cyclins and breast cancer

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Journal of Mammary Gland Biology and Neoplasia (JMGBN) pp1152-jmgbn-483567 March 30, 2004 22:56 Style file version June 22, 2002

Journal of Mammary Gland Biology and Neoplasia, Vol. 9, No. 1, January 2004 ( C© 2004)

Cyclins and Breast Cancer

Robert L. Sutherland1,2 and Elizabeth A. Musgrove1

The D-type and E-type cyclins control the G1 to S phase transition during normal cell cycleprogression and are critical components of steroid- and growth factor-induced mitogenesis inbreast epithelial cells. Mammary epithelial cell-specific overexpression of these genes leadsto mammary carcinoma, while in cyclin D1-deficient mice mammary gland development isarrested prior to lobuloalveolar development. Cyclin D1 null mice are resistant to mammarycarcinoma induced by the neu and ras oncogenes, indicating an essential role for cyclin D1 inthe development of some mammary cancers. Cyclin D1 and E1 are commonly overexpressedin primary breast cancer, with some evidence of an association with an adverse patient out-come. This observation may result in part from their ability to confer resistance to endocrinetherapies. The functional consequences of cyclin E overexpression in breast cancer are likelyrelated to its role in cell cycle progression, whereas that of cyclin D1 may also be a consequenceof a more recently defined role in transcriptional regulation.

KEY WORDS: cyclin D1; cyclin E; breast cancer; cell cycle; steroid hormones.

INTRODUCTION

Loss of normal growth control, includingaberrations in the homeostatic mechanisms thatensure integrity of cell cycle progression, is a hall-mark of cancer (1). A pivotal regulatory pathwaydetermining rates of cell cycle transition from G1

to S phase is the cyclin/cyclin-dependent kinase(Cdk)/p16Ink4A/retinoblastoma protein (pRb) path-way. Alterations to different components of thispathway through overexpression, mutation, andepigenetic gene silencing are almost universal inhuman cancer (2). Interestingly, there appears to bea degree of tissue specificity in the particular geneticabnormalities within the Rb pathway. In breastcancer these include the overexpression of cyclinsD1, D3, and E1, decreased expression of the p27Kip1

Cdk inhibitor and silencing of the p16Ink4A gene

1 Cancer Research Program, Garvan Institute of Medical Research,St. Vincent’s Hospital, Darlinghurst, Sydney, Australia.

2 To whom correspondence should be addressed at CancerResearch Program, Garvan Institute of Medical Research, 384Victoria Street, Darlinghurst, New South Wales Sydney, 2010,Australia; e-mail: [email protected].

through promoter methylation (2). These aberrationsoccur at relatively high frequency in breast cancer,i.e. each abnormality is present in 30–45% of primarytumors, implying a major role for loss of function ofthe Rb pathway in breast oncogenesis.

Cyclin D1 is the product of the CCND1 gene andwas first implicated in breast cancer following local-ization of the gene to chromosome 11q13, a regionof the genome that is commonly amplified in a rangeof human carcinomas, including about 15% of breastcancers (3). The subsequent demonstration that cy-clin D1 was overexpressed at the mRNA and proteinlevel in up to 50% of primary breast cancers identifiedcyclin D1 as one of the most commonly overexpressedoncogenes in breast cancer (4–7). Although amplifi-cation of the cyclin E1 locus is a relatively rare eventin breast cancer, the protein product is overexpressedin ∼40% of breast cancers (8,9). Interestingly, cyclinD1 is overexpressed predominantly in ER+ tumorswhile cyclin E overexpression is confined almost ex-clusively to ER− tumors (4–10). Since these proteins

Abbreviations used: Cdk; cyclin-dependent kinase; ER, estrogenreceptor; pRb, retinoblastoma protein; MMTV, mouse mammarytumor virus.

951083-3021/04/0100-0095/0 C© 2004 Plenum Publishing Corporation

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96 Sutherland and Musgrove

appear to have redundant functions in the mammarygland, as evidenced by the ability of cyclin E to substi-tute for cyclin D1 (11), it is tempting to speculate thatthey might be exerting similar roles in tumorigenesisin these two different breast cancer phenotypes.

