Cyclins and Breast Cancer

[Farman Ali, Z – 30] , [Abid Aslam , Z – 26] , [Hamayun Akbar, Z – 40] , [Umar Farooq, Z – 43]
Department of Zoology Govt, Superior Science college Peshawar. (Affiliated with University of Peshawar)

The D-type and E-type cyclins control the G 1 to S phase transition during normal cell cycle
progression and are critical components of steroid- and growth factor-induced mitogenesis in
breast epithelial cells. Mammary epithelial cell-speci c overexpression of these genes leads
to mammary carcinoma, while in cyclin D1-de cient mice mammary gland development is
arrested prior to lobuloalveolar development. Cyclin D1 null mice are resistant to mammary
carcinoma induced by theneuandrasoncogenes, indicating an essential role for cyclin D1 in
the development of some mammary cancers. Cyclin D1 and E1 are commonly overexpressed
in 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 endocrine
therapies. The functional consequences of cyclin E overexpression in breast cancer are likely
related to its role in cell cycle progression, whereas that of cyclin D1 may also be a consequence
of a more recently de ned role in transcriptional regulation.

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

INTRODUCTION through promoter methylation (2). These aberrations

Loss of normal growth control, including
aberrations in the homeostatic mechanisms that
ensure integrity of cell cycle progression, is a hall-
mark of cancer (1). A pivotal regulatory pathway
determining 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 this
pathway through overexpression, mutation, and
epigenetic gene silencing are almost universal in
human cancer (2). Interestingly, there appears to be
a degree of tissue speci city in the particular genetic
abnormalities within the Rb pathway. In breast
cancer these include the overexpression of cyclins
D1, 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 Cancer
Research Program, Garvan Institute of Medical Research, 384
Victoria Street, Darlinghurst, New South Wales Sydney, 2010,
Australia; e-mail: r.sutherland@garvan.org.au.
occur at relatively high frequency in breast cancer,
i.e. each abnormality is present in 30–45% of primary
tumors, implying a major role for loss of function of
the Rb pathway in breast oncogenesis.
Cyclin D1 is the product of theCCND1gene and
was rst implicated in breast cancer following local-
ization of the gene to chromosome 11q13, a region
of the genome that is commonly ampli ed in a range
of human carcinomas, including about 15% of breast
cancers (3). The subsequent demonstration that cy-
clin D1 was overexpressed at the mRNA and protein
level in up to 50% of primary breast cancers identi ed
cyclin D1 as one of the most commonly overexpressed
oncogenes in breast cancer (4–7). Although ampli -
cation of the cyclin E1 locus is a relatively rare event
in breast cancer, the protein product is overexpressed
in∼40% of breast cancers (8,9). Interestingly, cyclin
D1 is overexpressed predominantly in ER+tumors
while cyclin E overexpression is con ned almost ex-
clusively to ER−tumors (4–10). Since these proteins
Abbreviations used:Cdk; cyclin-dependent kinase; ER, estrogen
receptor; pRb, retinoblastoma protein; MMTV, mouse mammary
tumor virus.
appear to have redundant functions in the mammary
gland, as evidenced by the ability of cyclin E to substi-
tute for cyclin D1 (11), it is tempting to speculate that
they might be exerting similar roles in tumorigenesis
in these two different breast cancer phenotypes.
This review summarizes contemporary literature
addressing the functions of D- and E-type cyclins in
mammary epithelial cells and their potential roles in
the development and progression of human breast
cancer.
CYCLINS AND CELL CYCLE CONTROL
In eukaryotes, cell cycle progression is mediated
by the sequential activation and inactivation of the
Cdk serine/threonine kinases (12). These enzyme
complexes contain a Cdk catalytic subunit, the
expression of which remains relatively uniform
throughout the cell cycle, and a regulatory cyclin
subunit, the abundance of which is tightly regulated
by transcriptional regulation, subcellular localization
and protein degradation, thus allowing activation
at distinct phases of cell cycle progression (12,13).
