Modeling Cell Cycle Control and Cancer with pRB Tumor Suppressor

Kaldis: Cell Cycle RegulationDOI 10.1007/b136682/Published online: 1 September 2005 © Springer-Verlag Berlin Heidelberg 2005 Modeling Cell Cycle Control and Cancer with pRB Tumor Suppres

P Kaldis: Cell Cycle Regulation

DOI 10.1007/b136682/Published online: 1 September 2005

© Springer-Verlag Berlin Heidelberg 2005

Modeling Cell Cycle Control and Cancer

with pRB Tumor Suppressor

Lili Yamasaki

Department of Biological Sciences, Columbia University, New York, NY 10027, USA

Abstract Cancer is a complex syndrome of diseases characterized by the increased dance of cells that disrupts the normal tissue architecture within an organism Defining one universal mechanism underlying cancer with the hope of designing a magic bullet against cancer is impossible, largely because there is so much variation between vari- ous types of cancer and different individuals However, we have learned much in past decades about different journeys that a normal cell takes to become cancerous, and that the delicate balance between oncogenes and tumor suppressor is upset, favoring growth and survival of the tumor cell One of the most important cellular barriers to cancer de- velopment is the retinoblastoma tumor suppressor (pRB) pathway, which is inactivated

abun-in a wide range of human tumors and controls cell cycle progression via repression of the E2F/DP transcription factor family Much of the clarity with which we view tumor

suppression via pRB is due to our belief in the universality of the cell cycle and our tempts to model tumor pathways in vivo, nowhere so evident as in the multitude of data emerging from mutant mouse models that have been engineered to understand how cell cycle regulators limit growth in vivo and how deregulation of these regulators facilitates cancer development In spite of this clarity, we have witnessed with incredulity several stunning results in the last 2 years that have challenged the very foundations of the cell cycle paradigm and made us question seriously how important these cell cycle regulators actually are.

in RB or in genes encoding upstream regulators of pRB (i.e INK4A, CCND1,

CDK4) are found frequently in a mutually exclusive pattern in almost all

hu-man tumors (see Sect 3 below and Palmero and Peters 1996,Sherr 1996) Two

examples of human tumors that illustrate the impact of inactivating the pRB

tumor suppressor pathway include lung cancer and cervical cancer, and arehighlighted below

The most frequent types of cancer diagnosed in the United States in creasing incidence are breast cancer, prostate cancer, lung cancer and coloncancer; yet lung cancer is the deadliest form of cancer in the United States(163 500 deaths annually and 172 500 new cases annually) Twenty percent

de-of lung cancer is classified as SCLC (small-cell lung cancer; 32 700 deathsannually); the vast majority (∼ 90%) of cases carry mutations that directly

inactivate the RB locus (encoding pRB)(Kaye 2002, Minna et al 2004) It is

in-deed sobering to consider the number of deaths due to lung cancer that arelargely preventable with abstinence from or cessation of smoking, and the factthat while smoking is on the decline in the United States, smoking and its as-sociated lung cancer has been exported heavily to the developing world in thepast 30 years Moreover, the list of cancers for which smoking is a causativefactor has grown to include cancers of the respiratory, gastrointestinal andgenitourinary tracts (US Department of Health and Human Services, SurgeonGeneral’s Report on Smoking, http://www.surgeongeneral.gov)

Human cancer caused by viral infection remains a significant cause ofsuffering and death worldwide Human papillomavirus (HPV) infection is

a prominent sexually transmitted disease (20 million infected in the UnitedStates and 630 million infected worldwide), and its striking association(∼ 99%) with cervical cancer (15 000 new cases annually in the US and

470 000 new cases annually worldwide) is most prevalent in the ing world where screening (i.e Pap smear) is not routinely performed (zurHausen 2002 and World Health Organization, http://www.who.it) The asso-ciation of high-risk HPV (types 16 and 18) with cervical cancer is due to itsability to inactivate pRB growth suppression by direct binding of the HPV-E7oncoprotein to pRB Although a substantial time after primary viral infection

develop-is observed before cancer develops, the evidence that HPV causes cervicalcancer is strong enough to classify it as a carcinogen by IARC (InternationalAgency for Research on Cancer) and by the Federal Department of Health andHuman Services

