Qunyan Yu
Charles University Prague
1st Faculty of Medicine
Institute of Molecular Genetics
G1 cyclins and their role in oncogenesis
Thesis presented by
Qunyan Yu
Prague, 2006
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Contents
1. Introduction
1.1. G1 phase and its regulators in mammalian cell cycle
1.2. D-type cyclins
1.2.1. Ablation of D type cyclins
1.2.2. D-type cyclins in tumorigenesis
1.2.2.1. Cyclin D1 in breast cancer
1.3. E-type cyclins
1.3.1. Ablation of E cyclins
1.3.2. E-type cyclins in tumorigenesis
2. Papers
Paper 1: Specific protection against breast cancers by cyclin D1 ablation
Paper 2: Requirement for CDK4 kinase function in breast cancer
Paper 3: Ras and Myc can drive oncogenic cell proliferation through individual Dcyclins
Paper 4. Cyclin E ablation in the mouse
3. Discussion
3.1. Cyclins and breast cancer
3.2. Cyclins as tissue-specific recipients of oncogenic signals
4. Conclusions
5. References
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1. Introduction
1.1. G1 phase and its regulators in mammalian cell cycle
The cell cycle consists of four phases: G1 (gap 1), S (DNA synthesis), G2 (gap
2), and M (mitosis). The G1 phase separates mitosis from DNA synthesis. During
this phase the proliferation of mammalian cells is impacted by the extracellular
signals, such as mitogens or antimitogens. Once cells pass through the so-called
restriction point in the G1 phase, their cell cycle progression becomes mitogenindependent. Hence, passing the restriction point in the G1 phase commits cells to
undergo cell division (Pardee, 1974).
The proliferation of cells is driven by the core cell cycle machinery operating
in the cell nucleus. The key components of this machinery are proteins called cyclins.
Cyclins represent regulatory subunits that bind, activate and provide substrate
specificity for their associated cyclin-dependent kinases (CDKs) (Sherr and Roberts,
1999)
Cyclins and CDKs operating during the G1 phase serve as recipients of the
external mitogenic and oncogenic signals. Consistent with their critical role in cell
proliferation, these G1 cyclins and CDKs are often hyperactivated in human cancers
(Malumbres and Barbacid, 2001).
Two classes of cyclins operate in mammalian cells during G1 progression: Dtype (cyclin D1, D2 and D3) and E-type (cyclin E1 and E2) (Sherr and Roberts,
1999). D-type cyclins bind and activate CDK4, CDK6, and under certain conditions
CDK2 (Matsushime et al., 1992; Meyerson and Harlow, 1994; Morgan, 1997). Etype cyclins interact primarily with CDK2, but also with CDK1 and CDK3 (Geisen et
al., 2003; Koff et al., 1992; Lauper et al., 1998; Zariwala et al., 1998). Recent work
suggests that another cyclin, cyclin C, acting together with CDK3 may play role in G1
progression (Ren and Rollins, 2004). However, the G1 phase function of cyclin C
remains controversial, in contrast to the well-documented role for cyclin C-CDK8
holoenzyme in phosphorylating C terminal domain of the RNA polymerase II
(Rickert et al., 1996; Tassan et al., 1995).
The activity of cyclin-CDK complexes is negatively regulated by cell cycle
inhibitors. The inhibitors from the INK family (p16INK4a, p15INK4b, p18INK4c and
p19INK4d) inhibit the activity of cyclin D-CDK4 and cyclin D-CDK6 complexes (Sherr
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and Roberts, 1999). Specifically, these inhibitors bind CDK4 and CDK6 and displace
D-type cyclins, thereby extinguishing the catalytic activity of cyclin D-CDK4/6
kinase. The second group of inhibitors, the Kip/Cip family, consists of p21Cip1,
p27Kip1 and p57Kip1 (Sherr and Roberts, 1999). These molecules form ternary
complexes with cyclin-CDK2 and with cyclin-CDK1 complexes and block their
enzymatic activity (Sherr and Roberts, 1999).
1.2. D-type cyclins
Three D-type cyclins have been enumerated in mammalian cells: D1, D2 and
D3. The three D-type cyclins are encoded by different genes, localized on different
chromosomes. D-cyclin proteins show significant amino acid similarity: 57% across
the entire coding sequence, 78% within the most-conserved cyclin box domain (Inaba
et al., 1992; Xiong et al., 1992). D-cyclins are expressed in an overlapping fashion in
all proliferating cell types. For example, fibroblasts express all three D-cyclins (Tam
et al., 1994), T-lymphocytes express cyclins D2 and D3 (Ajchenbaum et al., 1993),
while ovarian granulosa cells only cyclin D2 (Sicinski et al., 1996).
