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Regular Article
LYMPHOID NEOPLASIA
Epstein-Barr virus latent membrane protein 2A enhances MYC-driven
cell cycle progression in a mouse model of B lymphoma
Kamonwan Fish, Jia Chen, and Richard Longnecker
Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois
Elevated expression of MYC is a shared property of many human cancers. Epstein-Barr
virus (EBV) has been associated with lymphoid malignancies, yet collaborative roles
between MYC and EBV in lymphomagenesis are unclear. EBV latent membrane protein
• Latent membrane protein
2A augments MYC oncogene 2A (LMP2A) functions as a B-cell receptor (BCR) mimic known to provide survival
signals to infected B cells. Co-expression of human MYC and LMP2A in mice (LMP2A/
in driving the cell cycle by
l-MYC) accelerates B lymphoma onset compared with mice expressing human MYC
increasing protein instability
kip1
alone (l-MYC mice). Here we show a novel role of LMP2A in potentiating MYC to
of a tumor suppressor p27 .
promote G1-S transition and hyperproliferation by downregulating cyclin-dependent
• Latent membrane protein 2A
kinase inhibitor p27kip1 in a proteasome-dependent manner. Expressing a gain-ofpotentiates MYC expression
function S10A mutant of p27kip1 has minor effect on tumor latency. However, pretumor
to overcome a cell cycle
B cells from l-MYC mice expressing homozygous S10A mutant show a significant
checkpoint without disrupting
decrease in the percentage of S-phase cells. Interestingly, LMP2A is able to counteract
p53 tumor suppressor
the antiproliferative effect of the S10A mutant to promote S-phase entry. Finally, we
pathway.
show that LMP2A expression correlates with higher levels of MYC expression and
suppression of p27kip1 before lymphoma onset. Our study demonstrates a novel function of EBV LMP2A in maximizing MYC expression, resulting in hyperproliferation and cellular transformation into cancer cells in
vivo. (Blood. 2014;123(4):530-540)
Key Points
Introduction
The cell cycle is a tightly regulated process governed by cyclins,
cyclin-dependent kinases (CDKs), and CDK inhibitors. Cell cycle
checkpoints are important cellular mechanisms that prevent uncontrolled proliferation caused by oncogenic stimuli. Retinoblastoma
(Rb) and p53 pathways are critical pathways preventing premature cell
cycle progression by inducing cell cycle arrest or apoptosis. Although pathways and mutations leading to malignancies are different
depending on cancer types, many routes converge to increase MYC
expression through chromosomal translocation, amplification of
c-MYC transcription, and protein stabilization. MYC is a basic helixloop-helix (bHLH) transcription factor regulating many target genes
in most, if not all, cell types. MYC heterodimerizes with a binding
partner, Max, and binds to CACGTG-containing DNA sequences.1,2
Recent studies suggest that the main function of MYC is to upregulate
transcription of its target genes which in turn indirectly lead to
repression of certain genes,3,4 including those encoding CDK
inhibitors. MYC is important for B-lymphocyte activation and
proliferation.5 Constitutive expression of MYC in murine B cells
leads to increases in the percentage of cells in S and G2/M phases of
the cell cycle.6,7 MYC activation increases activities of cyclinCDK complexes, resulting in the hyperphosphorylation of Rb8 and
release of E2F transcription factors to upregulate S-phase genes.
MYC promotes the expression of D-type cyclins9,10 and E2F,11,12
and it contributes to the transcription repression of genes encoding
CDK inhibitors p27Kip1, p21Cip1, and p15Ink4b, leading to the
progression into S-phase.13 MYC also induces apoptosis via the
upregulation of p19ARF and induction of the p53 pathway.14 Mice
lacking p53, Cdkn2a, and Cdkn1b show rapid onset of MYCinduced lymphomagenesis,15,16 suggesting importance of cell cycle
regulators in preventing excessive proliferation caused by MYC.
Latent infection of Epstein-Barr virus (EBV), a member of
gammaherpesviruses, is associated with cellular hyperproliferation
observed in posttransplant lymphoproliferative disorders (PTLD), as
well as malignancies including Hodgkin disease, and non-Hodgkin
lymphoma.17 EBV is linked to a role in suppressing apoptosis
induced by MYC in infected B cells and thus aids MYC in cell
growth and proliferation.18 Latent membrane protein 2A (LMP2A)
is encoded by EBV and found in most latency programs. LMP2A
contains an ITAM (immunoreceptor tyrosine activation motif) similar
to that of the host BCR19 and provides BCR-like survival signals to
B cells.20-22 Furthermore, a recent study suggests that LMP2A can
contribute to proliferation of B cells during an early phase of EBVinduced B-cell proliferation,23 suggesting a pro-proliferative role of
LMP2A in the cellular transformation process.
