1. Professor of Pathology
Hallmarks of Cancer (2)
Neoplasia 6
Dr. Faten Ghazal
Professor of Pathology

2. Intended Leaning Outcomes (ILOs)
By the end of this lecture YOU will be able to:
Define tumour suppressor genes
Describe the genetic properties of cancer cells (hallmarks of cancer) including insensitivity to growth inhibition signals, escaping cell death by apoptosis, DNA damage and defect in repair system, avoiding cellular aging, development of sustained angiogenesis and ability to invade & metastasize
Define angiogenesis
Explain how angiogenic switch occur in neoplasm

3. Hallmarks of Cancer (Major Genetic Properties)
Acquiring Excessive Autonomous Growth (Self Sufficiency in Growth Signals)
Acquiring Insensitivity to Growth Inhibition Signals
Escaping Cell Death by Apoptosis
Avoiding Cellular Aging
Developing Sustained Angiogenesis
Acquiring Ability to invade & metastasize
(- - = + )

4. Acquiring Insensitivity to Growth Inhibition
(Growth Suppressing Anti-Oncogenes)

5. The Regulatory Genes of Normal Cell Growth
Proto-oncogenes
Tumour suppressor genes
Apoptosis regulatory genes
DNA repair genes
Do you remember genes regulating cell division, growth and differentiation

6. Tumour Suppressor Genes
What is the function of the normal (non-mutated) tumour suppressor gene?
They normally prevent uncontrolled growth by several ways and when mutated they lose their function thus allowing a transformed phenotype (mutated cell) to develop.

7. Tumour Suppressor Genes
Are these gene dominant or recessive?
They are recessive (why?)
Usually both normal alleles of tumour suppressor genes must be damaged for transformation to occur (recessive)
They are placed into 2 general groups:
“governors” as RB gene and “guardians” as p53.

8. Tumour Suppressor Genes
What is the function of the governors? Example
Governors (as RB gene) stop the cell cycle at G1-S phase. They are classic tumour suppressor genes where their mutation leads to transformation of the cell by removing an important brake of cell proliferation.

9. RB Gene : Governor of the Cell Cycle
Active form:
stops (blocks) cell cycle at G1-S phase
Mutated or Inactive form with loss of function :
loss of the brake (uncontrolled proliferation)
Is it dominant or recessive?
It is recessive i.e. both alleles must be deactivated (mutated) for retinoblastoma to occur.
Normal RB
Mutated RB

10. Retinoblastoma (RB) Gene Governor of the Cell Cycle
The function of RB gene can be lost in 2 different ways:
Loss of function mutations involving both RB alleles as in patients developing retinoblastoma & other tumours
A shift from active form to inactive form (functional inactivation) by:
gain of function of mutations of CD Kinase/Cyclin D activity or loss of function mutations that stop the activity of CDK Inhibitor
*High level of CD Kinase/cyclin D complexes & the low level of CDI Inhibitor transform the RB gene to the inactive form
Why do patients with germline mutations preferentially develop few types of cancer as retinoblastoma & osteosarcoma?? Possible there are other family members of RB genes that complement the function of mutant RB in other cells other than the retinoblasts and osteoblasts.
Why aren’t acquired mutations of RB gene present or found in all kinds of cancer? Because there are other mutations that can mimic the loss of function of RB gene as mutated gain of function of CD Kinase / Cyclin D that transform the active (normal) RB gene into an inactive form by hyperphosphorylation and also the loss of function of CD Kinase Inhibitor leads to inactivation of RB gene

11. Sporadic Form 60 % Retinoblastoma Familial Form 40 %
2nd Mutation
60 %
1st Mutation
Retinoblastoma
Familial Form
One Mutation
40 %
Normal RB Gene
Mutant RB Gene

12. RB Gene : Governor of the Cell Cycle
In sporadic retinoblastoma 2 hits of mutations are acquired during life in both alleles after birth. (older age of children & unilateral)
In inherited/familial retinoblastoma, children inherit one defective copy of RB gene while the other one is normal. During life one hit occurs leading to mutation of the 2nd allele. (younger age, bilateral & other tumours as osteosarcoma)

13. P53
The Guardian Genome

14. Tumour Suppressor Genes
What is the function of the guardians? Example
Guardians (as p53) are responsible for:
Sensing (Monitoring) genomic damage
Arrest of Cell cycle (cessation of proliferation)
Stimulate DNA Repair
If the damage is too great to be repaired:
Induction of Apoptosis by activating BAX
Induction of Permanent Cell Arrest (cellular senescence/ Cellular Aging)