Once induced, D-cyclins bind and activate cyclin-dependent kinases CDK4
and CDK6. Surprisingly, very few targets for cyclin D-CDK4/6 kinase have been
described. Cyclin D-CDK4/6 enzyme phosphorylates the retinoblastoma tumor
suppressor protein (pRB), and pRB-related proteins p107 and p130 (Bates et al.,
1994; Matsushime et al., 1992, 1994; Meyerson and Harlow, 1994). Indeed, pRB
may represent the major target for cyclin D-CDK4/6 in cell cycle progression, as
evidenced by the observations that cells lacking pRB no longer require D-cyclins for
proliferation (Bruce et al., 2000; Koh et al., 1995; Lukas et al., 1995; Medema et al.,
1995; Okamoto et al., 1994; Tam et al., 1994). However, D-cyclins were also shown
to be dispensable in cells lacking both p107 and p130 (p107-/-p130-/-cells) (Bruce et
al., 2000; Koh et al., 1995; Lukas et al., 1995; Medema et al., 1995; Okamoto et al.,
1994; Tam et al., 1994), suggesting that all three proteins: pRB, p107 and p130
represent the essential targets for cyclin D-CDK4/6 kinase.
The retinoblastoma
protein, along with p107 and p130 binds and represses the activity of E2F
transcription factors (Dyson, 1998).
Phosphorylation of pRB, p107 and p130
functionally inactivates these proteins, and releases the pRB-, p107-, or p130-bound
E2Fs. These transcription factors then proceed to induce genes required for the S
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phase entry such as thymidine kinase, thymidilate synthase, dihydrofolate reductase
(DHFR) and cyclin E (Adams, 2001; Dyson, 1998).
Recently a transcription factor SMAD3 was shown to represent another target
for cyclin D-CDK4/6 kinase (Liu and Matsuura, 2005; Matsuura et al., 2004).
SMAD3 normally serves to mediate the antiproliferative signals downstream of the
TGFβ receptor (Liu et al., 2003). Phosphorylation of SMAD3 is thought to cancel
this function, thereby contributing to cell proliferation. It is very likely that additional
targets for cyclin D-CDK4/6 exist, but they are currently unknown.
Cyclin D-
CDK4/6 might phosphorylate tissue-specific transcription factors, and hence
preventing cell differentiation. Consistent with this notion are the observations that
ectopic overexpression of cyclins D2 or D3 prevented differentiation of granulocytes
(Kato and Sherr, 1993). Likewise, overexpression of cyclin D1 was shown to inhibit
myogenic differentiation by interfering with the action of MyoD transcription factor
(Skapek et al., 1995, 1996).
In addition to activating the kinase activities of CDK4 and CDK6, cyclin DCDK complexes contribute to cell cycle progression in a ‘kinase-independent’
mechanism. Thus, cyclin D-CDK complexes ‘titrate’ p27Kip1 and p21Cip1 cell cycle
inhibitors from cyclin E-CDK complexes (which are inhibited by p27Kip1 and p21Cip1)
to cyclin D-CDK4/6 complexes (which use these inhibitors as assembly factors). This
titration of p27Kip1 and p21Cip1 activates kinase activity of cyclin E-CDK2
holoenzyme, thereby triggering entry of cells into the S-phase (Sherr and Roberts,
1999; Tsutsui et al., 1999).
Lastly, D-cyclins were shown to interact with several transcription factors
such as the estrogen receptor, androgen receptor, thyroid hormone receptor, CEBPβ,
STAT3, DMP1, Sp1 and others (Bienvenu et al., 2001; Ganter et al., 1998;
Horstmann et al., 2000; Inoue and Sherr, 1998; Lee et al., 1999; Lin et al., 2002;
Neuman et al., 1997; Shao and Robbins, 1995; Zwijsen, 1997, 1998). D-cyclins are
thought to act as co-activators or co-repressors of these transcription factors by
bringing in histone acetylases or deacetylases. It should be noted, however, that the in
vivo, physiological relevance of these cyclin D-transcription factors interactions
remains unclear.
1.2.1. Ablation of D type cyclins
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Dr. Sicinski’s laboratory generated mice lacking individual D-type cyclins.
These mice are viable and display narrow, tissue-specific lesions.
Cyclin D1-null mice show a triad of symptoms indicating a developmental
neurological abnormality: decreased body size, a spastic ‘leg-clasping’ reflex, and
premature mortality with approximately 25% of cyclin D1-null animals dying during
the first month of their lives.
