CELL CYCLE CONTROL CELL CYCLE CONTROL ANDAND

CANCERCANCER

Prof T Prof T RangaRanga RaoRao MDMDBiochemistry DepartmentBiochemistry DepartmentBiochemistry DepartmentGunturGunturGuntur Medical Medical Medical College,GUNTURCollege,GUNTURCollege,GUNTUR

The steps of cellular growth The steps of cellular growth and division that and division that

we observe today we observe today in our own cells is there in in our own cells is there in the original eukaryotic cells the original eukaryotic cells

since time immemorial since time immemorial

OutlineOutline

Cell cycle controlCell cycle controlBiochemicalBiochemical

CancerCancerProgressing from biochemical to genetic Progressing from biochemical to genetic

to clinicalto clinical

Three biologists share 2001’s Nobel Prize in Physiology or Medicine for helping to unlock the secrets of the cell cycle,

Leland H. Hartwell,USA

Paul M. Nurse,UK

R. Timothy Hunt,UK

Main phases of a cell cycleMain phases of a cell cycle

M

G2

S

G1One One chromatidchromatidper chromosomeper chromosome

Two chromatids Two chromatids per chromosomeper chromosome

One One chromatidchromatidper chromosomeper chromosome

The alternative to cell cyclingThe alternative to cell cycling

M

G2

S

G1

G0

GG00 cells are not dividing, cells are not dividing, but instead will be but instead will be

differentiateddifferentiated

The major cell cycle checkpointsThe major cell cycle checkpoints

The GThe G11 checkpoint: checkpoint: Is the cell large enough?Is the cell large enough?

The GThe G22 checkpoint: checkpoint: Has all DNA Has all DNA

completed division?completed division?

M

G2

S

G1

The M checkpoint: The M checkpoint: Are chromosomes Are chromosomes

aligned in the equatorial aligned in the equatorial plane?plane?

All three All three checkpoints: checkpoints:

Is the environment Is the environment favorable? favorable?

The integration at The integration at CyclinCyclindependent dependent KinaseKinase (CDK)(CDK)

PP

Has this phosphate been added

Has this phosphate been removed

Is cyclin present

CDK activity is CDK activity is ““ONON”” only if the answer is only if the answer is ““yesyes”” to all questions to all questions

CDKCDK--molecules and molecules and cyclinscyclins drive the drive the cell from one phase to the next. cell from one phase to the next.

The CDKThe CDK--molecules can be molecules can be compared with an engine and the compared with an engine and the cyclinscyclins with a gear box controlling with a gear box controlling whether the engine will run in the whether the engine will run in the

idling state or drive the cell forward in idling state or drive the cell forward in the cell cyclethe cell cycle..

The importance of The importance of CyclinsCyclins

•• Different Different CyclinsCyclins increase in increase in abundance at different stages abundance at different stages of the cell cycle of the cell cycle

•• Each Each CyclinCyclin Dependent Dependent KinaseKinase (CDK) (CDK) interacts with one or two specific interacts with one or two specific Cyclin(sCyclin(s) )

CANCERCANCER

GrowthGrowthInvasionInvasionSpread

90 % sporadic90 % sporadic

10% familial10% familialSpread

Cancer: Cancer: General General

Etiology and Etiology and PathogenesisPathogenesis

Cancer • Cells in culture and in

vivo exhibit contact-inhibition through feedback mechanism.

• Cancer cells lack contact inhibition feedback mechanisms. Clumps or foci develop.

