Cancer 4: Hallmarks of cancer

and oncogenic drivers

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 objective
• Understand molecular basis underlying carcinogenesis
• Understand and describe Vogelstein's cascade
• Name and describe hallmarks of cancer
• Understand and describe how mutations of proto-oncogenes result in
uncontrolled proliferation of cells
• Describe major signalling pathways underlying uncontrolled cell proliferation

“Thirty years ago, cancer was a black box. Now, as a result of research around the
world, most of the major genes involved in cancer are known. The greatest
achievements are still to come as scientists use this information to help people.”
Bert Vogelstein, Howard Hughes Medical Institute investigator
Malignant carcinoma of
the breast
 Molecular basis of carcinogenesis
Genomic themes common to all cancers
• Nonlethal genetic damage
• Carcinogenesis results from the accumulation of complementary mutation in a stepwise
fashion over time
• Tumour: clonal expansion of a single precursor cell that has incurred genetic damage
• Principal targets of cancer-causing mutations
– growth-promoting oncogenes
– growth-inhibiting tumour suppressor genes
– genes that regulate programmed cell death (apoptosis)
– genes involved in DNA repair
• In addition to DNA mutation, epigenetic changes contribute to the malignancy
 Molecular basis of carcinogenesis
Neoplastic transformation of a cell results from non-lethal cumulative genetic damage, a process that
typically involves at least 6 mutations, or more
• as many as 20,000 mutations may be detected in cancer cells, however, of these typically only 6-20
mutations “drive” the cancer
The genetic damage may be a consequence of:
1. Inherited mutations (germline mutations)
2. Acquired mutations (somatic mutations):
• Environmental factors eg chemicals, radiation & viruses
• Spontaneous and stochastic mutations

Tumour arises by clonal expansion of a single precursor cell with genetic damage
 Vogelstein’s cascade
A molecular model of the proposed evolution of a colorectal cancer through the benign adenoma - carcinoma sequence.

Morphological Appearance Molecular Change

Normal Colonic epithelium

Mutation in APC gene
Loss of DNA methylation
Early benign adenoma
(pedunculated polyp)
Mutation of ras gene
Mutation of DCC gene
Late benign adenoma
(sessile polyp)

Mutation of p53 gene

Colonic carcinoma
 Cancer is heterogeneous disease
• Heterogeneous between patients and heterogeneity within the tumour
• Brain tumours: > 100 types
• Tumours derived from glial cells: gliomas => low-grade gliomas & high-
grade gliomas: astrocytoma and glioblastoma
• Glioblastoma subtypes
• classical: EGFR, p53wt, CDKN2Anull, PTENwt
• mesenchymal: cMet, p53mut, CDKN2Anull, PTENnull
• proneural: PDGFR, p53mut, CDKN2Anull, PTENnull
• neural: p53mut, CDKN2Anull, PTENwt

TCGA of GBMs
 Hallmarks of Cancer
• Sustained proliferative signalling: e.g.; oncogene activation
• Evading growth suppressors (insensitivity to growth-inhibition): e.g.; inactivation of tumour suppressor genes
• Resisting cell death (evasion of apoptosis): e.g.; inactivation of p53 or activation of bcl-2
• Enabling replicative immortality (limitless replicative potential): e.g.; active telomerase expression
• Sustained angiogenesis: e.g.; overexpression of VEGF
• Ability to invade and metastasise: e.g.; overexpression of proteases

Weinberg & Hanahan, Cell 2010

 Hallmarks of Cancer: The next generation
• Defects in DNA repair: e.g.; leading to genomic instability
• Altered cellular metabolism: e.g.; switch to aerobic glycolysis (Warburg effect)
• Avoiding immune destruction: number of mechanisms of immune escape
• Tumour-promoting inflammation: e.g.; release of cytokines promoting proliferation

Weinberg & Hanahan, Cell 2011

 Hallmark 1: Sustained Proliferative Signalling
Proto-oncogenes: normal cellular genes whose products promote cell proliferation
Oncogene: mutated/overexpressed proto-oncogene promoting autonomous cell growth
Oncoprotein: a protein encoded by oncogene driving increased cell proliferation
Mutation of proto-oncogenes creates oncogenes that encode oncoproteins which promote cell growth in the absence
of normal growth-promoting signals.