In addition, cyclin D1-null mice display
underdeveloped, hypoplastic retinas. Lastly, cyclin D1-null females develop normal
mammary glands during sexual maturation, but these mammary glands fail to undergo
the proliferative burst (the so-called lobuloalveolar development) during pregnancy
(Fantl et al., 1995; Sicinski et al., 1995).
Cyclin D2-deficient mice are also viable. Cyclin D2-null females are sterile
due to an inability of their ovarian granulosa cells to respond to follicle stimulating
hormone (FSH). Cyclin D2-/- males are fertile, but display reduced testicular sizes
and reduced sperm counts (Sicinski et al., 1996). Subsequent analyses revealed
additional phenotypes in cyclin D2-null mice, such as mild abnormalities in cerebellar
development and impaired proliferation of B-lymphocytes (Huard et al., 1999; Lam et
al., 2000; Solvason et al., 2000).
Cyclin D3-knockout mice are viable and display a defect in the expansion of
immature thymocytes (Sicinska et al., 2003). In addition, these animals are refractory
to the stimulation by granulocyte colony-stimulating factor (G-CSF), and
consequently develop a severe neutropenia (Sicinska et al. submitted).
As mentioned above, each of the three D-type cyclins is expressed in a
characteristic, cell type-specific fashion, and ablation of individual D-type cyclins led
to narrow, tissue-specific phenotypes.
An important question arising from these
analyses was whether the differences between the three D-cyclins lie in tissue-specific
pattern of their expression, or in different functions for each of these proteins.
Bradley Carthon in Dr. Sicinski’s laboratory addressed this question by generating
cyclin D2D1 ‘knock-in’ strain of mice. In these mice, the coding sequences of
cyclin D1 were replaced by that of cyclin D2, via the homologous recombination in
embryonal stem cells. The resulting cyclin D2D1 animals lack cyclin D1, but
instead express cyclin D2 in its place. Analyses of cyclin D2D1 mice revealed that
expression of cyclin D2 largely corrected the phenotypic abnormalities seen in cyclin
D1-null mice (Carthon et al., 2005). These results indicate that the major differences
between the D-cyclins lie in tissue-specific pattern of their expression (i.e. in the
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sequences of their promoters). However, Carthon et al. noted that cyclin D2 did not
fully correct the phenotypes of cyclin D1-null mice, and that the retinal and
neurological development of cyclin D2D1 mice remained slightly impaired
(Carthon et al., 2005). These findings suggest that subtle differences between the Dcyclins do exist, and these differences allow particular D-cyclins to drive optimal
development of various lineages.
Ciemerych et al. (2002) further probed the requirement for the D-type cyclins
in development by generating double-knockout mice (D1-/-D2-/-, or D1-/-D3-/-, or D2-/D3-/-). Surprisingly, these mice - expressing only a single, intact D-cyclin: cyclin D1
in case of D2-/-D3-/-animals, cyclin D2 in case of D1-/-D3-/-animals, or cyclin D3 in
case of D1-/-D2-/-animals - did not show any new phenotypes, but instead displayed
combined phenotypes seen in animals lacking individual D-cyclins (Ciemerych et al.,
2002).
More recently, Kozar et al. (2004) in Dr. Sicinski’s laboratory generated mice
lacking all three D-type cyclins. It was assumed in the cell cycle field that at least one
D-type cyclin must be present to allow cell proliferation. Very surprisingly, however,
Kozar et al. observed that cyclin D-null mice survived until day 15.5 of gestation.
Eventually, cyclin D-null embryos died due to hematopoietic abnormalities (Kozar et
al., 2004). These studies demonstrated very unexpectedly that the majority of cell
types in the developing embryo can proliferate in the absence of D-type cyclins. It
remains to be seen whether cells in the adult organism can also proliferate without Dcyclins.
1.2.2. D-type cyclins in tumorigenesis
Consistent with their growth-promoting functions, amplification of the cyclin
D genes and overexpression of cyclin D protein is seen in many human cancers. The
best documented of these is frequent overexpression of cyclin D1 in the majority of
human breast cancers (see below). Moreover, cyclin D1 gene is amplified and protein
overexpressed in cancers of head and neck, esophagus, bladder, small-cell lung and
hepatocellular carcinomas, and in many others (Cheung et al., 2001; Fujii et al., 2001;
Lammie et al., 1991; Reissmann et al., 1999; Rodrigo et al., 2000; Vielba et al., 2003).
