Cancer 5.

Tumour suppressors, apoptosis

and DNA damage
Professor Lenka Munoz
School of Medical Sciences
 Cancer Module
• 6 lectures
•Cancer 1. Definition, nomenclature and characteristics
•Cancer 2. Metastasis and epidemiology of cancer
•Cancer 3. Etiology, clinical features and diagnosis
•Cancer 4. Hallmarks of cancer and oncogenic drivers
•Cancer 5. Tumour suppressors, apoptosis and DNA damage
•Cancer 6. Targeted cancer therapy
• 3 Pracs
– Microscopy Practical 1: Benign tumours (4 May 2022)
– Microscopy Practical 2: Malignant tumours (11 May 2022)
– Museum tutorial: Macrospecimens of tumours (18 May 2022)
 Learning Objectives
• Understand the roles of tumour suppressor genes
• Name and describe tumour suppressor genes involved in cell cycle
• Name and describe tumour suppressor genes involved in apoptosis
• Name and describe tumour suppressor genes involved in DNA damage repair
• Understand the the role of telomerases in cancer

Weinberg & Hanahan, Cell 2010 Weinberg & Hanahan, Cell 2011
 Oncogene vs Tumour Suppressor
• Oncogenes drive proliferation • Tumour suppressor genes are breaks to proliferation
• Introduction of oncogene into a cell with functional
Deregulated Growth Factor Signalling
1. GF
tumour suppressor gene will lead to permanent cell
Oncogenes encode
1. Growth Factors
2. Growth Factors Receptors (RTK)
cycle arrest – oncogene-induced senescence
P P
• Introduction of oncogene into a cell lacking tumour
3. Signal Transducing G-proteins
2. RTK 4. Intracellular Kinases
5. Transcription Factors
3. G-protein 6. Cyclins and CDKs
suppressor gene will lead to uncontrolled cell
proliferation and tumour growth
4. Kinases
• Abnormalities in these genes lead to failure of
5. TF
6. Cyclins Proliferation
CDKs
growth inhibition => Hallmark 2: Evading tumour
suppression
 Tumour Suppressors
• Tumour suppressor: gene or protein that suppresses various hallmarks of cancers
• Some block uncontrolled proliferation
• Some activate growth-inhibitory pathways leading to apoptosis
• Some induce cell differentiation causing cells to enter a post-mitotic, differentiated
pool without replicative potential
• Protein products of tumour suppressor genes are
– transcription factors e.g.; Rb
– cell cycle inhibitors e.g.; p16
– Receptors e.g.; WNT
– regulator of cell response to DNA damage e.g.; p53, BRCA1/2
 Tumour Suppressors
• Usually two “hits” that inactivate both alleles are required
• Often the first is inherited, the second is a somatic mutation in “susceptible” tissue

Gene Protein Function Hallmark Familial syndromes Sporadic cancers

RB Retinoblastoma protein Inhibitor of G1/S Sustained proliferation Familial retinoblastoma Retinoblastoma,
transition syndrome osteosarcoma
APC Adenomatous polyposis Inhibitor of Wnt Sustained proliferation Familial colonic polyps and Stomach, colon,
coli protein signalling carcinomas pancreatic cancer,
melanoma
PTEN Phosphatase and tensin Inhibitors of PI3K/Akt Sustained proliferation Cowden syndrome (variety of Diverse cancers, e.g.;
homolog signalling benign tumours) glioblastoma
BRCA1, Breast cancer-1 and Repair of double- DNA repair Familial breast and ovarian rare
BRCA2 Breast cancer -2 proteins stranded breaks in DNA carcinoma
TP53 P53 protein Apoptosis in response Genomic instability Li-Fraumeni syndrome Most human cancers
to DNA damage (diverse cancers)
CDH1 E-cadherin Inhibition of cell Metastasis Familial gastric cancer Gastric carcinoma
motility
 Tumour Suppressors
1. Rb

Deregulated G1/S Checkpoint

2. NF1
3. E-cadherin/APC
4. PTEN
5. BRCA
6. p53

• G1/S is crucial checkpoint governing the proliferation rate

• Nearly all cancers have genetic lesion that disable G1/S checkpoint causing cells to continually enter S phase => cell is
forced to complete the cell cycle and divide
• Mutations activating cell cycle genes: predominantly affecting the CDK4/cyclin D complex (G1/S checkpoint)
• cyclin D amplification: breast, oesophagus, liver, some lymphomas
• CDK4 amplification: melanomas, sarcomas, glioblastomas
• Mutations inactivating negative cell cycle regulators
• CDKN2A (encoding p16) deletion: pancreatic carcinomas, glioblastoma, esophageal cancers