Mitogenic stimulation of growth-arrested cells results
initially in the induction of the D-type cyclins (cyclins
D1, D2, and D3), and these molecules are believed
to have the primary function of linking extracellular
signals to the cell cycle machinery. The D-type cyclins
bind preferentially to Cdks 4 and 6 and phosphorylate
key downstream substrates, predominantly pRb and
other members of the pocket protein family, p107 and
p130 (14). The partial phosphorylation of pRb results
in the liberation of bound transcription factors, par-
ticularly those of the E2F family, which in turn bind
to the upstream regulatory elements of genes whose
transcription and function are essential for S phase
progression. Interestingly, cyclin E1 is an E2F target
gene, and thus the initial partial phosphorylation of
pRb results in the induction of cyclin E protein in
mid- to late-G1 phase and the formation of active
cyclin E-Cdk2 complexes. Consequent complete
phosphorylation of pRb negates its inhibitory action
on G1 to S phase progression. In addition to this pre-
dominantly transcriptional mechanism of control of
G1 phase cyclin–Cdk complexes, the D cyclins titrate
the balance of the Kip inhibitors, p21 Waf1/Cip1 and
p27Kip1, between cyclin E–Cdk2 complexes, in which
they inhibit kinase activity, and cyclin D–Cdk4/6
complexes, in which they act as stabilizing assembly
factors (13,15). These mechanisms ensure close inter-
play between the D-type and E cyclins in the ordered
control of cell cycle progression following mitogenic
stimulation. Consequently, genetic changes that
result in perturbation of this homeostatic mechanism
at any level would be expected to contribute to loss
of normal growth control and oncogenesis.
CYCLINS AND BREAST EPITHELIAL
CELL PROLIFERATION
Early studies on the expression and regulation of
G1 cyclins in mammary epithelium were con ned to
studies on cultured “normal” human breast epithe-
lium and breast cancer cell lines (4,5,16). Interpre-
tation of these studies was complicated by the fact
that the “normal” cells were derived from basal ep-
ithelial cells, which at the time were not thought to
give 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 but
not D2, which appeared to be con ned to cultures
of “normal” basal epithelial cells (4,17). Stimulation
of breast cancer cells with mitogenic growth factors,
including members of the EGF, IGF, and heregulin
families, 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 formation
of active Cdk complexes and progression into S phase
(16,18), as has been reported in a broad spectrum of
cell types (13). By contrast, studies of the actions of
estrogens and progestins, which were known to regu-
late cell cycle progression through effects on G1 phase
progression, identi ed some novel mechanisms of cell
cycle regulation.
The central involvement of estrogen in the
genesis of breast cancer (19) has been an impetus for
studies linking estrogen action to the cell cycle ma-
chinery. Estrogen stimulation of breast cancer cells
arrested in G0/G1 by prior treatment with estrogen
antagonists resulted in the transcriptional activation
of c-mycand cyclin D1, formation and activation of
cyclin D1–Cdk4 and cyclin E–Cdk2 complexes, pRb
phosphorylation and cell cycle progression (20–23).
A pivotal role for cyclin D1 was evident from the
observation that these events could be inhibited
by the use of antisense oligomers or antibodies
to cyclin D1 (24) and that inducible expression of
cyclin D1 or c-myccould recapitulate the effects
of estrogen in this model (25). Activation of cyclin
E–Cdk2 occurred early in the response to estrogen,
in mid-G1 phase, and was not accompanied by major
changes in cyclin E protein levels. Rather, cyclin
E–Cdk2 appeared to be activated predominantly by
the depletion of p21Waf1 from these complexes. This
effect resulted from both sequestration of p21Waf1
into the newly formed cyclin D1–Cdk4 complexes,
at the expense of cyclin E–p21Waf1 –Cdk2 inhibitory
complexes induced by antiestrogen (20, 23), and as a
direct consequence of estrogen-mediated inhibition
of p21Waf1 transcription allowing newly synthesized
cyclin E to form active complexes with Cdk2 in
the absence of inhibitor (26). The latter process
may be the result of c-myc-mediated transcriptional
repression of p21Waf1 (27), since c-mycis induced
within the rst hour of estrogen stimulation (20).
