Cyclin D1, Cancer Progression and Opportunities in Cancer
Treatment
Shuo Qie and J. Alan Diehl
Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of
South Carolina
Abstract
Mammalian cells encode three D cyclins (D1, D2 and D3) that coordinately function as allosteric
regulators of cyclin dependent kinase 4 and 6 (CDK4/CDK6) to regulate cell cycle transition from
G1 to S phase. Cyclin expression, accumulation and degradation, as well as assembly and
activation of CDK4/CDK6 are governed by growth factor stimulation. Cyclin D1 is more
frequently dysregulated than cyclins D2 or D3 in human cancers and as such it has been more
extensively characterized. Overexpression of cyclin D1 results in dysregulated CDK activity, rapid
cell growth under conditions of restricted mitogenic signaling, bypass of key cellular checkpoints
and ultimately neoplastic growth. This review discusses cyclin D1 transcriptional, translational,
posttranslational regulation, and its biological function with a particular focus on the mechanisms
that result in its dysregulation in human cancers.
Keywords
Cyclin D1; CDK4/CDK6; Proteasome; Posttranslational Regulation; Cancer
Introduction
The cell cycle refers to the experimentally determined intervals during which cells prepare
for and subsequently duplicate their genome equally between two daughter cells. It is
divided into four consecutive phases: G1 phase, during which cells accumulate mass and
metabolites necessary for DNA replication; S phase, when DNA is replicated; G2, a gap
phase that is essential to ensure accurate DNA replication; and M phase, DNA segregation
and cell division. While the primary phases of cell division define states of proliferation and
division, the majority of adult cells are maintained in a quiescent state (known as G0 phase),
a resting state cells often enter post-mitotically or prior to terminal differentiation [1]. Unlike
cells many terminally differentiate cells however, quiescent cells can renter the cell cycle in
G1 phase when exposed to appropriate mitogenic stimuli [2].
Transitions through the cell cycle are driven by cyclins and cyclin-dependent kinases
(CDKs) [1]. Cyclins are the allosteric activators of cognate CDKs; their levels typically
oscillate across the cell cycle, hence gaining the name cyclins. The cyclin family shares a
homologous N-terminal 100-amino acid motif referred as the cyclin box that has a highly
conserved three-dimensional structure and provides the binding interface for the appropriate
CDKs [3]. CDKs define the partner kinases that can be activated only when they bind to
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their cognate cyclins. Due to their biological significance, CDK activity is stringently
regulated by the following mechanisms: the levels of cyclin partners; phosphorylation status;
and the abundance of CDK inhibitory proteins, such as, the INK4 family: p16
INK4A
,
p15
INK4B
, p18
INK4C
and p19
INK4D
and the CIP and KIP family: p21
CDKN1A
, p27
CDKN1B
and p57
CDKN1C
[4].
D cyclins, including cyclins D1, D2 and D3, form active complexes with either CDK4 or
CDK6, which in turn phosphorylate the retinoblastoma protein (Rb) and drive G1 to S phase
progression [5]. D cyclins coordinate cell cycle progression with the extracellular
stimulation (e.g. growth factor availability, nutrient availability and integrin-derived
adhesion signaling) [6]. Given the role of D cyclins in mediating extracellular cues with cell
proliferation, it is not surprising that overexpression of D cyclins or hyperactivation of their
cognate CDK directly contributes to neoplastic growth. More specifically, cyclin D1 has
attracted widespread attention due to the prevalence of its dysregulation in human cancers
[7]. This review focuses on and discusses cyclin D1 structure, transcriptional, translational
and posttranslational regulation, and its biological function. It also addresses the
dysregulation of cyclin D1 in human cancers and the advancement and impact of new
therapeutic inhibitors targeting CDK4/CDK6.