In a mouse model of EBV latent infection, mice expressing
LMP2A and human MYC transgene (LMP2A/l-MYC) demonstrate
Submitted July 25, 2013; accepted October 27, 2013. Prepublished online as
Blood First Edition paper, October 30, 2013; DOI 10.1182/blood-2013-07517649.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
There is an Inside Blood commentary on this article in this issue.
530
© 2014 by The American Society of Hematology
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A NOVEL LINK OF EBV AND MYC IN THE CELL CYCLE
accelerated lymphomagenesis compared with mice expressing
human MYC alone (l-MYC mice),24,25 which is similar to another
study showing that constitutive activation of the BCR leads to a
rapid onset of MYC-induced lymphoma.26 The p19ARF-p53 pathway
is an important mechanism controlling aberrant proliferation induced
by pathologic expression of MYC. Therefore, mutations and inactivation of the p53 pathway are frequently found in MYC-induced
tumorigenesis.27 However, p53 pathway inactivation is absent in
LMP2A/l-MYC tumors, suggesting that LMP2A uses a different
mechanism to promote MYC-driven lymphomagenesis.
In this study, we investigate a role of LMP2A in altering the cell
cycle regulation independent of the p53 pathway. We demonstrate that
LMP2A cooperates with MYC in disrupting the G1 checkpoint
through the downregulation of a CDK inhibitor p27kip1, leading to
premature S-phase entry. We also show that LMP2A expression leads
to high MYC levels, indicating a novel relationship between MYC
and LMP2A. Our study sheds new light onto an intricate collaboration
of EBV and host oncoproteins in malignant transformation.
531
was used. Real-time polymerase chain reaction (RT-PCR) was carried out on an
Applied Biosystems Step One Plus machine using primers specific for the
indicated genes. Fast SYBR green master mix from Applied Biosystems was
used in all reactions. The DDCt method was used to normalize gene expression
to Gapdh.
Immunoblots
Pretumor B cells or tumor cells were lysed in lysis buffer with protease and
phosphatase inhibitor cocktails (Roche). Lysates were electrophoretically
separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (BioRad). Protein was transferred to a nitrocellulose membrane (Whatmann) and
incubated with primary antibodies. Membranes were probed with IRDye
secondary antibodies (Li-Cor Biosciences). The protein bands were visualized
with an Odyssey Fc Western Blotting Imager and analyzed with Image Studio
version 2.0 (Li-Cor Biosciences).
Statistical analysis
A 2-tailed Student t test, 1-way and 2-way analysis of variance (ANOVA),
and survival analysis were analyzed using GraphPad (GraphPad Software,
Inc.). P , .05 was considered statistically significant.
Materials and methods
Mice
Results
and l-MYC mice have
The Tg6 lines of EmLMP2A transgenic mice
been described and are in the C57BL/6 background. Mice expressing an
S10A mutant of p27kip1 (Cdkn1btm2Jro mice) were constructed previously29
and obtained from The Jackson Laboratory and are in the 129 background.
Tumor mice were sacrificed when lymph node tumors could be observed
externally or mice were moribund. Animals were maintained at Northwestern
University’s Center for Comparative Medicine, in accordance with the
university’s animal welfare guidelines.
20,21
28
Tumor and spleen cell isolation
Pretumor spleens were isolated from 21- to 25-day-old mice. Magnetic
activated cell sorting using CD19 positive selection (Miltenyi Biotec) were
used to purify splenic B cells. Tumor-bearing lymph nodes were dissociated
and treated with 155 mM ammonium chloride to lyse red blood cells.
Approximately 90% of tumor cells in tumor-bearing lymph nodes were of
B-cell lineage and thus did not required further cell sorting.
Flow cytometry and cell cycle analysis
Five million purified pretumor B cells were fixed in ice-cold 70% ethanol.
Cells were stained with Ki-67-FITC antibody and DNA was stained with
PI/RNase staining buffer according to the manufacturer’s instructions
(BD Biosciences). For the viability assay, primary tumor cells were stained with
7-AAD (Invitrogen) and anti–CD19-APC antibody (eBioscience) after Nutlin-3
(CalBiochem) treatments. All samples were analyzed using the FACS-CantoII
flow cytometer (BD Biosciences) and FlowJo v 9.2 software (Tree Star, Inc.).