Cyclin D2 amplification and overexpression has been documented in human ovarian
and testicular tumors, as well as in some lymphoid malignancies such as chronic
lymphocytic leukemias (Delmer et al., 1995; Houldsworth et al., 1997; Motokura and
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Arnold, 1993; Sicinski et al., 1996). Lastly, cyclin D3 is overexpressed in lymphoid
malignancies, including multiple myelomas, as well as in pancreatic cancers (Filipits
et al., 2002; Shaughnessy et al., 2001).
1.2.2.1. Cyclin D1 in breast cancer
Cyclin D1 gene is amplified in approximately 15% of mammary carcinomas,
while the majority of human breast cancers overexpress cyclin D1 protein (Bartkova
et al., 1994; Dickson et al., 1995; Gillett et al., 1994; McIntosh et al., 1995). The
overexpression of cyclin D1 was reported in all histologic types of breast cancers
(Weinstat-Saslow et al., 1995). Cyclin D1 overexpression is seen at the earliest stages
of breast cancer progression, such as ductal carcinoma in situ (DCIS), but not in premalignant lesions (Weinstat-Saslow et al., 1995). Hence, elevated levels of cyclin D1
represent a molecular switch that can distinguish pre-malignant hyperplasia from
neoplastic lesions. Once acquired by the tumor cells, the overexpression of cyclin D1
is maintained at all stages of breast cancer progression, and it is preserved even in
metastases (Bartkova et al., 1994; Gillett et al., 1996; Weinstat-Saslow et al., 1995).
Consistent with the causative role for cyclin D1 overexpression in breast
tumorigenesis are the observations that transgenic mice engineered to overexpress
cyclin D1 in their mammary glands succumb to mammary adenocarcinomas (Wang et
al., 1994).
1.3. E-type cyclins
Mammalian cells express two E-type cyclins, E1 and E2. The two E-type
cyclins show significant amino acid similarity (47% across the entire coding
sequence, 75% within the conserved cyclin box domain). The two E-type cyclins are
expressed in all proliferating compartments (Geng et al., 1999; Gudas et al., 1999;
Lauper et al., 1998; Zariwala et al., 1998).
A described above, E-type cyclins
represent transcriptional targets of the E2F transcription factors (Duronio and
O'Farrell, 1995). Activation of the E-type cyclins is believed to trigger entry of cells
into the S-phase (Sherr and Roberts, 1999).
Cyclins E1 and E2 associate with CDK2. In addition, in human cells Ecyclins bind and activate CDK3 (Braun et al., 1998). However, most of mouse
laboratory strains lack CDK3 expressions (Ye et al., 2001). Lastly, recent report
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indicates that the E-type cyclins can also bind and activate CDK1 in certain settings
(Aleem et al., 2005).
Cyclin E-CDK complexes have much broader spectrum of targets than cyclin
D-CDK complexes.
Like cyclin D-associated kinases, cyclin E-CDK complex
phosphorylates, pRB, p107 and p130 (Harbour et al., 1999; Lundberg and Weinberg,
1998). This phosphorylation contributes to the functional inactivation of pRB and
pRB-related proteins by the G1 cyclins. However, pRB-null cells remain dependent
on E-cyclins for proliferation, indicating the presence of other targets for cyclin E,
besides pRB (Kelly et al., 1998; Ohtsubo et al., 1995). Indeed, several other targets of
cyclin E-CDK have been identified, and they include proteins involved in cell cycle
progression (p27Kip1, CDC25A, E2F5), centrosome duplication (nuclephosmin,
CP110) histone gene transcription (NPAT), and RNA splicing (Hinchcliffe et al.,
1999; Hoffmann et al., 1994; Ma et al., 2000; Okuda et al., 2000; Sheaff et al., 1997;
Zhao et al., 2000). Cyclin E-associated kinase is also required to trigger loading of
CDC45 protein onto origins of DNA replication, and is responsible together with
Dbf4-associated kinase for origin firing (Arata et al., 2000; Zou and Stillman, 2000).
1.3.1. Ablation of E cyclins
In order to study the functions of E-type cyclins in development, Dr. Sicinski
laboratory generated cyclin E-null mice. I contributed to analyses of mutant mice and
cells. The relevant paper is included into this Thesis.