15. P53: Guardian Genome
The p53 can be activated by hypoxia or DNA damage.
Activated p53 controls the expression and activity of genes involved in cell cycle, DNA repair, cellular senescence & apoptosis.
70% of tumours demonstrate biallelic loss p53

16. Activation of normal p53 by DNA-damaging agents or by hypoxia leads to 1. cell cycle arrest in G1 (keeps RB active and 2. induction of DNA repair
Successful repair of DNA allows cells to proceed with the cell cycle; if DNA repair fails, 3.p53 triggers apoptosis.
In cells with Mutated p53, DNA damage does not induce cell cycle arrest or DNA repair, and genetically damaged cells proliferate, giving rise eventually to malignant neoplasms.

17. Some Tumour Suppressor Genes & their functions & associated cancer
Normal Function
Mutation
Cancer RB
(Governor)
Inhibits G1 to S phase
Inherited or
Acquired
Retinoblastoma, osteogenic sarcoma,
Breast, lung, colon cancers
P53
(Guardian)
.Inhibits G1 to S phase
.Gives time to DNA Repair to occur
.Inhibits BCL2 anti-apoptosis gene (initiates apoptosis),++BAX
Inherited (Autosomal
Dominant)
Li-Fraumeni Syndrome
Breast Carcinoma, Brain Tumour
Most Human Cancers
APC
Regulates the destruction of B catenin thus prevents nuclear transcription
Inherited (AD)
Familial Polyposis colorectal carcinoma
70% of Sporadic colorectal carcinoma

18. Hallmarks of Cancer (Major Genetic Properties)
Self sufficiency in growth signals (Excessive & Autonomous Growth)
Insensitivity to Growth Inhibition
Escaping Cell Death by Apoptosis
Avoiding Cellular Aging
Development of Sustained Angiogenesis
Ability to Invade & Metastasize

19. Quiz EGFR 1 EGFR 2 P53 RB RAS It blocks the cell cycle at G1-S phase
It blocks the cell cycle at G1-S phase, senses DNA damage & stimulates BAX and inhibits BCL2
Its increase in breast cancer indicates poor prognosis but Herceptin can be used in treatment
Is considered the guardian of the genome
Is mutated in most tumours
Is increased in squamous cell carcinoma
Is considered the governor of the genome
Is increased in pancreatic & colonic carcinoma and is responsible by activating & increasing transduction signals
Is one of the oncogenes which is overexpressed in mantle cell lymphoma 3
EGFR 1
EGFR 2
P53
Cyclin D1 RB
RAS 2 3 3 1 5 6 4

20. Hallmarks of Cancer (Major Genetic Properties)
Acquiring Excessive Autonomous Growth (Self Sufficiency in Growth Signals)
Acquiring Insensitivity to Growth Inhibition Signals
Escaping Cell Death by Apoptosis
Avoiding Cellular Aging
Developing Sustained Angiogenesis
Acquiring Ability to Invade & Metastasize
(- - = + )

21. The regulatory genes involved in normal cell division:
Proto-oncogenes
Tumour suppressor genes
Apoptosis regulatory genes
DNA repair genes

22. Apoptosis Another mechanism of tumour growth is:
escaping cell death by apoptosis.
In normal cell, apoptosis is guided by:
Pro-apoptotic factors (BAX, BAK)
Anti apoptotic factors (BCL2, BCL-X).
Cell death receptor CD95 (Fas)
Normally p53 activates pro-apoptotic gene BAX,
But with mutated p53 there is loss of this function and thus the apoptotic activity is reduced that allows cell proliferation.
P53
Can you deduce (expect) the decrease or increase level of either of the BCL2 and CD95 to be present in cancer ?

23. Apoptosis
Additionally in cancer cells the function of apoptosis is interfered by mutations in these genes.
BCL2 gene (anti-apoptotic gene) (??):
Mutation (over expression) the transformed tumour cells escape from apoptosis. Its mutant form was described in B cell lymphoma (t14;18) translocation.
CD95 (Fas) receptors:
are depleted in hepatocellular carcinoma, hence escaping apoptosis (cell death)
( ? Or ?):
Since BCL2 is anti- apoptotic gene therefore, it is against the cell to be dead and against the command of p53, thus overexpression in some tumours as B cell lymphoma is detected. On the contrary the loss of CD 95 the receptor of apoptosis will lead to loss of apoptosis is seen in hepatocellular carcinoma

24. The different regulatory genes involved in normal cell division:
Proto-oncogenes
Tumour suppressor genes
Apoptosis regulatory genes
DNA repair genes

25. DNA Repair Genes
Normal cells during mitosis suffer from minor DNA damage which is detected & repaired before mitosis is completed. p53 (guardian of the cell) is responsible for monitoring & detection and giving time to repair such DNA damage.
However, if this system is defective as in some inherited mutations (Mutator genes) the defect in unrepaired DNA is passed to the next progeny of cells & cancer results.

