E6 2006 2007: “Molecular Biology ofE6 2006-2007: “Molecular Biology of Cancer”

DEREGULATION OF CELL CYCLE CONTROL IN ONCOGENESIS

D. KardassisDivision of Basic Sciences

University of Crete Medical School andyIMBB-FORTH

Literature* Schwartz, G.K. And Shah, M.A. (2005) Targeting the cell cycle: A new approach to cancer therapy. J. Clin. Oncology 23:9408-9421 py gy

* Malumbres, M. and Barbacid, M. (2001) To cycle or not to cycle: a critical decision in cancer. Nature Rev Cancer 1:222-231.

* Vermeulen, K., Van Bockstaele, D.R. and Berneman, Z.N. (2003) The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif.36,131–14936,131 149Weiss, R.H. (2003) p21WAF1/Cip1 as a therapeutic target in breast and other cancers. Cancer Cell 4, 425-429Vid l A d K ff A (2000) C ll l i hibit th f ili it d bVidal, A. and Koff, A. (2000) Cell cycle inhibitors: three families united by a common cause. Gene 247:1-15.Sherr, C.J. (2000) The Pezcoller Lecture: Cancer cell cycles revisited. Cancer Res. 60:3689-3695Coqueret, O. (2002) Linking cyclins to transcriptional control. Gene 299:35-55.

* Required reading

Topics to be discussedTopics to be discussedC ll l h d l l lCell cycle phases and molecular playersActivation and degradation of cyclinsMajor CDK regulatory mechanismsStructure, function and regulation of cdk inhibitorsRegulation of the cell cycle by subcellular localization of critical componentsSignaling pathways regulating the cell cycle: The paradigm of TGFβCheckpoints in the cell cycleCell cycle regulators and cancerCDK regulation and opportunities for therapeutic intervention

Summary of cell cycle and its regulationSummary of cell cycle and its regulationMitogenic, growth

signals

The Restriction point (R)The Restriction point (R)

The key molecular players in cellThe key molecular players in cell cycle controly

Cyclin-dependent kinases (cdks) CyclinsCDK substrates (pocket family: Rb, p107,p130) (p y , p ,p )Regulators of CDKs (CAK, wee, cdc25)Cyclin dependent kinase inhibitors (CKIs)Cyclin-dependent kinase inhibitors (CKIs)Regulators of CKIs

Summary of cell cycle and its regulationSummary of cell cycle and its regulationMitogenic, growth

signals

Coordination between cellCoordination between cellCoordination between cell Coordination between cell cycle progression and cell cycle progression and cell

growth through the growth through the g gg gregulation of regulation of cyclincyclin D D

expression.expression.

Inhibition ofubiquitination

translationalregulation (mRNA export)export)

Degradation of cyclinsDegradation of cyclins

C li A d B t i D t tiCyclins A and B contain a Destruction Box and cyclins D and E contain a PEST sequence [segment rich inPEST sequence [segment rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues](S) and threonine (T) residues].

These are protein sequences requiredThese are protein sequences required for efficient ubiquitin-mediated cyclin proteolysis at the end of a cell cycleproteolysis at the end of a cell cycle phase

The ubiquitination system for protein q y pdegradation

Ubiquitination of cyclinsUbiquitination of cyclins

The Cyclin-CDK complexes

Major CDK regulatory mechanisms

Regulation of CDK activity by phosphorylation-dephosphorylationdephosphorylation

DNA damage

Phosphorylation by CAK (Cdk Activating Kinase) which consists of Cyclin H and P P

Cyclin A/B

inactive

Chk1/2

ATM,ATR

Kinase) which consists of Cyclin H and cdk7: Conform. changes, enhance binding of cyclins

CDK1P P

PP

Chk1/2

Phosphorylation by Wee/Myt1 dual specificity kinases at consecutive Thr-Tyr residues: Inactivation of cdk

Wee1/Myt1

Cyclin A/B

cdc25 PP

residues: Inactivation of cdk

De-phosphorylation by cdc25 dual specificity CDK1

P

tip p y y p y

phosphatases at consecutive Thr-Tyr residues: Re-activation of cdk Cyclin H

CDK7

active

Cyclin-dependent kinase inhibitorsCyclin dependent kinase inhibitors

Regulation of CDK activity by CKIs

Domain structure of the Cip/Kipsp p

Cip/Kip proteins regulate cyclin–CDK complexes d h i i iand their activity

Ci /Ki   t i   t   Cip/Kip proteins act as assembly factors for cycD/CDK complexes in the cytoplasm (in complexes in the cytoplasm (in contrast to INK4 CKIs which dissociate cyc/CDK complexes)

