Specific tumor suppressor function for E2F2 in Myc-induced T cell lymphomagenesis.
ABSTRACT Deregulation of the Myc pathway and deregulation of the Rb pathway are two of the most common abnormalities in human malignancies. Recent in vitro experiments suggest a complex cross-regulatory relationship between Myc and Rb that is mediated through the control of E2F. To evaluate the functional connection between Myc and E2Fs in vivo, we used a bitransgenic mouse model of Myc-induced T cell lymphomagenesis and analyzed tumor progression in mice deficient for E2f1, E2f2, or E2f3. Whereas the targeted inactivation of E2f1 or E2f3 had no significant effect on tumor progression, loss of E2f2 accelerated lymphomagenesis. Interestingly, loss of a single copy of E2f2 also accelerated tumorigenesis, albeit to a lesser extent, suggesting a haploinsufficient function for this locus. The combined ablation of E2f1 or E2f3, along with E2f2, did not further accelerate tumorigenesis. Myc-overexpressing T cells were more resistant to apoptosis in the absence of E2f2, and the reintroduction of E2F2 into these tumor cells resulted in an increase of apoptosis and inhibition of tumorigenesis. These results identify the E2f2 locus as a tumor suppressor through its ability to modulate apoptosis.
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ABSTRACT: In a previous genome-wide expression profiling study, we identified E2F2 as a hyperexpressed gene in stem-like cells of distinct glioblastoma multiforme (GBM) specimens. Since the encoded E2F2 transcription factor has been implicated in both tumor suppression and tumor development, we conducted a functional study to investigate the pertinence of E2F2 to human gliomagenesis. E2F2 expression was knocked down by transfecting U87MG cells with plasmids carrying a specific silencing shRNA. Upon E2F2 silencing, in vitro cell proliferation was significantly reduced, as indicated by a time-course analysis of viable tumor cells. Anchorage-independent cell growth was also significantly inhibited after E2F2 silencing, based on cell colony formation in soft agar. Subcutaneous and orthotopic xenograft models of GBM in nude mice also indicated inhibition of tumor development in vivo, following E2F2 silencing. As expression of the E2F2 gene is associated with glioblastoma stem cells and is involved in the transformation of human astrocytes, the present findings suggest that E2F2 is involved in gliomagenesis and could be explored as a potential therapeutic target in malignant gliomas.Oncology letters 10/2014; 8(4):1487-1491. · 0.99 Impact Factor
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ABSTRACT: MicroRNAs play important roles in carcinogenesis by negatively regulating the expression of target genes. Here we explore the biological function of miR-155 and the underlying mechanism in colorectal carcinoma. We validate, for the first time, that E2F2 is a direct target of miR-155 using western blot and a luciferase reporter assay and that miR-155 regulates the proliferation and cell cycle of colorectal carcinoma cells by targeting E2F2 using siRNA technology. We also found, for the first, time that E2F2 acts as a tumor suppressor in colorectal carcinoma. Overall, miR-155 plays an important role in colorectal carcinoma tumorigenesis by negative regulation of its targets including E2F2 and may be a potential therapeutic target for colorectal carcinoma treatment.Biotechnology Letters 05/2014; 36(9). · 1.74 Impact Factor
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ABSTRACT: Erufosine is a new antineoplastic agent of the group of alkylphosphocholines, which interferes with signal transduction and induces apoptosis in various leukemic and tumor cell lines. The present study was designed to examine for the first time the mechanism of resistance to erufosine in malignant cells with permanently reduced expression of the retinoblastoma (Rb) protein. Bearing in mind the high number of malignancies with reduced level of this tumor-suppressor, this investigation was deemed important for using erufosine, alone or in combination, in patients with compromised RB1 gene expression. For this purpose, clones of the leukemic T-cell line SKW-3 were used, which had been engineered to constantly express differently low Rb levels. The alkylphosphocholine induced apoptosis, stimulated the expression of the cyclin dependent kinase inhibitor p27Kip1 and inhibited the synthesis of cyclin D3, thereby causing a G2 phase cell cycle arrest and death of cells with wild type Rb expression. In contrast, Rb-deficiency impeded the changes induced by eru-fosine in the expression of these proteins and abrogated the induction of G2 arrest, which was correlated with reduced antiproliferative and anticlonogenic activities of the compound. In conclusion, analysis of our results showed for the first time that the Rb signaling pathway is essential for mediating the antineoplastic activity of erufosine and its efficacy in patients with malignant diseases may be predicted by determining the Rb status.PLoS ONE 07/2014; 9(7):e100950. · 3.53 Impact Factor