1.3.2. E-type cyclins in tumorigenesis
Overexpression of cyclin E was reported in several human malignancies,
including cancers of the breast, lung, uterine cervix and endometrium (Donnellan and
Chetty, 1999; Keyomarsi et al., 1994; Nielsen et al., 1996). In several instances,
abnormally high levels of cyclin E are caused by the inappropriate proteolytic
degradation of this protein (Moberg et al., 2001; Strohmaier et al., 2001). Normal
degradation of cyclin E is mediated by an F-box protein Fbw7. Mutational
inactivation of Fbw7 was shown to result in excessive levels of cyclin E (Koepp et al.,
2001; Strohmaier et al., 2001).
Clinical studies, mostly involving breast cancer
patients revealed that cyclin E overexpression is correlated with poor clinical outcome
(Keyomarsi et al., 2002; Porter et al., 1997).
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2. Papers
Paper 1. Specific protection against breast cancers by cyclin D1 ablation.
(2001) Nature 411, 1017-1021
Paper 2. Requirement for CDK4 kinase function in breast cancer. (2006)
Cancer Cell 9, 23-32
Paper 3. Ras and Myc can drive oncogenic cell proliferation through
individual D-cyclins. (2005) Oncogene 24, 7114-7119
Paper 4. Cyclin E ablation in the mouse. (2003) Cell 114, 431-443
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3. Discussion
3.1. Cyclins and breast cancer
Breast cancer is the most common malignancy among women. In the United
States, one out of nine women will develop breast cancer during their lifetime. The
majority of mammary carcinomas overexpress cyclin D1, a component of the core
cell cycle machinery. Cyclin D1 overexpression represents an early molecular lesion
that is maintained during tumor progression, and it is preserved even in metastases
(Bartkova et al., 1994; Gillett et al., 1996). Importantly, overexpression of cyclin D1
plays a causative role in breast tumorigenesis, as evidenced by the observations that
transgenic mice engineered to overexpress cyclin D1 succumb to mammary
carcinomas (Wang et al., 1994). Collectively, these observations indicate that cyclin
D1 might represent a potential therapeutic target in human breast cancers.
Following the discovery of the three D-type cyclins in 1991 (Matsushime et al.,
1991; Xiong et al., 1991), it was assumed that cyclins D1, D2 and D3 perform distinct,
non-redundant roles in cell cycle progression. It was also assumed that each of the Dtype cyclins is critically required for cell proliferation. This notion was based on
several observations. First, even when co-expressed within the same cell, the three Dtype cyclins are induced with different kinetics following mitogen challenge. For
example, stimulation of T-lymphocytes with phytohemagglutinin leads to rapid
induction of cyclin D2 (Ajchenbaum et al., 1993). In contrast, cyclin D3 is
upregulated approximately 12 hours later, coincident with entry of cells into the S
phase (Ajchenbaum et al., 1993). During embryo development, the expression of the
three D-cyclins is highly orchestrated, with different cyclins being induced at distinct
developmental stages (Ciemerych et al., 2002). Moreover, introduction of anti-cyclin
D1 antibodies, or antisense anti-cyclin D1 oligonucleotides into cultured cells arrested
the proliferation of these cells in the G1 phase (Baldin et al., 1993). These
observations strongly suggested that cyclin D1 is critically required for normal, nononcogenic cell proliferation.
In 1995, Sicinski et al. and Fantl et al. generated mice lacking cyclin D1.
Very surprisingly, they observed that cyclin D1-null mice were viable and displayed
very narrow, tissue-specific abnormalities. Moreover, cyclin D1-/- mouse embryonal
fibroblasts proliferated normally in vitro. These observations revealed that - contrary
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to the prevailing notion - cyclin D1 is dispensable for normal, non-oncogenic
proliferation of the overwhelming majority of cell types (Fantl et al., 1995; Sicinski et
al., 1995).
The narrow phenotype of cyclin D1-null mice, together with the very well
documented role of cyclin D1 overexpression in human breast cancers suggested to
me that cyclin D1 might represent an excellent target for breast cancer therapy. As a
first step towards a potential anti-cyclin D1 therapy in human breast cancers, I set to
determine whether ablation of cyclin D1 would protect mice against mammary
carcinomas. To address this possibility, I crossed cyclin D1-null mice with four
strains of transgenic breast cancer-prone animals. These transgenic mice develop
mammary carcinomas due to targeted overexpression of oncogenes in their mammary
glands (Muller et al., 1988; Sinn et al., 1987; Stewart et al., 1984; Tsukamoto et al.,
1988). For my analyses I chose mice overexpressing Ras, ErbB-2 (Neu, HER2), Myc
and Wnt-1 oncogenes, as these proteins were postulated to signal through D-type
cyclins.