Specific Cellular Functions in Specific Cellular Functions in Cancer: Genetic AlterationsCancer: Genetic Alterations

DNA RepairDNA Repair

Tumor Suppressor Tumor Suppressor GenesGenes OncogenesOncogenes

Interstitial DeletionInterstitial DeletionInactivating MutationInactivating MutationHypermethylationHypermethylation

Gene AmplificationGene AmplificationGene Gene OverexpressionOverexpression

Activating MutationActivating Mutation

Genetic InstabilityGenetic Instability

CANCERCANCER

Pathways leading to cancerPathways leading to cancer

DNA RepairOncogenesOncogenes

ActivationActivation

Tumor Suppressor Tumor Suppressor GenesGenes

InactivationInactivationProliferation

Alterations of Specific Cellular Alterations of Specific Cellular Functions in CancerFunctions in Cancer

CANCERCANCER

DefinitionsDefinitions

•• A tumor suppressor gene has a normal A tumor suppressor gene has a normal function in the cell, but can be inactivated. function in the cell, but can be inactivated. The normal function is putting a brake on The normal function is putting a brake on the cell cycle in a regulated fashion.the cell cycle in a regulated fashion.

•• A protoA proto--oncogene has a normal function in oncogene has a normal function in the cell, but can be activated into an the cell, but can be activated into an oncogene. The normal function is oncogene. The normal function is to promote cell division in a regulated fashion.to promote cell division in a regulated fashion.

Point mutations are often the initial Point mutations are often the initial events in cancer developmentevents in cancer development

•• Point mutations lead to changes in Point mutations lead to changes in amino acid sequenceamino acid sequence

•• Point mutations occur as result of Point mutations occur as result of DNA replication or (repair of) DNA DNA replication or (repair of) DNA damagedamage

•• Especially DNA polymerases Especially DNA polymerases repairing DNA damage are repairing DNA damage are error proneerror prone

Some of the factors involved in GSome of the factors involved in G11checkpoint controlcheckpoint control

p16Apoptosis

M

G2

S

G1

p53

E2F pRB

p21 ??CDK4

CyclinD

CDKCDK--ActivationActivation&Cancer&Cancer

PapillomaPapilloma virusvirus--CancerCancer

PapillomaPapilloma virus virus ---- interaction with interaction with p53 and p53 and pRBpRB

M

G2

S

G1

p53

p16

E2F pRB

Apoptosis p21 ?

?CDK4

E6

E7

pRB inactivation relieves E2F of inhibition

CyclinD

p53 degradation

Papillomavirus proteins

Tumor suppressors include p53, Tumor suppressors include p53, p16, p21, and p16, p21, and pRBpRB

p16Apoptosis

M

G2

S

G1

p53

E2F pRB

p21 ??CDK4

CyclinD

The role of p53The role of p53

p16Apoptosis

p53

M

G2

S

G1

E2F pRB

p21 ??CDK4

CyclinD

P53 P53 and the and the

cell cyclecell cycle

P53 arrests the cell cycle primarily by P53 arrests the cell cycle primarily by upregulatingupregulating p21 p21

which inactivates CDK/which inactivates CDK/cyclincyclin..

P53 can also activate apoptosisP53 can also activate apoptosis

P21 is a CDK inhibitorP21 is a CDK inhibitor

p53 p53 -- the most commonly the most commonly mutated gene in cancersmutated gene in cancers

•• Mutations in p53 almost always Mutations in p53 almost always somatic, very rarely inheritedsomatic, very rarely inherited

•• If DNA is damaged, p53 gene If DNA is damaged, p53 gene product stops cell division through product stops cell division through the the pRBpRB pathwaypathway

•• If DNA and the If DNA and the pRBpRB gene is damaged, gene is damaged, p53 will induce apoptosis p53 will induce apoptosis (programmed cell death)(programmed cell death)

p53 p53 -- continuedcontinued

•• p53 mutated cell can therefore p53 mutated cell can therefore proceed through Sproceed through S--phase with phase with damaged DNAdamaged DNA

•• Radiation treatment of tumors Radiation treatment of tumors damages DNA.damages DNA.