Specific tumor suppressor function for E2F2
in Myc-induced T cell lymphomagenesis
Rene Opavsky*†, Shih-Yin Tsai*†, Martin Guimond*‡, Anjulie Arora*†, Jana Opavska*†, Brian Becknell*‡,
Michael Kaufmann*†, Nathaniel A. Walton*†, Julie A. Stephens§, Soledad A. Fernandez§, Natarajan Muthusamy‡,
Dean W. Felsher¶, Pierluigi Porcu‡, Michael A. Caligiuri*‡?, and Gustavo Leone*†?**
*Human Cancer Genetics Program, Department of Molecular Virology, Immunology, and Medical Genetics, College of Medicine and Public Health
and†Department of Molecular Genetics, College of Biological Sciences,§Center for Biostatistics,‡Division of Hematology and Oncology, Department of
Internal Medicine, and?The Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210; and¶Division of Oncology, Department of
Medicine, Stanford University, CCSR 1105B, 269 Campus Drive, Stanford, CA 94305-5151
Edited by Tak Wah Mak, University of Toronto, Toronto, ON, Canada, and approved August 10, 2007 (received for review July 6, 2007)
Deregulation of the Myc pathway and deregulation of the Rb
pathway are two of the most common abnormalities in human
malignancies. Recent in vitro experiments suggest a complex cross-
regulatory relationship between Myc and Rb that is mediated
through the control of E2F. To evaluate the functional connection
between Myc and E2Fs in vivo, we used a bitransgenic mouse
progression in mice deficient for E2f1, E2f2, or E2f3. Whereas the
targeted inactivation of E2f1 or E2f3 had no significant effect on
tumor progression, loss of E2f2 accelerated lymphomagenesis.
Interestingly, loss of a single copy of E2f2 also accelerated tumor-
igenesis, albeit to a lesser extent, suggesting a haploinsufficient
function for this locus. The combined ablation of E2f1 or E2f3,
along with E2f2, did not further accelerate tumorigenesis. Myc-
overexpressing T cells were more resistant to apoptosis in the
absence of E2f2, and the reintroduction of E2F2 into these tumor
cells resulted in an increase of apoptosis and inhibition of tumor-
through its ability to modulate apoptosis.
transcription ? cancer
expression in hematopoietic, mammary, and other cancer types
(1–3). Deregulation of the Rb/E2F gene networks also represents
common events in cancer (4). Like Myc, E2F can positively and
negatively regulate the expression of hundreds of targets whose
gene products are involved in a wide spectrum of biological
Based on amino acid sequence analysis and structure–function
activator (E2F1–3) and repressor (E2F4–8) subclasses (11). Be-
cause of the intense interest in E2Fs as major regulators of the cell
cycle, individual E2F family members have also been extensively
studied in vivo by gene-targeting approaches in mice. E2f1?/?mice
are viable and suffer from impaired thymocyte apoptosis, defective
negative selection, and testicular atrophy. E2f2?/?mice are also
viable and have a mild increase in hematopoietic and autoreactive
T cells. Much later in life, a portion of E2f1?/?and E2f2?/?mice
develop hematopoietic malignancies (12–15). These mutant phe-
notypes might reflect the particular bias for the expression of E2f1
and E2f2 in hematopoietic tissues. Although disruption of the E2f3
gene in a mixed genetic background yields viable mice, its disrup-
tion in pure strains results in embryonic lethality at around em-
bryonic day 12.5 (G.L., unpublished observation). Surprisingly,
embryos deficient for each of these E2Fs have no apparent defect
in cellular proliferation, raising the possibility of functional redun-
appears to be functional redundancy among members of the
YC is often amplified in human cancers, and mouse models
of cancer have demonstrated a causal role for MYC over-
has little consequence on the proliferative capacity of cells, but the
combined disruption of E2f4 and E2f5 or E2f4 and E2f6 results in
the inappropriate proliferation and expression of target genes in
response to specific antiproliferative signals (16, 17).
Recent observations in cell culture systems indicate extensive
cross-regulation between the action of Myc and E2Fs in coordi-
nating the control of cellular proliferation. Myc action can be
funneled by a number of concerted mechanisms to control E2F
activity (5, 18), including through the regulation of their expression
(7). Although Myc can certainly influence E2F activities, it is also
true that Myc can be influenced by E2Fs. How this complex
cross-regulatory relationship between Myc and E2F is effectively
orchestrated in vivo remains poorly understood. In this study, we
with mice deficient for each of the E2F activators to directly
E2f2 Locus Harbors Tumor Suppressor Function. To examine the
connection between Myc and E2F transcription factors in vivo, we
used a conditional bitransgenic mouse model of MYC-induced T
cell lymphomagenesis (19). In this system, expression of E?SR-tTA
mediates the transcription of the Teto-MYC transgene in B and T
cells and results in the development of predominantly immature T
cell lymphomas. These tumors invade the spleen, lymphatics, bone
marrow, and blood and eventually lead to the death of mice by 4
months of age.
