The Hallmarks of Cancer

Self-sufficiency in growth signals

Normal cells rely on positive growth signals from other cells
Cancer cells can reduce their dependence on growth signals by:
o Production of their own extracellular growth factors
o Overexpression of growth factor receptors
o Alterations to intracellular components of signaling pathways

Drugs that target this: Herceptin targets the EGF receptor, which is overexpressed in some breast cancers
Vemurafinib (Zelboraf), which targets mutated Raf that has a Valine glutamate mutation

Resistance to inhibitory growth signals

Normal cells rely on antigrowth signals to regulate cell growth
Cancer cells can become insensitive to these signals
One way that this can happen is by disruption of the retinoblastoma protein (pRb) pathway
o pRb prevents inappropriate transition from the G1 phase of the cell cycle to the synthesis (S) phase
In cancer cells, pRb may be damaged, allowing the cell to divide uncontrollably

Evasion of apoptosis
Drugs that target this: BH3 mimetic compounds e.g. obatoclax, AT-101, ABT-737 and its derivatives ABT-
263 and ABT- 199. Mimic the action of the BH3-only proteins, natural antagonists of BCL2 and its pro-
survival relatives

Limitless replicative potential

Limits of replication are usually set by the length of telomeres
In normal cells, telomeres get shorter with each cell division until they become so short that the cell can no
longer divide
In cancer cells, telomeres are maintained by expression of telomerase, allowing the cell to divide an unlimited
number of times

Angiogenesis
Growth of tumours requires a blood supply
o Limits the growth of avascular tumours
Angiogenesis is mediated principally through vascular endothelial growth factor (VEGF)
o Stimulates endothelial cells to secrete MMPs (Matrix Metallo-Proteinases), which degrade the extracellular
matrix
Other growth factors also play a role, e.g.:
o Fibroblast growth factor (FGF)
o Platelet-derived growth factor (PDG) secreted from activated endothelial cells and recruits smooth muscle
cells
Enhanced permeability and retention effect
o Neo-vasculature tumours are usually leaky and tortuous, with unstable blood flow patterns compared to
normal tissues
o Means that the centre can be hypoxic and form a necrotic core
Can be targeted by drugs, which pass through the lymphatics

Invasion and metastasis

Eventually, tumours may spawn pioneer cells that can invade adjacent tissues and travel to other sites in the
body to form new tumours (metastasis)
This capability allows cancerous cells to colonise new areas where oxygen and nutrients are not limiting
Tumour cells express proteases to degrade basement membrane of the cells in which they originated
Enter blood and lymphatic systems and extravate into other tissues
Surgical resection of primary tumour can accelerate growth of the metastases
o Suggests that primary tumour secretes an angiogenic inhibitor that acts on the metastases
Angiostatin produced by cleaving plasminogen
 Can be administrated after resection to prevent metastases

Emerging hallmarks

Evasion of the immune system

The immune system is responsible for recognising and eliminating cancer cells, and therefore preventing tumour
formation
Evasion of this immune surveillance by weakly immunogenic cancer cells is an important emerging hallmark of
cancer

Drugs which target this: Ipilimumab, which prevents inactivation of T cells

Altered energy metabolism

(1a) Warburg effect
Tumor cells preferentially use glycolysis over mitochondrial oxidative phosphorylation
In proliferating cancer cells, the majority of the pyruvate generated from glucose (>90%) is converted to lactate
by lactate dehydrogenase (LDH-A), where it is readily secreted into the extracellular environment rather than
oxidized to completion
By converting pyruvate to lactate, LDH-A recovers the NAD+ needed to maintain glycolysis
ATP production by glycolysis it is far less efficient in terms of ATP generated per unit of glucose consumed (2 vs
36)
This shift therefore demands that tumour cells implement an abnormally high rate of glucose uptake to meet
their increased energy, biosynthesis and redox needs.
o Therefore they express tons of GLUT1 transporters (because they have a higher Km)
The shift to aerobic glycolysis in tumour cells is driven by multiple oncogenic signaling pathways:
PI3K is upregulated by Ras
Provides strong growth and survival signals to tumour cells but also has profound effects on their metabolism
PI3K activates AKT, which stimulates glycolysis by activating mTOR leading to mRNA translation and ribosome
biogenesis:
o Activated mTOR stimulates protein and lipid biosynthesis and cell growth in response to sufficient nutrient
and energy conditions and is often constitutively activated during tumorigenesis
o Increasing the expression key glycolytic enzymes, such as hexokinase and phosphofructokinase 2, pyruvate
dehydrogenase kinase, isozyme 1 (PDK1), which blocks the entry of pyruvate into the tricarboxylic acid (TCA)
cycle.
Hypoxia also induces the expression of the transcription factor HIF1
o Further promotes glycolysis

