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91345
CANCER

Biochemistry, Genes and Disease

 Cancer is a disorder of uncontrolled cell
Lecture 5 growth [imbalance between cell growth and cell
death]

CANCER

 Cells are normally programmed to develop,

grow, differentiate and die

Dr Najah Nassif


 Cancer results when a cell escapes these
Medical and Molecular Biosciences

programmed constraints

N. NASSIF
*

The Cell
Normal Cell Growth and Division

Cycle and

 Eukaryotic cells have universal regulatory mechanisms
Normal Cell for controlling cell division

Growth

 Protein kinases and protein phosphorylation are central

 Occurs in 4 phases:
to the timing mechanism that determines entry into cell


G1
division and orderly passage through the cycle



S



G2

 In normal cells, cell replication is stopped to repair any


M
DNA damage

 If this does not occur, cell replication continues and

cells replicate with damaged DNA

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Control of Cell Division
Cell Cycle Checkpoints

 The cell cycle and Check point

 Checkpoints are mechanisms to halt the progress of
mitosis are subject to replication if chromosomal DNA is damaged or
many controls, all 
certain processes are aberrant

involving specific
proteins






 Checkpoints ensure that each stage of replication is

complete and accurate


 Cell cycle progression is
controlled by cyclin- 

cdk kinase complexes 

that phosphorylate
specific proteins to
progress the cell 

sequentially from one 

Major check point

phase to the next
,+

N. NASSIF
,
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-

Life and Dormant Cell division

(G0)

Apoptosis

Death of a
Cell

Growth


 Programmed cell death - a genetically controlled
method of regulating cell numbers

cell

 Pathway of cell death that is induced by a tightly

regulated intracellular program

Death

Necrosis
Autophagy

 Function in tissue homeostasis

Apoptosis
Senescence


 Plays a critical role in development by removing
unwanted cells

Extrinsic Granzyme

Intrinsic

receptor-
mediated

 Contrast to necrosis (cell death due to injury)

N. NASSIF
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Apoptosis
Characteristics of Apoptosis

 It is characterised by distinct morphological and Apoptosis
Necrosis

biochemical changes including:

 Active process

 Passive process (?)


 Cell shrinkage

 Progressive



- Membrane blebbing

 Membrane blebbing
breakdown of


 Chromatin condensation

cellular structure




- Cell shrinkage

 DNA fragmentation




- Nuclear Fragmentation

 Does not induce an




- Chromatin condensation

inflammatory response

- Chromosomal DNA fragmentation

 Defective apoptotic processes implicated in a variety of

diseases (including cancer)

N. NASSIF
!
N. NASSIF


Necrosis vs Apoptosis
Normal Cellular Controls

Signals from
environment
GROW
REST

Reductive
capacity
Energy state

Membrane
integrity

DIE

mitochondria

Level of
State of DNA
denatured
proteins

nucleus
cytoplasm

environment

N. NASSIF

N. NASSIF



 
 






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CANCER
Oncogenesis or Tumorigenesis
CANCER


 Cancer includes a class of diseases in which cells
 A TUMOUR is a population of cells resulting from
display uncontrolled growth, invasion and localised, unregulated growth and division of a
metastasis (spread to other body locations)
single cell

 Oncogenesis or tumorigenesis is the process
whereby normal cells become transformed into
cancer cells


 It is usually a multi-step process involving
genetic
mutations/cellular changes at each step


 Also called a neoplasm or a cancer (also carcinoma,
sarcoma, lymphoma, leukaemia, blastoma)


 Tumour cells are characterised by unregulated
mitotic activity, under conditions that would
normally restrict cell division

N. NASSIF
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Cancer Statistics Properties of Cancer Cells 

Cancer Related Deaths in USA (in 2008)
1. Loss of Contact Inhibition

Males

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Females

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 Cancer cells and normal cells can be cultured in the
laboratory

 When normal cells are grown on a tissue culture

dish, they proliferate until the surface of the dish is
covered by a single layer of cells just touching each
other. Then mitosis ceases. This phenomenon is
called contact inhibition

 Cancer cells DO NOT show contact inhibition. Once

the surface of the dish is covered, cells continue to
divide and grow over each other

N. NASSIF
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Properties of Cancer Cells  Properties of Cancer Cells 

1. Loss of Contact Inhibition
2. Cellular Immortality

Normal Cells
Transformed Cells

 Tumour cells placed in culture conditions, in the
laboratory, are often immortal

 They will grow and divide indefinitely as long

as nutrient conditions are provided

 Non-tumour cells have only a limited capacity

for cell division in culture

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N. NASSIF
+.
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Properties of Cancer Cells 