G1/S checkpoint
Decision whether or not to enter S phase and proliferate

G2/M checkpoint
Check DNA integrity and decision whether to undergo
mitosis/divide or activate apoptosis/die
 Tumour Suppressors
1. Rb

Rb and Regulation of G1/S Checkpoint

2. NF1
3. E-Cadherin/APC
4. PTEN
5. BRCA1/2
6. p53

• Hypophosphorylated RB in complex with E2F binds to

DNA and inhibits transcription of S-phase genes
• Upon GF stimulation: active RTKs => Ras => intracellular
kinases => transcription factors => cyclin/CDK activation
• cyclin D-CDK4/6 and cyclin E-CDK2 phosphorylate RB =>
Rb is inactivated => release E2F
• Free E2F activates transcription of S-phase genes
• Activity of cyclin-CDK complexes and thus Rb
phosphorylation is controlled (inhibited) by endogenous
CDK inhibitors
– Most important: p16 encoded by CDKN2A gene

Nat Rev Mol Cell Biol 2013 (14): 297-306

 Tumour Suppressors
1. Rb
2. NF1

Rb and Regulation of G1/S Checkpoint

3. E-cadherin/APC
4. PTEN
5. BRCA1/2
6. p53

Virtually all cancer cells show dysregulation of G1/S checkpoint as

a result of mutation in one of four genes that regulate
Retinoblastoma gene/protein
1. Loss-of-function mutations affecting Rb
2. Amplification of CDK4 and cyclin D genes
3. CDKN2A deletion => Loss of CDK inhibitor p16
4. Viral oncoproteins that bind and inhibit Rb (=> Lecture 3)
 Tumour Suppressors
1. Rb

Consequences of Rb inactivation 2. NF1

3. E-Cadherin/APC
4. PTEN
5. BRCA1/2
6. p53

Genes Dev. 2016;30:1492-1502

 Tumour Suppressors
1. Rb

Cyclin-Dependent Kinase Inhibitors 2. NF1

3. E-cadherin/APC
4. PTEN
5. BRCA1/2
6. p53

• CDKN2A gene encodes p16 protein => endogenous inhibitor

blocking CyclinD-CDK4 phosphorylation of Rb
• CDKN2A gene encodes p14 which activates p53 by inhibiting MDM2
and preventing destruction of p53
• Mutation/silencing CDKN2A silences p16 and p14 expression =>
unregulated cyclin/CDK activity and loss of p53
• CDKN2A Germline mutations associate with familial forms of
melanoma
• CDKN2A Sporadic mutations in bladder cancer, head and neck
tumours, glioblastomas
 Tumour Suppressors
1. Rb

Neurofibromin-1 (NF1) 2. NF1

3. E-cadherin/APC
4. PTEN
5. BRCA1/2
6. p53
• Neurofibromin-1: GTPase-activating protein (GAP) that increases the GTPase rate of G-proteins
• NF1 is a negative regulator of Ras
• Increasing the GTPase rate of Ras inactivates Ras faster, thus NF1 is a Tumour Suppressor
• Individuals with mutant allele of NF1 gene develop benign neurofibromas, inactivation of the second copy
of the NF1 leads to optic nerve gliomas – neurofibromatosis type 1

GF

Inactive Ras
P P
RTK

G-protein

Kinases
Active Ras

Cyclins
TF
CDKs
 Tumour Suppressors
1. Rb

E-Cadherin 2. NF1
3. E-cadherin/APC
4. PTEN
5. BRCA1/2
6. p53
• Non-malignant cells proliferate until confluence; cell-cell contacts suppress further proliferation
• E-cadherin: mediates cell-cell contact inhibition in epithelial cells
• E-cadherin controls transcription of cell cycle genes via b-catenin
• In cancer: E-cadherin/b-catenin axis is deregulated => contact inhibition is abolished => cancer cell
continue to proliferate and pile on top of one another
• Reduced E-cadherin expression: colon, breast, ovarian and colon cancer
• Germline loss-of-function mutations of E-cadherin gene (CDH1) cause familial gastric carcinoma
• Some sporadic cancers also have mutations in the E-cadherin gene on chromosome 16
 Tumour Suppressors
1. Rb

