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91345

Biochemistry,GenesandDisease
BiochemistryGenesandDisease

CANCER
MatthewVerheyden
Email:Matthew.Verheyden@uts.edu.au
DrNajahNassif
SchoolLifeSciences,FacultyofScience

L
LectureOverview
O
i
Thecellcycleandapoptosis
Propertiesofcancercells
P
i
f
ll
Theinterplayofgeneticandenvironmentalfactorsin
p y g

thedevelopmentofcancer
Classesofcancergenes(oncogenesandtumour
Cl
f
(
dt

suppressorgenes)
TheroleofPTEN genemutationsincoloncancer

(laboratoryproject)
N.NASSIF

WHATISCANCER?
Cancerisadisorderofuncontrolledcellgrowth

(imbalancebetweencellproliferation/growthandcell
(imbalance
between cell proliferation/growth and cell
death)
Cellsarenormallyprogrammed todevelop,grow,

differentiate and die


differentiateanddie
Cellularescape
Cellular escape fromtheseprogrammedconstraints
from these programmed constraints

leadstocancer
N.NASSIF

TheCell
Cycleand
NormalCell
Growth
Occursin4phases:
Occurs in 4 phases:
G1
S
G2
M

Link to Cell Cycle


N.NASSIF

TheLifeandDeathofaCell
NormalCellGrowthandDivision
Eukaryoticcellshaveuniversalregulatorymechanisms
for controlling cell division
forcontrollingcelldivision
Proteinkinasesandproteinphosphorylationevents
regulatethetimingmechanismsthatdetermineentry
l
h i i
h i
h d
i
intocelldivisionandorderlypassagethroughthecycle
Innormalcells,cellreplicationisstoppedtoallowthe
repairofanyDNAdamage
Ifthisdoesnotoccur,cellreplicationwillcontinueand
cells will replicate with damaged DNA
cellswillreplicatewithdamagedDNA
N.NASSIF

TheLifeandDeathofaCell
ControlofCellDivision
Checkpoints are
mechanismsto
p g
halttheprogress
ofreplication if
chromosomal
DNAisdamaged
g
orcertain
processesare
aberrant
Checkpoints
ensurethateach
stageof
replicationis
completeand
accurate
N.NASSIF

TheLife
andDeath
ofaCell

Dormant
((G0)

Cell division

Growth

cell

CellDeath

D th
Death
Necrosis

Autophagy

Apoptosis
Extrinsic
receptormediated
N.NASSIF

Intrinsic

Senescence
Granzyme

TheLifeandDeathofaCell
Apoptosis(ProgrammedCellDeath)
Programmedcelldeath ageneticallycontrolled

method of regulating cell numbers


methodofregulatingcellnumbers
Pathwayofcelldeaththatisinducedbyatightly
Pathway of cell death that is induced by a tightly

regulatedintracellularprogram
Playsacriticalroleindevelopmentbyremoving

unwantedcells
Contrasttonecrosis(celldeathduetoinjury)
N.NASSIF

CharacteristicsofApoptosis
Itischaracterisedbydistinctmorphological
h
db d
h l
l
andbiochemicalchanges
Apoptosis

Necrosis

Activeprocess
p
Passiveprocess(?)
Passive process (?)
Cellshrinkage
Progressive
Membraneblebbing
breakdownof
Chromatincondensation cellularstructure
DNAfragmentation
Doesnotinducean
inflammatoryresponse
N.NASSIF

Necrosisvs Apoptosis

N.NASSIF

10

NormalCellularControls
Signals from
environment

GROW
Reductive
capacity

REST

Energy state

Membrane
M
b
integrity

DIE

mitochondria
Level of
denatured
proteins

State of DNA

nucleus

cytoplasm
environment
N.NASSIF

11

CANCERDevelopment
Oncogenesis orTumorigenesis
Cancerincludesaclassofdiseasesinwhichcells

displayuncontrolledgrowth,invasion
display
uncontrolled growth invasion and
and
metastasis (spreadtootherbodylocations)
Oncogenesis ortumorigenesisistheprocess

wherebynormalcellsbecometransformed into
cancercells
Itisusuallyamultistepprocessinvolvinggenetic
It is usually a multi step process involving genetic