•• Tumor cells in which p53 is mutated Tumor cells in which p53 is mutated survives Radiation better giving the survives Radiation better giving the patient a poorer prognosis. patient a poorer prognosis.

p53 p53 -- continuedcontinued

•• p53 is most often classified as a tumor p53 is most often classified as a tumor suppressor genesuppressor gene

•• Unlike other tumor suppressors, p53 Unlike other tumor suppressors, p53 has been found with dominant negative has been found with dominant negative mutations. In these instances, one mutations. In these instances, one mutation in one of the alleles is enough mutation in one of the alleles is enough to produce a phenotypeto produce a phenotype

Tumor suppressor genesTumor suppressor genes

•• Normally, one functional allele in a cell Normally, one functional allele in a cell is enough to have full functionis enough to have full function

•• Therefore, Therefore, KnudsonsKnudsons twotwo--hit hypothesis hit hypothesis on inactivation of the second alleleon inactivation of the second allele

KnudsonsKnudsons twotwo--hit hypothesishit hypothesis

•• KnudsonsKnudsons twotwo--hit hypothesis relates to tumor hit hypothesis relates to tumor suppressor genes.suppressor genes.

•• It states that in some cancers, like the It states that in some cancers, like the retinoblastoma, two hits are necessary before retinoblastoma, two hits are necessary before the cells start uncontrolled divisions. In the the cells start uncontrolled divisions. In the inherited cases, hit number one is found in all inherited cases, hit number one is found in all cells. In sporadic cases, hit number one is cells. In sporadic cases, hit number one is found in one or few somatic cell, and hit found in one or few somatic cell, and hit number two happens at least in one cell.number two happens at least in one cell.

KnudsonsKnudsons twotwo--hit hypothesis (2)hit hypothesis (2)•• Familial cancer shows phenotype only if Familial cancer shows phenotype only if

a second hit does happen. a second hit does happen. •• Well known familial cancers show Well known familial cancers show

dominant inheritance with penetrance of dominant inheritance with penetrance of approx. 60approx. 60--90%.90%.

•• How can the penetrance be that high?How can the penetrance be that high?•• High penetrance means that loss of the High penetrance means that loss of the

second allele happens with a high probability second allele happens with a high probability in one or few of the many cells in the body.in one or few of the many cells in the body.

Tumor suppressors in summaryTumor suppressors in summary

•• TS gene products acts as brakes on the cell TS gene products acts as brakes on the cell cycle. Loss of both alleles leads to loss of the cycle. Loss of both alleles leads to loss of the brake action, therefore leading to increased brake action, therefore leading to increased cell divisions.cell divisions.

•• TS genes can also be involved in DNA repair TS genes can also be involved in DNA repair and apoptosis, with loss of both alleles leading and apoptosis, with loss of both alleles leading to increased cell divisions.to increased cell divisions.

Where Where mitogensmitogens enter the pictureenter the picture

p16Apoptosis

M

G2

S

G1

p53

E2F pRB

p21 ??CDK4

CyclinD

Mitogens

Examples of Examples of mitogensmitogens

•• InsulinInsulin

•• Epidermal growth factor (EGF), Epidermal growth factor (EGF), platelet derived GF (PDGF), platelet derived GF (PDGF), fibroblast GF (FGF), fibroblast GF (FGF), insulininsulin--like GF (IGF), etc.like GF (IGF), etc.

Response of the insulin receptor kinase (IRK) Response of the insulin receptor kinase (IRK) to to ligandligand bindingbinding

•• HeterotetramerHeterotetramer (2a, 2b)(2a, 2b)

•• Insulin binding leads to Insulin binding leads to change in structure (different change in structure (different from other from other RTKsRTKs))

•• Conformation change Conformation change activates bactivates b--subunit TK subunit TK activity activity

•• b subunit b subunit phosphorylatesphosphorylates TyrTyrresidues on cytoplasmic residues on cytoplasmic domains as well as domains as well as downstream substrates (IRS)downstream substrates (IRS)

ThreeThree--dimensional structures of the insulin dimensional structures of the insulin receptor tyrosine kinase (IRK)receptor tyrosine kinase (IRK)