The
3. Abnormal Karyotype

Hallmarks

 Normal cells ordinarily have the normal set of of Cancer

chromosomes of the species (i.e. have a
normal karyotype)

 Cancer cells almost always have an abnormal

karyotype (eg. abnormal chromosome number
or structure)

N. NASSIF
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Influence of Genetics and Environment Influence of Genetics and Environment

in the Development of Cancer
in the Development of Cancer


 Cancers result from the interaction of both
Some individuals
have a genetic
genetic and environmental factors leading to makeup that
accumulation of mutations in essential genes
makes them
more

 Exposure to certain agents know to increase the susceptible to
certain agents

risk of cancer (eg. cigarette smoke, asbestos,
radiation)

Others can
tolerate more of

 Levels of susceptibility differ between individuals
the carcinogen
before they will
develop cancer

N. NASSIF
32
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33

Occupational Exposure and The Relationship Between Age and Cancer

Cancer Risk

Occupation
Agent
Site of Disease

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International Variation in the Incidence of Involvement of Genes in Cancer

Various Types of Cancers

Development


 Subsets of genes in the genome found to be
important in the prevention, development and
progression of cancer

 These genes have been found to be either

malfunctioning, or non-functional in different
tumour types

 Categorised into 2 broad categories based on

cellular function

N. NASSIF
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"

Involvement of Genes in Cancer

Development
Classes of Genes in Cancer

1. Genes whose protein products stimulate or enhance
cell division and growth and viability (i.e. oncogenes)

2. Genes whose protein products directly or indirectly

inhibit or prevent cell division or promote cell
death (i.e. tumour suppressor genes)

 Major classes of genes identified:





3. Genes whose protein products are involved in the

1. Oncogenes

correction of acquired mutations in DNA (i.e. repair

2. Tumour suppressor genes

proteins)



3. DNA repair genes

N. NASSIF
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Classes of Cancer Genes Genes in Cancer

1. The Oncogenes


 Oncogenes = growth promoting (dominant gain of
function)


 Normal versions are termed proto-oncogenes and
their mutated counterparts are called oncogenes


 Protein products of proto-oncogenes stimulate cell
division and/or inhibit cell death


 Proto-oncogenes are usually growth factors, growth
factor receptors, signal transduction molecules or
nuclear transcription factors

N. NASSIF

Oncogenes and Their Functions


 Growth factor receptors – stimulate cell growth.
May function as transmembrane protein kinases


 Protein kinases – alter function of other proteins by
phosphorylation


 G-proteins – bind GTP and mediate cell signalling


 Transcription factors – proteins that bind to DNA
and activate transcription

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N. NASSIF


Classes of Cancer Genes Oncogenes Identified in Cancer

1. The Oncogenes (cont.)

 








 Mutated oncogenes can lead to unregulated cell

division

 Cells are able to grow in the absence of normal

growth signals

 Examples:
- ras: a signal transduction molecule

- myc: a transcription factor







- src: a protein tyrosine kinase

N. NASSIF

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The Ras Signalling

The ras Oncogene
Cascade


Ras transmits signals from receptor

 Ras subfamily is a family of small
tyrosine kinases (RTK to regulate

GTPases involved in cell signal
many biological functions


transduction

Activated Ras acts by regulating
the cellular response through

 Ras communicates and translates
distinct Ras effector proteins and
their complex signal transduction

signals from outside the cell to the
cascades


nucleus


The best-characterised signal
transduction pathway of Ras is by

 Activation of ras signalling causes cell growth, and the Raf kinases

survival

Another cascade of Ras-activated
signalling is by anti-apoptotic PI3-K


 Mutations of ras can permanently activate it and this can







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lead lead to cancer


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N. NASSIF
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Mutated Oncogenes Can Lead to Classes of Cancer Genes

Unregulated Cell Growth
2. The Tumour Suppressor Genes

Gene expression without

 Tumour Suppressor genes = anti-oncogenes
Normal Growth
correct signals
(recessive loss of function)

 Tumour suppressor gene products act to inhibit the

division of cells if conditions of growth are not met

 Conditions triggering the brakes of cell division

include: DNA damage, lack of growth factors

 It is the absence, or loss of function of a tumour

suppressor gene product that leads to tumour formation

N. NASSIF
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Classes of Cancer Genes  








2. The Tumour Suppressor Genes (cont.)

Tumour
Suppressor

 Some examples include:
Genes
Identified in


- p53 (TP53): a transcription factor regulating cell





division
Cancer



- Rb: controls cell division



- APC: controls availability of a transcription factor



- BRCA: Involved in repair of damaged DNA

N. NASSIF
#
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Genes in Cancer Genes in Cancer

The p53 Tumour Suppressor


 p53 (the guardian of the genome)