1.2

Modeling Human Cancer in the Mouse

The ability of researchers to model cancer has grown substantially in the pastdecade, particularly in the mouse, in which numerous models of tumor devel-opment are now available for study The eventual goals of such modeling are

to faithfully reproduce the complexity of human tumorigenesis in the modelorganism, and then exploit the model system to uncover the regulatory points

or Achilles heel of cancer, such that new therapies can be designed to tack the clinical disease The many engineered strains of mutant mice haveprovided an excellent genetic model system in which to pursue these goals,and in fact, our efforts to model human cancer in the mouse, are far fromexhaustive

at-Many transgenic mice overexpressing an oncogene of interest have beenmade using tissue-specific promoters (e.g MMTV LTR for breast, keratin 14promoter for skin, and CD4 promoter for T-cells) to model a particular form

of cancer Knockout mice lacking specific tumor suppressors have been gineered to define the requirement of these genes during development andfor inhibition of tumor development throughout life Multistep tumorigenesiscan be studied by treating these mutant mice with carcinogens, by combin-ing such transgenic mice with knockout mice or by using proviral tagging toenhance the frequency or severity of specific cancers Of course, the ability

en-to generate these mice from genetically pure or inbred backgrounds, and en-toexamine time points during embryogenesis or life after birth gives us greaterinsight into the different mechanisms of tumor development than that pos-sible from studying human tumors alone Additionally, mutation of tumorsuppressors in mice leads to a restricted set of tumor types analogous to thenarrow spectrum of tumors seen in inherited human tumor syndromes, themechanistic basis for which is still elusive

Nowhere has hope for modeling cancer in the mouse been as great as inthe large number of mutants engineered in genes encoding components ofthe cell cycle machinery and the pRB and p53 tumor suppressor pathways.The universality of the cell cycle in all eukaryotes strongly suggested that anti-cancer therapy aimed at regulators of the cell cycle would be of great clinicalbenefit This review will discuss the phenotypes of such mutant mice andthe surprising recent results that suggest the cell cycle paradigm may greatlyunderestimate the complexity of the wiring of the cell cycle at least duringembryonic development (see Sect 5 below)

While the existing mutant mouse models of cancer are both impressive andpowerful, they do not necessarily reflect the spectrum or complexity of hu-man cancer Only a small amount of human cancer can be attributed to theinheritance of dominantly acting mutant alleles of known tumor suppressors(Balmain et al 2003) It has been estimated that only 12% of human breast

cancer patients carry mutations in either the BRCA1 or the BRCA2 tumor

sup-pressor gene, and that the majority of human, cancer susceptibility is due tothe combined action of common, low penetrance cancer predisposing alleles

or genetic modifiers of tumor susceptibility that have not yet been

identi-fied (Pharoah et al 2002) Recently, the Stk6 locus encoding the Aurora-2

centrosome-associated kinase was identified as a weak modifier of skin morigenesis in mice, and a polymorphism in the human Aurora homologue,

tu-STK15 (Phe311), is found frequently amplified in human colon cancer

(Ewart-Toland et al 2003) Eventually, the hope is that as mutant mouse models ofcancer improve, we can pursue more of these weak tumor predisposing al-leles, for instance, by using our dominantly acting mutant mouse models ofcancer to screen for enhancers or suppressors of these phenotypes Perhapsonly then can more clinically relevant and beneficial information be forth-coming from mutant mouse models of cancer

The Universality of the Cell Cycle

The discovery of the cell cycle emerged from distinct studies in yeast,flies, clams, and frogs, and eventually the conclusions drawn were testedand found to be operative also in mice and humans The power of thecell cycle theory is that it unified eukaryotic biology, for which Hartwell,Nurse and Hunt were recognized with the Nobel Prize in Medicine in 2001(http://nobelprize.org/medicine/laureates/2001) The model of the cell cyclehas given researchers the chance to understand complex mammalian systemsand human disease, using genetic studies in evolutionarily lower organisms

From studying temperature sensitive cdc mutants of budding yeast

(Sac-charomyces cerevisiae) with abnormal budded morphology at the

non-permissive temperature, Hartwell and colleagues proposed that a simpleorder of dependent events (e.g budding, DNA synthesis, cytokinesis), com-pletion of which were necessary for cell division In this way, the many ofthe primary components of the cell division cycle were identified and placed

in a genetic pathway, in particular, Cdc28, which held precedence over all the cdc mutants as a master regulator in G1 Importantly, this work initiated

the concept of checkpoints, non-essential genes that ensure these tal processes were completed Nurse and co-workers built on these seminal

fundamen-concepts using fission yeast (S pombe), first identifying temperature tive wee1 mutants and then Cdc2 as a master regulator of the cell cycle in

sensi-G2/M Nurse showed that Cdc2 was required also in G1, and

complementa-tion experiments then showed that Cdc2 was the S pombe homologue of the

S cerevisiae Cdc28 regulator The identification of Cdc28 and then Cdc2 as

kinases, helped define the function of other cdc genes (e.g Cdc25 and Wee1)

that modify the kinase activity of these master switches Importantly, Masui’searly studies of MPF (maturation promoting factor) activation in frog oocyteswere crucial for understanding the commonality of these mechanisms even in

vertebrates Nurse and colleagues then cloned human Cdc2 through mentation of the yeast cdc2 mutant.