IRK conformational change upon IRK conformational change upon activation loop phosphorylation. activation loop phosphorylation. The NThe N--terminal lobe of IRK is terminal lobe of IRK is colored white and the Ccolored white and the C--terminal terminal lobe is colored dark grey. lobe is colored dark grey. The The activation loop (green) contains activation loop (green) contains autophosphorylationautophosphorylation sitessites Y1158, Y1158, Y1162 and Y1163, and the Y1162 and Y1163, and the catalytic loop (orange) contains catalytic loop (orange) contains the putative catalytic base, the putative catalytic base, D1132.D1132. Also shown are the Also shown are the unbound/bound ATP analog and unbound/bound ATP analog and tyrosinetyrosine--containing substrate containing substrate peptide (pink).peptide (pink). [Hubbard, EMBO J. 16, 5572 (1997)]

Once Once TyrTyr--PhosphorylatedPhosphorylated, the IRK activity , the IRK activity trigerrstrigerrs a number of signaling pathwaysa number of signaling pathways

•• PhosphatidylinositolPhosphatidylinositol 33--hydroxy kinase, makes hydroxy kinase, makes PIP2,PIP3PIP2,PIP3

•• Grb2, Grb2, SosSos, activates , activates RasRas

•• Activation of PIActivation of PI--PLCPLC

Fibroblast Growth Factor Fibroblast Growth Factor Receptor Tyrosine KinaseReceptor Tyrosine Kinase

•• The fibroblast growth factor The fibroblast growth factor receptor (FGFR) family have receptor (FGFR) family have been linked widely to the been linked widely to the development of cancer and development of cancer and disease. disease.

•• DimericDimeric assembly of 2 assembly of 2 FGF2:FGFR1 complexes.FGF2:FGFR1 complexes.FGF2 is colored orange,FGF2 is colored orange, IgIg--like domain 2 of FGFR1 is like domain 2 of FGFR1 is colored greencolored green, and , and IgIg--like like domain 3 of FGFR1 is domain 3 of FGFR1 is colored cyancolored cyan.. [Plotnikov et al.,Cell 98, 641 (1999)]

Growth factor signalingGrowth factor signaling

•• Binding to external parts of receptorsBinding to external parts of receptors•• activation of second messengers by activation of second messengers by

eithereither–– integral receptor integral receptor kinasekinase–– dissociation of dissociation of trimerictrimeric GG--proteinsproteins–– activation of other associated enzyme activation of other associated enzyme

(mostly a (mostly a kinasekinase) ) •• Further levels in the cascadeFurther levels in the cascade

Growth factor signaling (2)Growth factor signaling (2)

•• Activation of a transcription factor and Activation of a transcription factor and subsequent changes in gene regulation is the subsequent changes in gene regulation is the most important endmost important end--point of signaling.point of signaling.

•• Steroid hormones reach this level directly, as Steroid hormones reach this level directly, as they bind to receptors that themselves are they bind to receptors that themselves are transcription factors.transcription factors.

Any gene encoding any of these proteins have Any gene encoding any of these proteins have the potential for being a the potential for being a protooncogeneprotooncogene

ProtoProto--oncogenes oncogenes vsvs OncogenesOncogenes

ProtoProto--oncogenes are the normal cellular gene from oncogenes are the normal cellular gene from which the viral oncogenes are derived.which the viral oncogenes are derived.

Mechanism of Mechanism of oncogeniconcogenic activation:activation:

Mutation and/or OverexpressionMutation and/or Overexpression

Slow transforming retroviruses demonstrate that Slow transforming retroviruses demonstrate that overexpression of protooverexpression of proto--oncogenes also plays a role in oncogenes also plays a role in retroviral retroviral oncogenesisoncogenesis. .