- gene located on chromosome 17


- mutations lead to loss of function


- functions to prevent cell division of cells with

damaged DNA


- p53 mutations observed in approximately 50%

of all cancers

The p53 Tumour Suppressor


 p53 is a multifunctional protein
which plays a role in:

- modulating gene transcription

- policing cell cycle checkpoints

- activating apoptosis

- controlling DNA replication and

repair

- maintaining genomic stability

- responding to genetic insults

N. NASSIF
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Genes in Tumorigenesis  Knudsons 2-Hit Hypothesis for the

Tumour Specific Tumour Suppressor Genes
Development of Cancer


 Breast cancer
Gene mutations may be inherited or acquired during a
persons life time


- BRCA1 and BRCA2 genes


- Mutations increase risk of developing breast
Sporadic Cancer:


cancer
Both mutations are
acquired

Tumour


 Colon cancer
Inherited Cancer:

1 mutation inherited


- Adenomatous polyposis coli (APC) gene
1 mutation acquired
Tumour


- Deleted in colon cancer (DCC) gene

N. NASSIF

N. NASSIF


Cancer Normal Cell

Familial vs Sporadic Cancers
Development
is a First Mutation


 Familial cancers have younger age of onset Multistep
compared to their sporadic counterpart

Process
Second Mutation


 Usually a strong family history, with multiple family
members affected in 2 or more generations is
 Mutations Third Mutation

observed
are acquired
at every Fourth or

 Involvement of tumours in other organs (eg. breast step
later
Mutation

and ovary)

Malignant Cell

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Cancer Development is a Multistep The Stages of Colorectal

Process
Tumorigenesis

Loss of the tumour suppressor gene APC

Activation of ras oncogene

Loss of tumour suppressor gene DCC

Loss of tumour suppressor gene p53

Additional mutations

N. NASSIF
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!#

Colorectal Cancer

ROLE OF THE TUMOUR
 Cancer of the colon and/or rectum

SUPPRESSOR GENE PTEN IN
 Third most commonly diagnosed cancer in

men (8%), and the second in women (10%)

SPORADIC COLORECTAL
CANCER (CRC)

 Third most common cause of cancer death
(9%) (NSWCC 2007)

N. NASSIF
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Familial Colorectal Cancer
Sporadic Colorectal Cancer

(A)
Hereditary Non Polyposis Colorectal Cancer
 Accounts for approximately 80% of all CRC

(HNPCC)

• Makes up 5-6% of all CRC cases

 10-15% of tumours have a deficiency in the DNA
• DNA mismatch repair genes
mismatch repair pathway

(B) Familial Adenomatous Polyposis (FAP)

 Distinct pathway(s) from benign polyp to
malignant polyp to metastatic cancer have been
• Makes up 1% of all CRC cases
identified

• APC Tumour suppressor gene

N. NASSIF
"
N. NASSIF
!

Genetic Changes Identified in Non-Genetic Factors in Colon

Colorectal Cancer
Cancer


 Age – 90% of CRCs occur in people over 50


 Inflammatory bowel disease (IBD) – inflammatory
disorder of the colon can lead to ulcers

 Diet – diets high in red meat & fat, and low in fibre
increases risk

 Exercise – Low level increases risk

 Alcohol consumption & smoking – increase risk

N. NASSIF
!
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!


 
 






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Genes Involved in Colonic PTEN as a Candidate Gene in

Tumorigenesis
Colorectal Cancer


 Loss of PTEN gene region observed in 35% of
sporadic CRC


 Mutations / deletions of PTEN observed in many
sporadic cancers (eg. brain, prostate, endometrium)


 Mice heterozygous for PTEN develop CRC


 Germline (inherited) PTEN mutations give rise to
inheritable disorders (Cowden, Bannayan Riley
Ruvalcaba syndromes) characterised increased risk
of certain cancer types

N. NASSIF
"
N. NASSIF
"!

The PTEN Tumour Suppressor Gene
PTEN Gene and Protein Structure


 PTEN:
Phosphatase and tensin homolog
  
 



  
 




deleted on chromosome 10
 
  
 


