1. MOLECULAR BASIS OF CANCER Assoc. Prof. Işık G
MOLECULAR BASIS OF CANCER Assoc.Prof. Işık G. Yuluğ Bilkent University Department of Molecular Biology and Genetics

2. 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
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 uncontrolled cell growth or loss of a cell's ability to undergo "apoptosis."
Apoptosis, or programmed cell death, is the mechanism by which old or damaged cells normally self-destruct.

3. Cancer Cell Do Not Grow Faster Than Normal Cells
Rather, Their Growth is Just
Uncontrolled

4. 1 fertilized egg 50x1012 Proliferation Death Differentiation
1016 cell divisions/lifetime
Proliferation
Differentiation
Death

5. Cellular equilibrium Proliferation Death Differentiation Transit
Renewing
Transit
Proliferating
Exiting

6. Cancer: disruption of cellular equilibrium
Proliferation
Differentiation
Death

7. Stem cells as the target of carcinogens
Post mitotic
Stem cell
Normal senescent differentiated cell
Differentiated
Grade 3 or 4 malignancy
Grade 2 malignancy
Benign tumor

8. 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
Cancers are capable of spreading through the body by two mechanisms: invasion and metastasis.
Invasion refers to the direct migration and penetration by cancer cells into neighboring tissues.
Metastasis refers to the ability of cancer cells to penetrate into lymphatic and blood vessels, circulate through the bloodstream, and then invade normal tissues elsewhere in the body.

9. Malignant versus Benign Tumors
Benign tumors generally do not spread by invasion or metastasis
Malignant tumors are capable of spreading by invasion and metastasis
Depending on whether or not they can spread by invasion and metastasis, tumors are classified as being either benign or malignant.
Benign tumors are tumors that cannot spread by invasion or metastasis; hence, they only grow locally.
Malignant tumors are tumors that are capable of spreading by invasion and metastasis.
By definition, the term "cancer" applies only to malignant tumors.

10. What causes Cancer? Carcinogenic chemicals Radiation Some viruses
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%
Cancer is often perceived as a disease that strikes for no apparent reason. This is because scientists don't know all the reasons. But many of the causes of cancer have already been identified. Besides heredity, scientific studies point to the existence of three main categories of factors that contribute to the development of cancer: chemicals (e.g., from smoking or diet), radiation, and viruses or bacteria.
In some cases, we don't know.
We do know that there are chemical, physical and biological agents that trigger the cell mutations that cause cancer.
These are called carcinogens, and include tobacco, ultraviolet radiation and asbestos.
A number of cancers share risk factors. One in eight cancers and one in five cancer deaths are due to smoking and about 1 per cent of cancers are related to alcohol consumption. Many cancers occur as a direct result of dietary influences, from infectious agents or exposure to radiation (especially skin cancers from ultraviolet radiation), while a few result from inherited faulty or altered genes.
Myths: It is a common myth that injuries can cause cancer. Cancer is not caused by a fall, fracture, bruise or bump. Sometimes it is when a person seeks medical help for an injury that a tumour is discovered, but it may have been there for a long time and is not due to the injury. Some people believe that cancer may be caused by stress, but there is not yet any reliable evidence to support this.
Cancer is caused by mutations in somatic cells. They can be induced (by carcinogens, radiation and some viruses and bacteria), or some individuals have inherited genetic mutations that predispose trhem to develop specific types of cancer.

11. Oncogenes Cell cycle Apoptosis Tumor Suppressor Inv. and Mets
Angiogenesis
Cell cycle
Hanahan and Weinberg, Cell 100: 57, 2000

12. 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

13. 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)

14. CANCER AND GENETICS Cancer: genome disease Causes of genomic changes
Effects of genomic changes
Revolution in cancer treatment: ‘Smart Bullets Period’

15. Changes in nucleotides Epigenetic effects
CANCER: GENOME DISEASE
Loss of DNA
Gain of DNA
Changes in nucleotides
Epigenetic effects

16. Signs for Genomic Changes in Cancer
Changes in chromosome numbers
- Aneuploidy
Chromosomal changes
Increase in DNA copy number -15 different region
- Loss in chromosomal regions
Micro changes
- Microsatellite changes Mikrosatellite
- Nucleotide changes