My analyses revealed that cyclin D1-deficient mice were completely resistant
to mammary carcinomas triggered by the Ras and ErbB-2 oncogenes. I also observed
that mice lacking highly related D-type cyclins, cyclin D2-null and D3-null animals,
remained susceptible to Ras-induced mammary tumors. These results underscore a
specific requirement for cyclin D1 - but not D2 or D3 - in Ras-driven tumorigenesis.
My detailed molecular analyses revealed that in mammary epithelial cells the ErbB2 Ras pathway signals to the core cell cycle machinery through cyclin D1. These
observations are consistent with previous reports that oncogenic Ras upregulated the
activity of cyclin D1 promoter via the AP-1 and Ets binding sites (Albanese et al.,
1995; Filmus et al., 1994; Lee et al., 2000; Liu et al., 1995).
Importantly, cyclin D1-null mice remained sensitive to mammary carcinomas
triggered by Myc and Wnt-1. I interpreted this as an indication that Myc and Wnt-1
can signal to the core cell cycle machinery independently of cyclin D1. What are the
cell cycle targets for Myc and Wnt-1 in mammary epithelial cells?
The Myc
oncogene was postulated to induce cyclin D2 via an E-box element located in the
promoter of the cyclin D2 gene (Bouchard et al., 2001; Bouchard et al., 1999; Coller
et al., 2000; Dey et al., 2000). In addition, Myc was also postulated to increase the
rate of cyclin D1’s synthesis (Perez-Roger et al., 1999). Others proposed that Myc
upregulates the levels of CDC25A, a phosphatase that activates CDK2-containing
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complexes (Zornig and Evan, 1996). Also CDK4, a catalytic partner of the D-type
cyclins was reported to represent a Myc target (Hermeking et al., 2000; Mateyak et al.,
1999). Lastly, it was hypothesized that Myc may activate the core cell cycle
machinery through cyclin E (Muller et al., 1997; Steiner et al., 1995). Although my
work did not allow me to unequivocally identify the cell cycle target of Myc in
mammary epithelial cells, I observed that Myc-driven mammary carcinomas
expressed greatly elevated levels of cyclins E1 and E2.
Following up on these
observations, Yan Geng in Dr. Sicinski’s laboratory crossed transgenic MMTV-Myc
mice with animals lacking E-type cyclins. Since cyclin E1-/-E2-/- mice die in utero
(Geng et al., 2003), cyclin E1-/-E2+/- and cyclin E1+/-E2-/- animals were used for
analyses.
These mice displayed significantly reduced incidence of Myc-induced
mammary carcinomas, revealing that Myc oncogene signals to the cell cycle
machinery in mammary epithelial cells through cyclins E1 and E2 (Geng et al.,
unpublished).
The cell cycle targets for Wnt-1 in mammary epithelium remain unknown. I
observed high levels of cyclins D1 and D2 in mammary tumors triggered by Wnt-1.
Hence, I hypothesize that the Wnt-1-dependent pathways activate the core cell cycle
machinery through cyclins D1 and D2. In order to test this hypothesis, MMTVWnt1/cyclin D1-/-D2-/- females need to be generated and observed for breast cancer
incidence. However, cyclin D1-/-D2-/- mice die within three weeks after birth due to
cerebellar abnormalities (Ciemerych et al., 2002), well before the age when Wnt-1driven tumors start appearing. One way to circumvent this would be to transplant
mammary glands of MMTV-Wnt-1/cyclin D1-/-D2-/- females into wild-type hosts, and
to observe the recipients for breast cancer incidence. Alternatively, cyclin D1- and
D2-conditional knockout strains, which are currently being developed in Dr.
Sicinski’s laboratory, will allow one to perform analyses of Wnt-1 signaling when
cyclins D1 and D2 are specifically deleted in mammary glands.
As stated above, I found that cyclin D1-null mice were resistant to mammary
carcinomas triggered by Ras and ErbB-2 oncogenes. Similar conclusions regarding
ErbB-2-driven tumors were independently reached by Bowe et al. (Bowe et al., 2002).
I concluded that cyclin D1 function is critically required for Ras- and ErbB-2-driven
breast oncogenesis. However, these analyses did not allow me to determine which
exact molecular cyclin D1 function is required to mediate Ras- and ErbB-2-driven
neoplasia. Cyclin D1 was shown to interact with several different proteins, including
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CDK4, CDK6, p27Kip1, p21Cip1, the retinoblastoma protein, transcription factors such
as DMP1, CEBPβ, STAT3, and many others (Bienvenu et al., 2001; Ganter et al.,
1998; Horstmann et al., 2000; Inoue and Sherr, 1998; Lee et al., 1999; Lin et al., 2002;
Neuman et al., 1997; Shao and Robbins, 1995; Zwijsen, 1997, 1998). Given the
frequent involvement of CDK4 overexpression in human cancers (Malumbres and
Barbacid, 2001), I hypothesized that the rate-limiting function of cyclin D1 in
mammary neoplasia is mediated through its interaction with CDK4.