mutations/cellularchangesateachstep
N.NASSIF

12

CANCERDevelopment
p
Oncogenesis orTumorigenesis
ATUMOUR isapopulationofcellsresultingfrom

localised,unregulatedgrowthanddivisionofasingle
localised
unregulated growth and division of a single
cell
Tumour cellsarecharacterisedbyunregulatedmitotic

activity,underconditionsthatwouldnormallyrestrict
celldivision
Alsoreferredtoasaneoplasmora
Al
f
dt
l
cancer (also
( l

carcinoma,sarcoma,lymphoma,leukaemia)
N.NASSIF

13

CANCERDevelopment
Oncogenesis orTumorigenesis
Factorsnotedtocauseincreasedcancerincidence

include: viruses,chemicalexposure,UVlightexposure,
,
p
,
g
p
,
radiation,somaticmutationsandinheritedgenetic
abnormalities
Howdothesefactorscauseanormalcelltobecome

malignant?
Severaltheoriesincluded:

((a)abnormalcelldifferentiation
) b
l ll d ff
(b)humantumourviruses
(c) mutagens causing damage to cellular DNA
(c)mutagenscausingdamagetocellularDNA
N.NASSIF

14

CancerStatistics
CancerRelatedDeathsinUSA(in2008)
F
Females
l

Males
M
l
294,120CancerRelatedDeaths

271,530CancerRelatedDeaths

Lung and bronchus


Lungandbronchus

Lung and bronchus


Lungandbronchus

Prostate

Breast

Colonandrectum

Colonandrectum

N.NASSIF

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HallmarksofCancerCells
1.LossofContactInhibition
Cancercellsandnormalcellscanbecultured inthe

laboratory
Normalcellsgrownontissueculturedishesproliferate

untilthesurfaceofthedishiscoveredbyasinglelayer
il h
f
f h di h i
db
i l l
ofcellstouchingeachother.Thenmitosisceases.This
phenomenon is called contact inhibition
phenomenoniscalledcontactinhibition
CancercellsDONOTshowcontactinhibition.Oncethe

surfaceofthedishiscovered,cellscontinuetodivide
andgrowovereachother
N.NASSIF

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HallmarksofCancerCells
1.LossofContactInhibition

ContactInhibition
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HallmarksofCancerCells
2.CellularImmortality
Normal(nontumour)cellshaveonlyalimited

capacityforcelldivision inculture
Tumour cellsplacedincultureconditions,inthe

laboratory,areoftenimmortal
Theywillgrowanddivideindefinitelyaslongas

nutrientconditionsareprovided

N.NASSIF

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HallmarksofCancerCells
3.AbnormalKaryotype
Normal cellsordinarilyhavethenormalsetof
y

chromosomes ofthespecies(i.e.haveanormal
karyotype)
y yp )
Cancer cellsalmostalwayshaveanabnormal
cells almost always have an abnormal

karyotype (e.g.abnormalchromosomenumberor
structure)
structure)

N.NASSIF

19

HallmarksofCancerCells
3.AbnormalKaryotype

http://galleryhip.com/aneuploidy.html

N.NASSIF

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The
Hallmarks
ofCancer
1. Failuretostop
growing
2. Evasionof
apoptosis

3. Growth
without
appropriate
signals
4. Cellular
immortality
5. Angiogenesis
6. Tissue
Invasionand
metastasis

N.NASSIF

21

InfluenceofGeneticandEnvironmental
FactorsintheDevelopmentofCancer
Cancersresultfromtheinteractionofbothgenetic

andenvironmentalfactors
and
environmental factors leadingtoaccumulation
leading to accumulation
ofmutationsinessentialgenes
EnvironmentalInfluence: Exposuretocertainagents

nowknowntoincreasetherisk ofcancer(e.g.
cigarettesmoke,asbestos,radiation)
k
b
d
)
GeneticInfluence:
Genetic Influence: Levelsofsusceptibilitydiffer
Levels of susceptibility differ

betweenindividuals(determinedbygeneticmakeup)
N.NASSIF

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InfluenceofGeneticandEnvironmental
FactorsintheDevelopmentofCancer
Someindividuals
haveagenetic
makeup
k
th t
that
makesthem
more
susceptible to
certainagents

Otherscan
tolerate moreof
the carcinogen
thecarcinogen
beforetheywill
developcancer
N.NASSIF