GF Regulated Growth Factor Signalling

P P
RTK
Degradation
G-protein

Kinase

Cyclins Proliferation
TF
CDKs
 Sustained Proliferative Signalling
Most if not all cancers have molecular defects affecting one or more components of the growth factor
signalling pathway.

Regulated Growth Factor Signalling Deregulated Growth Factor Signalling

GF 1. GF Oncogenes encode
1. Growth Factors
2. Growth Factors Receptors (RTK)
P P P P 3. Signal Transducing G-proteins
RTK 2. RTK
Degradation
4. Intracellular Kinases
G-protein 3. G-protein
5. Transcription Factors
6. Cyclins and CDKs
Kinases 4. Kinases

Cyclins 6. Cyclins
TF
CDKs Proliferation 5. TF
CDKs Proliferation
 Oncogenes encode

Growth Factors
1. Growth Factors
2. Growth Factors Receptors (RTK)
3. Signal Transducing G-proteins
4. Intracellular Kinases
5. Transcription Factors
6. Cyclins and CDKs

• Mutations may result in over-expression of a normal growth factor, hence excess secretion by the tumour
cells, which feeds back onto itself, resulting in growth by an autocrine mechanism.
• While not sufficient to cause neoplastic transformation by itself, it increases the risk of mutation in
proliferating cells.

Growth factor Proto- Mechanism in cancer Associated tumour

oncogene
PDGF-β PDFGB Overexpresison Astrocytoma

FGF3 FGF3 Overexpression Osteosarcoma, Bladder cancer

Amplification Breast cancer, Melanoma
TGF-α TGFA Overexpression Astrocytoma

HGF HGF Overexpression Hepatocellular carcinomas

Thyroid cancer
VEGF* VEGF Overexpression Glioblastoma, Renal cancer
Ovarian cancer, Lung cancers

* VEGF promotes growth of blood vessels leading to extensive angiogenesis.

 Oncogenes encode

Growth Factors Receptors

1. Growth Factors
2. Growth Factors Receptors (RTK)
3. Signal Transducing G-proteins
4. Intracellular Kinases
5. Transcription Factors
6. Cyclins and CDKs
• Trans-membrane receptors with a kinase domain in the intracellular end
• Mutations may result in the over-expression of normal receptors
• Mutations may result in abnormal receptors which have their kinase activity permanently switched on =>
constitutively active kinase => unlimited activation of downstream pathways

Growth factor Proto-oncogene Mechanism in cancer Associated tumour

receptor
EGFR family ERBB1 (EGFR) Mutation Glioblastoma
ERBB2 (HER) Amplification Lung cancer
Breast carcinoma
FLT3 FLT3 Point mutations Leukemia

RET RET Point mutations Multiple endocrine

neoplasia 2A and 2B
PDGFR PDGFRB Overexpression Gliomas
Translocation Leukemia
KIT KIT Point mutations GIT tumours
Leukemia
ALK ALK Translocation Lung cancer
Fusion gene formation Lymphomas
Point mutation Neuroblastoma
 Oncogenes encode
1. Growth Factors

Regulated EGFR Signalling

2. Growth Factors Receptors (RTK)
3. Signal Transducing G-proteins
4. Intracellular Kinases
5. Transcription Factors
6. Cyclins and CDKs
• Epidermal Growth Factor Receptor (EGFR)
• Receptor-tyrosine kinase: activated by EGF binding => dimerisation and mutual phosphorylation => p-EGFR
• Expressed in most tissues; activity regulated via expression and tightly controlled degradation

EGF Regulated EGFR Signalling

P P
EGFR
Degradation
G-protein

Kinases

Cyclins Proliferation
TF
CDKs
14
 Oncogenes encode
1. Growth Factors

Deregulated EGFR Signalling 2. Growth Factors Receptors (RTK)

3. Signal Transducing G-proteins
4. Intracellular Kinases
5. Transcription Factors
6. Cyclins and CDKs

• EGFR over-expressed and/or mutated in solid tumours: lung, cervical, endometrial tumours, glioblastoma
• 1960s: one of the first growth factors isolated
• 1975: link between EGFR and malignant transformation established
• 1980s: proposal that EGFR inhibitors might be used to treat cancer
• 1983: first clinical trial with anti-AGFR antibody => today many cancers are treated with drugs targeting EGFR (Lecture 6)