To explore the possibility that E2f1, E2f2, and E2f3 play a role in
E2Fs are expressed in hematopoietic cell lineages. As assessed by
real-time RT-PCR assays, E2f1, E2f2, and E2f3 are expressed in all
of the main hematopoietic organs, including bone marrow, spleen,
thymus, and lymph nodes (Fig. 1A). Most other organs tested
expressed significantly lower levels of E2f1 and E2f3 and essentially
little or no E2f2. It is not clear whether the particular high
expression of E2f2 in hematopoietic organs is a reflection of the
high levels of E2f2 transcripts in these compartments or is just a
reflection of the low basal levels of E2f2 in other tissues. We also
examined the expression of these three E2fs in T cell tumors that
developed in E?SR-tTA;Teto-MYC mice. This analysis revealed a
Author contributions: R.O. and G.L. designed research; R.O., S.-Y.T., M.G., A.A., J.O., M.K.,
N.A.W., and G.L. performed research; B.B., D.W.F., and P.P. contributed new reagents/
analytic tools; R.O., J.A.S., S.A.F., N.M., M.A.C., and G.L. analyzed data; and R.O. and G.L.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviation: MS, median survival.
**To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
September 25, 2007 ?
vol. 104 ?
significant reduction in E2f2 expression in most tumors tested but
data not shown). The basis for the reduction in E2f2 expression
remains to be elucidated, but we speculated that it might be related
to its potential role in the MYC-induced tumorigenic process.
To rigorously assess the physiological role of E2F activators in
MYC-induced lymphomagenesis, we initially generated cohorts of
E?SR-tTA;Teto-MYC mice that lacked either one or both alleles of
a period of 1 year, as described (19). Time of death was noted and
plotted on Kaplan–Meier survival graphs. As shown previously,
with a median survival (MS) of 83 days [confidence interval (C.I.)
78–85 days; n ? 109]. The inactivation of E2f1 had no significant
effect on the MS of these tumor mice (Fig. 1C). Strikingly, the
inactivation of E2f2 significantly accelerated tumor onset and
65–70 days; n ? 128; P ? 0.001; Fig. 1C). Interestingly, loss of even
compared with the control wild-type cohort (E2f2?/?, P ? 0.001),
derived from E2f2 heterozygous animals retained the wild-type
levels of E2F2 protein could have a substantial impact in tumori-
genesis. Characterization of tumors by cell surface marker expres-
for CD3 and TCR? and negative for the B cell marker B220 or
myeloid marker CD11b (data not shown), indicating tumors
were of T cell origin. In each case, tumors consisted of either
CD4/CD8 double-positive cells or CD4 single-positive cells (SI
Fig. 6). Together, these results formally demonstrate a haplo-
Germ-line inactivation of E2f3 in an FVB strain background
results in embryonic lethality at around embryonic day 12.5,
precluding tumor studies with these mice (G.L., unpublished ob-
servations). To circumvent the problem of embryonic lethality, we
analyzed tumor development in E?SR-tTA;Teto-MYC mice con-
taining the Teto-Cre transgene and a conditional allele of E2f3
results in the expression of both the Teto-MYC and Teto-Cre
transgenes. Thus, MYC expression and Cre-mediated ablation of
As shown in Fig. 1C, there was no significant difference in the MS
time between the E?SR-tTA;Teto-MYC;E2f3LoxP/LoxPand E?SR-
tTA;Teto-MYC;Teto-Cre;E2f3LoxP/LoxPgroups of mice (P ? 0.375).
Southern blot analysis and PCR-based genotyping confirmed the
complete ablation of E2f3 in tumors arising in E?SR-tTA;Teto-
MYC;Teto-Cre; E2f3LoxP/LoxPmice (Fig. 1F and data not shown),
tumorigenesis. Moreover, Cre expression itself did not appear to
affect tumor outcome, because cohorts of mice containing the
Teto-Cre transgene had identical MS times as those lacking the
Teto-Cre transgene (data not shown). These results strongly suggest
that E2f3 does not significantly contribute to MYC-induced lym-
phomagenesis. Because E2f3 was conditionally deleted in hemato-
poietic lineages, as opposed to the global deletion of E2f1 or E2f2,
we cannot rule out the formal possibility that inactivation of E2f3
?;?/?;LoxP/LoxP]; red line). (F) Conditional deletion of the E2f3LoxPallele in T
cell lymphomas derived from E?SR-tTA;Teto-MYC;Teto-Cre;E2f3LoxP/LoxPor
E?SR-tTA;Teto-MYC;Teto-Cre;E2f3?/LoxPmice as analyzed by Southern blot-
ting using an E2f3-specific probe. Nondeleted E2f3LoxP/LoxPgenomic DNA
E2f3LoxP(LoxP), and E2f3 wild-type (?/?) alleles is indicated.
