MOLECULAR BASIS OF CANCER
Assoc.Prof. Işık G. Yuluğ Bilkent University Department of Molecular Biology and Genetics
yulug@fen.bilkent.edu.tr
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Cellular Basis of Cancer
• Cancer is a collection of diseases characterized by abnormal and uncontrolled growth
• Cancer arises from a loss of normal growth control
• In normal tissues, the rates of new cell growth and old cell death are kept in balance • In cancer, this balance is disrupted • This disruption can result from 1) uncontrolled cell growth or 2) loss of a cell's ability to undergo apoptosis
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Cancer Cell Do Not Grow Faster Than Normal Cells Rather, Their Growth is Just Uncontrolled
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1 fertilized egg
1016 cell divisions/lifetime
50x101
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Proliferation
Differentiation Deat
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Cellular equilibrium
Proliferation
Differentiation Deat h
Transit
Renewing
Proliferating
Exiting
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Cancer: disruption of cellular equilibrium
Proliferation
Differentiation
Deat
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Stem cells as the target of carcinogens Post mitotic
Stem cell
Differentiated Normal
senescent differentiate d cell
Benign tumor Grade 2 malignancy
Grade 3 or 4 malignancy
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Invasion and Metastasis
• Abnormal cells proliferate and spread (metastasize) to other parts of the body Invasion - direct migration and penetration into neighboring tissues Metastasis - cancer cells penetrate into lymphatic system and blood vessels
•
•
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Malignant versus Benign Tumors
• Benign tumors generally do not spread by invasion or metastasis • Malignant tumors are capable of spreading by invasion and metastasis
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What causes Cancer?
• Cancer is caused by alterations or mutations in the genetic code • Can be induced in somatic cells by:
– Carcinogenic chemicals – Radiation – Some viruses
• Heredity - 5%
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Oncogen es
Cell cycle
Apoptosi s
Tumor Suppressor
Angiogenesis
Inv. and Mets
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Hanahan and Weinberg, Cell 100: 57, 2000
• What is the molecular basis of cancer? • Cancer is a genetic disease.
• Mutations in genes result in altered proteins –During cell division –External agents –Random event • Most cancers result from mutations in somatic cells • Some cancers are caused by mutations in germline cells
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• Theories of cancer genesis
Standard Dogma • Proto-oncogenes (Ras – melanoma) • Tumor suppressor genes (p53 – various cancers) Modified Dogma • Mutation in a DNA repair gene leads to the accumulation of unrepaired mutations (xeroderma pigmentosum) Early-Instability Theory • Master genes required for adequate cell reproduction are disabled, resulting in aneuploidy (Philadelphia chromosome)
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CANCER AND GENETICS
• Cancer: genome disease • Causes of genomic changes • Effects of genomic changes •Revolution in cancer treatment: ‘Smart Bullets Period’
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CANCER: GENOME DISEASE
• Loss of DNA • Gain of DNA • Changes in nucleotides • Epigenetic effects
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Signs for Genomic Changes in Cancer
• Changes in chromosome numbers - Aneuploidy • Chromosomal changes
- Increase in DNA copy number -15 different
region
- Loss in chromosomal -200.000 regions
• Micro changes
- Microsatellite changes Mikrosatellite - 100.000 - Nucleotide changes
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Chromosomal changes in the genome of cancer cells: tip of the iceberg
Deletion Duplication Reciprocal translocation Ring Chromosome
Terminal Deletion
Insertion
Inversion
Robertsonian Translocation
Isochromosomes
http://www.tokyo-med.ac.jp/genet/cai-e.htm
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Nucleotide changes in the genome of cancer cells: unseen site of the iceberg
Nucleotide Deletions Nucleotide Insertions Nucleotide Substitutions
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http://www.tokyo-med.ac.jp/genet/cai-e.htm
DNA Loss in cancer cells
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DNA Loss in cancer cells: beyond coincidence ...
Early Brain Tumor (Astrocytoma Stage II) Advance Brain Tumor Glioblastoma Multiform (Stage IV)
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Chromosomal loss: Mostly, it is a sign for the loss of a tumor suppressor gene
CDKN2 locus PTEN locus RB1 locus ??? locus p53 locus
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Cancer: Genome Disease
Epigenetic effects
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Genetic and Epigenetic Silencing of Tumor Suppressor Genes
Plass - 2002
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THE CAUSES OF GENOMIC CHANGES IN CANCER
UV
Carcinogenic chemicals
Replication Errors Radiation
Normal cell Damaged DNA Point mutations
Viruses
Rearrangements (translocation, deletions, amplifications)
Alters DNA of genes controlling cell proliferation. (Proliferation becomes abnormal)
Cancer cell
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THE CAUSES OF GENOMIC CHANGES IN CANCER: Somatic Changes
Hasar Etken Türü
Hasar Etkeni
Kanser Riski
İşareti
Morötesi Işınlar Fiziksel
Radyasyon Benzopren Kimyasal Aflatoksin Oksidatif Stres Biyolojik HBV
Deri Ka., Melanoma
Tiroid Ka., Lösemi Akciğer Ka. Karaciğer Ka. Yaşlılık Kanserleri Karaciğer Ka.
