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

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

Knudson’s Two Hit Hypothesis

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”)

InheritedInherited SporadicSporadic

TumorigenesisTumorigenesis

TumorigenesisTumorigenesis

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

Genomic instability

Mismatch repair process

GGGG

parental strandparental strand

newly replicated strandnewly replicated strand

Mismatch recognized by Mismatch recognized by hMSH2/GTBP complexhMSH2/GTBP complex

hMLH1 & hPMS2 join repair hMLH1 & hPMS2 join repair complexcomplex

GGGG

GGGG

GGCC

Excision of mismatch and Excision of mismatch and resynthesis of correct baseresynthesis of correct base

Microsatellite instability (MIN)

general increase in mutation rate

specifically related to frameshift mutations in genes with repetitive sequences

TGF receptor II has polyA tract mutated in 90% of MIN+ colorectal ca

BAX gene involved in apoptosis mutated in 50% of MIN+ colorectal ca

Genomic instability

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 toxiceffects of methylating agents

MGMT-deficient cells surviving exposure often evolve tolerancedue 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 growth-promoting genes AND result in general methylation and (MMR) gene silencing

Mismatch repair and methylation tolerance

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 formationand chromosomal segregation; activates mitotic checkpoint (MCP)

Explanation: MCP deficiency may give growth advantage to cells exposedto BAF carcinogens

Chromosomal Instability and bulky adduct-forming

carcinogens

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

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 Ataxia telangiectasia

& Translocation Double-strand break repair Blm, Wrn Bloom’s & Werner’s syndromes

DNA cross-link repair FANCA-FANCG Fanconi’s anaemia

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

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 phosphoproteinEncodes p53 – 393 a.a. nuclear phosphoprotein

• DNA-binding protein: role in transcriptional DNA-binding protein: role in transcriptional regulationregulation

• Controls cell’s decision to replicate DNA at Controls cell’s decision to replicate DNA at G1/S checkpointG1/S checkpoint

• Causes cells with DNA damage to arrest at G1Causes cells with DNA damage to arrest at G1

• Exerts control over cell’s decision to undergo Exerts control over cell’s decision to undergo apoptosisapoptosis

P53 binding DNAP53 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 cancersAccounts 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 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)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 cancerMay also predispose to malignant melanoma, prostate, pancreatic, gall bladder, bile duct and stomach cancer

• Encodes 3418 a.a. nuclear proteinEncodes 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 High penetrance families have additional genetic and/or additional genetic and/or environmental factors present environmental factors present – many women in the family – many women in the family are affectedare 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

Promoter

Breakpoints

Promoter

Promoter

Gene A

Gene B

Fusion protein

Domain from A Domain from B

Fusion Gene A/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 proto-oncogene 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 liposarcomaTLS/FUS

ERG Myeloid leukaemia

FEV

FLI1EWS Ewing’s sarcoma

ETV1

E1AF

WT1 Desmoplastic small round cell tumour

ATF1 Clear cell sarcoma

TEC Extraskeletal myxoid chondrosarcoma

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

Oncogene amplifications in solid tumours

• 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

Oncogenes activated by locus amplification

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

Oncogene amplifications in solid tumours

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

Chromosomal Abnormality % of tumours Oncogene Suppressor gene location

1p deletion 45%1q deletion/amplification 60%3p deletion 40% FHIT6q deletion 40%7q deletion 0-80%8p deletion 50%8q amplification 15% MYC9p deletion 45%10q deletion rare PTEN11q amplification 40% CCND113q deletion 50% BRCA2, RB116q deletion 65% ECDH17p deletion 50% TP5317q deletion/amplification 30-50% HER2 BRCA118q deletion 40%20q amplification 15%22q deletion 40%

Molecular Genetics of Breast Cancer

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

Expression patterns of

different tumours can be compared

Van’t Veer. L.J. et al. Nature, Van’t Veer. L.J. et al. Nature, 415, 530-536 (2002) 415, 530-536 (2002)

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genesgenes

Red-upregulatedRed-upregulatedGreen-downregulatedGreen-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

• 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

Conclusions