In the same model systems progestins are growth
inhibitory and arrest cells in G 1 phase (28). Growth
arrest is accompanied by decreased expression of
both cyclin D1 and cyclin E and induction of the Cdk
inhibitor p18Ink4c. This INK4 inhibitor blocks the
formation of cyclin D–Cdk4 complexes, leading to
resorting of the cyclin–Cdk-inhibitor complexes and
increasing availability of p27Kip1 to form inhibitory
cyclin E–Cdk2-p27Kip1 complexes (29). Thus both
cyclin D–Cdk4 and cyclin E–Cdk2 activities are
inhibited following progestin treatment, resulting
in decreased pRb phosphorylation and arrest in
G1 phase (28,29). Together these data indicate that
steroids, the major regulators of cell proliferation
and differentiation in breast epithelial cells, stimulate
or inhibit cell cycle progression through effects on
multiple targets in the pRb pathway. Thus aberrations
in this pathway would be expected to have major
effects on these normal control mechanisms, with
consequent effects on steroid sensitivity and respon-
siveness. Such changes could, in turn, have signi cant
consequences for the development, progression, and
effective treatment of breast cancer.
Since antiestrogens remain the treatment of
choice for hormone-dependent breast cancer and
have been known for 20 years to arrest cells in the
G0/G1 phase of the cell cycle (30), there has been con-
siderable interest in their effects on speci c molecules
within the pRb pathway. Antiestrogen-mediated cell
cycle arrest is associated with decreased cyclin D1
gene expression, inactivation of cyclin D1–Cdk4 com-
plexes and decreased phosphorylation of pRb (31).
Inhibition of cyclin E–Cdk2 also occurred prior to a
decrease in the S phase fraction and is dependent on
recruitment of p21Waf1 to cyclin E–Cdk2 complexes, as
evidenced by the observation that treatment with an-
tisense oligonucleotides to p21Waf1 attenuates the ef-
fect (32,33). Recruitment of p21Waf1 to cyclin E–Cdk2
complexes is dependent on decreased cyclin D1 gene
expression, which is, in turn, dependent on antiestro-
gen inhibition of c-mycgene expression. Indeed anti-
sense oligonucleotide inhibition of c-mycexpression
to levels that mimic the decreased expression induced
by antiestrogens, is suf cient to initiate the same
cascade of events documented above for antiestrogen
inhibition of breast cancer cell proliferation (34). The
p27Kip1 inhibitor is also essential for cell cycle arrest
by antiestrogens, since antisense-mediated downreg-
ulation of p27Kip1 abrogates antiestrogen-induced cell
cycle arrest in MCF–7 cells (33). However, p21Waf1
antisense treatment is accompanied by decreased
p27Kip1 protein levels while the reverse is not the case
(32), arguing that p21Waf1 initiates inhibition of cyclin
E–Cdk2 and consequent accumulation of p27Kip1 .
More recent studies identify differences in the
effects of different classes of antiestrogens on cell
cycle arrest. While it has long been known that the
nonsteroidal antiestrogens, i.e. the selective estrogen
receptor modulators (SERMs), of which tamoxifen
is prototypic, arrest cells in early G1 phase (30), the
pure steroidal antiestrogens like ICI 182780 appear
to arrest cells in a quiescent G0 state (32). This state
is characterized by the formation of transcriptionally
inhibitory p130/E2F4 complexes, the accumulation of
hyperphosphorylated E2F4, and insensitivity to mito-
genic growth factors (32,35). Induction of p27Kip1 by
the pure antiestrogen appears critical for induction of
the G0 state, since transduction of p27Kip1 into SERM-
treated cells induces quiescence and resistance to
growth factor mitogens (35). Thus, upregulation
of p27Kip1 by pure antiestrogens may contribute
to their ef cacy in tamoxifen-resistant disease.