(1b) Pyruvate kinase

Cancer cells use enzyme pyruvate kinase M2 to catalyze the rate determining step of glycolysis
Also found in other fast proliferating cells, self renewing cells such as embryonic and adult stem cells
The dimeric form of M2-PK is characterized by a low affinity to its substrate PEP and is nearly inactive at
physiological PEP concentrations.
When M2-PK is mainly in the less active dimeric form, which is the case in tumor cells, all glycolytic
intermediates above pyruvate kinase accumulate and are channelled into synthetic processes, which branch off
from glycolytic intermediates, such as nucleic acid-, phospholipid-, and amino acid synthesis Nucleic acids,
phospholipids, and amino acids are important cell building-blocks, which are greatly needed by highly
proliferating cells, such as tumor cells.

Enabling characteristics
Genetic instability
Cancer cells achieve genome instability by increasing their mutability, or rates of mutation, through increased
sensitivity to mutagenic agents or breakdown of genomic maintenance machinery.

Tumour promoting inflammation

Immune cells infiltrate tumours and produce inflammatory responses, which can paradoxically enhance
tumourigenesis, helping tumours acquire the hal marks of cancer
Explanations for the Warburg effect
1 Biosynthesis
At first seems counter intuitive to have a glycolytic enzyme that inhibits ATP generation
Cancer cell, like any normal cell, must obtain the building blocks that are required for the synthesis of lipids,
nucleotides and amino acids. Without sufficient precursors available for this purpose, rapid cell proliferation will
halt, no matter how vast a supply of ATP is present.
PKM2 provides an advantage to cancer cells because, by slowing glycolysis, this isozyme allows carbohydrate
metabolites to enter other subsidiary pathways, including the hexosamine pathway, uridine diphosphate (UDP)
glucose synthesis, glycerol synthesis and the PPP, which generate macromolecule precursors, that are necessary
to support cell proliferation, and reducing equivalents such as NADPH

2 - Hypoxia
Due the high proliferation rate of cancerous cells, they form layers on top of each other
This means cells are hypoxic therefore oxidative phosphorylation cannot occur
The only solution is for cells to undergo glycolysis
This explanation is not fully supported because tumours will still use Warburg effect in presence of oxygen

(2) Glutamine
Rapidly consumed by cancer cells and all fast proliferating ones e.g. immune cells
Most abundant circulating amino acid but if demand is high can lead to increased proteolysis, causing cachexia in
patients
o Glutamine addiction
Taken up at much higher levels that in normal cells net uptake by tumour
As well as being taken up by cells, it needs to be transported into the mitochondria
Cancer cells have a specific mitochondrial transport system with high capacity for L-glutamine

Why do cancer cells need glutamine?

1) Blocks oxidative stress
Caused by excessive flux of electrons into the OP pathway
Leads to productions of superoxide which can damage DNA see leningher
Glutamine is both a precursor and a regenerating source of the major intracellular antioxidant glutathione
Can be used to get rid of free radicals allowing cells to evade oxidative stress
2) Buffers cell
A hallmark of the rapid proliferation of cancer cells is the metabolism of glucose and glutamine to lactate for
fuel.
Cells need to be kept at a minimum pH for survival, but with cancer cells dependent on the production of the
acidic lactate, the intracellular pH will be lowered to a detrimental level, unless a substrate is available to
effectively neutralise this excess acid production.
In the metabolism of glutamine ammonia is produced, and it has now being hypothesised that this ammonia
production is key in the cancer cell's acid resistance and for ensuring its survival.
3) Stops autophagy

Drugs that target this: Flurodeoxyglucose can be used to image cancerous cells because you know they take up
loads of glucose
 Oncogenes vs. Tumour suppressors

Oncogenes and tumour suppressors are cancer-causing genes

Proto-oncogenes
Genes that as a result of dysregulated activity or overexpression, cause cancer
o Their overactive forms are known as oncogenes
Can be involved in growth signalling pathways, transcription factors,
Isolated oncogenes can transform immortalised cell lines but not primary cells

Ras
Mutations can block GTPase signal, which leads to prolonged inappropriate signal

Abl
Protein kinase, which becomes overactive when its amino terminal is deleted
E.g. As a result Philadelphia chromosome t(9,22) that results in a chimaeric protein which have overactive
kinase activity
o Gives rise to chronic myeloid leukaemia

c-fos
A transcription factor that is an effector of the MAPK pathway (EGF pathway)
Associates with c-Jun leading to the formation of AP1 (a transcription factor) that promotes growth
Normally extremely unstable, therefore present in low levels
Overexpression is associated with fibrous dysplasia abnormal bone growth