18. Chromosomal changes in the genome of cancer cells: tip of the iceberg
Reciprocal
translocation
Ring
Chromosome
Deletion
Duplication
Terminal
Deletion
Insertion
Inversion
Robertsonian
Translocation
Isochromosomes

19. Nucleotide changes in the genome of cancer cells: unseen site of the iceberg
Deletions
Nucleotide
Insertions
Nucleotide
Substitutions

20. DNA Loss in cancer cells

21. DNA Loss in cancer cells: beyond coincidence ...
Early Brain Tumor
(Astrocytoma Stage II)
Advance Brain Tumor
Glioblastoma Multiform (Stage IV)

22. Mostly, it is a sign for the loss of a tumor suppressor gene
Chromosomal loss:
Mostly, it is a sign for the loss of a tumor suppressor gene
CDKN2
locus
PTEN
locus
RB1
locus
???
locus
p53
locus

23. Cancer: Genome Disease
Epigenetic effects

24. Genetic and Epigenetic Silencing of Tumor Suppressor Genes
Plass

25. THE CAUSES OF GENOMIC CHANGES IN CANCERUV
Replication Errors
Carcinogenic chemicals
Radiation
Normal cell
Viruses
Damaged DNA
Rearrangements (translocation, deletions, amplifications)
Point mutations
Alters DNA of genes controlling cell proliferation. (Proliferation becomes abnormal)
Cancer cell

26. Virus DNA İntegrasyonu
THE CAUSES OF GENOMIC CHANGES IN CANCER:
Somatic Changes
Hasar
Etken Türü
Etkeni
Kanser Riski
İşareti
Fiziksel
Morötesi Işınlar
Deri Ka., Melanoma
P53 (CC-TT)
Radyasyon
Tiroid Ka., Lösemi
Translokasyon
Kimyasal
Benzopren
Akciğer Ka.
p53 (G-T)
Aflatoksin
Karaciğer Ka.
p53 (249 G-T)
Oksidatif Stres
Yaşlılık Kanserleri
P53 (C-T)
Biyolojik
HBV
Virus DNA İntegrasyonu

27. THE CAUSES OF GENOMIC CHANGES IN CANCER: Hereditary Predisposition
Genes
Disease
Function
Inheretance
Cancer Risk
FA Genes
F-A
DNA Damage respose ? OR
Lösemi
XP Genes
X-P
NER Type
DNA Repair
Skin Ca.
BLM
Bloom
DNA Helicase ?
Various cancers
WRN
Werner
Sarcoma
RECQ4
Rothmund-Thomson
DNA Helicase
MLH1, MSH2,
PMS1, PMS2
MMR OD
Colon, Endometrium Ca.
Lösemi, NF1
BRCA1, BRCA2
Breast, Ovary,
Prostate, Pancreas Ca
ATM
A-T
DNA Damage sense ?
Lymphoma, Leukemia
Breast Ca. ?
p53
Li-Fraumeni
DNA Damage sense

28. CANCER AND GENETICS Approximately 90-95% of all cancers are sporadic.
5-10% are inherited.

29. GENES PLAYING ROLE IN CANCER DEVELOPMENT
• Oncogenes
• Tumor suppressor genes
• DNA repair genes

30. - What are the genes responsible for tumorigenic cell growth? + ++
Normal + -
Proto-oncogenes
Cell growth
and
proliferation
Tumor suppressor genes
Cancer
Mutated or “activated”
oncogenes ++
Malignant
transformation
Loss or mutation of
Tumor suppressor genes

31. ONCOGENES Oncogenes are mutated forms of cellular proto-oncogenes.
Proto-oncogenes code for cellular proteins which regulate normal cell growth and differentiation.

32. Five types of proteins encoded by proto- oncogenes 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

33. Functions of Cellular Proto-Oncogenes 2. Growth Factor Receptors
1. Secreted Growth Factors
2. Growth Factor Receptors
4. Nuclear
Proteins:
Transcription
Factors
3. Cytoplasmic
Signal Transduction
Proteins
5. Cell Growth
Genes

