture

1
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

2
(No Transcript)
3
(No Transcript)
4
(No Transcript)
5
Retinoblastomaautosomal 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

6
Knudsons Two Hit Hypothesis
Inherited
Sporadic
One normal allele One mutant allele (1st hit)
Two normal alleles
One normal allele One mutant allele (1st hit)
Two mutant alleles (2nd hit)
Two mutant alleles (2nd hit)
Tumorigenesis
Tumorigenesis
7
Genomic instability
An abnormal cell state associated with an
increased rate of heritable genomic alterations
including mutations chromosomal
rearrangements deletions inversions
8
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
9
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
10
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
11
Mismatch repair process
Mismatch recognized by hMSH2/GTBP complex
hMLH1 hPMS2 join repair complex
12
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
13
Mismatch repair and methylation tolerance
Paradox DNA damaging environment potentiates
growth advantage of repair deficiency Methylatin
g 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-defi
cient cells surviving exposure often evolve
tolerance due to defects in MMR pathway
14
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
growth- promoting genes AND result in general
methylation and (MMR) gene silencing
15
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
16
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
17
Genomic instability
18
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 Ataxia telangiectasia
Translocation Double-strand break repair Blm,
Wrn Blooms Werners syndromes DNA
cross-link repair FANCA-FANCG Fanconis anaemia
19
Ways to acquire genomic instability
Type Biological process Genes in
pathway Associated disorder Chromosomal
instability Sister chromatid cohesion
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
20
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

21
TP53 function

• Encodes p53 393 a.a. nuclear phosphoprotein
• DNA-binding protein role in transcriptional
  regulation
• Controls cells decision to replicate DNA at G1/S
  checkpoint
• Causes cells with DNA damage to arrest at G1
• Exerts control over cells decision to undergo
  apoptosis

P53 binding DNA
22
(No Transcript)
23
(No Transcript)
24
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

25
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

26
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

27
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
28
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

29
(No Transcript)
30
(No Transcript)
31
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

32
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

33
(No Transcript)
34
(No Transcript)
35
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
36
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
  proto-oncogene downstream of a strong
  constitutive promoter from another gene
  proto-oncogene is now expressed at inappropriate
  time/place
• Burkitt Lymphoma

37
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

38
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

39
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

40
Fusion Genes in Solid Cancers
CHOP Myxoid liposarcoma TLS/FUS ERG Myel
oid leukaemia FEV FLI1 EWS Ewings
sarcoma ETV1 E1AF WT1 Desmoplastic
small round cell tumour ATF1 Clear cell
sarcoma TEC Extraskeletal myxoid
chondrosarcoma
41
Oncogene amplifications in solid tumours
How do oncogenes amplify? Intrachromosomal tand
em 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
42
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

43
Oncogene amplifications in solid tumours
N-MYC originally identified as HSRs or DMs in
20 neuroblastoma less frequent in small
cell lung cancer retinoblastoma malignan
t gliomas peripheral neuroectodermal
tumours typically present as 50-100-fold
amplification co-amplification of DDX1 in
50 of N-MYC retinoblastomas
neuroblastomas
44
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
45
HER2 is amplified in many breast cancers

• Encodes transmembrane receptor tyrosine kinase,
  overexpression leads to homodimer formation-gt
  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

46
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

47
Molecular Genetics of Breast Cancer
Chromosomal Abnormality of tumours Oncogene Su
ppressor gene location 1p deletion
45 1q deletion/amplification
60 3p deletion 40 FHIT 6q deletion
40 7q deletion 0-80 8p deletion
50 8q amplification 15 MYC 9p deletion
45 10q deletion
rare PTEN 11q amplification
40 CCND1 13q deletion 50 BRCA2,
RB1 16q deletion 65 ECDH 17p deletion
50 TP53 17q deletion/amplification
30-50 HER2 BRCA1 18q deletion
40 20q amplification 15 22q deletion
40
48
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

49
Expression patterns of different tumours can be
compared
patients
Vant Veer. L.J. et al. Nature, 415, 530-536
(2002)
metastases
genes
Red-upregulated Green-downregulated
Identity of the genes is not important--
predictive profile is
50
Conclusions

• The three major classes of genes involved in
  cancer development are
• Oncogenes
• Tumour suppressors
• Genes involved in cellular and genomic
  maintenance

51
Conclusions

• Oncogenes can be activated in several ways
• Point mutations
• RAS
• Chromosomal translocation
• BCR/ABL - CML
• MYC/Ig - Burkitts Lymphoma
• Amplification
• HER2 Breast, ovarian cancers
• Telomerase can serve as an oncogene by postponing
  cell senescence

52
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