Myc
Transcription factor
Downstream activator of PI3K pathway
Regulates genes involved in many pathways:
Promotes: proliferation, translation, metabolism, stromal remodelling, angiogenesis, invasion and the immune
system
Inhibits: differentiation of cells
Tends to be deregulated in most cancers
Burkitts lymphoma is caused by a translocation of c-myc between chromosome 8 and 14

DNA tumour viruses

Hijack the host cells in order to replicate themselves
Express oncogenic proteins
o Simian virus Large T, Small T
o Human Papilloma Virus E6, E7
o Adenovirus 5 E15, p55E18, p19E18

Bcl-2
BCL-2 is an oncogene
o Allows tumours to evade apoptosis
In hematologic malignancies, this impairment of apoptosis is often caused by overexpression of the pro-survival
protein BCL2 which sustains these tumors
One approach to targeting BCL2 in hematologic malignancies is the development of BH3 mimetic compounds
e.g. obatoclax, AT-101, ABT-737 and its derivatives ABT-263 and ABT- 199.
o Small molecules that mimic the action of the BH3-only proteins, natural antagonists of BCL2 and its pro-
survival relatives
o Act by restoring the ability of a cell to undergo apoptotic cell death.
Tumour supressing genes
Genes that when under expressed can cause cancer
Phenotype is characterised as loss of function
o Two hit hypothesis A person has two copies of the tumour suppressor and requires a mutation in
both to get cancer
Therefore inheriting a mutation in one massively increases the risk of getting cancer and getting
it earlier
Loss of heterozygoty
p53
Guardian of the genome
p53 responds to DNA damage to induce:
o Cell cycle arrest
o DNA repair mechanisms
o As a final resort apoptosis (programmed cell death)
o Senescence
Unlike Rb, p53 isnt required for normal growth of cells
o Kept in low levels in the cell
o Expression increases as a result of:
UV light and gamma rays, which block its degradation
Abnormal expression of Ras and Myc
When telomeres get too short
Phosphorylation by CHK2, which is activated as a result of dsDNA damage
Phosphorylation by CHK1, as a result of ss breakage
Acts as a gene regulatory protein
Induces transcription of p21, which inhibits Cdks and prevents cell cycle progression
May also trigger apoptosis by upregulating expression of PUMA
Mutations in the p53 gene (TP53) often inactivate its DNA binding function and more than 50% of
human tumours have mutations in p53

Inheriting p53 mutation is known as Li-Fraumeni syndrome

Rb
Protein that binds to and inhibits expression of E2F protein
o Via chromatin remodelling, histone deacetylases etc.
E2F proteins bind DNA sequences and upregulate genes coding for G 1/S cyclins, S-cyclins, DNA
synthesizing proteins
Inactivated as a response of hypophosphorylation e.g. by Cdk2/cyclin E

NF1
Protein encoded by the NF1 gene, neurofibromin, contains a GTPase activating protein (GAP) domain
o Interacts with Ras to switch off signal transduction
In nerve sheath tumours from patients with NF1, editing has been detected
Editing of NF1 mRNA, converts a CGA to a UGA termination codon
Truncates the protein at the critical GAP domain, and thus to functionally inactivate the tumour
suppressor
Increases risk of nerve sheath tumours
WT1
A transcription factor, which recognizes GC- or TC-rich promoter sequences.
A two domain protein with:
o An N-terminal proline and glutamine-rich activation domain
o A C-terminal DNA-binding protein with 4 Zn2+ fingers
Essential for normal development of the kidney and gonads
o Its inactivation leads to cancer

Wilms tumour
Mutations in introns or exons of WT1
Leads to altered splicing pattern and to truncated forms of protein that lack the DNA-binding domain, or parts of
the activation domain.
o Truncated forms of the protein no longer function as tumour suppressor transcription factors.
Mutations in WT1 may lead to: Childhood kidney cancer (Wilms tumor), severe kidney disease or gonadal
dysgenesis (male-female sex reversal).
o Arises in 1/10,000 children, around the age of 5.
o Most common solid tumour of childhood
o Treated using surgical removal of the affected kidney and combined chemo- and radiation therapy
o Gives cure rates of >80%

Frasier syndrome
Also caused by mutated WT1
An intronic mutation, deletes 3 amino acids and disrupts the alternative splice site that normally leads to +KTS
isoform
Unlike Wilms tumor mutations, the Frasier mutation results in normal WT1 proteins, but in different ratios of
+/- KTS isoforms.
In normal cells, the +KTS/-KTS ratio is 2:1, in Frasier this is 1:2.
o KTS forms bind WT1 promoter DNA sequences and act as transcription factors
o +KTS forms do not bind DNA but function as RNA processing factors.
Leads to severe developmental defect that affects the kidney and gonads.
XY individuals have dysgenetic gonads, often showing female external features