Histology
Tumor-bearing lymph nodes were fixed in 10% buffered formalin phosphate
(Fisher Scientific) followed by paraffin embedding. Pretumor spleens were
frozen in tissue-embedding medium (Tissue-Tek). Tumors were sectioned
and stained with hematoxylin and eosin (tumor lymph nodes) or with antiB220 antibody (pretumor spleens) at the Mouse Histology and Phenotyping
Laboratory at Northwestern University. Stained tissue sections were imaged
using an EVOS XL Core digital inverted microscope (Advanced Microscopy
Group) using a 203 magnification lens.
Real-time polymerase chain reaction
Total RNA was isolated from purified pretumor B cells using the RNeasy RNA
extraction kit with DNase I digestion from QIAGEN. To generate cDNA,
a High-Capacity cDNA Reverse Transcription kit from Applied Biosystems
LMP2A couples with MYC to promote G1-S transition
Because previous studies suggested that LMP2A promotes cell cycle
progression in the context of deregulated MYC,24,25 we chose to
investigate mechanisms by which LMP2A disrupts the cell cycle
regulation. Because p53 pathway inactivation is frequent in MYCdriven lymphomagenesis, the status of the p53 pathway may interfere
with analysis of the cell cycle when comparing tumor cells from
LMP2A/l-MYC and l-MYC mice. Thus we chose to examine cell
cycle profiles of pretumor B cells because the p53 pathway is intact
at this stage in both LMP2A/l-MYC and l-MYC mice. Purified
B cells from 3-week-old pretumor LMP2A/l-MYC mice demonstrated high proliferative characteristics, with a significant increase in
the percentage of Ki-67–positive cells (Figure 1A-B) and percentage
of S-phase cells (Figure 1C-D) compared with those isolated from
l-MYC mice of the same age. The hyperproliferative phenotype of
LMP2A/l-MYC pretumor B cells was also evident histologically.
Although wild-type (WT) C57BL/6 and LMP2A and l-MYC transgenic mice formed B-cell follicles in secondary lymphoid organs
such as the spleen, LMP2A/l-MYC mice showed disorganized B-cell
follicles (Figure 1E) and an increase in B2201 population. WT,
LMP2A, and l-MYC mice had comparable percentages of immature
and mature B cells. LMP2A/l-MYC B2201 cells were IgM–IgD–,
likely as a result of LMP2A replacing the requirement for the
BCR20,30,31 (see supplemental Figures 1 and 2 available on the
Blood Web site for other detailed analyses). These data indicate
that LMP2A promotes S-phase entry and hyperproliferation of
B lymphocytes before malignant transformation.
LMP2A expression disrupts a cell cycle checkpoint during G1
We further delineated changes in cell cycle regulators of the G1
checkpoint in LMP2A– and LMP2A1 cells by western blot analyses.
Interestingly, of all CDK inhibitors important for preventing premature progression from G1 to S phase, p27kip1 was the only CDK
inhibitor significantly downregulated in pretumor LMP2A/l-MYC
B cells compared with l-MYC B cells (Figure 2A-B). There was also
a moderate increase in phosphorylated Rb and cyclin A (a marker for S
phase) in LMP2A/l-MYC B cells, suggesting that low levels of p27kip1
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FISH et al
BLOOD, 23 JANUARY 2014 x VOLUME 123, NUMBER 4
Figure 1. Pretumor B cells from LMP2A/l-MYC mice
demonstrate highly proliferative phenotypes. (A)
Histograms of Ki-671 purified B cells from 3-week-old
representative mice. The number in each histogram
indicates the percentage of Ki-671 cells in purified
B cells. (B) Combined Ki-67 data from 3 experiments
shown as mean 6 SD. The differences in the percentage of Ki-671 cells were analyzed by 1-way ANOVA.
***P , .001; ns, not significant. (C) Representative
histograms of propidium iodine staining for DNA content
in purified pretumor B cells. (D) Pie charts show
mean 6 SD of cells in different phases of the cell
cycle as analyzed in (C). WT: n 5 3, LMP2A: n 5 3,
LMP2A/l-MYC: n 5 4, l-MYC: n 5 4. (E) Immunohistochemistry of B2201 cells in representative 3-weekold mouse spleens (original magnification 320). No
1° Ab, negative control with secondary antibody only.
result in the disruption of the Rb pathway in G1 checkpoint and lead to
premature S-phase entry. However, quantitative RT-PCR (qRT-PCR)
revealed that Cdkn1b mRNA levels were similar between LMP2A/
l-MYC and l-MYC pretumor B cells (Figure 2C). These data agreed
with previous studies demonstrating that MYC can promote Cdk1b
transcriptional repression.32,33 Taken together, LMP2A does not contribute to Cdkn1b transcriptional repression mediated by MYC, but
rather downregulates p27kip1 at the protein level to promote cell cycle
progression.