comple-By studying changes in protein expression ongoing in the fertilization ofclam and sea urchin eggs, Hunt and colleagues identified the first cyclin pro-teins, the abundance of which fluctuates with the division of the eggs Theidentification of classes of yeast cyclins (Clns in G1 and Clbs in G2) thatcontrol Cdc28 and classes of vertebrate cyclins (D- and E-type cyclins in G1and A-and B-type cyclins in G2/M) that control a family of Cdks (cyclin-

dependent kinases) greatly enhanced our understanding of the complexityunderlying control of the cell cycle Control of Cdk activity through cyclinbinding and degradation, inhibitory and activating Cdk phosphorylationsand association of Cdk inhibitors outlined a range of regulatory mechanismsfor controlling cell cycle progression (Morgan 1997; Zachariae and Nasmyth1999) These studies and others demonstrated the universality of the cell

cycle, and strongly suggested that deregulation of the mechanisms controllingcell cycle progression could result in cancer.

3

The pRB Tumor Suppressor Pathway

The concept that chromosomes could suppress malignancy is attributed toBoveri’s writings from 1914 (Balmain 2001; Knudson 2001) In 1969, so-matic cell hybridization experiments between normal and transformed cellsresulted in phenotypically normal hybrids that often reverted to being trans-formed, indicating the existence of cellular genes that normally suppressedtransformation (Ephrussi et al 1969) Below is outlined the compelling evi-dence that the pRB tumor suppressor pathway is a crucial target that must beinactivated during the progression of normal cells into tumors

3.1

The Discovery of pRB

The path of discovery for the prototypic tumor suppressor, RB, has been

repeated many times with the identification of human tumor suppressors,mutation of which leads to cancer development In 1971, Knudson consid-ered the clear differences between the clinical presentation of inherited andsporadic retinoblastoma cases (i.e frequency, age of onset, unilateral vs bi-lateral, unifocal vs multifocal lesions), and proposed the “two-hit” hypothesisfor pediatric retinoblastoma development that put forward the following ex-planation for these clinical differences (Knudson 1971) Inherited retinoblas-toma patients must carry a germ-line loss-of-function mutation in a putative

retinoblastoma tumor suppressor gene (RB), and sometime during

develop-ment or shortly after birth, a somatically acquired mutation specifically in

a few retinal cells would inactivate the remaining normal RB allele, giving rise

to multiple retinoblastomas per patient In contrast, sporadic retinoblastoma

patients must acquire two somatic RB mutations within the same retinal cell,

an extremely rare event, giving rise to a single retinoblastoma per patient.The later identification of cytogenetic abnormalities involving deletions ofChr13q14 in normal blood cells from inherited retinoblastoma patients and

in sporadic retinoblastoma tumor samples, strongly suggested the location of

the RB gene, facilitating its subsequent positional cloning of the RB gene in

1986 Shortly thereafter, RB mutations were found frequently in

osteosarco-mas, small cell lung carcinomas and carcinomas of the prostate, bladder andbreast It has been estimated that 40–50% of human tumors contain direct

inactivation of the RB gene (Palmero and Peters 1996; Sherr 1996).

Importantly, re-expression of pRB in tumor cells can revert the formed phenotype (Huang et al 1988), giving support for cancer therapies

trans-that restore pRB function In 1988, interactions of pRB with viral teins from three distinct DNA tumor virus families (i.e adenovirus E1A,SV40-T and HPV-E7) were demonstrated to be necessary for cellular trans-formation by these viruses (DeCaprio et al 1988; Dyson et al 1989) Binding

oncopro-of viral oncoproteins requires a large central region oncopro-of pRB, known as the

“pocket” domain, and tumor-derived RB mutants often had sustained

dele-tions of exons encoding pieces of this “pocket” region of pRB Two pRB mologues (p107 and p130) have been identified that show extensive homologythrough the central “pocket” domain of pRB and also to each other (reviewed

ho-in Classon and Harlow 2002) Although both pRB homologues can ho-inhibitgrowth when overexpressed, mutations in genes encoding p107 or p130 areonly rarely found in human tumors, and thus, these pRB homologues are notgenerally considered to be human tumor suppressors Importantly, pRB sup-presses growth and promotes lineage-specific differentiation; yet p107 andp130 appear to act together to suppress growth It may be important to re-assess the status of p107 and p130 mutations in tumors bearing RB mutations,given the recent evidence that these pRB family members act as tumor sup-pressors in conjunction with pRB (see Sect 7)