Transcriptional, Post-transcriptional and Translational Regulation of cyclin
D1
1. β-catenin-dependent regulation of cyclin D1 transcription
Physiologically, Wnt/β-catenin pathway regulates the development of various tissues and
organs, including heart, liver, lung, brain, kidney and so forth [8]. Moreover, it also plays
important roles in pathological conditions including gastric cancer, colorectal carcinoma,
liver cancer and melanoma [9]. β-catenin mediates the canonical Wnt signaling pathway: the
binding of Wnt to its receptor suppresses the degradation of β-catenin, which is mediated by
the cytoplasmic β-catenin destruction complex. Reduced degradation and cytoplasmic
accumulation of β-catenin results in increased nuclear translocation, where it associates with
lymphoid enhancer factor/T-cell factor (LEF/TCF) and drives expression of key downstream
target genes. The CCND1 gene, which encodes cyclin D1, represents a key target,. β-
catenin/LEF-1 complexes target motifs at -75 and -15 within the CCND1 promoter [10].
Importantly, cyclin D1 is necessary for β-catenin to drive colon carcinoma development
[11]. It is also noteworthy that Wnt regulates cyclin D1 protein stability independent of β-
catenin much as Ras-signaling regulates cyclin D1 accumulation and activation through
multiple mechanisms [12, 13].
2. Epidermal Growth Factor Receptor (EGFR) and cyclin D1 expression
Cyclin D1 expression is responsive to a variety of growth factors [14], among which EGF is
a classic mediator [15]. EGFR overexpression and/or hyperactivation correlates with poor
prognosis in human cancers, including breast cancer, non-small cell lung carcinoma, and
colon carcinoma [16]. As a mitogenic growth factor, EGF regulates prostate cancer cell
proliferation at least partially through regulating cyclin D1 expression [17], and it regulates
cyclin D1 accumulation at both mRNA and protein levels. ErbB2, also known as Neu or
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Her2, is implicated in 20-30% of human breast cancers [18]. Here again, cyclin D1
expression is induced by Her2/Neu, Ras, Rac, Rho, c-Jun N-terminal kinase and p38 [19]; it
is of equal importance that cyclin D1-CDK4 function is required for Her2-driven mammary
carcinoma [19-21]. This work has contributing directly to the use and thus the success of
CDK4/6 inhibitors in patients with HER2 positive breast cancers [22].
3. Phosphatidylinositol 3-kinase (PI3K) regulates cyclin D1
PI3K catalyzes the phosphorylation of phosphatidylinositol 4,5-biphosphate (PIP2) to form
phosphatidylinositol 3,4,5-triphosphate (PIP3); PIP3 in turn recruits Akt/PKB to the cell
membrane, where it is phosphorylated and activated [23]. Activated Akt/PKB controls cell
growth, differentiation, proliferation, motility and metabolism. Previous work revealed a role
of PI3K in promoting G1/S cell cycle progression [24], suggesting a potential connection
with D-type cyclins. Indeed, PI3K/Akt regulate nuclear accumulation of cyclin D1 through
regulation of GSK3β [12]. Consistently, dominant-negative (DN) alleles of either subunit of
PI3K strongly suppress EGF-induced cyclin D1 accumulation [24]. Likewise, chemical
inhibition of PI3K also reduces cyclin D1 at both mRNA and protein levels upon EGF
stimulation, while rapamycin, a well-known mTORC1 inhibitor, exhibits no effect on EGF-
induced cyclin D1 regulation [24]. Cumulatively, this supports a model where PI3K is
indispensable for EGF-induced cyclin D1 upregulation. In glioma cells, cyclic-AMP
response element binding (CREB) protein acts as a critical hub that mediates PI3K-Akt-
induced cyclin D1 upregulation upon mitogenic stimulation [25]. Modulation of cyclin D1
by the PI3K-Akt signaling pathway represents one mechanism of growth factor-dependent
sensing by cyclin D1.
4. NF-κB-dependent control of cyclin D1
The NF-κB transcription factor family, including p65 (RelA), RelB, c-Rel, p50/p105 (NF-
κB1), and p52/p100 (NF-κB2), participates in various physiological and pathological
processes including inflammation, tumorigenesis and tumor progression [26]. Members of
the NF-κB family contain conserved Rel homology domain that mediates dimerization,
nuclear localization, DNA binding and their interaction with inhibitory IκB proteins (IκB).