Instability of p27kip1 in LMP2A/l-MYC and l-MYC pretumor
B cells
Because p27kip1 is largely controlled by posttranslational modification
and proteolysis,34-36 we further explored the possibility that LMP2A
could increase the instability of p27kip1 to promote cell cycle progression. Pretumor B cells of LMP2A/l-MYC and l-MYC littermates
were treated with the protein synthesis inhibitor cycloheximide (CHX)
for 1, 2, and 3 hours. LMP2A/l-MYC B cells showed a slightly shorter
half-life compared with l-MYC B cells after CHX treatment. However,
the reduction of p27kip1 was significantly diminished when LMP2A/
l-MYC B cells were co-treated with both CHX and MG-132, a
proteasome inhibitor (Figure 3A-B). Furthermore, when treated with
MG-132 alone, LMP2A/l-MYC B cells had twice the level of p27kip1
compared with vehicle-treated cells. In contrast, MG-132 had minimal
effect on p27kip1 expression in l-MYC B cells (Figure 3C). These data
indicate that LMP2A promotes p27kip1 degradation through a proteasome-dependent process.
Expression of S10A mutant form of p27kip1 (p27S10A) in
MYC-driven lymphomagenesis
To examine the importance of p27kip1 in LMP2A-mediated lymphomagenesis, we crossed our transgenic mice to knock-in mice in
which serine 10 of p27kip1 was replaced with alanine (p27S10A). We
chose the p27S10A knock-in mutant mice because previous studies
demonstrated a tumor-suppressing phenotype compared with WT
p27kip1 in a mouse model of lung cancer,29 a lymphoproliferative
disorder,37 and SV40-induced tumorigenesis.38 In addition, murine
embryonic fibroblasts (MEFs) from p27S10A/S10A mice have a nuclear
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Figure 2. P27Kip1 is downregulated at the protein
level, but not at the mRNA level, in pretumor B cells
from LMP2A/l-MYC mice compared with those in
l-MYC mice. (A) Representative western blots of cell
cycle regulators in purified B cells from 3-week-old mice
of indicated genotypes (C57BL/6 background). Each lane
represents a protein sample from one mouse. (B) P27kip1
protein expression in B cells from 3-week-old mice shown
as a ratio to WT expression from 5 independent experiments. (C) qRT-PCR analyses of Cdkn1b expression
from 2 independent experiments using a WT mouse as
a control for each experiment. All differences in mRNA
and protein expression were analyzed by 1-way ANOVA.
**P , .01, ***P , .001; ns, not significant. Data represent
the mean 6 SD.
localization phenotype29 and are more refractory to degradation
mediated by SV40-induced transformation compared with MEFs
from WT mice.38 Because the role of S10 phosphorylation of p27kip1
had not been tested in the context of deregulated MYC in vivo, the
S10A mutant could act as a gain-of-function form of p27kip1 and
would allow us to test whether the expression of this mutant would
influence lymphoma development in our model. We hypothesized
that expression of p27S10A would prevent LMP2A-mediated p27kip1
suppression and would delay lymphomagenesis to be similar to that of
l-MYC mice. However, we found the median survival time of all
LMP2A/l-MYC mice was very similar (36-38 days). We noted that
a small subset of LMP2A/l-MYC1/S10A and LMP2A/l-MYCS10A/S10A
mice were tumor-free at day 50 when all LMP2A/l-MYC1/1 developed tumors. One LMP2A/l-MYCS10A/S10A mouse lived tumor-free
beyond the median survival time of l-MYC1/1 (122 days). All
l-MYC mice regardless of p27kip1 genotypes had similar time-tolymphoma onset. This observation suggests that p27S10A has a minimal
effect on the latency time of lymphoma development in our model
(Figure 4A).