EGFR mutations => constitutive EGFR activity

EGFRvI (NH2-terminal del)
EGF glioblastoma
EGFRvII (exons 14-15 del)
EGFRvIII (exons 2-7 del)

R84K, A265V, P545L

P P
EGFR
T790M
L858R
G-protein
G791S
in-frame del in exon 19 NSCLC

Kinases

Cyclins
TF
CDKs
Proliferation
 Oncogenes encode
1. Growth Factors

Regulated Ras Signalling 2. Growth Factors Receptors (RTK)

3. Signal Transducing G-proteins
4. Intracellular Kinases
5. Transcription Factors
6. Cyclins and CDKs
• Stimulation of RTKs (EGFR, PDGFR etc.) leads to exchange of GDP for GTP in Ras => active Ras
• Active Ras-GTP activates intracellular kinases (RAF-MEK-Erk) which then activate TF => proliferation
• Ras has an intrinsic GTPase activity that is accelerated by GTPase Activating Proteins (GAPs)
• GAPs bind to active Ras and augments its GTPase activity, thus terminating signal transduction => GAPs
prevent uncontrolled Ras activity

GF
Regulated RAS activity = Regulated Proliferative Signalling

P P
RTK

G-protein

Kinases

Cyclins
TF
CDKs
 Oncogenes encode
1. Growth Factors

Deregulated Ras Signalling 2. Growth Factors Receptors (RTK)

3. Signal Transducing G-proteins
4. Intracellular Kinases
5. Transcription Factors
• 15-20% of all tumours express mutated Ras proteins: H-Ras, K-Ras, N-Ras 6. Cyclins and CDKs

• In most cases, the somatic missense Ras mutations found in cancer cells introduce amino-acid substitutions at
positions 12, 13 and 61
• These changes impair the intrinsic GTPase activity and confer resistance to GAPs, thereby causing Ras mutants to
accumulate in the active GTP-bound conformation
• G12V and G12R mutants have robust transformation phenotypes
GF Regulated RAS activity
De-regulated RAS activity

P P
RTK

G-protein

Kinases

Cyclins
TF
CDKs Proliferation
Nature Reviews Cancer 7, 295-308 (2007)
Nature Reviews Molecular Cell Biology 9, 517-531 (2008)
Nature Reviews Drug Discovery 13, 828–851 (2014)
 Oncogenes encode
1. Growth Factors

Deregulated B-RAF Signalling 2. Growth Factors Receptors (RTK)

3. Signal Transducing G-proteins
4. Intracellular Kinases
5. Transcription Factors
6. Cyclins and CDKs
• BRAF: intracellular kinase that phosphorylates/activates MEK1/2-Erk1/2
• BRAF mutations: melanoma, thyroid, lung and colon cancers, subset of hematological malignancies.
• The most common BRAF mutation BRAFT1799A transversion leads to onco-protein BRAF V600E: substitution of a
glutamic acid for valine at amino acid 600 (V600E) in the kinase domain of the protein.
• This substitution mimics phosphorylation of the activation loop => constitutive BRAF kinase activity

Regulated B-Raf Signalling

GF

P P
RTK

G-protein De-regulated BRAF activity

BRAF V600E (>50% melanomas)
CRAF S259F (rare)
B-Raf / MEK / Erk
ARAF S124C (rare)

Cyclins
TF
CDKs Proliferation
Nature Reviews Cancer 14, 455-67, 2014
 Oncogenes encode
1. Growth Factors

Deregulated PI3K Signalling 2. Growth Factors Receptors (RTK)

3. Signal Transducing G-proteins
4. Intracellular Kinases
5. Transcription Factors
6. Cyclins and CDKs

• PI3K is recruited to the membrane upon GF binding to RTK and activates cascades of intracellular kinases, most
important is AKT
• AKT has many substrates, e.g.; mTOR = sensor of cellular nutrient status, BAD = pro-apoptotic protein, FOXO =
transcription factor regulating apoptosis
• PI3K mutations affect the catalytic domain and result in increased catalytic activity => activation of downstream
substrates => proliferation (and survival)

Regulated PI3K Signalling

GF

P P
RTK
PI3K amplification
PI3K / Akt
Missense point mutations –
too many to be listed