Further support for this hypothesis comes from the
observation that MAP kinase activation, potentially
as a consequence of c-erbBreceptor overexpression,
may contribute to antiestrogen resistance through
downregulation of p27Kip1 (36). Similarly, dysregu-
lation of other cell cycle regulatory genes has been
associated with endocrine resistance. Overexpression
of c-mycin breast cancer cells results in resistance
to antiestrogens (37) and complete insensitivity to
progestins (our unpublished data). Cyclin D1 overex-
pression is accompanied by an initial insensitivity to
antiestrogen-mediated growth arrest, but this effect
is not maintained following long term treatment,
while overexpression of cyclin E1 has little effect
on antiestrogen sensitivity in vitro (38). In marked
contrast cyclin D1 confers almost complete resistance
to the growth inhibitory effects of progestins, while
cyclin E1 has a signi cant but less marked effect (39).
Together these data demonstrate that at least in vitro
overexpression of cyclins D1 and E1 and downregu-
lation of p21Waf1 and p27Kip1 modulate the sensitivity
of breast cancer cells to clinically relevant breast
cancer therapies for hormone-responsive disease.
CYCLINS IN MAMMARY GLAND
DEVELOPMENT AND CARCINOGENESIS
Deeper insight into the role of cyclins in mam-
mary carcinoma has come fromin vivostudies
employing genetically manipulated mice. Several lab-
oratories have studied mammary gland development
in transgenic mice expressing D- and E-type cyclins
under the control of mammary-epithelial-cell-speci c
promoters, particularly the mouse mammary tumor
virus long terminal repeat (MMTV-LTR). In cyclin
D1 transgenics, mammary gland development is
perturbed, with increased proliferation and preco-
cious lobuloalevolar development typical of early
pregnancy (40). These effects are followed by the
formation of adenocarcinomas with papillary and
cribriform elements as well as squamous differen-
tiation (40). Tumors appear in about 75% of mice,
but only after a relatively long latency period of
18 months, suggesting that cyclin D1 is a relatively
weak oncogene compared with activated c-neu,
Ha-ras, and c-myc, which induce tumors at ∼ 3, 6, and
11 months, respectively, when overexpressed under
the regulation of the MMTV-LTR (41–43). These
observations also suggest that additional genetic
events may be required for cyclin D1 to exert its
oncogenic potential. More recently MMTV-cyclin
D1 transgenics have been shown to lack the pulse of
p16Ink4A expression associated with normal mammary
gland involution, suggesting that this abnormality
may result in expansion of the stem cell population
responsible for sustained proliferation (44).
MMTV-driven cyclin D2 overexpression in the
mammary gland leads to increased cell proliferation
in the pregnant gland but a partial or complete block
of alveolar differentiation, in marked contrast with
the precocious lobuloalveolar development induced
by cyclin D1 (45). This effect was accompanied by re-
duction in the abundance of cyclin D1 isoforms and an
increase in p27Kip1 expression, which may explain the
phenotype given the necessity for cyclin D1 function
in normal alveologenesis. Overexpression of cyclin D2
resulted in a low frequency of tumor development,
with 19% of mice developing tumors (45). The rele-
vance of these ndings to breast cancer requires fur-
ther investigation, since the cyclin D2 gene is normally
methylated in breast cancer (46). Thus, these data may
not necessarily re ect an intrinsic role of cyclin D2,
but rather may indicate that when overexpressed in
the luminal epithelium cyclin D2 can mimic the ef-
fects of cyclin D1 in inducing tumorigenesis in this
experimental model. However, the degree to which
the functions of cyclin D1 and D2 overlap remains
to be fully de ned. Data from mice expressing only a
single-D-type cyclin argue for a signi cant degree of
redundancy (47). However, cyclin D2 has a different
selectivity for Cdk activation, preferentially binding
and activating Cdk2 in human breast epithelial cells
(48), and is ineffective in interacting with transcription
factors and activating gene expression, a property that
appears unique to cyclin D1 (see below). Thus the role
of cyclin D2 in mammary carcinoma requires further
investigation.