P53-mediated apoptosis is an important tumor-suppressive mechanism counteracting MYC oncogenic function. To examine whether
p27S10A expression would change the status of the p53 pathway,
primary tumor cells were treated with the MDM2 inhibitor Nutlin-3,
which leads to the release of p53 from its negative regulator. All
LMP2A/l-MYC tumor cells were sensitive to Nutlin-3 treatments,
resulting in a sharp decrease in viability compared with control at 3
hours post treatment, suggesting that these cells have an intact p53
pathway after activation of p53 upon Nutlin-3 treatment. All l-MYC
tumor cells were resistant to Nutlin-3 treatments (Figure 4B),
indicating that p53 is mutated or the pathway is inactivated, similar to
previous reports.24,27 Previous studies reported that tumors with
a defect in the p53-19ARF-MDM2 pathway show aberrant accumulation or stabilization of p19ARF and p53 caused by the disruption of
a p53 negative-feedback loop.24,27,39-41 Excessive accumulation of
p53 and p19ARF proteins in l-MYC tumor cells was evident but was
absent in all LMP2A/l-MYC tumors, regardless of genotypes of
p27kip1 (Figure 4C). These data indicate that LMP2A effectively
promotes lymphoma development without perturbing the p53 pathway, and tumor promotion by LMP2A is unaffected by p27S10A
expression. Despite the strong differences in tumor onset time and
p53 status, all tumors from LMP2A/l-MYC and l-MYC mice were
histologically similar, with the “starry sky” appearance resembling
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BLOOD, 23 JANUARY 2014 x VOLUME 123, NUMBER 4
Figure 3. LMP2A facilitates p27kip1 degradation in
a proteasome-dependent manner. (A) Representative western blot analyses of p27kip1 expression after
treatments with 25 mg/mL cycloheximide (CHX) with or
without 40 mM MG-132 at indicated time points. The
levels of p27kip1 were normalized to calnexin (a loading
control), and the fold reductions of proteins were
calculated using cells at 0 hour of each genotype as
a control. (B) Combined data from 3 experiments
represent the mean 6 SD of the p27kip1 level. Differences in p27kip1 protein expression were analyzed by
2-way ANOVA. *P , .05, **P , .01, ***P , .001. (C)
Representative western blot analyses of pretumor
B-cell treatment with vehicle control (dimethyl sulfoxide
[DMSO]) 40 mM, or 60 mM MG-132 for 2 hours. Gapdh
and calnexin were used as loading controls. The left
and right parts of the images shown are from the same
membrane, and the middle, irrelevant, lanes were
removed. Experiments were repeated at least 3 times.
Burkitt lymphoma (Figure 4D and supplemental Figure 3). Taken
together, these data indicate that p27S10A expression does not prevent
p53 pathway inactivation in l-MYC tumors and that the p53 pathway
is still functionally intact in all LMP2A/l-MYC tumors examined.
LMP2A antagonizes p27S10A function
To investigate whether the p27S10A mutant could alter the cell cycle
profile at earlier steps of lymphomagenesis, spleen mass was analyzed in mice at 3 weeks of age. P27S10A expression did not
significantly change the relative spleen mass or the ratios of B:
T cells, although there was a small decrease in relative spleen weight
of mice with homozygous p27S10A compared with those with WT
p27kip1 (supplemental Figure 4). We found that all LMP2A/l-MYC
pretumor B cells were .90% Ki-671 (Figure 5A). Interestingly,
there was a dose-dependent decrease in the percentage of Ki-671
B cells with the expression of heterozygous and homozygous
p27S10A in l-MYC pretumor B cells (Figure 5A). Furthermore, cell
cycle analyses showed a significant dose-dependent increase in G1
cells and a significant decrease in S-phase cells in l-MYC pretumor
B cells with the expression of 1 or 2 alleles of p27S10A. These data
indicate that p27S10A impedes S-phase entry in early events of
lymphoma development. However, the effect of p27S10A in blocking
G1-S transition was lost in all LMP2A/l-MYC pretumor B cells
(Figure 5B), indicating that LMP2A can counteract the antiproliferative
function of p27S10A to promote MYC-driven cell cycle progression.
To delineate the molecular mechanisms responsible for the failure
of p27S10A in blocking G1-S transition in LMP2A/l-MYC pretumor
B cells but successfully preventing S-phase entry in l-MYC pretumor
B cells, pretumor B cells were subjected to western blot analyses. We
found an opposite trend of increasing p27kip1 and decreasing MYC
levels in representative l-MYC1/S10A and l-MYCS10A/S10A compared
with l-MYC1/1. In addition, we found a decrease in cyclin A in
l-MYCS10A/S10A pretumor B cells, corresponding to a lower percentage
of B cells in S-phase shown in Figure 5B. LMP2A/l-MYCS10A/S10A
pretumor B cells did not show an increase in p27kip1 level compared
with LMP2A/l-MYC1/1 cells. Furthermore, MYC was expressed at
high levels in all LMP2A/l-MYC pretumor B cells, regardless of
p27S10A expression (Figure 6A-B).