Cyclins
TF
CDKs
 Oncogenes encode
1. Growth Factors

Transcription factors 2. Growth Factors Receptors (RTK)

3. Signal Transducing G-proteins
4. Intracellular Kinases
5. Transcription Factors
6. Cyclins and CDKs

Uncontrolled proliferation might also occur as a consequence of mutations affecting transcription factors that
regulate the expression of pro-growth genes and cyclins.
• Transcription factors bind specifically to DNA regulatory elements to stimulate or repress transcription within
the nucleus.
• Transcription factors act on a large bank of responder genes that orchestrate the cell’s entry and progression
through the cell cycle.
• Malfunction in transcription factor regulation, expression or binding leads to de-regulation of gene expression
and to neoplastic growth. GF

• TF regulating proliferation: MYC, MYB, JUN, FOS, REL P P

RTK

G-protein
• MYC is most commonly involved in tumour

Kinases

Cyclins
TF
CDKs
Proliferation
 Oncogenes encode
1. Growth Factors

Deregulated Myc Activity 2. Growth Factors Receptors (RTK)

3. Signal Transducing G-proteins
4. Intracellular Kinases
5. Transcription Factors
6. Cyclins and CDKs

• Myc proto-oncogene rapidly induced by RAS/MAPK signalling following growth factor stimulation of quiescent cells.
• Physiological MYC protein concentration is tightly regulated.
• In cancer, genetic mutations alter the function of enhancer elements that increase MYC expression.
• MYC overexpression activates expression of many genes that are involved in cell growth - MYC amplification occurs in
breast, colon, lung carcinomas, neuroblastoma.
• MYC upregulation is also caused by constitutive RAS/MAPK signalling (many cancers), Notch signalling (some
hematologic cancers), Wnt signalling (colon carcinoma), Hedgehog signalling (medulloblastoma).
 Oncogenes encode
1. Growth Factors

Myc translocation in Burkitt lymphoma 2. Growth Factors Receptors (RTK)

3. Signal Transducing G-proteins
4. Intracellular Kinases
5. Transcription Factors
6. Cyclins and CDKs

• All Burkitt lymphomas have translocation of the chromosome 8q24.

• Myc-containing segment of chromosome 8 is translocated to chromosome 14q32, placing it close to the the
immunoglobulin heavy chain (IgH) gene.
• The translocation removes the regulatory sequences of the Myc gene and replaces them with control regions of
the IgH gene, which is highly expressed in B-cells.
• The Myc coding sequence remain intact and Myc is expressed in high levels.
 Oncogenes encode
1. Growth Factors

Regulated Cyclin-Dependent Kinases 2. Growth Factors Receptors (RTK)

3. Signal Transducing G-proteins
4. Intracellular Kinases
5. Transcription Factors
6. Cyclins and CDKs
The ultimate outcome of all growth-promoting stimuli is the entry of quiescent cells into the cell cycle:
• Cell cycle is regulated by cyclins & cyclin-dependent kinases (CDKs)
• Cyclin proteins expressed transiently to allow cell to enter the next phase of cell cycle
• Cycling bind to and form complexes with CDKs => active CDKs
• The cyclin/CDK complexes drive the cell cycle by phosphorylating various substrates controlling 2 checkpoints:
• G1/S checkpoint: decision to enter S-phase and replicate
• G2/M checkpoint: check DNA integrity and decide to divide or die (apoptosis)
• Activity of cyclin/CDK complexes is controlled by CDK inhibitors - endogenous molecule which enforce cell cycle arrest
• 7 families of CDKi: p16, p21, p27, p57…
 Oncogenes encode

Deregulated Cyclin-Dependent Kinases

1. Growth Factors
2. Growth Factors Receptors (RTK)
3. Signal Transducing G-proteins
4. Intracellular Kinases
5. Transcription Factors
6. Cyclins and CDKs

• 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
 What do I need to know?
• Definitions of proto-oncogene, oncogene, oncoprotein
• Hallmarks of cancer
• Understand and briefly explain the Vogelstein cascade
• Briefly describe the main oncogenic signalling pathways: EGFR-Ras-Raf, CDK-cyclin
• Concept of oncogene addiction, gene translocation and links to uncontrolled proliferation
• Examples of genes translocation