Cip/Kip proteins enhance the p/ p pnuclear import of cycD/CDK complexes

Low Cip/Kip levels

High Cip/Kip levels

Mechanism of inhibition of CDK activity by Mechanism of inhibition of CDK activity by CiCi /Ki i/Ki iCiCip/Kip proteinsp/Kip proteins

p21 is an essential mediator of the tumor suppressor activity of p53suppressor activity of p53

The role of transcription factor Sp1 in the regulation of the human p21 The role of transcription factor Sp1 in the regulation of the human p21 WAF1/Cip1WAF1/Cip1 gene by tumor suppressor pathwaysgene by tumor suppressor pathwaysg y pp p yg y pp p y

UV, drugs

DNA damage

p21mycMithramycin A

Sp1

Sp1

Sp1

Sp1

p21Miz1p53 p53 human p21 promoter-2100

Koutsodontis et al, JBC, 2001; Koutsodontis et al, Biochemistry, 2002; Koutsodontis and Kardassis, Oncogene, 2004

CytoplasmicCytoplasmic CipCip/Kip /Kip relocalizationrelocalization and cell and cell transformationtransformation

p27 cytoplasmic localization is found in 40% of breast cancer cells or up to 35% of colon cancer cells and correlates with poor survival rates up to 35% of colon cancer cells and correlates with poor survival rates in Barrett’s associated adenocarcinoma. 

By contrast, nuclear expression of p27 in cancer cells gives a more favorable prognosis.p g

What are the consequences of Cip/Kip relocalizationWhat are the consequences of Cip/Kip relocalizationin tumor cells?

Nuclear localization of Cip/Kip proteins enhances their cytostatic propertiesp p

The cytoplasmic distribution of Cip/Kips is utilized by tumour cells for The cytoplasmic distribution of Cip/Kips is utilized by tumour cells for their advantage i.e.

Inhibition of apoptosisp pIncreased cytoskeletal reorganization (cell migration)

Normal cells Breast cancer cells

The role of p21/WAF1 in normal and cancer cellsNormal cells Breast cancer cells

Si li th l ti th llSignaling pathways regulating the cell cycle: The example of TGFβcycle: The example of TGFβ

Genes that are up- or down-l t d b TGF β i ith li l llregulated by TGF-β in epithelial cells

Reference: Massague, J. and Gomis (2006) FEBS Lett. 580, 2811-2820

The cytostatic program of TGFβ

Primary Induction of

TGFβ Receptors Smad3/4FoxO, Sp1,

E2F4,5p107 ATF3

repression a repressor

p21Cip1

INK4bc-myc Id1

, p ,C/EBPβ

p107,C/EBPβ

ATF3

Miz1

p15INK4by Id1

G1

CDKsSM Cell cycle

G2

M

Mutations that disrupt the cytostatic program G2Mutations that disrupt the cytostatic programFoxO: Mutated in glioblastomasC/EBPβ: mutated in breast cancer

Regulation of cell cycle progression by the intracellular localization of cell cycle regulatory proteinsy g y p

During DNA damage, or prior to prophase cyclin B and cdc25prophase, cyclin B and cdc25 phosphatase are exported from the nucleus by 14-3-3σ

The Wee and Myt1 CDK inactivating kinases are located in th l d G l i ti lthe nucleus and Golgi respectively and protect cells from premature mitosis

Regulation of G1 and the G1/S transitionRegulation of G1 and the G1/S transition

Animal studiesModels of redundant genetic relationships

between cell-cycle inhibitors.Pure redundancy

Absence of severe developmental defects in

y

most of the KO mouse strains (of CKIs or pocket proteins) suggests:

Compens. redundancyRedundant roles between different family

memebrs

C t h i

p y

Compensatory mechanisms

Phenotypic redundancy

Vidal and Koff (2000) Gene 247, 1-15

Mouse strain Phenotypic consequences

Cip/Kip

esp21 −/− No evident phenotype. MEFs show defective G1 checkpoint

p27 −/− Gigantism and organomegalia. Infertile females. Pituitary hyperplasia

p57 −/− Some embryonic and neonatal lethality. Altered cell proliferation and apoptosis in

tudi

e several tissues.

p21 −/− p57 −/− Altered lung development. Enhanced skeletal abnormalities. Skeletal muscle differentiation failure

27 / 57 / I d b i l th lit M l lt ti Pl t l d f t

al s

t p27 −/− p57 −/− Increased embryonic lethality. More severe lens alterations. Placental defects

INK4

p16 −/− Normal development. General tumor predisposition

nim

a

p15 −/− Viable. No tumor predisposition

p18 −/− Gigantism and organomegalia. Pituitary hyperplasia

p15 −/− p18 −/− Infertile animals

An p15 / p18 / Infertile animals.