This review summarizes contemporary literatureaddressing the functions of D- and E-type cyclins inmammary epithelial cells and their potential roles inthe development and progression of human breastcancer.

CYCLINS AND CELL CYCLE CONTROL

In eukaryotes, cell cycle progression is mediatedby the sequential activation and inactivation of theCdk serine/threonine kinases (12). These enzymecomplexes contain a Cdk catalytic subunit, theexpression of which remains relatively uniformthroughout the cell cycle, and a regulatory cyclinsubunit, the abundance of which is tightly regulatedby transcriptional regulation, subcellular localizationand protein degradation, thus allowing activationat distinct phases of cell cycle progression (12,13).Mitogenic stimulation of growth-arrested cells resultsinitially in the induction of the D-type cyclins (cyclinsD1, D2, and D3), and these molecules are believedto have the primary function of linking extracellularsignals to the cell cycle machinery. The D-type cyclinsbind preferentially to Cdks 4 and 6 and phosphorylatekey downstream substrates, predominantly pRb andother members of the pocket protein family, p107 andp130 (14). The partial phosphorylation of pRb resultsin the liberation of bound transcription factors, par-ticularly those of the E2F family, which in turn bindto the upstream regulatory elements of genes whosetranscription and function are essential for S phaseprogression. Interestingly, cyclin E1 is an E2F targetgene, and thus the initial partial phosphorylation ofpRb results in the induction of cyclin E protein inmid- to late-G1 phase and the formation of activecyclin E-Cdk2 complexes. Consequent completephosphorylation of pRb negates its inhibitory actionon G1 to S phase progression. In addition to this pre-dominantly transcriptional mechanism of control ofG1 phase cyclin–Cdk complexes, the D cyclins titratethe balance of the Kip inhibitors, p21Waf1/Cip1 andp27Kip1, between cyclin E–Cdk2 complexes, in whichthey inhibit kinase activity, and cyclin D–Cdk4/6complexes, in which they act as stabilizing assemblyfactors (13,15). These mechanisms ensure close inter-play between the D-type and E cyclins in the ordered

control of cell cycle progression following mitogenicstimulation. Consequently, genetic changes thatresult in perturbation of this homeostatic mechanismat any level would be expected to contribute to lossof normal growth control and oncogenesis.

CYCLINS AND BREAST EPITHELIALCELL PROLIFERATION

Early studies on the expression and regulation ofG1 cyclins in mammary epithelium were confined tostudies on cultured “normal” human breast epithe-lium and breast cancer cell lines (4,5,16). Interpre-tation of these studies was complicated by the factthat the “normal” cells were derived from basal ep-ithelial cells, which at the time were not thought togive rise to breast carcinomas, while the breast carci-noma cells arose from breast luminal epithelial cells.The latter cells expressed both cyclin D1 and D3 butnot D2, which appeared to be confined to culturesof “normal” basal epithelial cells (4,17). Stimulationof breast cancer cells with mitogenic growth factors,including members of the EGF, IGF, and heregulinfamilies, resulted in the expected transcriptional acti-vation of cyclin D1 in early G1 phase, cyclin D3 in mid-G1 and cyclin E in late G1, with concurrent formationof active Cdk complexes and progression into S phase(16,18), as has been reported in a broad spectrum ofcell types (13). By contrast, studies of the actions ofestrogens and progestins, which were known to regu-late cell cycle progression through effects on G1 phaseprogression, identified some novel mechanisms of cellcycle regulation.

The central involvement of estrogen in thegenesis of breast cancer (19) has been an impetus forstudies linking estrogen action to the cell cycle ma-chinery. Estrogen stimulation of breast cancer cellsarrested in G0/G1 by prior treatment with estrogenantagonists resulted in the transcriptional activationof c-myc and cyclin D1, formation and activation ofcyclin D1–Cdk4 and cyclin E–Cdk2 complexes, pRbphosphorylation and cell cycle progression (20–23).A pivotal role for cyclin D1 was evident from theobservation that these events could be inhibitedby the use of antisense oligomers or antibodiesto cyclin D1 (24) and that inducible expression ofcyclin D1 or c-myc could recapitulate the effectsof estrogen in this model (25). Activation of cyclinE–Cdk2 occurred early in the response to estrogen,in mid-G1 phase, and was not accompanied by majorchanges in cyclin E protein levels. Rather, cyclin