Real-time RT-PCR analysis of the expression of E2f1, E2f2, E2f3a, and E2f3b in
normal mouse tissues (b.m., bone marrow; l.n., lymph node). The relative
representative example of three independent experiments is shown. Error
the top of interrupted bars. (B) Real-time RT-PCR analysis of E2f1, E2f2, E2f3a,
and E2f3b expression in normal mouse thymocytes (controls) and MYC-
induced T cell lymphomas (tumors). (C) Kaplan–Meier survival curves (time
from birth to death) of the E?SR-tTA (tetracycline controlled transactivator);
Teto-MYC (?/?) and E?SR-tTA;Teto-MYC cohorts of mice containing het-
erozygous (?/?) or homozygous (?/?) mutations of indicated E2fs. Survival
curve for E?SR-tTA;Teto-MYC is shown by solid black line. Curves for the
cohorts of mice containing deletion of E2f2 allele are shown by red lines,
shown in blue. Dashed lines and solid lines indicate heterozygous and ho-
mozygous deletion of E2fs, respectively. The number of mice used in each
cohort is indicated by n. Student’s t test was used for statistical analyses as
described in Materials and Methods, and P values are shown. (D) Kaplan–
tTA;Teto-MYC;Teto-Cre;E2f1?/?;E2f3LoxP/LoxP([?/?;LoxP/LoxP]; red line),
E?SR-tTA;Teto-MYC;E2f1?/?;E2f2?/?([?/?;?/?]; red line), or E?SR-tTA;Teto-
(E) Kaplan–Meier survival curves of the E?SR-tTA;Teto-MYC ([?/?;?/?;?/?];
Loss of E2f2 accelerates Myc-induced T cell lymphomagenesis. (A)
Opavsky et al.PNAS ?
September 25, 2007 ?
vol. 104 ?
no. 39 ?
in the germ line would have a different phenotypic consequence on
T cell lymphomagenesis than observed here.
E2f1 and E2f3 Are Dispensable for MYC-Induced Lymphomagenesis.
Functional compensation among E2F family members, as demon-
strated in fibroblasts cultured in vitro (20), may explain the lack of
an effect on tumor outcome imparted by the loss of either E2f1 or
both E2f1 and E2f3. Surprisingly, E?SR-tTA;Teto-MYC;E2f1?/?;
as wild-type mice (Fig. 1D). Southern blot analysis of tumor DNA
confirmed the complete deletion of E2f3 in tumors derived from
mice expressing Cre (SI Fig. 7A and data not shown). From these
in vivo studies, we conclude that E2f1 and E2f3 do not play a
measurable role in MYC-induced lymphomagenesis.
E2f2’s Tumor Suppressor Function Is Independent of E2f1 and E2f3.
The tumor studies described above demonstrate specificity among
E2fs in the manifestation of tumor outcome; however, because of
the incredible functional plasticity among E2F family members
Although no significant change in the expression of other E2F
rerouted in E2f2-deficient cell to perform functions not normally
performed in cells containing all three activator E2Fs. These
‘‘acquired functions’’ could be responsible for diminishing or ac-
centuating the manifestation of tumor outcome resulting from the
loss of E2f2. To test this possibility, we created cohorts of E?SR-
tTA;Teto-MYC;E2f2?/?mice that were also deficient for either
E2f1, E2f3, or both. As shown in Fig. 1D, E?SR-tTA;Teto-
E2f3LoxP/LoxPcohorts had a similar MS time as E?SR-tTA;Teto-
MYC;E2f2?/?mice (66, 67, and 67 days, respectively). Surprisingly,
the simultaneous inactivation of all three E2F activators in E?SR-
sulted in a similar progression of disease as in E2f2-deficient mice
(MS time of 65 and 67 days, respectively; Fig. 1E). Once again,
Southern blot analysis confirmed the complete deletion of
C). Based on these data, we conclude that E2f2’s tumor suppressor
role in T cell lymphomagenesis is independent of E2f1 and E2f3.
