You are on page 1of 18

8Cu LecLure 3 - CAnCL8 9/2/10

n. nASSll (Sprlna 2010) 1
91345
Biochemistry, Genes and Disease
Lecture 5
CANCER
Dr Najah Nassif
Medical and Molecular Biosciences
CANCER
Cancer is a disorder of uncontrolled cell
growth [imbalance between cell growth and cell
death]
Cells are normally programmed to develop,
grow, differentiate and die
Cancer results when a cell escapes these
programmed constraints
N. NASSIF 2
The Cell
Cycle and
Normal Cell
Growth
N. NASSIF 3
Occurs in 4 phases:
G1
S
G2
M
hup://www.cellsallve.com/cell_cvcle.hLm
Normal Cell Growth and Division
Eukaryotic cells have universal regulatory mechanisms
for controlling cell division
Protein kinases and protein phosphorylation are central
to the timing mechanism that determines entry into cell
division and orderly passage through the cycle
In normal cells, cell replication is stopped to repair any
DNA damage
If this does not occur, cell replication continues and
cells replicate with damaged DNA
N. NASSIF 4
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 2
Control of Cell Division
N. NASSIF 3

8esLrlcuon
Major check point
Check point
p8b
p33
The cell cycle and
mitosis are subject to
many controls, all
involving specific
proteins
Cell cycle progression is
controlled by cyclin-
cdk kinase complexes
that phosphorylate
specific proteins to
progress the cell
sequentially from one
phase to the next

CrowLh
facLors
Cell Cycle Checkpoints
Checkpoints are mechanisms to halt the progress of
replication if chromosomal DNA is damaged or
certain processes are aberrant
Checkpoints ensure that each stage of replication is
complete and accurate
N. NASSIF 6
Life and
Death of a
Cell
N. NASSIF 7
Dormant
(G
0
)
Cell division
Growth
Necrosis
Extrinsic
receptor-
mediated
Intrinsic
Granzyme
cell
Death
Apoptosis Senescence
Autophagy
Apoptosis
Programmed cell death - a genetically controlled
method of regulating cell numbers
Pathway of cell death that is induced by a tightly
regulated intracellular program
Function in tissue homeostasis
Plays a critical role in development by removing
unwanted cells
Contrast to necrosis (cell death due to injury)
N. NASSIF 8
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 3
Apoptosis
It is characterised by distinct morphological and
biochemical changes including:
- Membrane blebbing
- Cell shrinkage
- Nuclear Fragmentation
- Chromatin condensation
- Chromosomal DNA fragmentation
Defective apoptotic processes implicated in a variety of
diseases (including cancer)
N. NASSIF 9
Characteristics of Apoptosis
N. NASSIF 10
Active process
Cell shrinkage
Membrane blebbing
Chromatin condensation
DNA fragmentation
Does not induce an
inflammatory response
Passive process (?)
Progressive
breakdown of
cellular structure
Apoptosis Necrosis
Necrosis vs Apoptosis
N. NASSIF 11
Normal Cellular Controls
N. NASSIF 12
nucleus cytoplasm
environment
mitochondria
GROW REST
DIE
State of DNA
Level of
denatured
proteins
Energy state
Reductive
capacity
Membrane
integrity
Signals from
environment
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 4
CANCER
Oncogenesis or Tumorigenesis
Cancer includes a class of diseases in which cells
display uncontrolled growth, invasion and
metastasis (spread to other body locations)
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
N. NASSIF 13
CANCER
A TUMOUR is a population of cells resulting from
localised, unregulated growth and division of a
single cell
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 14
Cancer Statistics
Cancer Related Deaths in USA (in 2008)
N. NASSIF 13
Males Females
294,120 Cancer-8elaLed ueaLhs 271,330 Cancer-8elaLed ueaLhs
Properties of Cancer Cells
1. Loss of Contact Inhibition
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 16
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 3
Properties of Cancer Cells
1. Loss of Contact Inhibition
N. NASSIF 17
hup://users.rcn.com/[klmball.ma.ulLraneL/8loloavÞaaes/C/CancerCellslnCulLure.hLml
Normal Cells Transformed Cells
Properties of Cancer Cells
2. Cellular Immortality
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
N. NASSIF 18
Properties of Cancer Cells
3. Abnormal Karyotype
Normal cells ordinarily have the normal set of
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 19
The
Hallmarks
of Cancer
N. NASSIF 20
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 6
Influence of Genetics and Environment
in the Development of Cancer
Cancers result from the interaction of both
genetic and environmental factors leading to
accumulation of mutations in essential genes
Exposure to certain agents know to increase the
risk of cancer (eg. cigarette smoke, asbestos,
radiation)
Levels of susceptibility differ between individuals
N. NASSIF 21 N. NASSIF 22
Some individuals
have a genetic
makeup that
makes them
more
susceptible to
certain agents
Others can
tolerate more of
the carcinogen
before they will
develop cancer
Influence of Genetics and Environment
in the Development of Cancer
Occupational Exposure and
Cancer Risk
N. NASSIF 23
Occupation Agent Site of Disease
uve manufacLurers, rubber workers Aromauc amlnes 8ladder
AsbesLos mlnlna and handllna AsbesLos Luna, mesoLhellum
Cadmlum workers Cadmlum ÞrosLaLe
uranlum mlners lonlslna radlauon Luna
Coal aas manufacLurers, asphalLers Þolvcvcllc hvdrocarbons Skln, luna
and oLhers
larmers, sallors uv llahL Skln, llp
Pardwood furnlLure manufacLurers unknown nasal slnuses
The Relationship Between Age and Cancer
N. NASSIF 24
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 7
International Variation in the Incidence of
Various Types of Cancers
N. NASSIF 23
Involvement of Genes in Cancer
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 26
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)
3. Genes whose protein products are involved in the
correction of acquired mutations in DNA (i.e. repair
proteins)
N. NASSIF 27
Involvement of Genes in Cancer
Development
Classes of Genes in Cancer
Major classes of genes identified:

1. Oncogenes
2. Tumour suppressor genes
3. DNA repair genes
N. NASSIF 28
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 8
Classes of Cancer Genes
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 29
Genes in Cancer
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
N. NASSIF 30
Classes of Cancer Genes
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 31
Oncogenes Identified in Cancer
N. NASSIF 32
Cncogene Iuncnon Cancer 1ype
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 9
The ras Oncogene
Ras subfamily is a family of small
GTPases involved in cell signal
transduction
Ras communicates and translates
signals from outside the cell to the
nucleus
Activation of ras signalling causes cell growth, and
survival
Mutations of ras can permanently activate it and this can
lead lead to cancer
N. NASSIF 33
The Ras Signalling
Cascade
N. NASSIF 34
Ras transmits signals from receptor
tyrosine kinases (RTK to regulate
many biological functions
Activated Ras acts by regulating
the cellular response through
distinct Ras effector proteins and
their complex signal transduction
cascades
The best-characterised signal
transduction pathway of Ras is by
the Raf kinases
Another cascade of Ras-activated
signalling is by anti-apoptotic PI3-K
Loulse van der Wevden , uavld !. Adams (2007) 8locblmlco et
8lopbvslco Acto.
Mutated Oncogenes Can Lead to
Unregulated Cell Growth
N. NASSIF 33
Normal Growth
Gene expression without
correct signals
Classes of Cancer Genes
2. The Tumour Suppressor Genes
Tumour Suppressor genes = anti-oncogenes
(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 36
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 10
Some examples include:
- p53 (TP53): a transcription factor regulating cell
division
- Rb: controls cell division
- APC: controls availability of a transcription factor
- BRCA: Involved in repair of damaged DNA
N. NASSIF 37
Classes of Cancer Genes
2. The Tumour Suppressor Genes (cont.)
Tumour
Suppressor
Genes
Identified in
Cancer
N. NASSIF 38
1umour
5uppressor
Iuncnon Cancer 1ype
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
N. NASSIF 39
Genes in Cancer
The p53 Tumour Suppressor
N. NASSIF 40
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
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 11
Genes in Tumorigenesis
Tumour Specific Tumour Suppressor Genes
Breast cancer
- BRCA1 and BRCA2 genes
- Mutations increase risk of developing breast
cancer
Colon cancer
- Adenomatous polyposis coli (APC) gene
- Deleted in colon cancer (DCC) gene
N. NASSIF 41
Knudsons 2-Hit Hypothesis for the
Development of Cancer
N. NASSIF 42
Gene mutations may be inherited or acquired during a
persons life time
Sporadic Cancer:
Both mutations are
acquired
Inherited Cancer:
1 mutation inherited
1 mutation acquired
Tumour
Tumour
Familial vs Sporadic Cancers
Familial cancers have younger age of onset
compared to their sporadic counterpart
Usually a strong family history, with multiple family
members affected in 2 or more generations is
observed
Involvement of tumours in other organs (eg. breast
and ovary)
N. NASSIF 43
Cancer
Development
is a
Multistep
Process
N. NASSIF 44
Normal Cell
First Mutation
Second Mutation
Third Mutation
Fourth or
later
Mutation
Malignant Cell
Mutations
are acquired
at every
step
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 12
Cancer Development is a Multistep
Process
N. NASSIF 43
The Stages of Colorectal
Tumorigenesis