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Cyclins and Breast Cancer 97

E–Cdk2 appeared to be activated predominantly bythe depletion of p21Waf1 from these complexes. Thiseffect resulted from both sequestration of p21Waf1

into the newly formed cyclin D1–Cdk4 complexes,at the expense of cyclin E–p21Waf1–Cdk2 inhibitorycomplexes induced by antiestrogen (20, 23), and as adirect consequence of estrogen-mediated inhibitionof p21Waf1 transcription allowing newly synthesizedcyclin E to form active complexes with Cdk2 inthe absence of inhibitor (26). The latter processmay be the result of c-myc-mediated transcriptionalrepression of p21Waf1 (27), since c-myc is inducedwithin the first hour of estrogen stimulation (20).

In the same model systems progestins are growthinhibitory and arrest cells in G1 phase (28). Growtharrest is accompanied by decreased expression ofboth cyclin D1 and cyclin E and induction of the Cdkinhibitor p18Ink4c. This INK4 inhibitor blocks theformation of cyclin D–Cdk4 complexes, leading toresorting of the cyclin–Cdk-inhibitor complexes andincreasing availability of p27Kip1 to form inhibitorycyclin E–Cdk2-p27Kip1 complexes (29). Thus bothcyclin D–Cdk4 and cyclin E–Cdk2 activities areinhibited following progestin treatment, resultingin decreased pRb phosphorylation and arrest inG1 phase (28,29). Together these data indicate thatsteroids, the major regulators of cell proliferationand differentiation in breast epithelial cells, stimulateor inhibit cell cycle progression through effects onmultiple targets in the pRb pathway. Thus aberrationsin this pathway would be expected to have majoreffects on these normal control mechanisms, withconsequent effects on steroid sensitivity and respon-siveness. Such changes could, in turn, have significantconsequences for the development, progression, andeffective treatment of breast cancer.

Since antiestrogens remain the treatment ofchoice for hormone-dependent breast cancer andhave been known for 20 years to arrest cells in theG0/G1 phase of the cell cycle (30), there has been con-siderable interest in their effects on specific moleculeswithin the pRb pathway. Antiestrogen-mediated cellcycle arrest is associated with decreased cyclin D1gene expression, inactivation of cyclin D1–Cdk4 com-plexes and decreased phosphorylation of pRb (31).Inhibition of cyclin E–Cdk2 also occurred prior to adecrease in the S phase fraction and is dependent onrecruitment of p21Waf1 to cyclin E–Cdk2 complexes, asevidenced by the observation that treatment with an-tisense oligonucleotides to p21Waf1 attenuates the ef-fect (32,33). Recruitment of p21Waf1 to cyclin E–Cdk2complexes is dependent on decreased cyclin D1 gene

expression, which is, in turn, dependent on antiestro-gen inhibition of c-myc gene expression. Indeed anti-sense oligonucleotide inhibition of c-myc expressionto levels that mimic the decreased expression inducedby antiestrogens, is sufficient to initiate the samecascade of events documented above for antiestrogeninhibition of breast cancer cell proliferation (34). Thep27Kip1 inhibitor is also essential for cell cycle arrestby antiestrogens, since antisense-mediated downreg-ulation of p27Kip1 abrogates antiestrogen-induced cellcycle arrest in MCF–7 cells (33). However, p21Waf1

antisense treatment is accompanied by decreasedp27Kip1 protein levels while the reverse is not the case(32), arguing that p21Waf1 initiates inhibition of cyclinE–Cdk2 and consequent accumulation of p27Kip1.

