Genetics of Cancer

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Genetics of Cancer
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Cancer is a genetic disease

• Cancers result from mutations in the genes that
  regulate cell growth.
• DNA damage increases the risk of developing
  cancer.
• Familial cancer syndromes are due to mutations in
  genes affecting DNA repair or genes that regulate
  cell growth.

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Experimental approaches

• Cytogenetics
• Chromosomes (metaphase) from dividing tumor cells
  are spread onto glass slides and stained with DNA
  dyes. Can only detect gross changes (chromosome
  gain, loss or rearragment)
• Insensitive and applicable to only a limited
  number of cancers. However, have been critical in
  pointing the way toward specific cancer genes.

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Experimental approaches

• FISH (Fluorescent in situ Hybridization)
• DNA from interphase tumor cells are spread onto
  glass slides. Hybridization with specific DNA
  sequences, labeled with fluorescent dyes. Detects
  translocation, loss or gain (amplification) of
  gene in question.
• Sensitive but applicable only to enquiry
  regarding the specific gene(s) in question.

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Experimental approaches

• PCR
• DNA extracted from tumor cells and PCR-amplified
  with gene-specific probes. Detects translocation.
  In combination with sequencing can identify point
  mutations in the gene in question.
• Highly sensitive but applicable only to enquiry
  regarding the specific gene(s) in question.

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Experimental approaches

• Viral carcinogenesis (Retrovirus)
• Identification of the gene activated by
  insertional mutagenesis (slow-transforming
  viruses).
• Identification of the transduced cellular gene
  for acutely transforming retroviruses.
• Experiment of nature. Requires independent
  confirmation but extremely rich source for
  identification of cellular genes involved in
  cancer. Does not identify tumor suppressor genes.

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Experimental approaches

• Viral carcinogenesis (DNA viruses)
• Identification of the proteins that are
  inactivated by interaction with viral proteins.
• Experiment of nature. Requires identification
  of the protein product (easier now) and testing
  in other model systems to determine function.
  Generally identifies tumor suppressor genes.

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Cancers are associated with mutations that
activate proteins that stimulate cell growth
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The Ph1 chromosome

• Chronic myelogenous leukemia (CML) is a malignant
  disorder of blood cells that ultimately evolves
  into acute leukemia, which is rapidly fatal.

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The Ph1 chromosome

• In 1960, Nowell demonstrated that the leukemic
  cells from CML patients had a characteristic
  chromosomal abnormality, t(922).
• This was not present in the patients normal
  cells. Thus, the Ph1 chromosome is an acquired
  genetic abnormality.
• This was the first example of a genetic
  abnormality consistently associated with cancer.

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Retroviral Oncogenesis

• Simple RNA viruses that transform cells by
• Integrating into DNA and activating transcription
  of a growth regulating gene (proto-oncogene).
  Slow transformation.
• Transducing a cellular oncogene. Acute
  transformation.

LTR gag pol env
LTR proto-oncogene
LTR gag pol env
abl LTR
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DNA is responsible for transforming normal cells
into cancer cells

• The demonstration that certain transduced
  cellular genes (oncogenes) cause cancer earned
  Varmus and Bishop the Nobel Prize in Medicine.
  This provided direct evidence that DNA was the
  element of cancerous transformation.
• Similarly, Weinberg demonstrated that DNA
  extracted from human bladder cancer could
  transform cultured cells, independently confirmed
  the that DNA was the element responsible for
  cancerous transformation.

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The Ph1 chromosome

• In CML the abnormal clone of cells (which possess
  Ph1) overgrow the normal blood cells.
• This implies that Ph1 provides a growth advantage
  to the blood cells that acquire it.

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Evidence that Ph1 provides a growth advantage

• Ph1 translocation creates a novel protein that
  fuses Bcr and Abl proteins
• Bcr Abl
• While the function of Bcr is unknown, Abl is a
  known retroviral oncogene and a tyrosine kinase

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Tyrosine kinases transmit growth signals
Growth Factor
Growth Factor Receptor
TyK
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Tyrosine kinases transmit growth signals
Growth Factor
Growth Factor Receptor
P
P
TyK
P
P
P
Signal transduction proteins
P
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Bcr/Abl expressing bone marrow cells recapitulate
CML

• Is Bcr/Abl responsible for the growth advantage
  of CML cells?
• Bcr/Abl

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Genes whose activated products promote cancer are
referred to as Oncogenes

• Growth Factors
• Oncogene Cancer
• Sis astrocytoma
• FGF stomach, bladder
• TGF? astocytomas, hepatomas
• HGF thyroid cancer
• Activation by overexpression. Autocrine
  stimulation.