Because p27S10A/S10A showed a nuclear translocation phenotype,29,37 the low level of total p27kip1 in LMP2A/l-MYC pretumor
B cells suggests that another degradation process plays a role in
LMP2A-mediated p27kip1 degradation. Previous studies showed that
Cks1 and Skp2, both are constituents of SCFSkp2 complex regulating
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Figure 4. Expression of S10A mutant confers minor effect on time-to-tumor onset of LMP2A/l-MYC mice and has no effect on the p53 pathway. (A) Kaplan-Meier
curves indicating the percent survival of indicated genotypes. (B) Viability of primary tumor cells (7-AAD–, CD191) at 3 hours after 5 mM or 10 mM Nutlin-3 treatments from 3
independent experiments. Percentage of viability in vehicle control in each genotype was set at 100%. (C) Western blot analyses of representative primary tumor cells showed
aberrant stabilization of p53 and/or p19ARF in tumors from l-MYC mice. (D) Hematoxylin and eosin (H&E) staining of tumor-bearing lymph nodes demonstrating a “starry sky”
appearance in all tumor genotypes (original magnification 320).
nuclear p27kip1 degradation, were increased in precancerous B cells
from Em–Myc mice compared with WT B cells.42 We found similar
Skp2 levels in both LMP2A/l-MYC and l-MYC pretumor B cells,
whereas there was an increase in Cks1 protein level in LMP2A/
l-MYC B cells. There was also an elevated Cks1 mRNA in LMP2A/
l-MYC compared with l-MYC B cells, but the difference was not
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Figure 5. Homozygous p27S10A expression significantly decreases S-phase pretumor B cells from
l-MYC mice but has no effect in LMP2A/l-MYC
pretumor B cells. (A) Percentage of Ki-671 pretumor
B cells of each genotype. Data were combined from 5
experiments and represent the mean 6 SD. (B) Pie
charts and bar graphs demonstrating the cell cycle
profile of pretumor B cells. Data were analyzed with
1-way ANOVA and represent the mean 6 SD. *P , .05,
**P , .01. The inset shows pie charts representing
mean percentages of pretumor B cells in each phase of
the cell cycle.
statistically significant (supplemental Figure 5). These data suggest
that high MYC levels in LMP2A/l-MYC B cells promotes further
p27kip1 degradation, at least in part by upregulating Cks1 in the
SCFSkp2 complex, which is active in the late G1 and S phases.
Overall, these results indicate that LMP2A expression can antagonize
the function of p27S10A by maintaining MYC at a high level, leading
to continued downregulation of p27kip1 levels. In the absence of
LMP2A, the gain-of-function p27S10A is able to increase total p27kip1
level, which in turn inversely correlates with MYC expression.
Discussion
This study reports the collaborative efforts of MYC and EBV LMP2A
in disrupting the cell cycle before progression to malignancy. The
complex relationship of a tumor virus and a human oncogene is
exemplified in our model, which demonstrates that EBV provides an
optimal intracellular environment conducive to malignant transformation. Our studies confirm previous studies on how 2 important
tumor suppressor pathways counteract MYC and reveal how
EBV changes the equation to favor the pro-proliferative function
of MYC.
Low levels of p27kip1 are observed in several human cancers,43
and murine models of cancers indicate that p27kip1 is a dosedependent tumor suppressor.44-47 Mice hemizygous for p27kip1
(p272/1) display an intermediate phenotype in body weight and cell
proliferation between p272/2 and p271/1 mice.48 The loss of 1 or 2
copies of p27kip1 demonstrates a dose-dependent decrease in tumor
latency in g-irradiation and carcinogen-induced tumor models.49,50
Our studies are compatible with these studies because expression of 1
or 2 copies of the gain-of-function S10A mutant in l-MYC pretumor
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Figure 6. LMP2A expression associates with high
MYC level, which inversely correlates with low
p27kip1 levels. (A) Western blot analyses of cell cycle
regulators in purified B cells from 3-week-old WT,
LMP2A/l-MYC1 /1 , LMP2A/l-MYC1 /S10A , LMP2A/
l-MYC S10A/S10A , l-MYC 1 /1 , l-MYC 1 /S10A , and
l-MYCS10A/S10A mice. Cyclin A was used as an S-phase
marker. Normalized levels of p27kip1 and MYC are shown
as percentages to Gapdh (loading control). (B) Bar
graphs demonstrating MYC and p27kip1 protein expression in B cells from 3-week-old mice from 2 independent experiments. Each bar represents combined
data from 4 experiments, with data represented as the
mean 6 SD.
B cells results in a dose-dependent decrease in the percentage of cells
in S-phase and that are Ki-671.