N. NASSIF 46
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
ROLE OF THE TUMOUR
SUPPRESSOR GENE PTEN IN
SPORADIC COLORECTAL
CANCER (CRC)
Colorectal Cancer
Cancer of the colon and/or rectum
Third most commonly diagnosed cancer in
men (8%), and the second in women (10%)
Third most common cause of cancer death
(9%) (NSWCC 2007)
N. NASSIF 48
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 13
Familial Colorectal Cancer
(A) Hereditary Non Polyposis Colorectal Cancer
(HNPCC)
• Makes up 5-6% of all CRC cases
• DNA mismatch repair genes
(B) Familial Adenomatous Polyposis (FAP)
• Makes up 1% of all CRC cases
• APC Tumour suppressor gene
N. NASSIF 49
Sporadic Colorectal Cancer
Accounts for approximately 80% of all CRC
10-15% of tumours have a deficiency in the DNA
mismatch repair pathway
Distinct pathway(s) from benign polyp to
malignant polyp to metastatic cancer have been
identified
N. NASSIF 30
Genetic Changes Identified in
Colorectal Cancer
N. NASSIF 31
Non-Genetic Factors in Colon
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 32
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 14
Genes Involved in Colonic
Tumorigenesis
N. NASSIF 33
PTEN as a Candidate Gene in
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 34
The PTEN Tumour Suppressor Gene
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 33
PTEN Gene and Protein Structure
N. NASSIF 36
Lncodes N-term|na| Doma|n Lncodes C-term|na| Doma|n
£ncodes Phosphotose uomoin
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 13
Cellular Functions 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 37
The Cellular Role(s) of PTEN
N. NASSIF 38
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 39
Multiple Roles of PTEN in Tumour Suppression
N. NASSIF 60
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 16
Detection
of PTEN
Gene
Mutations
in Sporadic
Colorectal
Cancer
N. NASSIF 61
Tumour DNA
B A
Germline DNA
Tumour DNA
B A
Germline DNA CRC 1
Exon 5
A>G
CRC 2
Exon 5
G>A
PTEN Mutation Detected in Sporadic
CRC
N. NASSIF 62
319X
k123L
k628
E1 E2 E3 E4 E5 E6 E7 E8 E9
D153N
D153Y
V217A ?63C N323K
E150Q
K125X
C124S
G129E
Results
41 paired tumour samples were screened
Mutations detected in 8/41 (19.5%) primary
sporadic colorectal tumours
Overall, alterations (mutations and deletions) of
the PTEN gene were present in 15/41 (37%)
primary sporadic colorectal tumours
N. NASSIF 63 N. NASSIF 64
Project
Evaluation of the Functional
Significance of the Detected
PTEN Mutations
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 17
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
N. NASSIF 63
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 66
Effect of Mutation on PTEN
Subcellular Localisation
N. NASSIF 67
C124S C>N C124S C>N
K62R C=N Y65C C=N
K125E
C>N
Punctate
G129E C>N WT PTEN C=N
WT PTEN is
evenly distributed
between the
cytoplasm and
the nucleus
Some mutants
have increased
cytoplasmic
localisation
N. NASSIF
C=N D153Y
V217A C=N V217A C=N
319X
C>N
Punctate N323K C=N
D153Y C>N K125X
C>N
Punctate E150Q C=N
Punctate 319X
C=N
Effect of
Mutation on
PTEN
Subcellular
Localisation
68
8Cu LecLure 3 - CAnCL8 9/2/10
n. nASSll (Sprlna 2010) 18
The Effect of PTEN Mutations on
Cell Cycle Distribution
N. NASSIF 69
The Effect
of PTEN
Mutations
on Cell
Proliferation
N. NASSIF 70
WT PTEN is
able to cause a
slowing of cell
proliferation
Aim to
determine the
effect of PTEN
mutation on
cell
proliferation