NF-κB directly binds to cyclin D1 promoter and controls cyclin D1 transcription [27]. Other
related studies implicated c-Rel, RelB and p52 in the regulation of cyclin D1 transcription in
mammary tumors of transgenic mice [28], suggesting a key role of NF-κB-dependent
regulation of cyclin D1 during mammary gland tumorigenesis.
Post-transcriptional Control (Alternative Splicing) of cyclin D1
The gene encoding cyclin D1, CCND1, contains five coding exons, from which two
transcripts are derived (cyclins D1a and D1b) (Figure 1) [29, 30]. Cyclin D1a is transcribed
from an mRNA transcript derived from all five exons. The N-terminal region of cyclin D1a
has a conserved Rb binding LXCXE motif; the middle contains cyclin box with the greatest
homology between D Cyclins (Cyclin box is the domain that interacts with CDKs and CDK
inhibitors: p21, p27 and p57); the C-terminal domain regulates protein stability. As
discussed subsequently, this domain contains a threonine residue (Thr-286) that is
phosphorylated by glycogen synthase kinase-3β (GSK-3β) [12]; phosphorylation of this
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residue is both necessary and sufficient for ubiquitylation-dependent degradation. In contrast
to D1a, cyclin D1b is encoded by an mRNA where intron 4 is not spliced, resulting in a
unique C-terminus. Alternative splicing of CCND1 occurs primarily in the context of cancer
and splicing factors implicated in its generation include ASF/SF2 and Sam68 [31, 32]. As a
result of this alternative splicing, cyclin D1b losses its key regulatory motif encoded by exon
5 that directs its ubiquitylation-dependent degradation; the consequence is cyclin D1b
accumulation in the nucleus and ultimately tumorigenesis [30, 33].
Posttranslational Regulation of cyclin D1
Cyclin D1 is highly labile, with a half-life of 10-30 minutes, and its degradation depends on
cell cycle phases [12, 34]. Protein degradation is directed by polyubiquitylation, and
thereafter, destruction via the 26S proteasome. Cyclin D1 degradation requires site-specific
phosphorylation by GSK-3β at a conserved threonine residue, Thr-286. Mutation of this
threonine to a non-phosphorylatable residue dramatically stabilizes cyclin D1, inhibits its
nuclear export and triggers the constitutive activation of CDK4/CDK6 within the nuclear
compartment [12, 35, 36]. This nuclear dysregulation ultimately drives p53 inactivation,
rampant genomic instability and neoplastic transformation in vitro and tumorigenesis in vivo
[35, 37-40]. Although transcriptional regulation of cyclin D1 is complicated and is likely
responsive to an underappreciated number of transcriptional regulators, post-transcriptional
control ultimately dictates the overall accumulation of cyclin D1 in both normal and tumor
cells due to its relative instability.
Protein ubiquitylation requires the concerted and coordinated function of three enzymes: E1
ubiquitin activating enzyme, E2 conjugating enzyme, and E3 ubiquitin ligase. The E3 ligase
directs substrate specificity; it contains the largest family members and is generally the key
regulatory component in this pathway. E3 ligases are classified into three categories:
Homologous to E6-Associated Protein C-Terminus (HECT), Really Interesting New Gene
(RING) and U-box [41]. Among these, cyclin D1 ubiquitylation is directed by the RING
family E3 ligases. As discussed below, the SKP1-Cullin 1-F-box (SCF) is the primary
subclass that directs cyclin D1 ubiquitylation [42]. Within this subclass, SKP1 and Cullin 1
are core components; while the F-box proteins, composed of ~ 80 family members,
determine the substrate specificity. F-box proteins are defined by an F-box motif that is so
coined for its homology with cyclin F [43]. F-box proteins are divided into three classes:
Fbxw (with WD40 repeats as substrate binding domain), Fbxl (with Leucine-rich repeats as
substrate binding domain) and Fbxo (with “other” substrate binding domains) [44]. The
following section discusses the E3 ligases that have been implicated in regulating cyclin D1
ubiquitylation and degradation.