Germline deletion or mutation of the p27kip1 gene, CDKN1B, in
human cancer is rare, suggesting that other mechanisms play a major
role in regulating p27kip1 in cancer. MYC promotes transcriptional
repression of the p27kip1 gene,32 but p27kip1 is largely controlled at
the protein level. MYC promotes p27kip1 protein degradation by
upregulating transcription of negative regulators of p27Kip1,42,51-53
including Cks1 and Skp2. Furthermore, phosphorylation of p27kip1 at
different sites controls degradation and subcellular localization of
p27kip1 at different time points in the cell cycle. There are at least 2
pathways regulating p27kip1 degradation. In G0 and early G1, p27kip1
is phosphorylated at serine 10 and is exported from the nucleus.
Cytoplasmic p27kip1 is targeted for proteasomal degradation mediated by the Kip1 ubiquitination-promoting complex (KPC).54 In
the late G1 and S phases, p27kip1 is phosphorylated at threonine 187
and recognized by the SCFSkp2 complex, which ubiquitinates p27kip1
and targets it for the degradation by the proteasome in the
nucleus.55,56 Mice lacking Csk1 or Skp2 showed an increase in
p27kip1 level and delayed MYC-induced tumorigenesis.42,52 We
found that p27S10A fails to increase total p27kip1 levels in LMP2A/
l-MYC1/S10A and LMP2A/l-MYCS10A/S10A pretumor B cells,
indicating that LMP2A still efficiently promotes nuclear degradation of p27kip1 mediated by SCFSkp2 complex in late G1 and
S-phase and may do so via Cks1 upregulation. Another possibility
is that high MYC levels in pretumor B cells from LMP2A/l-MYC
mice make the degradation of p27kip1 very rapid (Figure 6). However, both cytoplasmic and nuclear degradation pathways may be
required to act in concert. Future studies will further examine the
role of the SCFSkp2 complex in LMP2A-mediated lymphoma
development.
It is important to note that regulatory processes of p27kip1 in
cancers can be different in different cell types and oncogenic stimuli.
Immortalized MEFs from p27S10A/S10A mice are more resistant to
in vitro transformation by K-Ras but show similar transformation
ability induced by MYC compared with WT MEFs.50 These data
suggest that K-Ras and MYC suppress p27kip1 through different
mechanisms during cellular transformation and could explain the
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538
FISH et al
BLOOD, 23 JANUARY 2014 x VOLUME 123, NUMBER 4
Figure 7. The proposed relationship of MYC,
LMP2A, and p27kip1 in lymphoma development.
(Top) In an l-MYC mouse model, overexpression of
MYC results in repression of Cdkn1b transcription and
increases p27kip1 degradation, leading to an increase in
proliferation. The remaining pool of p27kip1 partially
impedes MYC-induced cell cycle progression and
downregulates MYC. Abundant expression of MYC
also induces p53 pathway activation. Both p27kip1 and
p53 are able to prevent uncontrolled proliferation for
a certain length of time. At a later step, selection
pressure forces deregulated MYC cells to overcome
the apoptosis mechanism mediated by p53, and a
lymphoma develops. (Bottom) In the presence of
LMP2A, p27kip1 turnover is more rapid and weakens
the MYC repression network, resulting in a high level of
MYC. Although the p53 pathway is induced as a result
of overexpression of MYC, cell cycle progression and
proliferation outpaces the rate of apoptosis. A severely
low level of p27kip1 may relieve the selection pressure,
and a lymphoma develops without the need to inactivate the p53 pathway.
difference in the tumor-suppressing function of p27S10A in our MYC
model and K-Ras–induced tumorigenesis in previous studies.29,38
Furthermore, the localization of p27kip1 in quiescent or activated
B cells is different from that of T cells and MEFs. P27kip1 is mainly
localized in the cytoplasm of resting B cells and is translocated to the
nucleus upon BCR activation. Defects in the suppression of serine
10 phosphorylation on p27kip1 inhibit the nuclear translocation
process and thus contribute to autoimmunity and a lymphoproliferative disorder, and expression of p27S10A corrects this anomaly.37
Although homozygous p27S10A expression significantly reduced the
percentage of S-phase pretumor B cells from l-MYC mice (Figure 6B),
it had only a small effect in tumor latency (Figure 4A). Therefore,
p27S10A may be a mild gain-of-function mutant in an MYC-driven
lymphoma model.