 PTEN gene is located at on chromosome 10q23.3

 The gene product is a protein and lipid phosphatase

 Regulates cell growth survival, cell adhesion, cell

differentiation and death (apoptosis)

N. NASSIF
""
N. NASSIF
"#


 
 

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Cellular Functions of PTEN
The Cellular Role(s) of PTEN

 The major substrate of PTEN is the second messenger lipid
PIP-3 (phosphoinositol-3,4,5-triphosphate)


 PTEN dephosphorylates PIP-3 to PIP-2, thus keeping
PIP-3 levels low


 Loss of PTEN leads to increased levels of PIP-3 and
consequent activation of Akt/PKB and the PI3K cell
survival/anti-apoptic pathway (growth stimulatory effect)


 Hyperactivation of Akt leads to protection from apoptotic
stimuli and increased cell growth and survival


 PTEN is therefore a negative regulator of cell survival

N. NASSIF
!#
N. NASSIF
!$

Multiple Roles of PTEN in Tumour Suppression

Additional Functions of PTEN


 PTEN has also been shown to be:





- a regulator of cell adhesion




- an inhibitor of cell migration




- a regulator of cell size




- an inhibitor of angiogenesis




- an inhibitor of tumour metastasis

 The effect is dependent upon the substrate of

PTEN dephosporylation

N. NASSIF
!%
N. NASSIF
"


 
 

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A B PTEN Mutation Detected in Sporadic

Detection
Germline DNA Tumour DNA CRC 1
CRC

of PTEN Exon 5

Gene A>G

E150Q

Mutations
D153N

in Sporadic
%!

K125X
D153Y
319X

A B
Colorectal %$

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V217A
N323K

Cancer
Germline DNA Tumour DNA
CRC 2

Exon 5

G>A
E1
E2
E3
E4
E5
E6
E7
E8
E9

G129E

C124S

N. NASSIF
%
N. NASSIF
%!

Results


 41 paired tumour samples were screened

Project

 Mutations detected in 8/41 (19.5%) primary Evaluation of the Functional

sporadic colorectal tumours

Significance of the Detected

 Overall, alterations (mutations and deletions) of PTEN Mutations

the PTEN gene were present in 15/41 (37%)
primary sporadic colorectal tumours

N. NASSIF
%"
N. NASSIF
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!


% 

Functional Analysis of the Detected

PTEN Gene Mutations


 The wild type (WT) and each of the mutant PTENs
engineered into a mammalian expression vector


 The effect of the WT and mutant PTEN on cell
cycle distribution and cell proliferation (your project
aim) was determined after transfection


 The effects of PTEN mutation on PTEN subcellular
localisation was also determined

Engineering The PTEN Mutants


 Point Mutations were generated from the WT
PTEN clone using in vitro site directed
mutagenesis


 Truncating mutants were generated by PCR
amplification from the WT PTEN clone

N. NASSIF
"!
N. NASSIF
""

Effect of Mutation on PTEN

Effect of
Subcellular Localisation

Mutation on

WT PTEN is K125X
C>N
Punctate E150Q C=N D153Y C>N
PTEN
evenly distributed Subcellular
between the
cytoplasm and Localisation

the nucleus
C>N
WT PTEN C=N C124S C>N G129E
C=N

Some mutants D153Y C=N V217A C=N
C=N 319X Punctate

have increased
cytoplasmic
localisation

C>N
K125E Punctate C>N
K62R C=N Y65C C=N
319X Punctate N323K C=N
N. NASSIF
"#
N. NASSIF
"$


 
 

#




!


% 

The Effect of PTEN Mutations on The Effect

of PTEN
Cell Cycle Distribution
Mutations


on Cell


Proliferation




 WT PTEN is 

able to cause a
slowing of cell
proliferation




 Aim to
determine the
effect of PTEN
mutation on
cell
proliferation

N. NASSIF
"%
N. NASSIF
#

Conclusions
References


 WT PTEN brings about cell cycle arrest in cultured
 Nelson and Cox (2008) Lehninger Principles of Biochemistry
cells
5th Edition


 Mutant PTEN is unable to bring about a cycle arrest
that is equivalent to that of WT PTEN. The level is
 G. Karp (2008) Cell and Molecular Biology: Concepts and
lower for the PTEN mutants.
Experiments. 5th Edition

 WT PTEN is able to bring about a slowing of cell
 MH. Lodish et al. Molecular Cell Biology

proliferation of cultured cells

 Is mutant PTEN able to slow cell proliferation as

effectively as the WT PTEN? (You will find out in
your project)

N. NASSIF
#
N. NASSIF
#


 
 

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