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Genes whose activated products promote cancer are
referred to as Oncogenes

• Growth Factor Receptors
• Oncogene Cancer
• ERBB1 lung, gliomas
• HER2/neu breast
• RET MEN 2A 2B
• PDGFR Gliomas
• KIT GIST and other sarcomas
• Activated either by point mutations that activate
  the kinase or by overexpression (amplification)

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Genes whose activated products promote cancer are
referred to as Oncogenes

• Signal transduction molecules
• Oncogene Cancer
• Ras proteins colon, lung, breast, bladder,
  kidney,
• Abl CML, ALL
• Raf melanoma
• WNT liver
• Myc proteins Burkitt lymphoma, neuroblastoma
• lung cancer (SCLC)
• Generally activated by point mutations.

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Genes whose activated products promote cancer are
referred to as Oncogenes

• Cell Cycle Regulators
• Oncogene Cancer
• Cyclin D Lymphoma, Breast, Esophagus
• Cyclin E Breast
• CDK4 Brain, melanoma, sarcoma
• Activation by overexpression for cyclins. Point
  mutation for CDK4.

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TyK inhibition can be exploited to treat human
cancers

• GI stromal tumors (GISTs) are routinely
  associated with activating mutations in the c-kit
  TyK (growth factor receptor).
• Imitanib inhibits c-kit TyK
• Can imitanib be used to therapeutic advantage?

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(No Transcript)
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Signal transduction inhibitors as cancer therapy

• Imitanib - Abl, Kit, /- PDGFR CML, GIST
• Dasatinib - same as above
• Erlotinib/gefitinib - ERBB1 lung cancer, /-
  pancreas cancer, gliomas
• Lapatinib - ERBB2 Breast cancers that
  overexpress ERBB2

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Signal transduction inhibitors as cancer therapy

• Traztuzumab - monoclonal antibody targeting
  ERBB2 Breast Cancer, /- endometrial cancer
• Cetuximab - monoclonal antibody targeting ERBB1
  colon cancer, oropharyngeal cancers, /- pancreas
  cancer

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Cancers are associated with mutations that
inactivate proteins that inhibit cell growth
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DNA Tumor viruses identify proteins involved in
limiting cell growth

• SV40 T-Ag binds to a normal cellular protein,
  p53.
• Oncogenic strains of human papillomavirus bind
  p53 and RB.
• Binding of viral proteins to p53 RB lead to
  their inactivation.

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p53

• Activated in cells with damaged DNA
• Activates transcription of the CDK inhibitor, p21
  and the pro-apoptotic protein, Bax.
• p21 arrests cells in the G1 phase of the cell
  cycle (inhibits cdk4/cyclin D complexes)
  (checkpoint control).
• Bax causes apoptosis of cells with extensive DNA
  damage
• p53 is mutated in over 50 of human cancers.

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Retinoblastoma

• A (pediatric) cancer with a strong genetic
  component.
• Genetic mapping identified RB as the mutant gene
  in individuals with inherited susceptibility to
  retinoblastoma. Retinoblastoma occurs when the
  second RB allele becomes inactivated.
• Somatic mutations in RB occur in a large number
  of cancers.

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G1 to S progression
RB
G1
S
CDK4/D Cyclins
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G1 to S progression
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G1 to S progression
Inhibitors of Cdk4 p16INK4 p21 p27
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APC/?-catenin
WNT
APC
??Catenin
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APC/B-catenin
WNT
??Catenin
APC
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APC/?-catenin
WNT
APC
??Catenin
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APC/?-catenin
WNT
??Catenin
APC
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APC/?-catenin
WNT
APC
??Catenin
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APC/?-catenin
WNT
APC
??Catenin
c-myc Cyclin D
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APC/?-catenin
Mutation of APC results in multiple colonic
polyps, with increased risk of colon CA
WNT
APC
??Catenin
c-myc Cyclin D
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PTEN is a Tumor Suppressor
PTEN
PI-3K
TyK
PI-3K
TyK
PDK1
PTEN
AKT/PKB
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NF-1 is a tumor suppresor
RAS
NF-1
GDP
SOS
TyK
Grb2
TyK
shc
SOS
shc
Grb2
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Tumor suppressor genes promote cancer when
inactivated