3.2

Upstream Regulators of pRB

Cell cycle-dependent phosphorylation of pRB occurs in G1 by dependent kinases that sequentially inactivate the tumor suppressive prop-erties of pRB Hyper-phosphorylation of pRB prevents binding of viraloncoproteins and cellular proteins to the central “pocket” region of pRB.Non-phosphorylatable pRB mutants suppress growth more efficiently thanwild-type pRB Normally, D-type cyclin/Cdk4 or cyclin/Cdk6 complexes

cyclin-phosphorylate pRB in early G1, while cyclin E1–2/Cdk2 complexes

phos-phorylate pRB at the G1/S transition, stimulating S-phase entry and cell

cycle progression Overexpression of D- and E-type cyclins and Cdk4 is served frequently in human tumors (see Sect 5), supporting the notion thatthese cell cycle regulators are critical for cell cycle progression Inactiva-

ob-tion of the complex locus at Chr9p21 containing the INK4A gene encoding

p16, the cyclin-dependent kinase inhibitor specific for Cdk4 or Cdk6, is monly seen in about half of human tumors (for discussion of the ARF tumorsuppressor also residing at Chr9p21, see Sect 6) Mutation of genes encod-ing these upstream regulators of pRB is observed in approximately 50% ofall human tumors, in a mutually exclusive pattern to those tumors carry-

com-ing RB mutations (Palmero and Peters 1996; Sherr 1996) Thus, inactivation

of the pRB tumor suppressor pathway directly (RB mutations) or indirectly (CCND1, INK4A or CDK4 mutations) occurs in almost all human tumors, em-

phasizing the importance of overcoming pRB-mediated growth control fortumor progression

Two classes of human tumors that do not contain mutations of RB or tions in genes encoding upstream regulators of pRB have derived other ways

muta-to circumvent the pRB tumor suppressor pathway Colon carcinomas increasetranscription of CCND1 via increases in β-catenin/TCF signaling resulting

from loss of the APC tumor suppressor (Tetsu and McCormick 1999)

Neu-roblastomas increase transcription of Id2 via amplification of N-MYC thatantagonizes pRB-mediated tumor suppression (Lasorella et al 2000, and seeSect 3.4)

3.3

Phenotype of Mice Lacking pRB Family Members

Mice lacking pRB die in mid-gestation with extensive defects in the centraland peripheral nervous systems, fetal liver and lens (Clarke et al 1992; Jacks

et al 1992; Lee et al 1992) (Table 1) Chimaeras made with Rb-deficient ES

cells develop surprisingly well, demonstrating that many tissues do not quire pRB for normal function (Maandag et al 1994; Williams et al 1994b)

re-Bypass of the mid-gestational lethality in Rb-deficient embryos allowed

de-fects in muscle differentiation to be observed later in development

(Zacksen-haus et al 1996) The extensive apoptosis evident in the Rb-deficient embryos

has now been shown to be due to placental insufficiency, specifically due

to the hyperproliferation of the spongiotrophoblast layer of the placenta atE11.5 (Wu et al 2003) Hyperproliferative and/or differentiation defects in

the CNS, lens, and muscle are still present in Rb-deficient embryos once the placental requirement for Rb is circumvented through the use of chimaeras or through conditional deletion of Rb (Lipinski et al 2001; Ferguson et al 2002;

de Bruin et al 2003; MacPherson et al 2003) Similarly, erythropoietic

de-fects are apparent in the fetal liver of Rb-deficient embryos, and Rb-deficient

erythroblasts fail to fully mature in vitro or reconstitute irradiated wild-typedonors (Iavarone et al 2004; Spike et al 2004)

Rb+/- mice develop neuroendocrine tumors of the pituitary, thyroid, and

adrenals (Jacks et al 1992; Hu et al 1994; Harrison et al 1995) (Table 2)

Neuroendocrine tumorigenesis in Rb+ /- mice is dependent on LOH of the

wild-type Rb allele similar to the retinoblastomas and osteosarcomas

de-veloping in germ-line retinoblastoma patients This system has been usedextensively to test the functional significance of numerous cell cycle regula-

tors and interactors of pRB, including E2F family members and CKIs (Table 1

and Sects 3.4 and 4) Interestingly, the spectrum of neuroendocrine tumors

observed in Rb+ /- mice is dependent on strain-specific modifiers, and

specif-ically, inherent abnormalities of the 129Sv strain enhance the development oftumors in the intermediate lobe of the pituitary (Leung et al 2004)

In contrast to phenotypes of the Rb mutant mice, p107-deficient or

p130-deficient mice live to be viable adults without tumor predisposition on

a mixed genetic background with the C57LB/6 strain (Cobrinik et al 1996;

Table 1 Phenotypes of mice lacking Rb family members and rescue of Rb deficiency

Rb– /– Mid-gestational lethality at (Clarke et al 1992;

E13.5-E15.5 with widespread Jacks et al 1992;

Placental bypass required for (Maandag et al 1994; survival to late gestation Williams et al 1994b;

Wu et al 2003;

Zacksenhaus et al 1996) Defects then found in CNS/PNS, (de Bruin et al 2003; fetal liver, muscle and lens Ferguson et al 2002;

Lipinski et al 2001; MacPherson et al 2003; Spike et al 2004)

p107–/– Myeloid hyperplasia and growth (LeCouter et al 1998a;

deficiency on Balb/c background Lee et al 1996)