More recent studies identify differences in theeffects of different classes of antiestrogens on cellcycle arrest. While it has long been known that thenonsteroidal antiestrogens, i.e. the selective estrogenreceptor modulators (SERMs), of which tamoxifenis prototypic, arrest cells in early G1 phase (30), thepure steroidal antiestrogens like ICI 182780 appearto arrest cells in a quiescent G0 state (32). This stateis characterized by the formation of transcriptionallyinhibitory p130/E2F4 complexes, the accumulation ofhyperphosphorylated E2F4, and insensitivity to mito-genic growth factors (32,35). Induction of p27Kip1 bythe pure antiestrogen appears critical for induction ofthe G0 state, since transduction of p27Kip1 into SERM-treated cells induces quiescence and resistance togrowth factor mitogens (35). Thus, upregulationof p27Kip1 by pure antiestrogens may contributeto their efficacy in tamoxifen-resistant disease.Further support for this hypothesis comes from theobservation that MAP kinase activation, potentiallyas a consequence of c-erbB receptor overexpression,may contribute to antiestrogen resistance throughdownregulation of p27Kip1 (36). Similarly, dysregu-lation of other cell cycle regulatory genes has beenassociated with endocrine resistance. Overexpressionof c-myc in breast cancer cells results in resistanceto antiestrogens (37) and complete insensitivity toprogestins (our unpublished data). Cyclin D1 overex-pression is accompanied by an initial insensitivity toantiestrogen-mediated growth arrest, but this effectis not maintained following long term treatment,while overexpression of cyclin E1 has little effecton antiestrogen sensitivity in vitro (38). In markedcontrast cyclin D1 confers almost complete resistanceto the growth inhibitory effects of progestins, whilecyclin E1 has a significant but less marked effect (39).Together these data demonstrate that at least in vitro

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overexpression of cyclins D1 and E1 and downregu-lation of p21Waf1 and p27Kip1 modulate the sensitivityof breast cancer cells to clinically relevant breastcancer therapies for hormone-responsive disease.

CYCLINS IN MAMMARY GLANDDEVELOPMENT AND CARCINOGENESIS

Deeper insight into the role of cyclins in mam-mary carcinoma has come from in vivo studiesemploying genetically manipulated mice. Several lab-oratories have studied mammary gland developmentin transgenic mice expressing D- and E-type cyclinsunder the control of mammary-epithelial-cell-specificpromoters, particularly the mouse mammary tumorvirus long terminal repeat (MMTV-LTR). In cyclinD1 transgenics, mammary gland development isperturbed, with increased proliferation and preco-cious lobuloalevolar development typical of earlypregnancy (40). These effects are followed by theformation of adenocarcinomas with papillary andcribriform elements as well as squamous differen-tiation (40). Tumors appear in about 75% of mice,but only after a relatively long latency period of18 months, suggesting that cyclin D1 is a relativelyweak oncogene compared with activated c-neu,Ha-ras, and c-myc, which induce tumors at∼3, 6, and11 months, respectively, when overexpressed underthe regulation of the MMTV-LTR (41–43). Theseobservations also suggest that additional geneticevents may be required for cyclin D1 to exert itsoncogenic potential. More recently MMTV-cyclinD1 transgenics have been shown to lack the pulse ofp16Ink4A expression associated with normal mammarygland involution, suggesting that this abnormalitymay result in expansion of the stem cell populationresponsible for sustained proliferation (44).

MMTV-driven cyclin D2 overexpression in themammary gland leads to increased cell proliferationin the pregnant gland but a partial or complete blockof alveolar differentiation, in marked contrast withthe precocious lobuloalveolar development inducedby cyclin D1 (45). This effect was accompanied by re-duction in the abundance of cyclin D1 isoforms and anincrease in p27Kip1 expression, which may explain thephenotype given the necessity for cyclin D1 functionin normal alveologenesis. Overexpression of cyclin D2resulted in a low frequency of tumor development,with 19% of mice developing tumors (45). The rele-vance of these findings to breast cancer requires fur-ther investigation, since the cyclin D2 gene is normally

methylated in breast cancer (46). Thus, these data maynot necessarily reflect an intrinsic role of cyclin D2,but rather may indicate that when overexpressed inthe luminal epithelium cyclin D2 can mimic the ef-fects of cyclin D1 in inducing tumorigenesis in thisexperimental model. However, the degree to whichthe functions of cyclin D1 and D2 overlap remainsto be fully defined. Data from mice expressing only asingle-D-type cyclin argue for a significant degree ofredundancy (47). However, cyclin D2 has a differentselectivity for Cdk activation, preferentially bindingand activating Cdk2 in human breast epithelial cells(48), and is ineffective in interacting with transcriptionfactors and activating gene expression, a property thatappears unique to cyclin D1 (see below). Thus the roleof cyclin D2 in mammary carcinoma requires furtherinvestigation.