26. Family History of Breast & Ovarian Cancer
Angelina Jolie
Family History of Breast & Ovarian Cancer
Bilateral Mastectomy
Bilateral Salpingo-oophorectomy

27. DNA Repair Genes Examples of Mutator genes in the inherited disorders:
BRCA1 & BRCA2 are mutated in 80% of familial breast cancer & ovarian cancer.
Hereditary non-polyposis colon cancer
Xeroderma Pigmentosum is an inherited disorder in which there is a defect in DNA repair mechanism. Upon exposure to sunlight, the UV radiation damage to DNA cannot be repaired. Such patients are more prone to skin cancers.

28. Hallmarks of Cancer (Major Genetic Properties)
Self sufficiency in growth signals (Excessive & Autonomous Growth)
Insensitivity to Growth Inhibition
Escaping Cell Death by Apoptosis
Avoiding Cellular Aging
Development of Sustained Angiogenesis
Ability to Invade & Metastasize

29. Avoiding Cellular Aging
Telomere Complexes:
Are repetitive sequences nontranscribed DNA at ends of chromosomes.
They determine the longevity of a cell.
They shorten with each round of replication & eventually when becoming unstable, signals for apoptosis occur.
Telomerase:
is an enzyme that helps to maintain normal length of telomere. It is active only in stem cells & inactive in normal somatic cells.

30. Avoiding Cellular Aging
Malignant tumours have upregulated telomerase activity which prevents the shortening of telomeres with cell replication.
In 95% of cancers, the tumour cells avoid aging by activation of telomerase that maintains telomere length.
Thus avoiding aging, leads to immortalizing the cancer cells. .

31. Hallmarks of Cancer (Major Genetic Properties)
Acquiring Excessive Autonomous Growth (Self Sufficiency in Growth Signals)
Acquiring Insensitivity to Growth Inhibition Signals
Escaping Cell Death by Apoptosis
Avoiding Cellular Aging
Developing Sustained Angiogenesis
Acquiring Ability to invade & metastasize

32. Robbins Basic Pathology 9th Edition p. 191-192
Angiogenesis
Please refer to
Robbins Basic Pathology 9th Edition p

33. Angiogenic Inhibitors
Any tumour (benign or malignant) needs blood supply for oxygen and nutrients.
Early there is a balance between angiogenic inducers and angiogenic inhibitors
Angiogenic Switch
Angiogenic Inducers
Angiogenic Inhibitors
HIF
Mutated P53 ++
- - -
++++
Thrombospondin 1
Angiostatin
Endostatin
VEGF
Basic FGF
PDGF
Thrombospondin 1
Angiostatin
Endostatin
VEGF
Basic FGF
PDGF

34. Angiogenic Inhibitors
Angiogenic Switch
Angiogenic Inducers
Angiogenic Inhibitors
HIF
Mutated P53 ++
- - -
++++
Thrombospondin 1
Angiostatin
Endostatin
VEGF
Basic FGF
PDGF
Thrombospondin 1
Angiostatin
Endostatin
VEGF
Basic FGF
PDGF

35. Angiogenic Factors e.g. VEGF, bFGF & PDGF=
Angiogenic Factors
Antiangiogenic Factors
Tumour Cells
Stromal Cells
Inflammatory Cells
Proteases
Angiostatin
Endostatin
Vasculostatin
Normal p53 induces synthesis of Thrombospondin-1
Extracellular Matrix
Angiogenic Factors e.g. VEGF, bFGF & PDGF
Proteolytic Cleavage Products:
Antiangiogenic Products

36. Angiogenic Factors e.g. VEGF, bFGF & PDGF= =
Angiogenic Factors
Antiangiogenic Factors
Tumour Cells
Stromal Cells
Inflammatory Cells
Proteases
Angiostatin
Endostatin
Vasculostatin
Normal p53 induces synthesis of Thrombospondin-1
Extracellular Matrix
Angiogenic Factors e.g. VEGF, bFGF & PDGF
Proteolytic Cleavage Products:
Antiangiogenic Products