P53 (CC-TT)
Translokasyon p53 (G-T) p53 (249 G-T) P53 (C-T)
Virus DNA İntegrasyonu
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THE CAUSES OF GENOMIC CHANGES IN CANCER: Hereditary Predisposition
Genes
FA Genes XP Genes BLM
Disease
F-A
Function
DNA Damage respose ?
Inheretance
OR
Cancer Risk
Lösemi
X-P
Bloom
NER Type DNA Repair
DNA Helicase ?
OR
OR
Skin Ca.
Various cancers
WRN
RECQ4 MLH1, MSH2, PMS1, PMS2
Werner
RothmundThomson
DNA Helicase ?
DNA Helicase
OR
OR OD
Sarcoma
Sarcoma Colon, Endometrium Ca.
MMR DNA Repair
OR
OD
Lösemi, NF1
Breast, Ovary, Prostate, Pancreas Ca Lymphoma, Leukemia Breast Ca. ? Various cancers
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BRCA1, BRCA2
DNA Repair OR DNA Damage sense ? OD
ATM
A-T
p53
Li-Fraumeni
DNA Damage sense
OD
CANCER AND GENETICS
• Approximately 90-95% of all cancers are sporadic. • 5-10% are inherited.
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GENES PLAYING ROLE IN CANCER DEVELOPMENT
• Oncogenes • Tumor suppressor genes • DNA repair genes
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What are the genes responsible for tumorigenic cell growth?
Normal
Proto-oncogenes Tumor suppressor genes
+
++
Cell growth and proliferation
Cancer
Mutated or “activated” oncogenes Loss or mutation of Tumor suppressor genes
Malignant transformation
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ONCOGENES
• Oncogenes are mutated forms of cellular proto-oncogenes. • Proto-oncogenes code for cellular proteins which regulate normal cell growth and differentiation.
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Five types of proteins encoded by protooncogenes participate in control of cell growth:
Class I: Growth Factors Class II: Receptors for Growth Factors and Hormones Class III: Intracellular Signal Transducers
Class IV: Nuclear Transcription Factors Class V: Cell-Cycle Control Proteins
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Functions of Cellular Proto-Oncogenes
1. Secreted Growth Factors
2. Growth Factor Receptors
3. Cytoplasmic Signal Transduction Proteins
4. Nuclear Proteins: Transcription Factors 5. Cell Growth Genes
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A generic signalling pathway
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Oncogenes
proto-oncogene = ras Oncogene = mutated ras Always activated Always stimulating proliferation
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Amino acid substitutions in Ras family proteins (inactivates GTPase)
amino acid position Ras gene 12 59 61 Tumor
c-ras (H, K, N) H-ras
K-ras
Gly Gly Val Cys Arg Val Gly Gly
Ala Ala Ala Ala Ala Ala Ala Ala
Gln Leu Gln Gln Gln Gln Lys Arg
normal cells lung carcinoma bladder carcinoma lung carcinoma lung carcinoma colon carcinoma neuroblastoma lung carcinoma Murine sarcoma virus
N-ras
H-ras K-ras
Arg Ser
Thr Thr
Gln Gln
Harvey strain Kirsten strain
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Activation mechanisms of proto-oncogenes
proto-oncogene --> oncogene
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CHROMOSOMAL REARRANGEMENTS OR TRANSLOCATIONS
Neoplasm Burkitt lymphoma Translocation t(8;14) 80% of cases t(8;22) 15% of cases t(2;8) 5% of cases Proto-oncogene c-myc1
Chronic myelogenous leukemia Acute lymphocytic Leukemia
t(9;22) 90-95% of cases
bcr-abl2
t(9;22) 10-15% of cases
bcr-abl2
1c-myc
is translocated to the IgG locus, which results in its activated expression fusion protein is produced, which results in a constitutively active abl kinase
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2bcr-abl
GENE AMPLIFICATION
Oncogene Amplification Source of tumor
c-myc
N-myc
~20-fold
5-1,000-fold
leukemia and lung carcinoma
neuroblastoma retinoblastoma
L-myc
c-abl c-myb
10-20-fold
~5-fold 5-10-fold
small-cell lung cancer
chronic myoloid leukemia acute myeloid leukemia colon carcinoma epidermoid carcinoma colon carcinoma adrenocortical carcinoma
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c-erbB K-ras
~30-fold 4-20-fold 30-60-fold
Oncogenes are usually dominant (gain of function)
• cellular proto-oncogenes that have been mutated (and “activated”)
• cellular proto-oncogenes that have been captured by retroviruses and have been mutated in the process (and “activated”)
• virus-specific genes that behave like cellular protooncogenes that have been mutated to oncogenes (i.e., “activated”)
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The result:
• Overproduction of growth factors • Flooding of the cell with replication signals • Uncontrolled stimulation in the intermediary pathways • Cell growth by elevated levels of transcription factors
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Tumor suppressor genes
• • • Normal function - inhibit cell proliferation Absence/inactivation of inhibitor --> cancer Both gene copies must be defective
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KNUDSON TWO HIT HYPOTHESIS IN FAMILIAL CASES
Familial RB (%30)
RB
rb
Normal cells
RB LOH
rb RB
rb
Inactivation of a tumor suppressor gene requires two mutations, inherited mutation and somatic mutation.