23

EnvironmentalFactors:
OccupationalExposureandCancerRisk
Occupation

Agent

Site of Disease

Dye manufacturers rubber workers


Dyemanufacturers,rubberworkers

Aromatic amines
Aromaticamines

Bladder

Asbestosminingandhandling

Asbestos

Lung,mesothelium

Cadmiumworkers

Cadmium

Prostate

Uraniumminers

Ionisingg radiation

Lungg

Coalgasmanufacturers,asphalters
andothers

Polycyclichydrocarbons

Skin,lung

Farmers,sailors,sportsmen

UVlight

Skin,lip

Hardwood furniture manufacturers


Hardwoodfurnituremanufacturers

Unknown

Nasal sinuses
Nasalsinuses

N.NASSIF

24

EnvironmentalFactors:
CancerRisk
Cigarettesmokingislinkedwithanincreasedriskofthe
following cancer types:
followingcancertypes:
Lungg
Larynx(voicebox)
Oralcavity(mouth,tongue)
Noseandsinuses
Pharynx(throat)
Esophagus
Stomach
N.NASSIF

Pancreas
Cervix
Kidney
Bladder
Ovary
Colorectum
l
Acutemyeloidleukemia
25

NonGeneticFactors:
AgeandCancerRisk

N.NASSIF

26

EnvironmentalFactors:
InternationalVariationinCancerIncidence
*
*

* *

*
*

N.NASSIF

27

GeneticFactors:
InvolvementofGenesinCancerDevelopment
Subsetsofgenesinthegenomehavebeenfound

tobeimportantintheprevention,development
to
be important in the prevention development
andprogressionofcancer
Thesegeneshavebeenfoundtobeeither

f
g,
f
malfunctioning,ornonfunctionalindifferent
tumour types
Categorised
d into3broadcategoriesbasedontheir
b d
b d
h

cellularfunction(s)
N.NASSIF

28

GeneticFactors:
InvolvementofGenesinCancerDevelopment
1. Geneswhoseproteinproductsstimulateorenhance
cell division, growth and viability (i.e. oncogenes)
celldivision,growthandviability(i.e.oncogenes)

2. Geneswhoseproteinproductsdirectlyorindirectly
p
p
y
y
inhibitorpreventcelldivisionorpromotecelldeath
(i.e.tumour suppressorgenes)

3. Geneswhoseproteinproductsareinvolvedinthe
correctionofacquiredmutationsinDNA(i.e.DNA
i
f
i d
i
i
(i
repairgenesandtheirproteinproducts)
N.NASSIF

29

Cl
ClassesofGenesinCancer
fG
i C

Majorclassesofgenesidentified:
Major classes of genes identified:

1. Oncogenes
2. Tumour suppressorgenes
3. DNArepairgenes
p g
N.NASSIF

30

ClassesofCancerGenes
1.TheOncogenes
Oncogenes =growthpromoting(dominantgainof

function)
Normalversionsaretermedprotooncogenesandtheir

mutated counterparts are called oncogenes


mutatedcounterpartsarecalledoncogenes
Proteinproductsofprotooncogenesstimulatecell

divisionand/orinhibitcelldeath
Protooncogenesareusuallygrowthfactors,growth
Proto oncogenes are usually growth factors growth

factorreceptors,signaltransductionmoleculesor
nucleartranscriptionfactors
p
N.NASSIF

31

ClassesofCancerGenes
1.TheOncogenes (cont.)
Mutatedoncogenes canleadtounregulatedcell

division
Cellsareabletogrowintheabsenceofnormal

growthsignals
Examples:
E
l
ras: asignaltransductionmolecule
i l
d i
l l

myc:
y atranscriptionfactor
p
src:aproteintyrosinekinase
N.NASSIF

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Oncogenes IdentifiedinCancer
Oncogene

Function

N.NASSIF

CancerType

33

Theras Oncogene
Ras subfamilyisafamilyofsmall

GTPases involvedincellsignal
t
transduction
d ti
Ras communicatesandtranslates
communicates and translates

signalsfromoutsidethecelltothe
nucleus
Activationofras signalling causescellgrowth,andsurvival
Mutationsofras canpermanentlyactivateitandthiscan

leadtocancer
N.NASSIF

34

MutatedOncogenes CanLeadto
UnregulatedCellGrowth
Geneexpressionwithout
correctsignals
i
l