34. A generic signalling
pathway

35. Oncogenes proto-oncogene = ras Oncogene = mutated ras Always activated
Always stimulating
proliferation

36. Amino acid substitutions in Ras family proteins (inactivates GTPase)
amino acid position
Ras gene Tumor
c-ras (H, K, N) Gly Ala Gln normal cells
H-ras Gly Ala Leu lung carcinoma
Val Ala Gln bladder carcinoma
K-ras Cys Ala Gln lung carcinoma
Arg Ala Gln lung carcinoma
Val Ala Gln colon carcinoma
N-ras Gly Ala Lys neuroblastoma
Gly Ala Arg lung carcinoma
Murine sarcoma virus
H-ras Arg Thr Gln Harvey strain
K-ras Ser Thr Gln Kirsten strain

37. Activation mechanisms of proto-oncogenes
proto-oncogene --> oncogene

38. CHROMOSOMAL REARRANGEMENTS OR TRANSLOCATIONS
Neoplasm Translocation Proto-oncogene
Burkitt lymphoma t(8;14) 80% of cases c-myc1
t(8;22) 15% of cases
t(2;8) % of cases
Chronic myelogenous t(9;22) 90-95% of cases bcr-abl2
leukemia
Acute lymphocytic t(9;22) 10-15% of cases bcr-abl2
Leukemia
1c-myc is translocated to the IgG locus, which results in its activated expression
2bcr-abl fusion protein is produced, which results in a constitutively active abl kinase

39. GENE AMPLIFICATION Oncogene Amplification Source of tumor
c-myc ~20-fold leukemia and lung carcinoma
N-myc ,000-fold neuroblastoma
retinoblastoma
L-myc fold small-cell lung cancer
c-abl ~5-fold chronic myoloid leukemia
c-myb fold acute myeloid leukemia
colon carcinoma
c-erbB ~30-fold epidermoid carcinoma
K-ras fold colon carcinoma
30-60-fold adrenocortical carcinoma

40. 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 proto-oncogenes that have been mutated to oncogenes (i.e., “activated”)

41. 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

42. Tumor suppressor genes
Normal function - inhibit cell proliferation
Absence/inactivation of inhibitor --> cancer
Both gene copies must be defective

43. KNUDSON TWO HIT HYPOTHESIS IN FAMILIAL CASES
Familial RB (%30) RB rb
Normal cells rb RB rb RB
LOH
Inactivation of a tumor suppressor gene requires two mutations, inherited mutation and somatic mutation.
Tumor cells
Normal cells

44. KNUDSON TWO HIT HYPOTHESIS IN SPORADIC CASESRB
LOH
Mutation
Normal
Cells RB RB
Inactivation of a tumor suppressor gene requires two somatic mutations.
Tumor cells

45. TUMOR SUPPRESSOR GENES
Disorders in which gene is affected
Gene (locus) Function Familial Sporadic
DCC (18q) cell surface unknown colorectal
interactions cancer
WT1 (11p) transcription Wilm’s tumor lung cancer
Rb1 (13q) transcription retinoblastoma small-cell lung
carcinoma
p53 (17p) transcription Li-Fraumeni breast, colon,
syndrome & lung cancer
BRCA1(17q) transcriptional breast cancer breast/ovarian
tumors
BRCA2 (13q) regulator/DNA repair

46. CELL CYCLE S CELL CYCLE Daugther cell Gateway Mitosis Growth Factors
DNA replication
Control Point
Gateway
Growth
Factors
Cell cycle inhibitors
CELL CYCLE S

47. 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

48. 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

49. 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 proto-oncogenes 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.

50. Molecular mechanisms of DNA double strand break repair
BRCA1/2
Van Gent et al, 2001

51. IMPORTANCE OF DNA REPAIR

52. Tumor Progression
Cellular
Multiple mutations lead to colon cancer Genetic changes --> tumor changes

53. Revolution in cancer treatment: ‘Smart Bullets Period’

54. Summary of 30 years of research (1971-2001)
Hanahan & Weinberg 2000

55. Bilimsel Araştırmaların Kanserle Savaşa Katkısı
HERCEPTIN
STI-571
Bilimsel Araştırmaların Kanserle Savaşa Katkısı
HERCEPTİN

56. Translocation and Bcr-Abl fusion in CML

57. STI-571 against Bcr-Abl

58. Smart bullet STI-571 lockes itself to the target molecule

59. Thousands of Targets ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? STI-571
HERCEPTIN ? ?
STI-571 ? ? ? ? ? ? ?

60. MOLECULAR BIOLOGY & INFORMATICS
Biyoinformatik
~ bp
DNA
~ genes
~ protein
~ interaction
1 human cell