Mechanisms of oncogene activationMechanisms of oncogene activation

Oncogenes of Acutely Transforming Oncogenes of Acutely Transforming RetroviriRetrovirisrcsrc RousRous sarcoma virussarcoma virus ChickenChickenmycmyc Avian Avian myelocytomatosismyelocytomatosis virus virus ChickenChickenerberb A, A, erberb B B Avian Avian erythroblastosiserythroblastosis virus virus ChickenChickenmybmyb Avian Avian myeloblastosismyeloblastosis virus virus ChickenChickenetsets Avian Avian erythroblastosiserythroblastosis virusvirus ChickenChickenrelrel Avian Avian reticuloendotheliosisreticuloendotheliosis virus virus TurkeyTurkeyHH--rasras Harvey rat sarcoma virus Harvey rat sarcoma virus RatRatKK--rasras Kirsten murine sarcoma virusKirsten murine sarcoma virus MouseMouseablabl AbelsonAbelson murine leukemia virusmurine leukemia virus MouseMouserafraf Murine sarcoma virusMurine sarcoma virus MouseMousefosfos Mouse Mouse osteosarcomaosteosarcoma virusvirus MouseMousefmsfms Feline sarcoma virus CatFeline sarcoma virus Catfesfes Feline sarcoma virusFeline sarcoma virus CatCatsis sis Simian sarcoma virus Simian sarcoma virus MonkeyMonkey

Types of Types of proteins proteins

encodes by encodes by oncogenesoncogenes

The classical pathwayThe classical pathwaygrowth factor receptor > growth factor receptor >

Grb2 > Grb2 > SOS > SOS > RAS > RAS >

MAPKKK (RAF) > MAPKKK (RAF) > MAPKK (MEK) > MAPKK (MEK) >

MAPK MAPK

The RAS proteinThe RAS protein

•• RAS binds either GTP or GDPRAS binds either GTP or GDP•• RAS containing GTP is active, RAS containing GTP is active,

RASRAS--GDP is inactiveGDP is inactive•• RAS can hydrolyze GTP to RAS can hydrolyze GTP to

GDP when triggered by GDP when triggered by GTPaseGTPase--Activating Proteins Activating Proteins ((GAPsGAPs))

•• RAS exchanges GDP for GTP RAS exchanges GDP for GTP when interacting with a GDPwhen interacting with a GDP--release protein (GNRP)release protein (GNRP)

The RAS exampleThe RAS example

•• Several Several mitogenicmitogenic proteins (growth factors) proteins (growth factors) bind to receptors with tyrosine bind to receptors with tyrosine kinasekinase activity, activity, thereby causing receptor thereby causing receptor autophosphorylationautophosphorylation

•• The The phosphophospho--tyrosine gets bound by the tyrosine gets bound by the adapter GRB2 which binds GTPadapter GRB2 which binds GTP--exchange exchange protein SOS. protein SOS.

•• SOS thereafter is active in exchanging GDP to SOS thereafter is active in exchanging GDP to GTP on RASGTP on RAS

The RAS example (2)The RAS example (2)

•• Active GTPActive GTP--RAS binds temporarily to RAS binds temporarily to RAF(MAPKKK), releasing it from RAF(MAPKKK), releasing it from inhibitory proteins.inhibitory proteins.

•• Free RAF protein binds to and Free RAF protein binds to and phosphorylatesphosphorylates MEK (MAPKK).MEK (MAPKK).

•• PP--MEK binds to and MEK binds to and phosphorylatesphosphorylatesMAP MAP KinaseKinase on both tyrosine and on both tyrosine and threoninethreonine thereby activating it thereby activating it

WordsWords

•• MEK is MAPMEK is MAP--ERK ERK KinaseKinase,,it it phospholylatesphospholylates both tyrosine and serineboth tyrosine and serine

•• MAP is MAP is MitogenMitogen Activated ProteinActivated Protein

•• ERK is ERK is ExtracellularExtracellular signalsignal--Regulated Regulated KinasesKinases

MAP MAP KinaseKinase

•• PP--MAP MAP KinaseKinase phosphorylatesphosphorylates multiple multiple targets differing among cell types. targets differing among cell types.

•• The targets include transcription factors The targets include transcription factors such as SRF, indirectly increasing the such as SRF, indirectly increasing the levels of FOS and JUN (together = APlevels of FOS and JUN (together = AP--1).1).

•• APAP--1 increases 1 increases CyclinCyclin--D production.D production.

Parallel pathwaysParallel pathways•• A number of parallel pathways exist. A number of parallel pathways exist.