NormalGrowth

N.NASSIF

35

ClassesofCancerGenes
2.TheTumour SuppressorGenes
TumourSuppressorgenes=antioncogenes (recessive

lossoffunction)
Tumour suppressorgeneproductsacttoinhibitthe

division ofcellsifconditionsofgrowtharenotmet
of cells if conditions of growth are not met
Conditionstriggeringthebrakesofcelldivision
gg
g

include:DNAdamage,lackofgrowthfactors
Itistheabsence,orlossoffunctionofatumour
It is the absence or loss of function of a tumour

suppressorgeneproductthatleadstotumour
formation
N.NASSIF

36

ClassesofCancerGenes
2.TheTumour SuppressorGenes
Someexamplesinclude:

p53(TP53):atranscriptionfactorregulatingcell
division
Rb: controlscelldivision
APC: controlsavailabilityofatranscriptionfactor
BRCA: InvolvedinrepairofdamagedDNA
N.NASSIF

37

Tumour
Suppressor
Genes
Identifiedin
Cancer

Tumour
Suppressor

Function

N.NASSIF

CancerType

38

GenesinCancer
Thep53Tumour Suppressor

p53(theguardianofthegenome)
functionstopreventcelldivisionofcellswith

damagedDNA
mutationsofp53
mutations of p53 leadtolossoffunction
lead to loss of function
p53mutationsobservedinapproximately50%of
p
f

allcancers(ofvarioustypes)

N.NASSIF

39

GenesinCancer
Thep53Tumour Suppressor
p53isamultifunctionalprotein
which plays a role in:
whichplaysarolein:
modulatinggenetranscription
modulating gene transcription
policingcellcyclecheckpoints
activatingapoptosis
g p p
controllingDNAreplication
andrepair
maintaininggenomicstability
respondingtogeneticinsults
N.NASSIF

40

GenesinCancer
TumourSpecificTumourSuppressorGenes
Breastcancer(inheritedform)

BRCA1andBRCA2genes
Mutatedinahighpercentageofearlyonsetinherited
breast and ovarian cancer cases
breastandovariancancercases
Mutationsincreaseriskofdevelopingbreastand
ovariancancer

N.NASSIF

41

Cancer
Development
isaMultistep
Process

Normal Cell

First Mutation

Second Mutation

Mutations
are
acquired
att every
step

Third Mutation
Fourth or
later
Mutation

Malignant Cell
N.NASSIF

42

Th R l fPTEN Gene
TheRoleofPTEN
G

MutationsinSporadic
ColorectalCancer

ColorectalCancer
l
l
Cancerofthecolon

and/or rectum
and/orrectum
Thirdmostcommonly

diagnosedcancerinmen
(8%),andthesecondin
women(10%)
Thirdmostcommoncause
Third most common cause

ofcancerdeath(9%)
N.NASSIF

44

GeneticChangesIdentifiedin
ColorectalCancer

N.NASSIF

45

GenesInvolvedinColonCancer
Development

N.NASSIF

46

Th PTEN TumourSuppressorGene
ThePTEN
T
S
G

PTEN: Phosphataseandtensin homologdeletedon

chromosome 10
chromosome10
PTEN geneislocatedatonchromosome10q23.3
gene is located at on chromosome 10q23.3
Thegeneproductisaproteinandlipidphosphatase
g
p
p
p p p
Regulatescellgrowth,survival,celladhesion,cell
g
g

differentiationanddeath(apoptosis)

N.NASSIF

47

PTENGeneandProteinStructure
EncodesNterminalDomain

EncodesCterminalDomain

EncodesPhosphataseDomain

N.NASSIF

48

CellularFunctionsofPTEN
ThemajorsubstrateofPTENisthesecondmessengerlipid
Th
j
b
f PTEN i h
d
li id

PIP3(phosphoinositol3,4,5triphosphate)
PTENdephosphorylatesPIP3toPIP2 (keepsPIP3levelslow)
LossofPTENleadstoincreasedlevelsofPIP3and

consequentactivationofAkt/PKBandthePI3Kcell
survival/anti apoptotic pathway
survival/antiapoptoticpathway
Subsequentprotectionfromapoptoticstimuliandincreased
q
p
p p

cellgrowthandsurvival
PTENisthereforeanegativeregulatorofcellsurvival
PTEN i th f
ti
l t
f ll
i l
N.NASSIF