Since cyclin D3 is also frequently overexpressed
in cancer and is associated with high grade breast
cancers (49), studies of its effects on mammary tu-
morigenesis have been eagerly awaited. In a recent
publication, MMTV-cyclin D3 mice, in marked con-
trast to their cyclin D1 counterparts, demonstrated
normal mammary gland development and involution
following lactation. However, these mice went on to
develop mammary carcinoma at high frequency, i.e.
in 73% of mice after multiple pregnancies. Further-
more, expression of the Cdk inhibitors, p16Ink4A and
p27Kip1 were modulated in a similar manner to that
described in other MMTV-cyclin D models (50). In-
terestingly, these mice developed squamous cell carci-
noma as opposed to the predominant development of
adenocarcinoma in the other two models (40,45,50).
Together these data demonstrate that overexpression
of each of the three D-type cyclins in the mammary
gland can result in the development of carcinoma, but
that gene-speci c differences are apparent. These are
manifest in the differential effects on development of
the normal mammary gland and in the different phe-
notypes of the cancers, such that cyclin D1 and D2
produce adenocarcinoma while cyclin D3 induces a
predominantly squamous phenotype. Perhaps these
differences can be explained in part by the three D-
type cyclin genes having similar effects on mammary
epithelial cell proliferation but distinct effects on
differentiation.
The consequences of cyclin E1 overexpression
have been investigated in transgenic mice in which the
human gene was expressed under the control of the
ovine -globulin promoter, resulting in mammary-
speci c expression during pregnancy and lactation
(51). This expression resulted in papillary projections
of hyperplastic cells during the rst pregnancy, the
majority of which were eliminated during subsequent
mammary gland involution following weaning. How-
ever∼10% of female mice developed adenocarcino-
mas after 8–13 months, and these tumors had signi -
cantly elevated levels of cyclin E mRNA and protein
and of cyclin E-associated kinase activity. Thus, like
the D-type cyclins, cyclin E1 is a “weak” oncogene in
mammary epithelium.
The ability of cyclin D1 overexpression to induce
mammary carcinoma and the necessity for cyclin
D1 function for cell cycle progression raises the
question of whether cyclin D1 is necessary for tumor
formation. Mammary gland development is impaired
in cyclin D1 null mice such that, although the basic
ductal structure forms normally at puberty, alveoli do
not develop during pregnancy and lactation does not
occur (52,53). This defect does not appear to re ect
a necessity for cyclin D1 per se, since epithelium
lacking both cyclin D1 and p27Kip1 can form a normal
mammary gland (54,55), as can epithelium in which
cyclin D1 is replaced by cyclin E1 (11). Instead it
appears that the speci c requirement is for timely
epithelial cell proliferation. Interestingly, cyclin D2
and D3 are not required for normal mammary gland
development, since gland development is normal in
cyclin D2 and D3 null mice (47) although, as noted
above, overexpression of these genes induces mam-
mary carcinoma. Crosses of cyclin D1 null mice with
mice expressing different mammary oncogenes under
the control of the MMTV promoter has provided
important insights into the role of cyclin D1 in dif-
ferent oncogenic pathways. The demonstration that,
in the absence of cyclin D1, mammary tumorigenesis
is compromised in c-neuand Ha-rastransgenics, but
not in c-mycandWnt-1 MMTV transgenics, identi es
cyclin D1 as a critical component of some pathways
of mammary tumorigenesis (56). Further mechanistic
studies into the molecular basis of these effects
provides deeper insight into the interactions between
different oncogenes in a tissue-speci c context. What
is clearly apparent from the earlier studies in cyclin
D1 null mice is that cyclin D1 plays a very speci c
role in mammary gland development, since the
defect in lobuloalveolar development occurs in the
presence of potentially redundant D2 and D3 cyclins.