Our study is the first to show an underlying mechanism of EBV
LMP2A in promoting cell cycle progression via p27kip1 downregulation. A recent study showed that LMP2A is important for cell
proliferation and efficient long-term growth after the transformation
of primary B cells.23 LMP2A may be dominant in promoting MYCinduced p27kip1 downregulation in latency program I, where EBNA1
and LMP2A appear to be uniquely expressed.57,58 Furthermore, in
vitro BCR stimulation in primary normal B cells results in the
downregulation of p27kip1, G1-S transition, and proliferation of
B cells.37 Given that LMP2A is a BCR mimic, pathways leading to
p27kip1 degradation in this study may be similar to a stimulated BCR,
which involves the Erk and/or Akt pathway.37 Inhibition of Akt
induces apoptosis in B cells from LMP2A transgenic mice.59
Furthermore, the inhibition of the Lyn kinase or the mammalian
target of rapamycin (mTOR) pathway leads to decreases in spleen
and tumor sizes in LMP2A/l-MYC mice compared with l-MYC
mice,60,61 suggesting that LMP2A is able to mimic the BCR signal
through the Akt/mTOR pathway to promote lymphomagenesis.
The dramatic downregulation of p27kip1 in pretumor B cells and
the accelerated lymphomagenesis in LMP2A/l-MYC mice correspond
with a previous study showing that Em–Myc mice lacking p27kip1 have
rapid tumor onset compared with those with WT p27kip1,16 suggesting
the importance of p27kip1 in suppressing MYC-driven lymphomagenesis. However, p53 status in tumors of Em–Myc; p272/2 was not
assessed. The relationship of how MYC represses p27kip1 is better
documented than how p27kip1 can negatively regulate MYC. It is
possible that p27kip1 is a part of a negative-feedback network
controlling the MYC level. Because LMP2A expression associates
with the continued p27kip1 repression, even when homozygous
p27S10A is present, the importance of p27kip1 in LMP2A-mediated
lymphomagenesis is still unclear and future studies should address
this issue.
In l-MYC B cells, our data suggest that the downregulation of
the p27kip1 and p53 pathway inactivation occurs at different time
points—p27kip1 downregulation precedes p53 inactivation. We propose that MYC downregulates p27kip1 both at the transcription and
protein levels, leading to cell cycle progression. The remaining pool of
p27kip1 then functions as part of an MYC repression network to control
MYC. MYC can also induce apoptosis through the induction of the
p53 pathway. Both p53 and p27kip1 act in concert or independently
to prevent uncontrolled proliferation. However, selection pressure
promotes the survival of only cells that contain a disruption in one or
more components of the p53 pathway. P27kip1 may act dominantly at
an early stage of lymphomagenesis, and the downregulation of p27kip1
may be less important after p53 pathway inactivation. In the presence
of LMP2A, such as in EBV-infected B cells, p27kip1 is downregulated
and MYC may be able to drive proliferation to a greater extent, thus
outpacing apoptosis. Therefore, B cells expressing both deregulated
MYC and LMP2A expand more readily without the need to mutate or
inactivate the p53 pathway (Figure 7).
In summary, we demonstrated for the first time the cooperative
role of LMP2A and MYC in disrupting the cell cycle through
promoting p27kip1 degradation at the early stage of lymphomagenesis. LMP2A perturbs the MYC negative feedback loop, partially
mediated by p27kip1, to accentuate MYC expression and accelerate
lymphomagenesis without altering the p53 pathway. Our data shed
a new light on how EBV infection aids MYC in counteracting with
antiproliferative cellular mechanisms, and our results also emphasize
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BLOOD, 23 JANUARY 2014 x VOLUME 123, NUMBER 4
A NOVEL LINK OF EBV AND MYC IN THE CELL CYCLE
critical roles of EBV infection in creating an intracellular state
favorable for malignant transformation.
539
(R.L.); and the Training Program in Viral Replication (T32AI060523-09)
(K.F.).
Authorship
Acknowledgment
The authors thank members of the Longnecker laboratory for help
in completion of this study. J.C. is a fellow of the American Heart
Association. R.L. is Dan and Bertha Spear Research Professor.
This work is supported by the Northwestern University Mouse
Histology and Phenotyping Laboratory and a Cancer Center Support
Grant, National Institutes of Health (NCI CA060553); the National
Cancer Institute, National Institutes of Health (CA073507, CA133063)
Contribution: K.F. and R.L. designed research, analyzed data,
and wrote the manuscript; and K.F. and J.C. performed the
research.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Richard Longnecker, Northwestern University,
303 East Chicago Ave, Chicago, IL 60611; e-mail: r-longnecker@
northwestern.edu.
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2014 123: 530-540
doi:10.1182/blood-2013-07-517649 originally published
online October 30, 2013
Epstein-Barr virus latent membrane protein 2A enhances MYC-driven
cell cycle progression in a mouse model of B lymphoma
Kamonwan Fish, Jia Chen and Richard Longnecker
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