49

TheCellularRole(s)ofPTEN
MultipleRolesofPTENinTumourSuppression
(PIP 3)
(PIP3)

(
(PIP2)
2)

N.NASSIF

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ProjectResults
Pairedtumourandcorrespondingnormaltissue

samplesfromsporadicCRCpatientswerescreenedfor
PTEN
PTENgenemutationsanddeletions
t ti
d d l ti
Mutationsweredetectedinapproximately20%of
Mutations were detected in approximately 20% of

primarysporadiccolorectaltumours
Overall,alterations(mutationsanddeletions)ofthe

PTEN genewerepresentin40%ofprimarysporadic
colorectaltumours

N.NASSIF

51

Detection
ofPTEN
Gene
Mutations
inSporadic
Colorectal
Cancer

NormalDNA

TumourDNA

NormalDNA

TumourDNA

N.NASSIF

Patient 1
Exon 5
A>G

Patient 2
Exon 5
G>T

52

PTEN MutationsDetectedinSporadicCRC
E150Q
D153N

K62R

K125X D153Y
K125E

Y65C
E1

E2

E3

E4

E5

E6

319X
N323K

V217A
E7

E8

E9

G129E
C124S

N.NASSIF

53

Project:
Evaluating the Functional
Significance of the Detected
PTEN Mutations

FunctionalAnalysisoftheDetected
PTEN GeneMutations
Wildtype(WT)PTENhasbeencloned,andeachofthe

mutantPTENshavebeenengineeredinmammalian
mutant
PTENs have been engineered in mammalian
expressionvectors
TheWTandmutantPTEN hasbeenexpressedinselected

cellslines (e.g.U87MGglioblastomacells)aftertransfection
DeterminedeffectofWTPTEN andeachPTEN mutanton:

(a)cellproliferation(yourprojectaim)and
(b)PTENsubcellulardistribution
N.NASSIF

55

TheEffectof
PTEN
Mutationson
U87MGCell
Proliferation

*
*

WTPTENisable
tocausea
slowing of cell
slowingofcell
proliferation

AIMofPROJECT:
Todetermine
the effect of
theeffectof
PTEN mutation
oncell
proliferation
N.NASSIF

56

EffectofPTENMutationonPTEN
S b ll l Di t ib ti
SubcellularDistribution
WTPTEN
WT PTEN
expressionis
evenly
di t ib t d
distributed
betweenthe
cytoplasmand
y p
thenucleusin
U87MGcells

SomePTEN
mutantshave
increased
cytoplasmic
localisation
N.NASSIF

57

Conclusions
WTPTENisabletobringaboutaslowingofcell

proliferation ofculturedU87MGcells
of cultured U87MG cells
IsMUTANTPTENabletoslowcellproliferationas
Is MUTANT PTEN able to slow cell proliferation as

effectivelyastheWTPTEN?
(Youwillfindthisoutaspartofyourpractical
project!)

N.NASSIF

58

UsefulWebsitesandContentLinks
Cellcycleanimation
C ll
l
i
i

http://www.cellsalive.com/cell_cycle.htm
Howthecellcycle

workshttp://www.youtube.com/watch?v=diXoAgH3LMk
Cellcyclecheckpointsandtheroleofp53

http://www.youtube.com/watch?v=1EB8q9aR8Hk&feature=related
Howcancerdevelops

http://www.youtube.com/watch?v=j_wRpa2b5XI&feature=related
LiteraturearticledescribingthediscoveryofPTENmutationsin

colorectalcancer(downloadablepdf fileavailableatthissite)
h //
http://www.nature.com/onc/journal/v23/n2/full/1207059a.html
/ /
l/ / /f ll/
h l
N.NASSIF

59

R f
References
NelsonandCox(2008)Lehninger PrinciplesofBiochemistry,6th

Edition
G.Karp(2008)CellandMolecularBiology:Conceptsand

Experiments.5
Experiments
5th Edition
MH.Lodish etal.MolecularCellBiology
ReviewarticlecoveringPTENanditsroleincancer(downloadable

pdf fileavailableatthissite)
file available at this site)
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2710138/?tool=p
ubmed
N.NASSIF

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