Adenomatous Polyposis Coli

2. NF1
3. E-Cadherin/APC
4. PTEN
5. BRCA1/2
6. p53

Loss of Adenomatous Polyposis Coli (APC) gene causes deregulation of the E-cadherin/b-catenin axis
• APC protein: forms a destruction complex promoting b-catenin via sequestration and destruction => down-
regulates growth => tumour suppressor
• Germ-line mutations of one allele of the APC gene cause familial adenomatous polyposis => thousands of
adenomatous colonic polyps by adulthood
• Colon cancer due to somatic mutation in the other allele of a polyp cell
• Homozygous loss of APC also occurs in 80% of non-familial colorectal cancers and adenomas (mechanism
unknown)
 Tumour Suppressors
1. Rb

Phosphatase and Tensin (PTEN) homologue

2. NF1
3. E-cadherin/APC
4. PTEN
5. BRCA1/2
6. p53

• Growth factor receptors (RTKs) induce proliferation and block

apoptosis via PI3K activation
• Active PI3K phosphorylates the lipid second messenger
phosphatidylinositol biphosphate (PIP2) to PIP3
• PIP3 recruits and activates PDK1
• PDK1 activates PKB (=Akt)
• PKB inhibits activities of FOXO transcription factors => block
apoptosis => cells survive and proliferate
• PTEN negatively regulates PI3K signalling by
dephosphorylating PIP3
• If PTEN is lost, PI3K pathway is constitutively active
• PTEN gene is lost in many cancers through deletion
• PTEN point mutations or epigenetic silencing also common
Nature Reviews Cancer 6, 184-192, 2006
 Tumour Suppressors

Breast Cancer Protein 1 and 2

1. Rb
2. NF1
3. E-cadherin/APC
4. PTEN
5. BRCA1/2
6. p53

• BRCA1 and BRCA2 normally expressed in the breast Mechanism of DNA damage repair governed by BRCA1/2

• BRCA1/2 Physiological function: DNA repair by

homologous recombination or destroy cells if DNA
damage cannot be repaired
• If BRCA1 or BRCA2 are mutated, damaged DNA is not
repaired properly => carcinogenesis
• Women with BRCA1/2 mutations have 80% chance
breast cancer, >50% chance of ovarian Ca
• BRCA1/2 mutation also related to prostate cancer

Nature Reviews Cancer 2012, 12, 68-78

Nature Reviews Cancer 2016, 16, 110 - 120
 Tumour Suppressors
1. Rb

p53: Guardian Angel

2. NF1
3. APC
4. PTEN
5. BRCA1/2
6. p53

• p53 protein (encoded by TP53 gene): regulates cell cycle

progression, cellular senescence, apoptosis in response to
DNA damage and oncogenic stress
• In healthy cells p53 is undetectable
• In stressed cells, p53 is activated via 2 mechanisms:
1. DNA damage, radiation and stress activate ATM/ATR,
which phosphorylate i.e.; activate p53
2. MDM2 degrades p53 (keeps p53 undetectable).
Oncogenic stress increases expression of p14/ARF
(encoded by CDKN2A). p14/ARF binds to MDM2,
displaces p53 and increased levels of free (active) p53.

Nature Reviews Cancer 9, 862-873, 2009

 Tumour Suppressors
1. Rb

p53: Guardian Angel

2. NF1
3. APC
4. PTEN
5. BRCA1/2
6. p53

• Upon DNA damage/oncogenic stress and p53 activation 3 events can occur
• Transient p53-induced cell cycle arrest
– p53-dependent transcription of CDKN1A gene encoding CDK inhibitor p21
– p21 inhibits cyclin D-CDK4 complexes, thereby maintaining Rb in active hypophosphorylated state
and blocking progression from G1 to S phase
– Cell repairs the DNA damage and returns to normal state

• p53-induced senescence
– Senescence: permanent cell cycle arrest with specific changes in morphology
– Senescent cells are metabolically active, but not proliferating – tumour does not grow

• p53-induced apoptosis – if DNA cannot be repaired

– Activated p53 increases transcription of BAX and PUMA which induce apoptosis

However, if p53 function is lost, DNA damage goes unrepaired, driver mutations accumulate and
cells become malignant.
 Tumour Suppressors
1. Rb