Pocket proteins

Rb −/− Embryonic lethality between d. 13.5 and 15.5. Defects in neurogenesis and hematopoiesishematopoiesis

p130 −/− Viable and fertile.

p107 −/− Viable and fertile.

130 / 10 / ffp130 −/− p107 −/− Neonatal lethality. Impaired chondrocyte differentiation

Vidal and Koff (2000) Gene 247, 1-15

Cell cycle has checkpointsCheckpoint controls (also known as surveillance mechanisms)

ensure the dependency of cell-cycle transitions on the completion of li tearlier events.

They consist of three distinct sets of functions:They consist of three distinct sets of functions: sensors (which look out for defects and emit a signal); signal-transduction cascades (checkpoint signals need to be g ( p g

transmitted throughout the nucleus or cell); andeffectors (a target is regulated to delay cell-cycle

i )progression)

The DNA damage checkpoint

The mitotic spindle assembly checkpoint

The DNA damage checkpoints

DNA damagecheckpointcheckpoint

The mitotic spindle assembly checkpoint

APC: Anaphase Promoting Complex. An E3 ubiquitin ligase that regulates degradation of Securin

Ub: Ubiquitin

Separase: Caspase-like protease needed for the separation of the sister chromatids

BUB (Budding Uninhibited by PLKBUB (Budding Uninhibited by Benomyl), MAD2 (Mitotic Arrest Deficient 2): Proteins participating in the spindle assembly checkpoint by recruiting cdc20

cdc20: Positive regulator of the APC

PLK: Polo-Like Kinase

CELL CYCLE DEREGULATION IN CANCER:CELL CYCLE DEREGULATION IN CANCER:MUTATIONS IN PROTEINS IMPORTANT AT DIFFERENT LEVELS OF THE CELL CYCLEDIFFERENT LEVELS OF THE CELL CYCLE

Cell cycle regulators and cancerInactivation

CyclinsCheckpoint proteins (p53)

Overexression, Amplificationtranslocations

Inactivation

CDKs CDK targets (Rb)CDK modulators (CKIs)

Mutational inactivation,Intracellular localization(promoter methylation)

Overexression, Amplification,Mutations in CKI binding

Mutational Inactivation(promoter

th l ti )(promoter methylation) Mutations in CKI binding methylation)

Cell cycle regulators and cancerCell cycle regulators and cancer: good prognostic value

: characterized genetic or epigenetic alterations: no mechanistic explanation

ororSmall molecule InhibitorsGene therapy,

Demethylating agentsiti

es fo

ities

fon.n.

Gene transferTxnalupregulation

port

unpo

rtun

vent

ion

vent

ion p g

siRNAs

nd o

ppnd

opp

c in

terv

c in

terv

tion

antio

n an

peut

icpe

utic

siRNAs

regu

lat

regu

lat

ther

apth

erap Phosphorylation

Proteasomal inhibitors

CD

K r

CD

K r

Phosphorylation

peptidomimeticssiRNAs

Phosphorylation

Small molecule CDK inhibitors

Flavopiridol: an anti-cancer drugs that targets the cell cycle

ororSmall molecule InhibitorsGene therapy,

Demethylating agentsiti

es fo

ities

fon.n.

Gene transferTxnalupregulation

port

unpo

rtun

vent

ion

vent

ion p g

siRNAs

nd o

ppnd

opp

c in

terv

c in

terv

tion

antio

n an

peut

icpe

utic

siRNAs

regu

lat

regu

lat

ther

apth

erap Phosphorylation

Proteasomal inhibitors

CD

K r

CD

K r

Phosphorylation

peptidomimeticssiRNAs

Phosphorylation

The p53 responseThe p53 response

StressOncogenes

Microtubule inhibitors

Ribonucleotide depletion

DNA damageHypoxia

Chemotherapy

Telomere erosionLoss of survival signalsg

p53 activation

Response

pIrreparable damageReparable damage

NucleusNucleus CytoplasmCytoplasm

Cell cycle arrestCell cycle arrest ApoptosisApoptosis

p21p21Cip1Cip1Cyclin/cdkPCNA

y py p

DNA repairpp

p21 KO, p21 siRNAp21antisense, c-myc

ororSmall molecule InhibitorsGene therapy,

Demethylating agentsiti

es fo

ities

fon.n.

Gene transferTxnalupregulation

port

unpo

rtun

vent

ion

vent

ion p g

siRNAs

nd o

ppnd

opp

c in

terv

c in

terv

tion

antio

n an

peut

icpe

utic

siRNAs

regu

lat

regu

lat

ther

apth

erap Phosphorylation

Proteasomal inhibitors

CD

K r

CD

K r

Phosphorylation

peptidomimeticssiRNAs

Phosphorylation