No obvious phenotype on mixed genetic background

p130– /– Embryonic lethality E11-E13 (Cobrinik et al 1996;

on a Balb/c background LeCouter et al 1998b)

No obvious phenotype on mixed genetic background

p107– /–; p130–/– Perinatal lethality with (Cobrinik et al 1996)

endochondral bone defects

on mixed background

Rb– /–; p107–/– Embryonic death prior to E12.5 (Lee et al 1996)

Rb– /–; E2f1–/– Rescue of mid-gestational (Tsai et al 1998)

lethality until late gestation

Rb– /–; E2f3–/– Rescue of mid-gestational (Ziebold et al 2001)

lethality until late gestation

Rb– /–; Id2–/– Rescue of mid-gestational (Iavarone et al 2004;

lethality until late gestation Lasorella et al 2000) and RBC enucleation in fetal liver

Rb– /–; p53–/– Reduction of cell death in CNS (Macleod et al 1996;

and lens, but not PNS Morgenbesser et al 1994)

Rb– /–; p19–/– No rescue of p53-dependent (Tsai et al 2002a)

apoptosis observed

Lee et al 1996) (Table 1) Combining p107 deficiency with p130 deficiency,

results in perinatal death with defects in endochondral bone development(Cobrinik et al 1996) On a 129Sv Balb/c background, p130-deficient em-

bryos die and p107-deficient animals exhibit growth and myeloproliferative

defects (LeCouter et al 1998a,b), again suggesting that strain-specific

mod-Table 2 Phenotypes of Rb+ /– mice lacking various cell cycle regulators

Rb+ /– Neuroendocrine tumorigenesis in (Harrison et al 1995;

intermediate lobe of the pituitary Hu et al 1994;

Jacks et al 1992) Additional tumorigenesis in thyroid, (Williams et al 1994a; anterior lobe of the pituitary, Nikitin et al 1999) and adrenal gland

Rb+ /–; p107–/– Neuroendocrine and non-endocrine (Dannenberg et al 2004)

tumorigenesis in chimaeras

Rb+ /–; p130–/– No pituitary or thyroid (Dannenberg et al 2004)

tumorigenesis but other endocrine tumors at low frequency in chimaeras

Rb+ /–; E2f1–/– Decreased neuroendocrine (Yamasaki et al 1998)

tumorigenesis and increased survival

Rb+ /–; E2f3–/– Decreased pituitary (Ziebold et al 2003)

tumorigenesis, but worsened thyroid tumors

Rb+ /–; E2f4–/– Decreased neuroendocrine (Lee et al 2002)

tumorigenesis and increased survival

Rb+ /–; p21–/– Increased neuroendocrine (Brugarolas et al 1998)

tumorigenesis, including pheochromacytomas, and decreased survival

Rb+ /–; p27–/– Increased neuroendocrine (Park et al 1999)

tumorigenesis with worsened thyroid tumors and decreased survival

Rb+ /–; p53–/– Increased neuroendocrine (Williams et al 1994a)

tumorigenesis and decreased survival

Rb+ /–; p19–/– Increased neuroendocrine (Tsai et al 2002b)

tumorigenesis and decreased survival

Rb+ /– Increased tumorigenesis (Leung et al 2004) (129Sv) in the intermediate lobe

of the pituitary, and greatly decreased survival

Rb+ /– Increased neuroendocrine (Leung et al 2004) (C57BL/6) tumorigenesis in the anterior

pituitary and thyroid glands with increased survival

ifiers regulate the severity of phenotypes resulting from inactivation of Rb family members While the constitutive inactivation of multiple Rb family

members is required for the immortalization of primary mouse embryonicfibroblasts (MEFs) (Dannenberg et al 2000; Sage et al 2000; Peeper et al

2001), the spontaneous loss of only Rb is sufficient to reverse cellular cence (Sage et al 2003) Conditional loss of Rb impairs the development of the cerebellum on a p107-deficient background (Marino et al 2003) and pro- duces medulloblastomas on a p53-deficient background (Marino et al 2000) Chimaeras generated with ES cells that are Rb-deficient and either p107- or

senes-p130-deficient are highly tumor prone, demonstrating that pRB family

mem-bers act in concert to suppress tumorigenesis in a wide variety of tissues inthe mouse (Dannenberg et al 2004) (Table 2) Chimaeras generated with EScells that are Rb+/- and either p107- or p130-deficient develop tumors, but

suggest that p107 is a more effective tumor suppressor that p130 berg et al 2004) The absence of retinoblastoma in Rb+ /- mice prompted

(Dannen-criticism of modeling human cancer in the mouse, but continued efforts to

generate inherited models of mouse retinoblastoma with Rb deficiency have

been successful recently (see Sect 7 below)