Since cyclin D3 is also frequently overexpressedin cancer and is associated with high grade breastcancers (49), studies of its effects on mammary tu-morigenesis have been eagerly awaited. In a recentpublication, MMTV-cyclin D3 mice, in marked con-trast to their cyclin D1 counterparts, demonstratednormal mammary gland development and involutionfollowing lactation. However, these mice went on todevelop mammary carcinoma at high frequency, i.e.in 73% of mice after multiple pregnancies. Further-more, expression of the Cdk inhibitors, p16Ink4A andp27Kip1 were modulated in a similar manner to thatdescribed in other MMTV-cyclin D models (50). In-terestingly, these mice developed squamous cell carci-noma as opposed to the predominant development ofadenocarcinoma in the other two models (40,45,50).Together these data demonstrate that overexpressionof each of the three D-type cyclins in the mammarygland can result in the development of carcinoma, butthat gene-specific differences are apparent. These aremanifest in the differential effects on development ofthe normal mammary gland and in the different phe-notypes of the cancers, such that cyclin D1 and D2produce adenocarcinoma while cyclin D3 induces apredominantly squamous phenotype. Perhaps thesedifferences can be explained in part by the three D-type cyclin genes having similar effects on mammaryepithelial cell proliferation but distinct effects ondifferentiation.

The consequences of cyclin E1 overexpressionhave been investigated in transgenic mice in which thehuman gene was expressed under the control of theovine β-globulin promoter, resulting in mammary-specific expression during pregnancy and lactation(51). This expression resulted in papillary projections

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of hyperplastic cells during the first pregnancy, themajority of which were eliminated during subsequentmammary gland involution following weaning. How-ever ∼10% of female mice developed adenocarcino-mas after 8–13 months, and these tumors had signifi-cantly elevated levels of cyclin E mRNA and proteinand of cyclin E-associated kinase activity. Thus, likethe D-type cyclins, cyclin E1 is a “weak” oncogene inmammary epithelium.

The ability of cyclin D1 overexpression to inducemammary carcinoma and the necessity for cyclinD1 function for cell cycle progression raises thequestion of whether cyclin D1 is necessary for tumorformation. Mammary gland development is impairedin cyclin D1 null mice such that, although the basicductal structure forms normally at puberty, alveoli donot develop during pregnancy and lactation does notoccur (52,53). This defect does not appear to reflecta necessity for cyclin D1 per se, since epitheliumlacking both cyclin D1 and p27Kip1 can form a normalmammary gland (54,55), as can epithelium in whichcyclin D1 is replaced by cyclin E1 (11). Instead itappears that the specific requirement is for timelyepithelial cell proliferation. Interestingly, cyclin D2and D3 are not required for normal mammary glanddevelopment, since gland development is normal incyclin D2 and D3 null mice (47) although, as notedabove, overexpression of these genes induces mam-mary carcinoma. Crosses of cyclin D1 null mice withmice expressing different mammary oncogenes underthe control of the MMTV promoter has providedimportant insights into the role of cyclin D1 in dif-ferent oncogenic pathways. The demonstration that,in the absence of cyclin D1, mammary tumorigenesisis compromised in c-neu and Ha-ras transgenics, butnot in c-myc and Wnt-1 MMTV transgenics, identifiescyclin D1 as a critical component of some pathwaysof mammary tumorigenesis (56). Further mechanisticstudies into the molecular basis of these effectsprovides deeper insight into the interactions betweendifferent oncogenes in a tissue-specific context. Whatis clearly apparent from the earlier studies in cyclinD1 null mice is that cyclin D1 plays a very specificrole in mammary gland development, since thedefect in lobuloalveolar development occurs in thepresence of potentially redundant D2 and D3 cyclins.This effect is thought to result from the failure ofupregulation of other cyclins in the mammary glandrather than to functional diversity of the cyclins(56). Similarly, MMTV-neu-induced and MMTV-ras-induced tumorigenesis was not impaired in cyclin D2and D3 null mice, and cyclin E1 could substitute for

cyclin D1 in tumorigenesis as well as in mammarygland development (56). These observations implythat it is the tissue-specific regulation and timing ofcyclin D1 expression that is critical to these processes.In support of this concept, increased expression ofcyclin D1, but not cyclins D2 and D3, was observedin MMTV-neu-induced and MMTV-ras-inducedmammary tumors, while Wnt-1 and c-myc-inducedtumors expressed both cyclins D1 and D2 (56).