•• Several of these have a Jun NSeveral of these have a Jun N--terminal terminal KinaseKinase(JNK) as the final activated (JNK) as the final activated kinasekinase

•• Each of these Each of these phosphorylatesphosphorylates isoformsisoforms of Jun, of Jun, the partner of the partner of FosFos in APin AP--1. P1. P--Jun is more Jun is more stable than native Jun, thereby increasing the stable than native Jun, thereby increasing the effective concentration of this transcription effective concentration of this transcription factor.factor.

OncogenesOncogenes –– RAS ProteinsRAS Proteins•• RAS is a protein binding either GDP or RAS is a protein binding either GDP or

GTP, hydrolyzing the latter to the GTP, hydrolyzing the latter to the former when induced by interaction with former when induced by interaction with a a GTPaseGTPase Activating Activating Protein(GAPProtein(GAP).).

•• Mutations in RAS often impair the Mutations in RAS often impair the ability to hydrolyze GTP, freezing ability to hydrolyze GTP, freezing RAS in the active state.RAS in the active state.

•• HRAS, KRAS and NRAS are three HRAS, KRAS and NRAS are three examples of RAS genes.examples of RAS genes.

OncogenesOncogenes –– RAS Proteins(2)RAS Proteins(2)

•• HRAS involved in carcinoma of colon, lung, HRAS involved in carcinoma of colon, lung, pancreas and others.pancreas and others.

•• KRAS involved in melanoma, thyroid KRAS involved in melanoma, thyroid carcinoma, acute carcinoma, acute myeloblasticmyeloblastic leukemia leukemia (AML), etc..(AML), etc..

•• NRAS involved in NRAS involved in myelomamyeloma and leukemia, and and leukemia, and in in neuroblastomaneuroblastoma. Less commonly activated . Less commonly activated than KRAS in than KRAS in myelomamyeloma and leukemia cancers.and leukemia cancers.

OncogenesOncogenes in summaryin summary

•• OncogenesOncogenes are very rarely inheritedare very rarely inherited•• OncogenesOncogenes are the activated, cancerare the activated, cancer--

causing forms of protocausing forms of proto--oncogenesoncogenes•• Any gene performing one of the steps Any gene performing one of the steps

from growth factor binding to gene from growth factor binding to gene transcription that leads to cell division transcription that leads to cell division could become an could become an oncogeneoncogene

Clinical observationsClinical observations•• Inherited cancers occur earlier in life than Inherited cancers occur earlier in life than

sporadic cancerssporadic cancers•• Inherited cancers show more foci Inherited cancers show more foci

(independent (independent neoplasmsneoplasms) than sporadic ) than sporadic cancerscancers–– E.g., breast cancer with bilateral occurrence E.g., breast cancer with bilateral occurrence

indicates an inherited caseindicates an inherited case•• More than one case of a rare cancer in a More than one case of a rare cancer in a

family indicates segregation of a familial family indicates segregation of a familial cancer mutationcancer mutation

Knowledge of cancer mutations: Knowledge of cancer mutations: any help?any help?

•• Yes! Recent news have claimed that cheap, Yes! Recent news have claimed that cheap, fast genotyping of patient tumors fast genotyping of patient tumors combinedcombinedwith knowledge of which medicine work best with knowledge of which medicine work best for which combination of mutations, will for which combination of mutations, will become the next big step forward in cancer become the next big step forward in cancer treatment. treatment.

•• The cost of such a tailored treatment will be The cost of such a tailored treatment will be even higher than current costs.even higher than current costs.

SummarySummary

•• Tumor suppressors are inactivatedTumor suppressors are inactivated–– Both alleles are inactivated in the cancer cellBoth alleles are inactivated in the cancer cell

•• OncogenesOncogenes are activatedare activated•• Inherited cancer involves inheritance of Inherited cancer involves inheritance of

one mutated tumor suppressor alleleone mutated tumor suppressor allele•• Inherited cancer shows dominant Inherited cancer shows dominant

inheritance with reduced inheritance with reduced penetrancepenetrance

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