Conclusions
WT PTEN brings about cell cycle arrest in cultured
cells
Mutant PTEN is unable to bring about a cycle arrest
that is equivalent to that of WT PTEN. The level is
lower for the PTEN mutants.
WT PTEN is able to bring about a slowing of cell
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 71
References
Nelson and Cox (2008) Lehninger Principles of Biochemistry
5th Edition
G. Karp (2008) Cell and Molecular Biology: Concepts and
Experiments. 5
th
Edition
MH. Lodish et al. Molecular Cell Biology
N. NASSIF 72

all involving specific proteins Cell cycle progression is controlled by cyclincdk kinase complexes that phosphorylate specific proteins to progress the cell sequentially from one phase to the next Check point Cell Cycle Checkpoints Checkpoints are mechanisms to halt the progress of replication if chromosomal DNA is damaged or certain processes are aberrant Checkpoints ensure that each stage of replication is complete and accurate Major check point N. NASSIF Autophagy Senescence Granzyme Function in tissue homeostasis Plays a critical role in development by removing unwanted cells Contrast to necrosis (cell death due to injury) N.a genetically controlled method of regulating cell numbers Pathway of cell death that is induced by a tightly regulated intracellular program cell Death Necrosis Apoptosis Extrinsic receptormediated N. NASSIF Life and Death of a Cell Dormant (G0) Growth Cell division Apoptosis Programmed cell death . NASSIF Intrinsic .Control of Cell Division The cell cycle and mitosis are subject to many controls. NASSIF N.

NASSIF Characteristics of Apoptosis Apoptosis Active process Cell shrinkage Membrane blebbing Chromatin condensation DNA fragmentation Does not induce an inflammatory response Necrosis Passive process (?) Progressive breakdown of cellular structure N.Chromatin condensation . NASSIF N.Apoptosis It is characterised by distinct morphological and biochemical changes including: . NASSIF .Membrane blebbing .Chromosomal DNA fragmentation Defective apoptotic processes implicated in a variety of diseases (including cancer) N.Nuclear Fragmentation . NASSIF Necrosis vs Apoptosis Normal Cellular Controls Signals from environment Reductive capacity Membrane integrity Energy state GROW REST mitochondria State of DNA Level of denatured proteins DIE nucleus cytoplasm environment N.Cell shrinkage .

NASSIF . NASSIF N. unregulated growth and division of a single cell Also called a neoplasm or a cancer (also carcinoma. they proliferate until the surface of the dish is covered by a single layer of cells just touching each other. This phenomenon is called contact inhibition Cancer cells DO NOT show contact inhibition. invasion and metastasis (spread to other body locations) 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 N. Then mitosis ceases. leukaemia. cells continue to divide and grow over each other N. Loss of Contact Inhibition Cancer cells and normal cells can be cultured in the laboratory When normal cells are grown on a tissue culture dish. sarcoma. lymphoma. NASSIF Cancer Statistics Cancer Related Deaths in USA (in 2008) Males Females Properties of Cancer Cells 1.CANCER Oncogenesis or Tumorigenesis Cancer includes a class of diseases in which cells display uncontrolled growth. Once the surface of the dish is covered. blastoma) Tumour cells are characterised by unregulated mitotic activity. NASSIF CANCER A TUMOUR is a population of cells resulting from localised. under conditions that would normally restrict cell division N.

abnormal chromosome number or structure) The Hallmarks of Cancer N. Loss of Contact Inhibition Normal Cells Transformed Cells Properties of Cancer Cells 2. NASSIF N. NASSIF Properties of Cancer Cells 3. NASSIF N. 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 N. in the laboratory.Properties of Cancer Cells 1. NASSIF . have a normal karyotype) Cancer cells almost always have an abnormal karyotype (eg.e. Cellular Immortality Tumour cells placed in culture conditions. Abnormal Karyotype Normal cells ordinarily have the normal set of chromosomes of the species (i.

Influence of Genetics and Environment in the Development of Cancer Cancers result from the interaction of both genetic and environmental factors leading to accumulation of mutations in essential genes Exposure to certain agents know to increase the risk of cancer (eg. NASSIF Levels of susceptibility differ between individuals N. NASSIF N. cigarette smoke. NASSIF Occupational Exposure and Cancer Risk Occupation Agent Site of Disease The Relationship Between Age and Cancer N. asbestos. NASSIF . radiation) Influence of Genetics and Environment in the Development of Cancer Some individuals have a genetic makeup that makes them more susceptible to certain agents Others can tolerate more of the carcinogen before they will develop cancer N.