Cell-Autonomous Tumor Suppressor Function of E2F2. The global
inactivation of E2f2 precluded us from making any conclusions
relating to where the critical action of E2F2 for suppressing
lymphomagenesis might reside. To examine whether loss of E2f2
accelerated the tumorigenic process in a cell-autonomous manner,
we used an adoptive transfer strategy to introduce E2f2-deficient
fetal liver cells into wild-type irradiated animals. To this end,
lethally irradiated recipient FVB mice were injected with fetal liver
cells isolated from E?SR-tTA;Teto-MYC and E?SR-tTA;Teto-
MYC;E2f2?/?15.5-day-old embryos. Injected mice were then mon-
itored for tumor formation over a period of 1 year (25 mice per
genetic group; see SI Fig. 8A). Approximately 60% of mice (14/25)
from the group that received E?SR-tTA;Teto-MYC cells developed
T cell lymphomas, as confirmed by FACS-based immunopheno-
succumbed to tumors with a MS time of 160 days. The other 40%
contrast, most mice (22/25) that received E?SR-tTA;Teto-
MYC;E2f2?/?fetal liver cells developed T cell lymphomas and had
a MS time of 110 days (SI Fig. 8A). Lymphomas that lacked E2f2
were immunophenotypically indistinguishable from those that had
E2f2. Statistical comparison between these two groups showed a
significant difference in their MS (P ? 0.019). These studies
demonstrate that the tumor suppressor function of E2f2 resides
within the hematopoietic compartment and is likely cell-
Tumor Analysis in E2f2-Deficient Mice. Loss of E2f2 has been shown
to lead to autoimmune disease in older mice due to enhanced
effector/memory T lymphocytes (14). We therefore explored
causally related to a T cell differentiation defect, which could be
divided into four substages depending on the expression of CD25
and CD44 markers: DN1 (CD44?CD25?), DN2 (CD44?CD25?),
we analyzed the expression of CD44, CD25, CD4, and CD8 in
thymocytes derived from 21-day-old wild-type E2f2?/?, E?SR-
tTA;Teto-MYC, and E?SR-tTA;Teto-MYC;E2f2?/?mice. Loss of
E2f2 in either a nontumor or tumor setting had no appreciable
effect on the distribution of DN1–DN4 cells or the proportion of
data not shown). We did observe, however, that in contrast to the
monoclonal nature of tumors derived from E?SR-tTA;Teto-MYC
mice (19), the vast majority of tumors in the E2f2?/?and E2f2?/?
background were oligoclonal, with homozygously deleted mice
having tumors composed of up to five different clones that ex-
pressed different TCR receptor isoforms (Fig. 2B).
In view of the well established role of E2Fs in the control of
cellular proliferation and apoptosis, we investigated whether
changes in these two processes may represent the basis for the
tumor suppressor function of E2F2. To this end, we measured
proliferation and apoptotic indices in control and E2f2-deficient
littermate animals at early and late stages of tumor development.
Analysis of precancerous (21 days of age) or terminally sick mice
(A) Development of double-negative thymocytes in 21-day-old E2f2?/?(non-
transgenic; white bars), E2f2?/?(nontransgenic; black bars), E?SR-tTA;Teto-
MYC;E2f2?/?(E?SR-tTA;Teto-MYC; white bars), E?SR-tTA;Teto-MYC;E2f2?/?
(E?SR-tTA;Teto-MYC; black bars) as assessed by FACS. Staining with anti-CD4
and -CD8 antibody was used to determine CD4?CD8?double-negative pop-
ulation within the live lymphoid gate. Double-negative populations were
subsequently analyzed for CD44 and CD25 surface expression. Data are pre-
sented as an average percentage ? SD for DN1 (CD44?), DN2 (CD44?CD25?),
DN3 (CD44?CD25?), and DN4 (CD44?CD25?). (B) Graphic representation of
tumor clonality in E?SR-tTA;Teto-MYC mice of the following genotypes:
E2f2?/?, E2f2?/?, and E2f2?/?, as determined by analysis of different tissues
using a panel of monoclonal antibodies recognizing different TCR V? chains.
(C) BrdU incorporation and apoptosis assays of E?SR-tTA;Teto-MYC;E2f2?/?
disease as determined by FACS using anti-BrdU or anti-Annexin V antibodies.
used for statistical analyses, and P values are shown.
Decreased apoptosis in mice deficient for E2f2 during tumorigenesis.
www.pnas.org?cgi?doi?10.1073?pnas.0706307104Opavsky et al.
failed to reveal any substantial difference in the proliferation of
control and E2f2-deficient T cells (Fig. 2C and data not shown). In
contrast, AnnexinV staining revealed that E2f2-deficient T cells
confer a selective advantage for the expansion of MYC-expressing
Molecular Characterization of T Cell Lymphomas in E2f2-Deficient
Mice. To identify relevant downstream activities that might be
responsible for the observed acceleration of tumorigenesis in
E2f2-deficient mice, we analyzed the expression of cell cycle inhib-
itors that have previously been implicated in cancer biogenesis.