37. Synthesis of Angiogenic factors and antiangiogenic factors
Both the angiogenic and the antiangiogenic factors are synthesized by tumour cells, macrophages & other inflammatory cells and also stromal cells.
Proteases secreted by tumour cells or stromal cells share in this balance by releasing angiogenic factors as basic fibroblast growth factor (FGF) stored in the stromal cells and other antiangiogenic factors as angiostatin, endostatin & vasculostatin.
Normal p53 stimulate synthesis of antiangiogenic factor (thrombospondin-1)
Role of Proteases
Role of p53 (tumour suppressor gene)

38. Both the angiogenic and the antiangiogenic factors are synthesized by tumour cells, macrophages & other inflammatory cells and also stromal cells.
Proteases secreted by tumour cells or stromal cells share in this balance by releasing angiogenic factors as basic fibroblast growth factor (FGF) stored in the stromal cells and other antiangiogenic factors as angiostatin, endostatin & vasculostatin.
Normal p53 stimulate synthesis of antiangiogenic factor (thrombospondin-1)

39. Angiogenic Inhibitors
Angiogenic Switch
Angiogenic Inducers
Angiogenic Inhibitors
HIF
Mutated P53 ++
- -
+++
Thrombospondin 1
Angiostatin
Endostatin
VEGF
Basic FGF
PDGF
Thrombospondin 1
Angiostatin
Endostatin
VEGF
Basic FGF
PDGF -

40. Normoxic Hypoxia Nucleus VEGF vHL: von Hippel Lindau protein (gene)
HIF: Hypoxia Inducible Factor
Normoxic
HIF-1alpha
vHL
HIF-1alpha
vHL
Destructed
Hypoxia
vHL
Transcription of VEGF
Nucleus
HIF-1alpha
VEGF
angiogenic factor

41. Normoxic Hypoxia Nucleus VEGF vHL: von Hippel Lindau protein (gene)
HIF: Hypoxia Inducible Factor
Normoxic
HIF-1alpha
vHL
HIF-1alpha
vHL
Destructed
Hypoxia
vHL
Transcription of VEGF
Nucleus
HIF-1alpha
VEGF
angiogenic factor

42. Transcription of VEGF
Von Hippel Lindau (vHL) gene binds to the Hypoxic Inducible Factor (HIF) in the presence of normal oxygen then it becomes destructed.
In presence of hypoxia von Hippel Lindau will not be able to bind to HIF thus it will be free to give orders (stimulate) the nucleus to form vascular endothelial growth factor (transcription of VEGF).

43. The 3 Functions of Angiogenic Factors are:1 3 2
Nutrients
Metastases
Growth of tumour
The increase in angiogenic factors lead to migration & proliferation of endothelial cells then organization & canalization of the cords of endothelial cells to form new capillaries with lumena.

44. Proliferation
Migration
VBM Assembly
Organization

45. To Revise and Summarize The Hallmarks of Cancer,
Genes Regulating Growth,
&
Carcinogenesis

46. Mutations in the Genome of Somatic Cells
Acquired (Environmental) DNA damaging agents as chemicals, radiation & Viruses
Normal Cell
DNA Damage
Mutations in Genes Affecting DNA Repair, Genes affecting cell growth (protooncogene& tumour suppressor genes), or apoptosis
BRCA1, BRCA2, HNPCC, XP
DNA Repair Gene
Failure of DNA Repair
Mutations in the Genome of Somatic Cells GF
GFR
Transduction
Transcription Cyclin D
Activation of Growth Promoting Oncogenes
Pro-apoptotic (BAX)
Anti-apoptotic (BCL2)
Receptor (CD95)
Inactivation of Tumour Suppressor Genes
Alteration in Genes Regulating Apoptosis
RB,p53,APC
Unregulated Cell Proliferation
Decreased Apoptosis
This is a BEAUTIFUL chart! Another way of understanding the development of malignancy in a logical way!
P53 & HIF
Angiogenic & antiangiogenic Fs
Clonal Expansion
Escape of aging by high expression of Telomerase
Angiogenesis
Tumour Progression
Additional Mutations
Escape from Immunity
Malignant Neoplasm
Invasion & Metastases

47. Suggested Textbook Robbin’s Basic Pathology 9th Edition p191-192
Textbook of Pathology Harsh Mohan 7th Edition pages