p53 Lost in Cancer 2. NF1

3. APC
4. PTEN
5. BRCA1/2
6. p53

• TP53 Deletion or Loss-of-function found in more than >50% cancers

• TP53 Mutations occur with some frequency in every tumour
• In most cases, mutations present in both alleles and are somatic (not inherited in the germline)
• Rare: germline inheritance of a mutation in one allele causes Li-Fraumeni syndrome: 25-fold
increased risk of multiple primary cancers in early adulthood
• Cancers in Li-Fraumeni syndrome are a consequence of a somatic mutation in the remaining normal
allele of the TP53 gene: most common breast cancer, leukemias, brain tumours
• TP53 point mutations lead to p53 proteins e.g.; p53 R175, G245, R248, R249, R273, R282 with
oncogenic properties!
• Mutation often also occur in proteins regulating p53; e.g.; MDM2 gene amplified in 33% of sarcomas,
leading to functional deficiency of p53 (although the tumour is p53wt)
 Resisting Cell Death
• Apoptosis/Programmed Cell Death: orderly dismantling of cells into
pieces that can be consumed and disposed of by neighboring cells
• Extrinsic apoptotic pathway initiated by activation of death receptors
by e.g.; tumour necrosis factor (TNF)
• Intrinsic (mitochondrial) apoptotic pathway is triggered by stress,
injury, cytokines/growth factors deprivation
• Activation of intrinsic pathway leads to permeabilization of the
mitochondrial membrane => release of cytochrome c => activation of
caspases that chop up cellular organelles/proteins
• Integrity of mitochondrial membrane regulated by BCL-2 protein family
Pro-survival/Anti-apoptotic: BCL-2, BCL-XL, MCL-1
Pro-apoptotic: BAX, BAK
BH3-only: BAD, BID, PUMA (tilt the balance in favour of the
pro-apoptotic molecules)
• In cancer, many sites of the apoptotic pathway are compromised to
enable cells to resist cell death
Nature Reviews Molecular Cell Biology 15, 49–63 (2014)
 Resisting Cell Death
• Best established: role of BCL2 in protecting cancer cells from apoptosis
• 85% of Follicular B cell lymphoma: chromosomal translocation of the
BCL-2 gene from chromosome 18 onto the IgH region of chromosome
14 => over-expression of BCL-2 protein
• Excessive quantities of BCL-2 => cell resist apoptosis => cell will
survive and proliferate (and potentially accumulate further mutations)
• Abnormalities in apoptotic pathways, predominantly in the
intrinsic/mitochondrial pathway found in numerous cancers
• Many tumours over-express anti-apoptotic BCL-2 proteins, e.g.;
glioblastoma

Nature Reviews Molecular Cell Biology 15, 49–63 (2014)

 Replicative immortality
• Evasion of senescence
• Most human cells become senescent after 60-70 division
• Senescence is induced by p53, p16/INK4 and Rb activation
• Senescence – permanent G1 cell cycle arrest
• Virtually all cancers have mutations in the Rb-dependent G1/S arrest and thus escape senescent state and
replicate

• However, immortality is given by telomerases

• Telomerase discovered by Elizabeth Blackburn and Carol Greider: Nobel Prize in Physiology and Medicine
2009
 Telomeres and telomerase expression
• Cellular Aging is regulated by the telomeres at the end of each chromosome undergoing shortening
(degenerating) with each round of cell division. Thus, telomere shortening is believed to be a “clock” that
counts the number of cell divisions.
• Once telomere shortening reaches a certain point, chromosomes undergo end-to-end fusion, resulting in
cell death by apoptosis.
• Telomerase is a ribonuclear enzyme that makes and repairs telomeres, by adding nucleotide repeats to the
ends of chromosomal DNAs to compensate for these losses - cellular immortalizing enzyme.
• Telomerase is only normally expressed early in cell life, very low expression in most somatic cells.
• Cancer cells often re-activate the expression of telomerase, thus avoiding cell senescence and cell death.
Alternatively, cancers arise from stem (long-lived) cells that continuously express telomerase.

Telomere maintenance/telomerase activity detected

in >85% tumours
 Hallmarks of Cancer Increased expression/activity of oncogenes
1. Growth Factors: VEGF amplification
2. Growth Factors Receptors (RTK): EGFR ampl/mutations
3. Signal Transducing G-proteins: Ras mutations
4. Intracellular Kinases: B-Raf, Jak mutations, Abl-Bcr chimera
5. Transcription Factors: Myc amplification
6. Cyclins and CDKs: Cyclin/CDK amplification

Up-regulation of anti-apoptotic
proteins; Bcl2, Bcl-XL
p53 Deletion/Mutations Loss of Tumour Suppressors
Rb, NF1, APC, PTEN deletion/mutations

VEGF amplification
VEGFR ampl/mutations

Upregulation/ re-activation of telomerases

 Hallmarks of Cancer: Next generation

p53 deletion/mutations
BRCA1/2 mutations
 What do I need to learn ?

• Understand and describe the concept of tumour suppressor genes

• Describe how major tumour suppressors control carcinogenesis
• Understand and describe genes that regulate apoptosis
• Understand and describe pathway involved in DNA damage repair
• Understand epigenetic changes in cancer
• Describe senescence and telomerase expression is linked to cancer