I tested this hypothesis using CDK4-deficient mice. My analyses revealed that
CDK4-null animals were completely resistant to ErbB-2-driven mammary carcinomas,
like mice lacking cyclin D1. I concluded that cyclin D1-CDK4 function is critically
required to mediate ErbB-2-driven mammary neoplasia.
Cyclin D1-CDK4 complexes have at least two different functions. In the
catalytic, kinase-dependent function, cyclin D1-CDK4 complexes phosphorylate
cellular proteins, such as the retinoblastoma protein (Sherr and Roberts, 1999). In
addition, cyclin D1-CDK4 complexes ‘titrate’ cell cycle inhibitors p27Kip1 and p21Cip1
from cyclin E-CDK2 complexes (which are inhibited by p27Kip1 and p21Cip1) to cyclin
D1-CDK4 complexes (which use these inhibitors as assembly factors), thereby
triggering kinase activity of cyclin E-CDK2 holoenzyme (Cheng et al., 1999; Sherr
and Roberts, 1999; Tsutsui et al., 1999). The titration of cell cycle inhibitors
represents the kinase-independent function of cyclin D1-CDK4.
My analyses did not allow me to determine which of the two functions of
cyclin D1-CDK4 is required for mammary tumorigenesis. Landis et al. (Landis et al.,
2006) addressed this question using a point mutant version of cyclin D1, called
K112E. This cyclin D1 mutant normally binds CDK4, but it is unable to activate
CDK4 kinase. Importantly, cyclin D1 K112E mutant fully retains the ability to titrate
p27Kip1 and p21Cip1. Landis et al. (Landis et al., 2006) used homologous
recombination in embryonal stem cells and generated a knock-in strain of mice
expressing kinase-deficient cyclin D1 K112E mutant in place of wild-type cyclin D1.
Landis et al. (Landis et al., 2006) observed normal development of cyclin D1dependent compartments (retinas, mammary glands) in homozygous cyclin D1
K112E knock-in mice, revealing that the kinase-dependent function of cyclin D1CDK4 complexes is dispensable for normal development. Strikingly, Landis et al.
(Landis et al., 2006) found that knock-in mice expressing kinase-deficient cyclin D1-
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CDK4 complexes were completely resistant to ErbB-2-driven mammary carcinomas,
like mice lacking cyclin D1 and CDK4. Collectively, these results reveal that the
kinase function of cyclin D1-CDK4 complexes is critically required for ErbB-2driven breast tumorigenesis.
The experiments described above addressed the role of cyclin D1-CDK4
kinase in the initiation of mammary carcinomas. My subsequent analyses revealed
that the continued presence of cyclin D1-CDK4 kinase is also required to maintain
tumor cell proliferation. In addition, my collaborators, Drs. Lyndsay Harris from the
Dana-Farber Cancer Institute, and Marie Ahnström and Olle Stål from Lynkoping
University in Sweden analyzed nearly one hundred human mammary carcinomas
overexpressing ErbB-2. They observed that approximately 25% of these tumors
greatly overexpressed cyclin D1. Ahnström et al. also found that patients bearing
these ErbB-2-positive cyclin D1-overexpressing tumors have particularly poor
prognosis, with only 13% of patients surviving seven years (Ahnstrom et al., 2005).
My work validated cyclin D1-CDK4 kinase as a potential target for
therapeutic intervention in mammary carcinomas.
Importantly, at the same time
Landis et al. (2006) observed that the kinase activity of cyclin D1-CDK4 is
dispensable for normal development. Based on this work, the Dana-Farber Cancer
Institute initiated a Phase I clinical study in which patients with ErbB-2-positive
breast cancers are being treated with a CDK-inhibitor flavopiridol.
However,
flavopiridol is not specific to CDK4 kinase, as it also inhibits other CDKs
(Senderowicz, 1999). More specific CDK4 inhibitors are currently being developed
by pharmaceutical companies. Based on my work, I predict that anti-CDK4 therapy
might be most beneficial in a subset of ErbB-2-positive breast cancers that
overexpress cyclin D1 (25% of ErbB-2-overexpressing cases).