The lack of dependence of c-myc-induced tumorson cyclin D1 is perhaps not surprising given evidencethat in breast cancer cells these two oncogenes can ac-tivate cyclin E–Cdk2 by separate pathways (25) andthat c-myc can induce cyclin D2 expression and se-questration of p27Kip1 into Cdk 4/6 complexes (57).More recent data from transcript profiles of differentoncogene-induced mammary cancers in mice confirmupregulation of cyclin D1 in MMTV-neu and -ras-induced tumors, whereas in MMTV-myc-induced tu-mors cyclins D2, E1, and E2 were upregulated (58).The fact that Wnt-1 can induce precocious mammarygland development and tumorigenesis in cyclin D1null mice implies that Wnt-1 signaling via β-cateninand increased cyclin D1 gene expression is not themajor pathway of activation, as proposed in other celltypes. While this pathway is probably intact in themammary gland and breast cancer, these data sup-port a role for Wnt-1 induction of cyclin D2 as a sig-nificant downstream effector in mammary epithelium(56). Taken together, these data provide compellingevidence for a requirement for appropriate regulationof cyclin D1 gene expression in the development ofthe mouse mammary gland and in the induction ofspecific groups of mammary carcinoma. By contrastcyclins D2, D3, E1, and E2 are not required for nor-mal mammary gland development; whether they arenecessary downstream of some mammary oncogenes,e.g., c-myc has yet to be determined.

CYCLIN OVEREXPRESSIONIN BREAST CANCER

Following the initial discovery that cyclin D1 wasone of the most commonly overexpressed oncogenesin breast cancer (4), an increasing body of literaturehas addressed the relationships between cyclin D1 ex-pression and various clinicopathological features ofbreast cancer (reviewed in (59)). Cyclin D1 is over-expressed in 30–60% of primary ductal adenocarci-nomas and is expressed early in the disease process,as evidenced by significantly increased expression

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in ductal hyperplasia (7) and ductal carcinoma insitu (60). Interestingly, cyclin D1 is almost universallyoverexpressed in lobular carcinomas (61), consistentwith an essential role for cyclin D1 in normal lobu-lar development. Early studies established a strongrelationship between cyclin D1 expression and ERstatus (4–6,59), which was confirmed at the mRNAlevel (10). A potential functional link between ERexpression and cyclin D1 expression is supported byevidence that cyclin D1 is a major downstream targetof estrogen action and plays a pivotal role in estrogen-induced mitogenesis in breast cancer cells (20–24).

The relationship between overexpression ofcyclin D1 and breast cancer outcome has been con-troversial, with studies reporting both positive andnegative findings (62–64). This controversy is perhapsnot surprising given the confounding issues of therelationship with ER status, itself an independentmarker of prognosis, and the interrelationships withother molecules in the pRb pathway, most of whichwere not measured concurrently. Subgroup analyseswithin relatively small patient cohorts have also beena major impediment to drawing any definitive conclu-sions. Notwithstanding these significant limitations,there appears to be a relationship between CCND1gene amplification and poor disease outcome in ER+patients (65–67). In contrast, cyclin D1 protein ex-pression is associated with a good prognosis in somestudies (62,68), potentially as a consequence of itspositive relationship with ER expression and negativerelationship with Rb mutations. Other studies failedto confirm this relationship (59,63). Interpretation isfurther complicated by suggestions that under somecircumstances cyclin D1 overexpression may lead toa worse clinical outcome by conferring resistance toendocrine treatments (69,70). Consistent with thispossibility, one small clinical study suggested that theduration of response to tamoxifen was significantlylonger in ER+ patients with low cyclin D1 thanthose with high cyclin D1 (69). Resolution of theseissues must await more detailed analysis of cyclinD1 expression and patient outcome in the context ofprospective randomized clinical trials.