First, we performed real-time RT-PCR expression analysis of the
MYC-transgene itself and two known MYC-target genes, Ornithine
decarboxylase (Odc) and Nucleolin, in thymic tumor masses from
terminally sick E?SR-tTA;Teto-MYC and E?SR-tTA;Teto-
MYC;E2f2?/?mice. This analysis confirmed that the expression of
the MYC transgene and its two target genes was similarly activated
in all tumors examined when compared with T cells from aged-
matched normal control mice (Fig. 3A and data not shown).
Previous work using the E?-MYC model of B cell lymphoma
demonstrated that MYC-induced B cell lymphomagenesis requires
the inactivation of apoptotic checkpoints, and that this is frequently
and E?SR-tTA;Teto-MYC;E2f2?/?mice. Direct sequencing of p53
cDNA prepared from 10 tumors did not reveal any mutation in its
coding sequence (SI Text). We then analyzed the expression of
p19ARF, whose induction had been shown to be associated with the
overexpression of MYC (23). As might have been expected, we
observed a dramatic induction of p19ARFexpression in all tumors
analyzed, but this induction was independent of the status of E2f2
differentially impacted by the presence or absence of E2f2, the
expression of one of its target genes, p21Cip1, did not change in
response to the loss of E2f2 (Fig. 3A). Because the expression of
p73, a proapoptotic member of the p53 family, has been implicated
death of peripheral T cells (24), we examined its expression in
normal and tumor tissues from E2f2-deficient mice. We found that
p73 transcripts were significantly decreased in tumor samples, but
this decrease did not depend on the status of E2f2 (Fig. 3A). In
contrast, the expression of the antiapoptotic version of p73,
deltaNp73, was unchanged between normal and tumor samples (SI
is independent of the p53 apoptotic axis.
The ability of Myc to impact the cell cycle at least partially
depends on its ability to down-regulate p27Kip1(25), a cyclin-
dependent kinase inhibitor known to contribute to the regulation
of the cell cycle and to be down-regulated in a number of tumor
settings (26, 27). Consistent with a posttranscriptional mechanism
of regulation, p27Kip1protein levels but not its mRNA levels, were
MYC or E?SR-tTA;Teto-MYC;E2f2?/?mice (Fig. 3 A and B).
Although this down-regulation of p27Kip1likely represents an
important event in MYC-induced T cell lymphomagenesis, its
regulation would appear not to be linked to E2F2’s tumor suppres-
Reintroduction of E2f2 Activity into E2F2-Deficient Cells Inhibits
Tumorigenesis. To further examine the role of E2f2 in lym-
phomagenesis, we evaluated T cells isolated from late-stage tumors
that were reconstituted with exogenous E2F2. Tumor cells derived
from E?SR-tTA;Teto-MYC;E2f2?/?mice readily adapted to in vitro
growth and could be efficiently infected with retroviral expression
vectors (Fig. 4 A and B). Flow cytometric analysis in multiple
experiments showed that 20–80% of tumor cells infected with a
control or E2F2-expressing MSCV-IRES-GFP vector were GFP-
positive (Fig. 4B). Subcutaneous injection of these cells resulted in
GFP-negative (?99% GFP-negative; Fig. 4 B and C). In contrast,
tumors that emerged from the injection of control-transduced cells
retained the same percentage of GFP-positive cells as observed
before the injection. These experiments reveal a profound bias
against the formation of tumors originating from E2F2 overex-
pressing T cells.
Alternatively, control and E2F2-transduced cells were plated on
tissue culture plates, and their growth was monitored over the
course of ?8 days. In these assays, we could measure a moderate
but consistent reduction in the proliferation of cells infected with
E2F2-expressing vectors (Fig. 4D). Importantly, comparison of
GFP-positive and -negative cells by FACS analysis revealed a
profound decrease over time in the percentage of GFP-positive
cells transduced with E2F2-vectors (Fig. 4E). In contrast, GFP-
positive and -negative cells in control-treated samples proliferated
equally well. BrdU incorporation assays indicated that the number
of GFP-positive cells entering S phase was not influenced by the
over-expression of E2F2 (Fig. 4F and data not shown). AnnexinV
assays, however, revealed that the ratio of GFP-positive/-negative
apoptotic cells was markedly increased in populations transduced
These results suggest a strong bias against the proliferation of cells
expressing the E2F2 protein that is based on its ability to potently
Real-time RT-PCR analysis of MYC, ODC, p19ARF, p73, p21Cip1, and p27Kip1
expression in normal thymocytes (E2f2?/?), E2f2-deficient thymocytes
(E2f2?/?), tumors derived from E?SR-tTA;Teto-MYC;E2f2?/?or E?SR-
tTA;Teto-MYC;E2f2?/?mice, as indicated. Sequences of primers used for
tested genes are shown in SI Table 1. (B) Western blot analysis of p27Kip1
expression in normal thymocytes (N) and E?SR-tTA;Teto-MYC tumors nonde-
leted or deleted for E2f2 as indicated.