NASSIF . Genes whose protein products stimulate or enhance cell division and growth and viability (i. Genes whose protein products directly or indirectly inhibit or prevent cell division or promote cell death (i. NASSIF 3. or non-functional in different tumour types Categorised into 2 broad categories based on cellular function N. development and progression of cancer These genes have been found to be either malfunctioning. repair proteins) N. NASSIF Involvement of Genes in Cancer Development 1. Genes whose protein products are involved in the correction of acquired mutations in DNA (i.e. tumour suppressor genes) Major classes of genes identified: 1.e.e.International Variation in the Incidence of Various Types of Cancers Involvement of Genes in Cancer Development Subsets of genes in the genome found to be important in the prevention. Oncogenes 2. DNA repair genes N. NASSIF N. oncogenes) Classes of Genes in Cancer 2. Tumour suppressor genes 3.

NASSIF . growth factor receptors. NASSIF Classes of Cancer Genes 1.myc: a transcription factor .src: a protein tyrosine kinase N.) Mutated oncogenes can lead to unregulated cell division Cells are able to grow in the absence of normal growth signals Examples: . The Oncogenes Oncogenes = growth promoting (dominant gain of function) Genes in Cancer Oncogenes and Their Functions Growth factor receptors – stimulate cell growth. NASSIF Oncogenes Identified in Cancer N. signal transduction molecules or nuclear transcription factors N. The Oncogenes (cont. NASSIF 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. 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 N.ras: a signal transduction molecule .Classes of Cancer Genes 1.

The Tumour Suppressor Genes Tumour Suppressor genes = anti-oncogenes (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. and survival Mutations of ras can permanently activate it and this can lead lead to cancer N. lack of growth factors It is the absence. NASSIF The Ras Signalling Cascade Ras transmits signals from receptor tyrosine kinases (RTK to regulate many biological functions Activated Ras acts by regulating the cellular response through distinct Ras effector proteins and their complex signal transduction cascades The best-characterised signal transduction pathway of Ras is by the Raf kinases Another cascade of Ras-activated signalling is by anti-apoptotic PI3-K N. or loss of function of a tumour suppressor gene product that leads to tumour formation N. NASSIF . NASSIF N.The ras Oncogene Ras subfamily is a family of small GTPases involved in cell signal transduction Ras communicates and translates signals from outside the cell to the nucleus Activation of ras signalling causes cell growth. NASSIF Mutated Oncogenes Can Lead to Unregulated Cell Growth Normal Growth Gene expression without correct signals Classes of Cancer Genes 2.

activating apoptosis .responding to genetic insults N. NASSIF Genes in Cancer The p53 Tumour Suppressor p53 (the guardian of the genome) .) Some examples include: . NASSIF .mutations lead to loss of function . NASSIF Genes in Cancer The p53 Tumour Suppressor p53 is a multifunctional protein which plays a role in: .policing cell cycle checkpoints .BRCA: Involved in repair of damaged DNA Tumour Suppressor Genes Identified in Cancer N. The Tumour Suppressor Genes (cont.maintaining genomic stability . NASSIF N.functions to prevent cell division of cells with damaged DNA .modulating gene transcription .APC: controls availability of a transcription factor .controlling DNA replication and repair .Rb: controls cell division .p53 mutations observed in approximately 50% of all cancers N.gene located on chromosome 17 .Classes of Cancer Genes 2.p53 (TP53): a transcription factor regulating cell division .

NASSIF Familial vs Sporadic Cancers Familial cancers have younger age of onset compared to their sporadic counterpart Usually a strong family history.Genes in Tumorigenesis Tumour Specific Tumour Suppressor Genes Breast cancer . breast and ovary) N. with multiple family members affected in 2 or more generations is observed Involvement of tumours in other organs (eg. NASSIF Cancer Development is a Multistep Process Mutations are acquired at every step Normal Cell First Mutation Second Mutation Third Mutation Fourth or later Mutation Malignant Cell N. NASSIF Knudson s 2-Hit Hypothesis for the Development of Cancer Gene mutations may be inherited or acquired during a person s life time Sporadic Cancer: Both mutations are acquired Inherited Cancer: 1 mutation inherited 1 mutation acquired Tumour Tumour N. NASSIF .BRCA1 and BRCA2 genes .Deleted in colon cancer (DCC) gene N.Mutations increase risk of developing breast cancer Colon cancer .Adenomatous polyposis coli (APC) gene .