This effect is thought to result from the failure of
upregulation of other cyclins in the mammary gland
rather than to functional diversity of the cyclins
(56). Similarly, MMTV-neu-induced and MMTV-ras-
induced tumorigenesis was not impaired in cyclin D2
and D3 null mice, and cyclin E1 could substitute for
cyclin D1 in tumorigenesis as well as in mammary
gland development (56). These observations imply
that it is the tissue-speci c regulation and timing of
cyclin D1 expression that is critical to these processes.
In support of this concept, increased expression of
cyclin D1, but not cyclins D2 and D3, was observed
in MMTV-neu-induced and MMTV-ras-induced
mammary tumors, whileWnt-1 and c-myc-induced
tumors expressed both cyclins D1 and D2 (56).
The lack of dependence of c-myc-induced tumors
on cyclin D1 is perhaps not surprising given evidence
that in breast cancer cells these two oncogenes can ac-
tivate cyclin E–Cdk2 by separate pathways (25) and
that c-myccan induce cyclin D2 expression and se-
questration of p27Kip1 into Cdk 4/6 complexes (57).
More recent data from transcript pro les of different
oncogene-induced mammary cancers in mice con rm
upregulation of cyclin D1 in MMTV-neuand-ras-
induced tumors, whereas in MMTV-myc-induced tu-
mors cyclins D2, E1, and E2 were upregulated (58).
The fact thatWnt-1 can induce precocious mammary
gland development and tumorigenesis in cyclin D1
null mice implies thatWnt-1 signaling via -catenin
and increased cyclin D1 gene expression is not the
major pathway of activation, as proposed in other cell
types. While this pathway is probably intact in the
mammary gland and breast cancer, these data sup-
port a role forWnt-1 induction of cyclin D2 as a sig-
ni cant downstream effector in mammary epithelium
(56). Taken together, these data provide compelling
evidence for a requirement for appropriate regulation
of cyclin D1 gene expression in the development of
the mouse mammary gland and in the induction of
speci c groups of mammary carcinoma. By contrast
cyclins D2, D3, E1, and E2 are not required for nor-
mal mammary gland development; whether they are
necessary downstream of some mammary oncogenes,
e.g., c-mychas yet to be determined.
CYCLIN OVEREXPRESSION
IN BREAST CANCER
Following the initial discovery that cyclin D1 was
one of the most commonly overexpressed oncogenes
in breast cancer (4), an increasing body of literature
has addressed the relationships between cyclin D1 ex-
pression and various clinicopathological features of
breast 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 signi cantly increased expression
in ductal hyperplasia (7) and ductal carcinoma in
situ (60). Interestingly, cyclin D1 is almost universally
overexpressed in lobular carcinomas (61), consistent
with an essential role for cyclin D1 in normal lobu-
lar development. Early studies established a strong
relationship between cyclin D1 expression and ER
status (4–6,59), which was con rmed at the mRNA
level (10). A potential functional link between ER
expression and cyclin D1 expression is supported by
evidence that cyclin D1 is a major downstream target
of estrogen action and plays a pivotal role in estrogen-
induced mitogenesis in breast cancer cells (20–24).
The relationship between overexpression of
cyclin D1 and breast cancer outcome has been con-
troversial, with studies reporting both positive and
negative ndings (62–64). This controversy is perhaps
not surprising given the confounding issues of the
relationship with ER status, itself an independent
marker of prognosis, and the interrelationships with
other molecules in the pRb pathway, most of which
were not measured concurrently. Subgroup analyses
within relatively small patient cohorts have also been
a major impediment to drawing any de nitive conclu-
sions. Notwithstanding these signi cant limitations,
there appears to be a relationship betweenCCND1
gene ampli cation and poor disease outcome in ER +
patients (65–67). In contrast, cyclin D1 protein ex-
pression is associated with a good prognosis in some
studies (62,68), potentially as a consequence of its
positive relationship with ER expression and negative
relationship with Rb mutations. Other studies failed
to con rm this relationship (59,63). Interpretation is
further complicated by suggestions that under some
circumstances cyclin D1 overexpression may lead to
a worse clinical outcome by conferring resistance to
endocrine treatments (69,70). Consistent with this
possibility, one small clinical study suggested that the
duration of response to tamoxifen was signi cantly
+
longer in ER patients with low cyclin D1 than
those with high cyclin D1 (69). Resolution of these
issues must await more detailed analysis of cyclin
D1 expression and patient outcome in the context of
prospective randomized clinical trials.