Tumor cells
Normal cells
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KNUDSON TWO HIT HYPOTHESIS IN SPORADIC CASES
Normal Cells
RB
RB
RB
RB
RB LOH
RB Mutation
Tumor cells
Inactivation of a tumor suppressor gene requires two somatic mutations.
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TUMOR SUPPRESSOR GENES
Disorders in which gene is affected
Gene (locus)
DCC (18q)
Function
cell surface interactions
Familial
unknown
Sporadic
colorectal cancer
WT1 (11p)
transcription
Wilm‟s tumor
lung cancer
Rb1 (13q)
transcription
retinoblastoma
small-cell lung carcinoma breast, colon, & lung cancer
breast/ovarian tumors
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p53 (17p)
transcription
Li-Fraumeni syndrome
breast cancer
BRCA1(17q)
transcriptional
BRCA2 (13q)
regulator/DNA repair
CELL CYCLE
Daugther cell
Mitosis
Gateway Growth Factors
S
DNA replication
CELL CYCLE
Cell cycle inhibitors Control Point
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Rb gene
• • • • • • • Rb protein controls cell cycle moving past G1 checkpoint Rb protein binds regulatory transcription factor E2F E2F required for synthesis of replication enzymes E2F - Rb bound = no transcription/replication Growth factor --> Ras pathway --> G1Cdk-cyclin synthesized Active G1 Cdk-cyclin kinase phosphorylates Rb Phosphorylated Rb cannot bind E2F --> S phase
– – Disruption/deletion of Rb gene Inactivation of Rb protein
--> uncontrolled cell proliferation --> cancer
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p53
• • Phosphyorylated p53 activates transcription of p21 gene p21 Cdk inhibitor (binds Cdk-cyclin complex --> inhibits kinase activity) • Cell cycle arrested to allow DNA to be repaired • If damage cannot be repaired --> cell death (apoptosis)
• Disruption/deletion of p53 gene • Inactivation of p53 protein --> uncorrected DNA damage --> uncontrolled cell proliferation --> cancer
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DNA REPAIR GENES
These are genes that ensure each strand of genetic information is accurately copied during cell division of the cell cycle. Mutations in DNA repair genes lead to an increase in the frequency of mutations in other genes, such as protooncogenes and tumor suppressor genes. i.e. Breast cancer susceptibility genes (BRCA1 and BRCA2) Hereditary non-polyposis colon cancer susceptibility genes (MSH2, MLH1, PMS1, PMS2) have DNA repair functions. Their mutation will cause tumorigenesis.
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Molecular mechanisms of DNA double strand break repair
BRCA1/2
Van Gent et al, 2001
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IMPORTANCE OF DNA REPAIR
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Tumor Progression
Cellular
Multiple mutations lead to colon cancer Genetic changes --> tumor changes
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Revolution in cancer treatment: „Smart Bullets Period‟
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Summary of 30 years of research (1971-2001)
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Hanahan & Weinberg 2000
HERCEPTIN
ERCEPTİN
Bilimsel Araştırmaların Kanserle Savaşa Katkısı
STI-571
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Translocation and Bcr-Abl fusion in CML
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STI-571 against Bcr-Abl
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Smart bullet STI-571 lockes itself to the target molecule
STI-571
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Thousands of Targets
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?
? ? ?
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HERCEPTIN
? ?
?
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STI-571
?
?
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?
?
?
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MOLECULAR BIOLOGY & INFORMATICS
Biyoinformatik
~3.000.000.000 bp DNA
~30.000 genes ~300.000 protein ~3.000.000 interaction 1 human cell
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