3.4

pRB Regulates Growth and Differentiation

In the following section, the best characterized effector of pRB, the E2F/DP

transcription factor family, is reviewed (see Sect 4) The ability of E2F/DP

complexes to control the expression of most if not all cell cycle related genesstrongly suggests that the E2F/DP family is a crucial, downstream pRB target

for controlling growth and thereby suppressing tumorigenesis However, yond the preponderance of reports on E2F and the significance of E2F for thegrowth suppressive function of pRB, there are numerous (∼ 110) other inter-actors of pRB that have been identified (Morris and Dyson 2001) While none

be-of the E2F family members contains an LCE motif, a number be-of these pRBinteractors (e.g RBP1, RBP2, HDAC) do contain this motif or one similar to it.The existence of low penetrance retinoblastoma mutations that encodepRB mutants capable of E2F interaction, demonstrate that repression of E2Factivity alone is insufficient for tumor suppression (Sellers et al 1998) SuchpRB mutants fail to interact with and activate transcription factors importantfor differentiation, suggesting that differentiation is an important component

of pRB’s ability to suppress tumor formation Additionally, Rb deficiency

in-hibits the differentiation of particular lineages (e.g adipogenesis, myogenesisand osteogenesis) due to the inability of lineage-specific transcription factors(e.g C/EBPα, MyoD, CBFA1) to be activated by pRB (Gu et al 1993; Chen

et al 1996; Thomas et al 2001) These studies suggest that it is the uniqueability of pRB to coordinate cell cycle exit with the induction of differentiationthat confers upon pRB its tumor suppressor function

The interaction of pRB with Id2 (inhibitor of differentiation) is especiallycompelling (Table 1) Id2 normally acts as a dominant negative inhibitor

of helix–loop–helix transcription factors; however, it is also able to

antag-onize pRB family function by direct interaction Loss of Id2 rescues the mid-gestational lethality of Rb-deficient embryos, minimally by rescuing the

defects seen in neurogenesis and erythropoiesis (Lasorella et al 2000)

Res-cue of erythropoiesis in Rb-deficiency occurs with loss of Id2, because Id2

normally inhibits PU.1, a transcription factor that stimulates the macrophagelineage, upon which developing erythroblasts are dependent for enucleation(Iavarone et al 2004)

4

The E2F /DP Transcription Factor Family

In 1992, researchers identified a critical downstream effector of pRB, the E2F1transcription factor, binding sites for which were found in the adenovirus E2promoter and the promoters of many cell cycle regulated genes (reviewed inTrimarchi and Lees 2002; Attwooll et al 2004) E2F1 stimulates entry into S-

phase and cooperates with activated Ras for cellular transformation Multiple

E2F family members (E2F1–6) have been identified that recognize E2F ing sites in target promoters following hetero-dimerization to a DP familymember (DP1 and DP2) More recently E2F7 has been identified that bindsE2F sites independently of association with a DP subunit E2F1, E2F2 andE2F3 interact preferentially with pRB, while E2F4 and E2F5 interact well withp107 and p130 E2F4 can interact with pRB at lower affinity E2F6 and E2F7 donot interact with pRB family members, and are not competent for activatingtranscription

bind-4.1

E2F Target Genes and Repression

Interaction of E2F1–3/DP complexes with pRB converts these transcriptional

activators to repressor complexes that are known to bind to target promotersand inhibit interaction with the basal transcription machinery Interactionwith pRB blocks the ability of E2F/DP complexes to induce its many target

genes that promote numerous cellular processes (Ishida et al 2001; Kalma

et al 2001; Ma et al 2002; Ren et al 2002; Stevaux and Dyson 2002; Weinmann

et al 2002; Wells et al 2002) Classical E2F target genes encode products thatpromote S-phase entry (e.g Orc1 and Mcm2–7) and DNA replication (e.g.Dhfr, Rnr, TK, TS and Polα) Many E2F target genes encode products in-

volved in cell cycle progression (e.g cyclins A and E, pRB, p107, E2F1–3) and

apoptosis (e.g p73, Apaf1, caspase) Recently however, E2F target genes havebeen identified, the products of which act in DNA repair (e.g Msh2, Mlh1,

Rad51), cell cycle checkpoints (e.g Mad2, Chk1, Bub3) and mitotic events(e.g cyclin B, Cdc2, and Smc2) These more recently discovered targets help

in understanding the aneuploidy found in human tumors, because

follow-ing loss of RB, the deregulation of E2F activity increases expression of Mad2,

a mitotic checkpoint gene that contributes to genomic instability (Hernando

et al 2004)

pRB binds HDAC family members while tethered through E2F to targetpromoters, inhibiting gene expression via chromatin remodeling Interest-ingly, pRB and E2F family members are known to be acetylated in vivo, whichchanges the ability of these proteins to bind DNA, suggesting that HDACassociation may modify pRB and E2F directly as well as histones in neigh-boring nucleosomes (Martinez-Balbas et al 2000; Marzio et al 2000; Chan

et al 2001; Brown and Gallie 2002; Nguyen et al 2004) While the pRB tumorsuppressor pathway is widely inactivated in human tumors, the E2F or DPgenes themselves are not, an observation that may be attributed to at least twounderlying causes First, the frequent inactivation of pRB and its upstreamregulators in human tumors leads in effect to deregulation of the E2F activ-ity Second, the bifunctional nature of E2F/DP complexes to act as activators

and/or repressors and the wide range of E2F target genes suggests that

dereg-ulation of E2F or DP may have pleiotropic and opposing effects that do notfavor tumor cell survival