In addition to breast cancers, amplification of the ErbB-2 gene and
overexpression of ErbB-2 protein were reported in several human malignancies
including ovarian cancers, pancreatic adenocarcinomas, non-small cell lung cancers,
biliary tract cancers, invasive bladder cancers, squamous cell carcinoma of the
esophagus, gastric cancers, uterine serous papillary carcinomas, and many others
(Berchuck et al., 1990; Eltze et al., 2005; Hansel et al., 2005; Mimura et al., 2005;
Nakazawa et al., 2005; Santin et al., 2005; Tanner et al., 2005; Ugocsai et al., 2005).
Moreover, mutational activation of ErbB-2 was documented in 10% of lung
adenocarcinomas (Stephens et al., 2004). Some of these tumors were also shown to
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contain elevated levels of cyclin D1-CDK4 complexes (Cheung et al., 2001; Dong et
al., 2001; Igarashi et al., 1999; Masaki et al., 2003). In addition to ErbB-2 positive
tumors, amplification of the cyclin D1 gene, and overexpression of cyclin D1 protein
is seen in many other human cancers (see: Introduction). Patients bearing these
tumors might also benefit from pharmacological inhibition of the CDK4 kinase.
3.2. Cyclins as tissue-specific recipients of oncogenic signals
One of the important outcomes of my work is the demonstration that the
wiring of the oncogenic pathways to the core cell cycle machinery operates differently
in various cell types. Thus, my analyses revealed that in mammary epithelial cells the
ErbB-2Ras pathway signals to the core cell cycle machinery through cyclin D1, and
consequently cyclin D1-null mice are completely resistant to mammary carcinomas
triggered by Ras and ErbB-2. On the other hand, I found that cyclin D1-null mice
remained fully susceptible to Ras-induced salivary gland tumors. Also Robles et al.
(Robles et al., 1998) reported that cyclin D1-/- mice were susceptible to Ras-induced
skin papillomas. These findings raised a possibility that the Ras signaling is wired to
the core cell cycle machinery in a cell type-specific fashion.
I analyzed this possibility using fibroblasts derived from mice lacking
particular D-type cyclins. I found that fibroblasts lacking cyclin D1 were fully
susceptible to the oncogenic transformation by Ras. Also, double-knockout fibroblasts,
expressing only a single D-type cyclin: D2-/-D3-/- (only cyclin D1 expressed), D1-/-D3/-
(only cyclin D2 expressed), and D1-/-D2-/- (only cyclin D3 expressed) remained
susceptible to the oncogenic action of Ras. Importantly, Kozar et al. (2004) observed
that cells lacking all three D-type cyclins (D1-/-D2-/-D3-/-) were resistant to Ras-driven
transformation. These results reveal that – in contrast to mammary epithelial cells - in
fibroblasts the Ras oncogene can signal to the core cell cycle machinery through all
three D-type cyclins. As stated above, the molecular pathway leading from the Ras
oncogene to cyclin D1 has been well described. My work indicates that novel,
currently unknown pathways exist that link Ras signaling to cyclins D2 and D3. The
elucidation of these molecular pathways may help to interfere with the oncogenic
action of Ras oncogene in human cancers.
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4. Conclusions
4.1. Cyclin D1-null mice are resistant to ErbB-2- and Ras-driven mammary
carcinomas, while being fully sensitive to Myc- and Wnt-1-induced breast
tumors.
4.2. The requirement for cyclin D1 in Ras-driven breast cancers is cyclin D1-specific,
as mice lacking cyclins D2 or D3 remain susceptible to Ras-driven mammary
carcinomas.
4.3. This requirement is mammary-specific, as mice lacking cyclin D1 remain
susceptible to Ras-driven salivary tumors.
4.4. The ability of cyclin D1 to interact with CDK4 kinase underlies the critical
requirement for cyclin D1 in breast cancer initiation and maintenance.
4.5. In mammary epithelial cells, the ErbB-2Ras pathway signals to the core cell
cycle machinery through cyclin D1, explaining the critical requirement for cyclin
D1 in Ras- and ErbB-2-driven mammary neoplasia.
4.6. In fibroblasts, the Ras oncogene can signal through all three D-type cyclins,
revealing that the wiring of the Ras-dependent pathways operates in a cell typespecific fashion.
4.7. In fibroblasts, the Myc oncogene can impact the core cell cycle machinery
through all three D-type cyclins.
4.8. E-type cyclins are dispensable for continuous proliferation of fibroblasts, but they
are critically required for oncogenic cell proliferation.
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