• Gene Tumor (somatic mutations)
• TGFß-receptor colon, stomach
• E-cadherin stomach
• NF-1 neuroblastoma
• NF-2 schwannoma, meningioma
• APC stomach, colon ,pancreas
• PTEN endometrial, prostate
• SMAD2, 4 colon pancreas
• RB retinoblastoma, breast, lung, colon,
  osteosarcoma
• p53 all
• WT-1 Wilms tumor
• INK4a kidney, pancreas, breast
• KFL6 prostate

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Tumor suppressor genes promote cancer when
inactivated

• Gene Tumor (germline mutation)
• E-cadherin stomach
• NF-1 neurofibromatosis type 1
• NF-2 neurofibromatosis type 2
• APC colon (familial adenomatous
  polyposis)
• RB retinoblastoma, osteosarcoma
• p53 multiple
• WT-1 Wilms tumor
• INK4a melanoma
• BRCA1 2 breast, ovary
• KFL6 prostate

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Cancer risk and DNA repair

• Cancer results from activating mutations in
  proto-oncogenes and inactivating mutations in
  tumor suppressor genes.
• Therefore, the risk of developing cancer should
  be increased with increasing risk of mutation.

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Li-Fraumeni syndrome

• Due to mutation in a single p53 allele
• Therefore, loss of p53 function requires only
  mutation in the single normal allele
• 25-fold increase in cancer development by age 50.

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Li-Fraumeni syndrome

• p53 causes G1 arrest in cells with DNA damage.
  (This allows for repair to occur prior to DNA
  replication). Causes apoptosis of cells with
  extensive DNA damage Guardian of the genome.
• Loss of p53 function Increased risk of mutation
  Significant increase risk in development of
  cancers.

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HNPCC (Lynch syndrome)

• Mutation in one of several enzymes involved in
  mismatch repair.
• DNA proofreading function is abnormal
• Increased risk of colon and endometrial cancers.

G
TCAGAG TACCTGG
AGTCTCGATGGACC
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Others DNA repair defects

• Syndrome Defect Cancer Risk
• Xeroderma NER (UV light) Skin
• Pigmentosum
• Ataxia-Telangiectasia Homologous Various
• Blooms Syndrome Recombination
• Fanconi Anemia
• BRCA-1, BRCA-2 DS-DNA breaks Breast, Ovary

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Carcinogenesis

• Animal studies identify two separate steps in
  carcinogenesis
• Initiation
• Promotion

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Initiation

• An initiator is a carcinogen that causes a
  permanent and irreversible change in a cell that
  increases the risk for cancerous transformation.
• Initiators are mutagens.

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Initiators

• Alkylating agents
• Direct-acting (do not require metabolic
  activation)
• Weak carcinogens
• Therapeutic agents
• Polycyclic aromatic hydrocarbons
• Indirect-acting (require metabolic activation)
• Tobacco smoke
• (risk to all tissue)

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Initiators

• Aromatic amines and azo dyes
• Indirect-acting
• primary effect in activating organ - liver
• secondary effect in bladder (reactivated by
  glucuronidase)
• -had previously been used as food dyes
• Nitrosamines and amides
• Converted to nitrites by gut bacteria
• food preservatives

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Initiators

• The fact that many initiators require metabolic
  activation raises the possibility that
• Genetic differences in activity of metabolizing
  enzymes may be an independent influence on cancer
  susceptibility.
• Some promoters may influence activity of
  initiators by inducing activity of the
  metabolizing enzymes (I.e., EtOH)

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Promotion

• Promoters are agents that can cause cancerous
  transformation of a previously initiated cell. As
  opposed to initiators, promoters do not cause
  stable change and their effects on cells are
  reversible.
• Promoters enhance proliferation of initiated
  cells.
• Expand population of initiated cells
• Increase risk of additional mutations (during DNA
  synthesis).