4.2

Mice Deficient in E2F Family Members

Mice lacking various E2F family members have been generated, and in

gen-eral, the viable phenotypes of individual E2f -deficient mice probably reflect

substantial functional redundancy of many E2F family members (Table 3).Loss of multiple E2F family members results in stronger phenotypes, occa-sionally in embryonic death, consistent with subsets of E2Fs having similar

function For example, while inactivation of E2f 1 leads to reduced adult

survival with broad-range of tumors and tissue atrophy (Field et al 1996;

Ya-masaki et al 1996), the simultaneous inactivation of E2f 1 and E2f 2 results

in high penetrance phenotypes that result in premature death, including abetes, exocrine pancreatic failure, hematopoietic failure and leukemias (Zhu

di-et al 2001; Li di-et al 2003a,b; Iglesias di-et al 2004) This is a similar situation to

that seen in MEFs, where loss of E2f 1 or E2f 2 does not result in a cell cycle defect and loss of E2f 3 leads to defective induction of numerous E2F target genes (Humbert et al 2000b); however, the simultaneous inactivation of E2f 1,

E2f 2 and E2f 3 is required to block MEF proliferation (Wu et al 2001)

Inac-tivation of Dp1 leads to embryonic lethality prior to E12.5, a sharp contrast

to the weaker phenotypes resulting from inactivation of individual E2F

fam-ily members (Kohn et al 2003) Dp1 deficiency cripples the development of

all extra-embryonic lineages (visualized as early as E6.5), leading to

placen-Table 3 Phenotypes of mice lacking E2F or DP family members

E2f1– /– Tumor predisposition and (Field et al 1996;

decreased thymocyte apoptosis Yamasaki et al 1996) Atrophy of testes and thyroid

gland

E2f2– /– Enhanced T cell proliferation (Murga et al 2001)

and auto-immunity

E2f3– /– Decreased viability and (Cloud et al 2002;

congestive heart failure Humbert et al 2000b)

on mixed background Inviable on 129Sv strain.

Decreased E2F target induction

and MEF proliferation

E2f4–/– Erythropoietic and craniofacial (Humbert et al 2000a;

defects resulting in juvenile death Rempel et al 2000) E2f5–/– Abnormal choroids plexus and (Lindeman et al 1998)

hydrocephaly leading to premature death

E2f6– /– Homeotic transformations of the (Storre et al 2002)

axial skeleton without premature death

Dp1– /– Embryonic lethality prior (Kohn et al 2003, 2004)

to E12.5 Placental bypass required for late gestational E17.5 survival without obvious defects

E2f1– /–; E2f2–/– Hematopoietic abnormalities (Zhu et al 2001;

(e.g., tumors and megaloblastic Li et al 2003a,b;

Insulin-dependent diabetes with exocrine pancreatic failure and auto-immunity

E2f1– /–; E2f3–/– No enhanced tumor (Cloud et al 2002)

predisposition, but worsened testicular atrophy and viability

E2f4– /–; E2f5–/– Embryonic lethality between (Gaubatz et al 2000)

E13.5 and birth

E2f1– /–; E2f2–/–; No organismal phenotype (Wu et al 2001)

E2f3– /– reported, but MEFs do

not proliferate

tal insufficiency that is responsible for the death of the embryo secondarily

and that occurs well before the requirement for Rb in the placenta Bypassing the extra-embryonic requirement for Dp1 with the construction of chimaeras surprisingly allows Dp1-deficient ES cells to develop into most tissues of the

embryo proper (Kohn et al 2004)

E2F family members represent direct targets of pRB as judged by the

re-versal of Rb-deficient phenotypes with inactivation of E2Fs Combination

of E2f 1-, or E2f 3-deficiency rescues the mid-gestational lethality of

Rb-deficiency and reduces the penetrance of neuroendocrine tumorigenesis in

Rb+/- mice (Tsai et al 1998; Yamasaki et al 1998; Ziebold et al 2001, 2003).