Expression of cell cycle-regulated genes in E2f2-deleted cells. (A)
Opavsky et al. PNAS ?
September 25, 2007 ?
vol. 104 ?
no. 39 ?
In parallel experiments, we could show that overexpression of
E2F1 and E2F3a in E2f2-deficient cells could also induce apoptosis
and preclude the growth and tumorigenicity of tumor cells (SI Fig.
10 A–F and data not shown). These results suggest that using
overexpression approaches, any of the three E2F activators can
these results to indicate that gene ablation strategies can reveal
functional specificity with greater fidelity than by overexpression
The E2f2 Locus Harbors Tumor Suppressor Function. Overexpression
of the MYC oncogene and inactivation of the RB tumor suppressor
pathway are hallmarks of human cancers. Recent in vitro experi-
we used a bitransgenic mouse model of MYC-induced T cell
lymphomagenesis and mice deficient for E2f1, E2f2, or E2f3 to
evaluate the functional relationship between Myc and E2Fs in vivo.
E2F2 in T cell lymphomagenesis. Adoptive transfer experiments
show that E2f2’s tumor suppressor function resides within the
hematopoietic compartment and is therefore likely to be cell-
autonomous. Loss of even one allele significantly accelerated
tumorigenesis, indicating that tumor progression is sensitive to
small changes in total E2F2 protein. This raises the possibility that
polymorphisms in the genome that result in lower levels of E2F2
protein, directly or indirectly, may place individuals at a higher risk
for cancer development.
E2f1 and E2f3 Are Not Required for Lymphomagenesis.Basedontheir
shared abilities to control cell proliferation and apoptosis, a func-
tional connection between the Myc and E2F pathways has been
long speculated. Most recently, work in mouse embryo fibroblasts
suggested that two important functions of Myc in the control of
proliferation and apoptosis are mediated, at least in part, by E2Fs
(28). This work showed that in fibroblasts, the execution of Myc’s
proliferative arm requires E2f2 and E2f3, and the execution of its
apoptotic arm requires E2f1. This bifurcation of Myc’s function at
the level of E2F suggested that E2f1 could have tumor suppressor
function and E2f2 and E2f3 could have oncogenic functions. These
predictions were not born out by the in vivo studies of T cell
lymphomagenesis presented here. In fact, mice lacking both E2f1
and E2f3 developed T cell lymphomas with similar kinetics as mice
containing a full complement of E2Fs, suggesting that E2f1 and
esis. The observation that T cells devoid of E2f1, E2f2, and E2f3
previous work showing that fibroblasts deficient for these E2Fs
this difference between E2f1/2/3-deficient T cells and fibroblasts
reflects a tissue-specific requirement for E2Fs or a consequence of
MYC overexpression in T cells. Clearly, important differences must
exist between fibroblasts and T cells, and a generalized outcome
be difficult to predict.
The observation that loss of E2f1 had little bearing on MYC-
induced T cell lymphomagenesis is also in direct contradiction to a
of E2f1 dramatically delayed MYC-induced B cell lymphomagen-
esis. These two different tumor outcomes resulting from the
of B and T cells. Alternatively, differences in the two E2f1-null
alleles (12, 13) or in genetic backgrounds (30, 31) housing envi-
ronments, methods of tumor analysis, and size of genetic cohorts
could account for the observed discrepancies.
Tumor Suppressor Role of E2F2 in Apoptosis. Two observations
indicate that the underlying basis for E2f2’s tumor suppressor
function is in the control of apoptosis rather than in cell prolifer-
ation. First, we observed a decreased number of apoptotic tumor
cells in E2f2-null mice. Second, reexpression of E2F2 in E2f2-
deficient tumor cells resulted in an increase of apoptosis and
abrogation of tumor cell expansion, demonstrating that the critical
components required to signal and execute apoptosis remain intact
in these tumor cells. In contrast, these loss-of-function and over-
expression studies failed to reveal any E2f2-dependent differences
The role of E2F2 in apoptosis early on during tumorigenesis
could be important in limiting the number of cells susceptible to
MYC-induced oncogenesis. This hypothesis would predict that loss
of E2f2 might increase the tumor-prone T cell population respon-
sive to MYC overexpression and thus facilitate tumor development.