As noted above, cyclin D2 is expressed in nor-mal human mammary epithelial cells, but detectableexpression is rare in breast cancer (4,17). This obser-vation is due to promoter hypermethylation in the ma-jority of cancers (46), but the functional significance,if any, in breast oncogenesis has yet to be elucidated,although potential roles of cyclin D2 in terminal dif-ferentiation and senescence of human breast epithe-lium have been proposed. Cyclin D3 has also been

reported to be overexpressed in breast cancers, butthere are limited data on its relationship to pheno-type and disease outcome (49).

As expected from studies on breast cancercell lines (4,5), cyclin E1 is abnormally expressedin ∼40% of breast cancers, in which the protein isoverexpressed as a series of isoforms ranging in sizefrom 35–50 kDa (8). The level of expression increaseswith increasing tumor stage and grade and appearsto be associated with high proliferation rates (8,9).In contrast to cyclin D1, cyclin E is overexpressedpredominantly in the ER- phenotype, in which it isassociated with a significantly increased risk of breastcancer relapse and death (9,71,72). Furthermore,about 40% of breast cancers overexpressing cyclin Ehave mutant pRb and high p16Ink4A levels, suggestingthat cyclin E expression may be linked to dysregula-tion of the pRb pathway (9). With the knowledge ofthe close interplay between cyclin E and p27Kip1 incell cycle regulation, it has been demonstrated thatcyclin E overexpression coupled with low p27Kip1

expression was of greater prognostic significancethan cyclin E alone (73).

The prognostic significance of cyclin E1 overex-pression is further influenced by the relative ratios ofwild type and truncated forms of the protein (71). In-deed, when outcome was related to overexpression ofthe low molecular weight forms of cyclin E, the prog-nostic power exceeded that of positive lymph nodestatus, the most important clinical marker of breastcancer outcome. Potential explanations for this effectreside in the observation that these low molecularweight, N-terminally truncated variants are tumor-cell-specific and are more efficient in mediating G1

to S phase transition (5). Further studies are requiredto verify these findings, since relationships betweencyclin E expression and outcome are dependent onthe parameters measured, i.e. cyclin E protein levelsby immunohistochemistry or Western blot (which al-lows assessment of both total- and isoform-specificexpression) and cyclin E mRNA. A recent study em-ploying the latter method failed to establish a relation-ship between cyclin E mRNA levels and relapse-freeor overall survival. However, high cyclin E mRNAlevels were associated with poor relapse-free survivalonly in patients treated with adjuvant endocrine ther-apy (72), supporting the view that cyclin E may conferendocrine resistance.

Together these data provide support for the viewthat cyclins D1 and E1 are intimately involved in theevolution of ER+ and ER− breast cancer, respec-tively. They also provide preliminary evidence that

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Table I. Potential Functional Roles for G1 Cyclins

Cyclin Binding partner Function

D1/D2/D3 Cdk4/6 Cell cycle progressionD2 Cdk2 Cell cycle progressionE Cdk2 Cell cycle progressionD1 ER, C/EBPβ Transcriptional activationD1 AR, Beta2/Neuro D, DMP1, Myb, Transcriptional repression

MyoD, SP1, STAT3, TRD1 AIB-1. GRIP-1, SRC1a CoactivationD1 CBP/p300, P/CAF Chromatin remodelingD1 TAF5250 Formation of the initiation complex

and recruitment of RNA polymerase IIE AR Coactivation

these genes may be useful markers of disease pro-gression and potential therapeutic responsiveness, butfurther studies are required to clarify these issues.

FUNCTIONAL CONSEQUENCES OF CYCLIND1 AND E1 OVEREXPRESSION

Since overexpression of D-type cyclins and cyclinE in a number of cell types, including breast epithe-lial cells (74), shortens G1 phase and accelerates Sphase entry, it was initially assumed that their overex-pression would result in increased proliferation ratesin breast cancer. Support for this hypothesis exists inthe case of cyclin E where overexpression is associ-ated with ER− negativity, high tumor grade, and ahigh proliferative index, as outlined above. However,when a panel of normal, immortalized, and neoplasticbreast epithelial cell lines was employed to determinerelationships between cyclin gene expression, cyclin–Cdk complex formation and Cdk activity, no corre-lations between overexpression of cyclin D1 and Eand the respective activities of Cdk4 and cyclin E–Cdk2 were evident (75). Similarly, several studies as-sessing the relationship between cyclin D1 expressionand markers of cell proliferation, e.g. S phase fractionand Ki67 staining, failed to demonstrate a relation-ship (59). Indeed there is now conclusive evidencethat cyclin D1 overexpression is associated with theER+, slow-growing, more differentiated phenotypeof breast cancer.