Surprisingly, E2f 4 deficiency is able to fulfil the latter function, by ing p107 and p130 repressive function to E2F1-3 (Lee et al 2002) It is also quite likely that E2f 2 loss will rescue Rb-deficient lethality Taken together, these studies demonstrate the functional significance of E2F family mem-

divert-bers as downstream effectors of pRB functions in development and genesis

tumori-5

Cyclin-dependent Kinases and their Inhibitors

5.1

Deregulation of Cyclins, Cdks and CKIs in Human Tumors

As predicted from the early studies of the cell cycle, deregulation of cellcycle components (cyclins, Cdks and CKIs) is observed in human cancer In-deed, direct genetic alterations and overexpression of numerous cyclins arecommon events in human neoplasia, and correlate with a poor prognosis ofsurvival (Reed 2003) Cyclin D1 overexpression occurs in mantle (centrocytic)cell lymphomas and parathyroid adenomas following chromosomal translo-

cation of the CCND1 (also known as the BCL1 or PRAD1) locus at Chr11q13

into the immunoglobulin heavy chain locus on Chr14 and chromosomal

in-version, respectively (Motokura et al 1991) CCND1 amplification also occurs

frequently in cancer of the breast, esophagus, bladder and pancreas CyclinD2 overexpression occurs in ovarian and testicular tumors, and cyclin D3

overexpression resulting from CCND3 rearrangements occurs in lymphoid

malignancies (Sicinska et al 1996, 2003) Cyclin E overexpression or

amplifi-cation of the CCNE locus on Chr19 is commonly observed in uterine, ovarian

and breast cancer, as well as an array of other tumor types Cyclin A

overex-pression resulting from random integration of HBV in the CCNA locus has been reported in a liver tumor (Wang et al 1990) Finally, CDK4 at Chr12q13

is amplified in a number of different tumor types, and mutation of CDK4

rendering insensitive to p16INK4inhibition has been reported in melanomas(Zuo et al 1996)

Inhibitors of Cdks or CKIs (cyclin-dependent kinase inhibitors) would bepredicted to act as tumor suppressors that slow cell cycle progression There

are two families of CKIs, the INK4 family (p16 INK4A, p15INK4B, p18INK4C andp19INK4D ) and the CIP /KIP family (p21 CIP1, p27KIP1and p57KIP2 ) INK4A and

INK4B at Chr9p21 are frequently mutated in human cancer (see Sect 6

be-low for the discussion of overlap between the INK4A and ARF locus), while

INK4C and INK4D show no such pattern (Kamb et al 1994) CIP1 (also known

as WAF1 /SDI1) and KIP1 are infrequently mutated in human cancer; however,

decreased expression of p27 through increased ubiquitin-mediated tion, commonly occurs in human tumors and also correlates with a poor

degrada-prognosis (Pagano and Benmaamar 2003) KIP2 is one of the imprinted genes

at Chr 11p15 frequently deleted in Beckwith–Wiedemann syndrome (BWS),characterized overgrowth, midline-defects and increased predisposition to

pediatric cancer, and a small subset of BWS patients have KIP2 mutations

(Hatada et al 1996; O’Keefe et al 1997)

Overexpression of other cell cycle components, such as the Cdc25A andCdc25B phosphatases that are important for Cdk activation in G1, is also ob-served in human tumors (Kristjansdottir and Rudolph 2004) It is of note thatoverexpression of Cdk1, Cdk2 or Cdc25C is not commonly seen in humancancer, perhaps because of the requirement to decrease mitotic Cdk1 activityfor mitotic exit Nevertheless, these reports strongly suggest that deregulation

of the cell cycle facilitates tumorigenesis in humans

5.2

Mice Deficient in Cyclins, Cdks and CKIs

Surprisingly, most individual G1 and G1/S regulators of the cell cycle are

largely (if not completely) dispensable for normal mouse embryonic ment (Gladden and Diehl 2003; Roberts and Sherr 2003; Pagano and Jackson2004; Sherr and Roberts 2004) (Table 4) Individual D-type cyclins are dis-pensable for development (Fantl et al 1995; Sicinski et al 1995, 1996; Sicinska

develop-et al 2003) Similarly, mice lacking individual E-type cyclins are viable (Geng

et al 2003; Parisi et al 2003) Cdk2-deficient mice are viable and are sterile due to meiotic defects (Berthet et al 2003; Ortega et al 2003) Cdk4-deficient

mice are viable, but runted and develop diabetes (Rane et al 1999; Tsutsui

et al 1999) Cdk6-deficient mice are viable and develop hematopoietic defects

(Malumbres et al 2004)

Mice lacking p21 are viable (Brugarolas et al 1995; Deng et al 1995), whilemice lacking p27 are viable, but display organomegaly and pituitary ade-nomas (Fero et al 1996; Kiyokawa et al 1996; Nakayama et al 1996) Micelacking p57 die in late gestation with placental and mid-line closure defects(Yan et al 1997; Zhang et al 1997) The absence of p21 or p27 accelerates the

development of neuroendocrine tumorigenesis in Rb+ /- mice, demonstrating

the importance of these factors as negative regulators of neoplasia