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Promoters

• Hormones
• Estrogen enodmetrial hyperplasia -gt endometrial
  cancer
• Inflammation
• Cell death - increased proliferation
• Chronic hepatitis -gt hepatocellular carcinoma
• Colitis -gt colon cancer
• Osteomyelitis sinus tracts -gt squamous cancer
• Chronic bronchitis -gt lung cancer

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Viral carcinogenesis

• Mutation via viral infection
• Frequently both initiation and promotion
• DNA viruses
• HPV
• EBV
• KSHV
• HBV
• RNA viruses
• HTLV-1
• HCV

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Viral carcinogenesis

• DNA viruses
• All are integrated into the host cell DNA and
  remain as a latent infection
• Mechanisms of transformation differ for each
  virus
• RNA viruses
• Chronic infection with cell proliferation and
  predisposition to mutations

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HPV

• Integration into DNA at E1/E2 ORF results in
  overexpression of E6 and E7 viral proteins
• E6 causes degradation of p53
• E7 causes degradation of RB
• Interferes with p53 transcriptional activation
• Inactivates p21
• High risk HPV subtypes differ from low risk by
  the efficiency of the above effects
• Additional genetic changes are also required for
  complete cervical carcinogenesis

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EBV

• African Burkitt lymphoma
• Non-Hodgkin Lymphoma in transplant patients
• Hodgkin Disease
• Nasopharyngeal carcinoma

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EBV

• Latent infection as nuclear episome
• EBV LMP-1 protein appears responsible for
  immortalizing infected B cells by activating
  normal cell pathways utilized by helper T-cells
  to activate B-cells
• EBNA-2 transcriptionally activates cyclin D and
  Src
• These changes are insufficient for tumorigenesis

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EBV Burkitt lymphoma

• Malaria may be an important co-factor for Burkitt
  lymphoma
• All Burkitt lymphomas acquire characteristic
  translocations that result in activation of myc
  oncogene expression.
• Other mutations in p53, p16INK4a also common

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c-myc translocations in Burkitt lymphoma
IgH c-myc
814
kappa c-myc
822
lambda c-myc
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EBV Lymphoma in transplant patients

• Polyclonal proliferation of B-cells in the
  absence of T-cell surveillance/suppression
• Development of monoclonal lymphomas in background
  of continued proliferation
• Burkitt-like translocations uncommon

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EBV Nasopharyngeal Cancer

• All tumors contain EBV DNA
• Mechanisms of tumorigenesis uncertain
• Endemic occurrence (Southern China, Intuits,
  parts of Africa) suggests important co-carcinogens

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HTLV

• Integration into DNA
• no evidence of insertional mutagenesis
• Clonal pattern of insertion
• Viral Tax protein transactivates
• FOS, IL-2, IL-2R
• Inactivates p16INK4a
• And inhibits DNA repair and ATM-mediated
  cell-cycle checkpoints
• Thus, both initiator and promoter

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Cancer genetic mechanisms

• Translolcation with novel fusion gene product
  (e.g., bcr/abl)
• Translocation with dysregulation (e.g. c-myc in
  Burkitt lymphoma, Bcl-2 in follicular lymphoma)

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Cancer genetic mechanisms

• Activating point mutations (e.g., c-kit kinase
  domain in GIST). These are generally in tyrosine
  kinases.
• Inactivating mutations, including point mutation
  or deletion/chromosome loss. (e.g., RB). These
  affect tumor suppressors.

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Cancer genetic mechanisms

• Gene amplification. Identified as double minutes
  or HSRs by cytogenetic studies. (e.g., HER2/neu,
  n-myc, cyclin D1).

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Cancer genetic mechanisms

• Epigenetic. Acquired suppression of gene
  expression, without mutation by DNA methylation
  (e.g., VHL, p14Arf, p16INK4a).

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Conclusions

• Cancer is a multigenic disease, due to mutations
  in genes that regulate cell growth in both a
  positive and negative manner
• Inherited cancer genes increase risk by
• Mutation of a tumor suppressor gene
• Increasing risk of mutation (DNA repair defects)

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Conclusions

• Most cancers are genetically unstable. This
  contributes to heterogeneity of cells within
  cancers and progression of disease.

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• Characteristics of Cancer
• Molecular Mechanisms of Growth Control
• Cancer Genetics
• Tumor/Host Interactions
• Clinical Application Breast Cancer