NASSIF N. NASSIF .Cancer Development is a Multistep Process The Stages of Colorectal 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. and the second in women (10%) Third most common cause of cancer death (9%) (NSWCC 2007) N. NASSIF Colorectal Cancer ROLE OF THE TUMOUR SUPPRESSOR GENE PTEN IN SPORADIC COLORECTAL CANCER (CRC) Cancer of the colon and/or rectum Third most commonly diagnosed cancer in men (8%).

and low in fibre increases risk Exercise – Low level increases risk Alcohol consumption & smoking – increase risk N. NASSIF N. NASSIF Genetic Changes Identified in Colorectal Cancer Non-Genetic Factors in Colon 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. NASSIF .Familial Colorectal Cancer (A) Hereditary Non Polyposis Colorectal Cancer (HNPCC) • Makes up 5-6% of all CRC cases • DNA mismatch repair genes (B) Familial Adenomatous Polyposis (FAP) • Makes up 1% of all CRC cases • APC Tumour suppressor gene N. NASSIF Sporadic Colorectal Cancer Accounts for approximately 80% of all CRC 10-15% of tumours have a deficiency in the DNA mismatch repair pathway Distinct pathway(s) from benign polyp to malignant polyp to metastatic cancer have been identified N.

NASSIF PTEN Gene and Protein Structure N.Genes Involved in Colonic Tumorigenesis PTEN as a Candidate Gene in Colorectal Cancer Loss of PTEN gene region observed in 35% of sporadic CRC Mutations / deletions of PTEN observed in many sporadic cancers (eg. NASSIF N. NASSIF The PTEN Tumour Suppressor Gene PTEN: Phosphatase and tensin homolog deleted on chromosome 10 PTEN gene is located at on chromosome 10q23. brain. prostate.3 The gene product is a protein and lipid phosphatase Regulates cell growth survival. endometrium) Mice heterozygous for PTEN develop CRC Germline (inherited) PTEN mutations give rise to inheritable disorders (Cowden. cell differentiation and death (apoptosis) N. Bannayan Riley Ruvalcaba syndromes) characterised increased risk of certain cancer types N. NASSIF . cell adhesion.

a regulator of cell adhesion . 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.an inhibitor of angiogenesis . NASSIF The Cellular Role(s) of PTEN N.a regulator of cell size .Cellular Functions of PTEN The major substrate of PTEN is the second messenger lipid PIP-3 (phosphoinositol-3. NASSIF Multiple Roles of PTEN in Tumour Suppression N.an inhibitor of cell migration .4. NASSIF .an inhibitor of tumour metastasis The effect is dependent upon the substrate of PTEN dephosporylation N. NASSIF Additional Functions of PTEN PTEN has also been shown to be: .5-triphosphate) PTEN dephosphorylates PIP-3 to PIP-2.

Detection of PTEN Gene Mutations in Sporadic Colorectal Cancer A Germline DNA B Tumour DNA CRC 1 Exon 5 A>G PTEN Mutation Detected in Sporadic CRC E150Q D153N A Germline DNA B Tumour DNA K125X D153Y CRC 2 Exon 5 G>A V217A E1 E2 E3 E4 E5 E6 E7 319X N323K E8 E9 G129E C124S N. NASSIF N.5%) primary sporadic colorectal tumours Overall. NASSIF . alterations (mutations and deletions) of the PTEN gene were present in 15/41 (37%) primary sporadic colorectal tumours Project Evaluation of the Functional Significance of the Detected PTEN Mutations N. NASSIF N. NASSIF Results 41 paired tumour samples were screened Mutations detected in 8/41 (19.

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 N. NASSIF . NASSIF 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 Subcellular Localisation WT PTEN is evenly distributed between the cytoplasm and the nucleus Some mutants have increased cytoplasmic localisation K62R C=N Y65C C=N K125E C>N Punctate K125X C>N Punctate E150Q C=N D153Y C>N Effect of Mutation on PTEN Subcellular Localisation WT PTEN C=N C124S C>N G129E C>N D153Y C=N V217A C=N C=N 319X C=N Punctate 319X C>N Punctate N323K C=N N.

Molecular Cell Biology N. 5th Edition MH. Lodish et al. WT PTEN is able to bring about a slowing of cell 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. The level is lower for the PTEN mutants. NASSIF Conclusions WT PTEN brings about cell cycle arrest in cultured cells Mutant PTEN is unable to bring about a cycle arrest that is equivalent to that of WT PTEN. NASSIF N. NASSIF . NASSIF References Nelson and Cox (2008) Lehninger Principles of Biochemistry 5th Edition G.The Effect of PTEN Mutations on Cell Cycle Distribution The Effect of PTEN 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. Karp (2008) Cell and Molecular Biology: Concepts and Experiments.