Subsequent studies demonstrating that cyclinD1 can bind to ERα in a Cdk-independent mannerand enhance ER-mediated transcription forced a re-evaluation of the role of cyclin D1 in oncogenesis(76,77). It is now clear that cyclin D1 can form poten-tially functional interactions with a variety of other

molecules (Table I) including cellular transcriptionfactors, e.g. ER, androgen receptor, DMP1, STAT3,BETA2/NeuroD, and most recently C/EBPβ(78), aswell as with both histone acetylases and deacetylases(reviewed in (79)). These interactions are indepen-dent of association with, and activation of, Cdk4 and−6 and point to a role for cyclin D1 in transcriptionalregulation. That these interactions are likely to be ofimportance in a physiological context is supportedby evidence that the functional interaction betweencyclin D1 and ERα is regulated by a signal trans-duction pathway involving cAMP (80). Thus PKA-dependent extracellular signals appear to be requiredfor the functional interaction between ER and cyclinD1 in mammary epithelial cells, resulting in enhancedligand-independent, ER-mediated transcription.

In a more recent development, Lamb et al. haveemployed adenoviral infection of MCF-7 cells withwild type cyclin D1 and the K112E mutant, which isincapable of activating Cdk4, to establish a pattern ofgene expression associated with the Cdk-independentactions of cyclin D1 (78). This gene expression profilewas represented across a broad spectrum of humancancers that overexpressed cyclin D1, includingbreast cancers, and the transcription factor C/EBPβemerged as a potential effector molecule. Functionalanalysis demonstrating that cyclin D1 antagonizesa repressive function of C/EBPβ on cyclin D1target genes provided compelling evidence for theinvolvement of C/EBPβ in cyclin D1-overexpressingtumors (78). Importantly, earlier experiments iden-tifying that changes in the relative expression ofthe two C/EBPβ isoforms, LAP and LIP, in themouse mammary gland led to the developmentof invasive carcinomas (81), provided precedencefor the proposed mechanisms. Together these dataidentify modulation of transcription by cyclin D1 as

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a likely primary event in breast oncogenesis. Recentevidence that cyclin E is a coactivator of the androgenreceptor (82) and that the oncogenic activity of cyclinE in vitro is independent of Cdk2 activation (83)suggests that Cdk-independent mechanisms may alsobe important in cyclin E-overexpressing cells.

CONCLUSIONS

Extensive investigation over the past decadehave identified potentially important functionalroles for the D- and E-type cyclins in the evolutionof human breast cancers. These genes are amongthe most commonly overexpressed genes in breastcancer, they are overexpressed in the early phases ofdisease development and they have proven oncogeniceffects on mammary epithelial cells both in vitroand in vivo. Their established role in Cdk activationand regulation of the Rb pathway focused initialattention on aberrant cell cycle regulation as thebasis of their oncogenic potential. More recent dataon the role of different G1 cyclins in differentiation,chromosome stability, and transcriptional regulationmake it clear that their role in breast cancer ismuch more complex than initially envisaged. Furtherinvestigation is likely to yield a deeper understandingof the role of these cyclins in the pathophysiology ofbreast cancer, with potential clinical benefits throughthe identification of new markers of prognosis andtherapeutic responsiveness and potential new targetsfor innovative therapeutic intervention.

ACKNOWLEDGMENTS

Work in our laboratories is supported by grantsfrom the National Health and Medical ResearchCouncil of Australia (NHMRC), The Cancer CouncilNSW, U.S. Army Medical Research and MaterielCommand (DAMD17-00-1-0252 and DAMD17-99-1-9184), Association for International CancerResearch (AICR), and Susan G Komen BreastCancer Foundation.

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