As noted above, cyclin D2 is expressed in nor-
mal human mammary epithelial cells, but detectable
expression 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 signi cance,
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, but
there are limited data on its relationship to pheno-
type and disease outcome (49).
As expected from studies on breast cancer
cell lines (4,5), cyclin E1 is abnormally expressed
in∼40% of breast cancers, in which the protein is
overexpressed as a series of isoforms ranging in size
from 35–50 kDa (8). The level of expression increases
with increasing tumor stage and grade and appears
to be associated with high proliferation rates (8,9).
In contrast to cyclin D1, cyclin E is overexpressed
predominantly in the ER- phenotype, in which it is
associated with a signi cantly increased risk of breast
cancer relapse and death (9,71,72). Furthermore,
about 40% of breast cancers overexpressing cyclin E
have mutant pRb and high p16Ink4A levels, suggesting
that cyclin E expression may be linked to dysregula-
tion of the pRb pathway (9). With the knowledge of
the close interplay between cyclin E and p27Kip1 in
cell cycle regulation, it has been demonstrated that
cyclin E overexpression coupled with low p27Kip1
expression was of greater prognostic signi cance than
cyclin E alone (73).
The prognostic signi cance of cyclin E1 overex-
pression is further in uenced by the relative ratios of
wild type and truncated forms of the protein (71). In-
deed, when outcome was related to overexpression of
the low molecular weight forms of cyclin E, the prog-
nostic power exceeded that of positive lymph node
status, the most important clinical marker of breast
cancer outcome. Potential explanations for this effect
reside in the observation that these low molecular
weight, N-terminally truncated variants are tumor-
cell-speci c and are more ef cient in mediating G1 to
S phase transition (5). Further studies are requiredto
verify these ndings, since relationships between
cyclin E expression and outcome are dependent on
the parameters measured, i.e. cyclin E protein levels
by immunohistochemistry or Western blot (which al-
lows assessment of both total- and isoform-speci c
expression) 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-free
or overall survival. However, high cyclin E mRNA
levels were associated with poor relapse-free survival
only in patients treated with adjuvant endocrine ther-
apy (72), supporting the view that cyclin E may confer
endocrine resistance.
Together these data provide support for the view
that cyclins D1 and E1 are intimately involved in the
evolution of ER +and ER − breast cancer, respec-
tively. They also provide preliminary evidence that
 Table I.Potential Functional Roles for G1 Cyclins
Cyclin Binding partner
D1/D2/D3 Cdk4/6
D2 Cdk2
E Cdk2
D1 ER, C/EBP
D1 AR, Beta2/Neuro D, DMP1, Myb,
MyoD, SP1, STAT3, TR
D1 AIB-1. GRIP-1, SRC1a
D1 CBP/p300, P/CAF
D1 TAF5 250
E AR
these genes may be useful markers of disease pro-
gression and potential therapeutic responsiveness, but
further studies are required to clarify these issues.
FUNCTIONAL CONSEQUENCES OF CYCLIN
D1 AND E1 OVEREXPRESSION
Since overexpression of D-type cyclins and cyclin
E in a number of cell types, including breast epithe-
lial cells (74), shortens G1 phase and accelerates S
phase entry, it was initially assumed that their overex-
pression would result in increased proliferation rates
in breast cancer. Support for this hypothesis exists in
the case of cyclin E where overexpression is associ-
ated with ER − negativity, high tumor grade, and a
high proliferative index, as outlined above. However,
when a panel of normal, immortalized, and neoplastic
breast epithelial cell lines was employed to determine
relationships between cyclin gene expression, cyclin–
Cdk complex formation and Cdk activity, no corre-
lations between overexpression of cyclin D1 and E
and the respective activities of Cdk4 and cyclin E–
Cdk2 were evident (75). Similarly, several studies as-
sessing the relationship between cyclin D1 expression
and markers of cell proliferation, e.g. S phase fraction
and Ki67 staining, failed to demonstrate a relation-
ship (59). Indeed there is now conclusive evidence
that cyclin D1 overexpression is associated with the
ER + , slow-growing, more differentiated phenotype
of breast cancer.