1. Fbxo4
Fbxo4 and alpha αB-crystallin, identified through the purification of cyclin D1 under
conditions that favor stabilization of substrate-E3 ligase binding, were subsequently
implicated as the major F-box protein binding to Thr-286 phosphorylated cyclin D1 [34, 45].
It was also noted that αB-crystallin is indispensable for Fbxo4-dependent binding to
phosphorylated cyclin D1. Fbox4-mediated cyclin D1 degradation involves the following
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steps: i) cyclin D1 phosphorylation; 2) CRM1-dependent nuclear export, and 3) cytoplasmic
polyubiquitylation and degradation (Figure 2) [46]. Phosphorylation of cyclin D1 at Thr-286
by GSK-3β is required for both binding to CRM1, which in turn directs nuclear export and
recognition by Fbxo4 [47]. GSK-3β also phosphorylates Fbxo4; this phosphorylation
generates a 14-3-3ε binding site and it is necessary for Fbxo4 homodimerization [48], a
regulatory event required for efficient cyclin D1 ubiquitylation. The importance of
phosphorylation and dimerization is emphasized by the identification of mutations in human
cancers that directly abrogate phosphorylation/dimerization, which in turn lead to cyclin D1
accumulation in human esophageal squamous cell carcinoma and melanoma [34, 47]. In
tumor cells, the overexpression and/or hyperactivation of mitogenic signaling pathways
activate PI3K-Akt signaling, which phosphorylates and inactivates GSK3β. This
“hypersignaling” directly impacts the Fbxo4-cyclin D1 axis, resulting in dysregulation of
nuclear cyclin D1-CDK4, and finally tumorigenesis [46, 48, 49].
While Fbxo4 is subject to point mutations in certain cancers, findings in hepatocellular
carcinoma (HCC) reflect a different mechanism. In HCC, sequencing analysis revealed four
Fbxo4 isoforms: Fbxo4α (full length), Fbxo4β (with 7 amino acids encoded by a read
through from intron 5, thus causing a sequence replacement for exon6), Fbxo4γ (missing
168-245 nt of exon1) and Fbxo4δ (missing exon6) [50]. Only Fbxo4α regulates cyclin D1
ubiquitylation-dependent degradation. The mechanisms that regulate the alternative splicing
and generation of different isoforms, and their impacts on cancers remain to be clearly
established.
2. Fbxo31
Cellular senescence can be triggered by the attrition of chromosomal telomeric ends or via
stress conditions that include low nutrient levels, oncogene activation, reactive oxygen
species, and radiation treatment. Among these, oncogene-induced senescence is considered
as an important mechanism for tumor suppression. Fbxo31 was identified in screening for
factors that regulate senescence. Fbxo31 levels can be induced by DNA-damage, and
interestingly, elevated Fbxo31 levels reversely correlate with cyclin D1 levels. Follow-up
investigation suggested that Fbxo31 is a checkpoint protein that arrests cells upon genotoxic
stress treatment [51]. Another work has revealed that Fbxo4 is also a major regulator of
cyclin D1 stability following DNA damage [52]. In fact, Fbxo4 is subject to hemizygous
mutations in human melanoma; moreover, Fbxo4 knockout mice overexpress cyclin D1 in
all tissues, including melanocytes. Of equal importance, Fbxo4 loss cooperates with
BRAF
V600E
to promote the development of metastatic melanoma in a cyclin D1-dependent
manner [47].
3. SKP2
The Cullin 1-SKP2-SKP1 E3 ligases make significant contribution to the regulation of the
G1/S transition. Key substrates include the CDK inhibitors p21 and p27 [53-57], which have
been validated biochemically and in cells through loss of function experiments. Cyclin D1
has also been suggested to be a substrate [58]. This conclusion was based on SKP2 loss of
function analysis and its binding to cyclin D1 in co-immunoprecipitation experiments.
However, SKP2 E3 ligase has not been shown to ubiquitylate cyclin D1, suggesting this
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