Subsequent studies demonstrating that cyclin
D1 can bind to ER in a Cdk-independent manner
and 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
Function
Cell cycle progression
Cell cycle progression
Cell cycle progression
Transcriptional activation
Transcriptional repression
Coactivation
Chromatin remodeling
Formation of the initiation complex
and recruitment of RNA polymerase II
Coactivation
molecules (Table I) including cellular transcription
factors, e.g. ER, androgen receptor, DMP1, STAT3,
BETA2/NeuroD, and most recently C/EBP (78), as
well 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 transcriptional
regulation. That these interactions are likely to be of
importance in a physiological context is supported
by evidence that the functional interaction between
cyclin D1 and ER is regulated by a signal trans-
duction pathway involving cAMP (80). Thus PKA-
dependent extracellular signals appear to be required
for the functional interaction between ER and cyclin
D1 in mammary epithelial cells, resulting in enhanced
ligand-independent, ER-mediated transcription.
In a more recent development, Lambet al.have
employed adenoviral infection of MCF-7 cells with
wild type cyclin D1 and the K112E mutant, which is
incapable of activating Cdk4, to establish a pattern of
gene expression associated with the Cdk-independent
actions of cyclin D1 (78). This gene expression pro le
was represented across a broad spectrum of human
cancers that overexpressed cyclin D1, including
breast cancers, and the transcription factor C/EBP
emerged as a potential effector molecule. Functional
analysis demonstrating that cyclin D1 antagonizes
a repressive function of C/EBP on cyclin D1
target genes provided compelling evidence for the
involvement of C/EBP in cyclin D1-overexpressing
tumors (78). Importantly, earlier experiments iden-
tifying that changes in the relative expression of
the two C/EBP isoforms, LAP and LIP, in the
mouse mammary gland led to the development
of invasive carcinomas (81), provided precedence
for the proposed mechanisms. Together these data
identify modulation of transcription by cyclin D1 as
a likely primary event in breast oncogenesis. Recent
evidence that cyclin E is a coactivator of the androgen
receptor (82) and that the oncogenic activity of cyclin
E in vitro is independent of Cdk2 activation (83)
suggests that Cdk-independent mechanisms may also
be important in cyclin E-overexpressing cells.
CONCLUSIONS
Extensive investigation over the past decade
have identi ed potentially important functional
roles for the D- and E-type cyclins in the evolution
of human breast cancers. These genes are among
the most commonly overexpressed genes in breast
cancer, they are overexpressed in the early phases of
disease development and they have proven oncogenic
effects on mammary epithelial cells both in vitro
andin vivo. Their established role in Cdk activation
and regulation of the Rb pathway focused initial
attention on aberrant cell cycle regulation as the
basis of their oncogenic potential. More recent data
on the role of different G1 cyclins in differentiation,
chromosome stability, and transcriptional regulation
make it clear that their role in breast cancer is
much more complex than initially envisaged. Further
investigation is likely to yield a deeper understanding
of the role of these cyclins in the pathophysiology of
breast cancer, with potential clinical bene ts through
the identi cation of new markers of prognosis and
therapeutic responsiveness and potential new targets
for innovative therapeutic intervention.
ACKNOWLEDGMENTS
Work in our laboratories is supported by grants
from the National Health and Medical Research
Council of Australia (NHMRC), The Cancer Council
NSW, U.S. Army Medical Research and Materiel
Command (DAMD17-00-1-0252 and DAMD17-
99-1-9184), Association for International Cancer
Research (AICR), and Susan G Komen Breast
Cancer Foundation.
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