analysis of T cells derived from E?SR-tTA;Teto-MYC;E2f2?/?tumors infected
with MSCV-IRES-EGFP empty vector (con) or MSCV-E2F2-IRES-EGFP (E2F2)
using anti-E2f2 antibody. Tubulin served as loading control. (B) FACS analysis
of unselected cells infected with the indicated retroviruses before injection
into nude mice (Upper). Representative examples of FACS analysis of individ-
ual tumors that developed in nude mice (Lower). The percentage of GFP-
of tumors that developed in mice injected with E?SR-tTA;Teto-MYC;E2f2?/?
tumor T cells that were infected either with control or E2F2 retroviruses. The
individual tumor relative to the percent of GFP-positive cells before injection.
n indicates a number of tumors analyzed for each group. (D) In vitro prolif-
eration assay of unselected cells infected with the indicated retroviral con-
structs. Cells were plated at a concentration of 0.2 ? 106per ml (day 0) and
of GFP-positive cells was determined by FACS. The infection efficiency at day
0 (28% for the MSCV-IRES-EGFP vector control and 39% for MSCV-E2F2-IRES-
EGFP) was set to 100%. The values obtained for percentage of GFP-positive
G) The percentage of BrdU- and Annexin V-positive cells infected with the
indicated retroviruses was measured 2 and 4 days after infection. For D–G,
representative examples from three independent experiments are shown.
Proapoptotic tumor suppressor function of E2f2. (A) Western blot
www.pnas.org?cgi?doi?10.1073?pnas.0706307104Opavsky et al.
Consistent with this notion, the vast majority of E2f2?/?and
E2f2?/?mice developed tumors that were oligoclonal in nature,
with homozygously deleted tumors expressing up to five different
TCR receptor isoforms (Fig. 2B). This is in contrast to the typical
monoclonal nature of tumors found in E2f2?/?mice. These data
suggest a role for E2f2 in both the early and late stages of tumor
Specificity of Function Among E2Fs. The tumor analysis in E2f-
deficient mice clearly demonstrates a specific role for E2f2 in T cell
lymphomagenesis. The molecular basis for this tumor suppressor
protein levels but not in E2F1 and E2F3 through the targeted
inactivation of E2f2 is sufficient to suppress apoptosis and permit
tumor progression. On the other hand, overexpression of E2F2 or
E2F1 and, to lesser degree, E2F3a, is able to induce apoptosis in
MYC-overexpressing tumor cells. Thus, loss-of-function and over-
expression studies appear to lead to contradictory results. Because
overexpression of E2Fs can potentially compete with and alter the
binding of other E2Fs to target promoters and/or cofactors, we
believe that gene ablation approaches are more adept at revealing
physiological differences between the function of E2F family
What is the underlying reason for the unique tumor suppressor
in vivo? One possibility is that endogenous levels of E2F2 protein
regulate the expression of a specific set of apoptotic-related genes
that can be similarly achieved only by other E2Fs when overex-
pressed at supraphysiological levels. The alternative explanation is
that E2f2’s tumor suppressor role is not intrinsic to the function of
its protein product but rather depends on the magnitude of
expression imparted by its locus. In other words, the size of the
‘‘total pool’’ of E2F activity may be the critical variable that
determines whether an apoptotic response can be surmounted in
face of an oncogenic insult. Although it is possible that the basis for
the unique role of E2f2 in MYC-induced lymphomagenesis may
stem from quantitative differences in the expression of ‘‘activator’’
E2Fs in T cells, three main reasons argue against this latter
possibility. First, if ‘‘total’’ E2F activity was determining tumor
suppression, it would be expected that loss of all three E2F
activators would accelerate lymphomagenesis further than that
observed by the simple loss of E2f2. This prediction was not
realized; rather, a deficiency of E2f1/E2f2/E2f3 accelerated lym-
phomagenesis to the same extent as by loss of E2f2 (Fig. 1 D and
E). Second, the absence of ‘‘enhanced’’ tumorigenesis in T cells
deficient for all three E2F activators is not simply because E2f1/3
are not expressed to sufficient levels to have a function in T cells.
In fact, E2F1 has been shown, as has E2F2, to contribute to normal
hematopoiesis in a number of settings. For example, it has been
previously shown that in contrast to E2f1?/?E2f2?/?or
E2f1?/?E2f2?/?cells, E2f1?/?E2f2?/?bone marrow cells are un-
able to contribute to development of multiple hematopoietic lin-
eages (33), suggesting that a single allele of E2f1 or E2f2 contribute
significantly to this process. Third, in unpublished work from our
laboratory, we have observed that loss of E2f2 in tissues that
normally express much lower levels of it than in T cells also
accelerates tumorigenesis (G.L., unpublished observations). It
due to its abundance in T cells but rather is likely a reflection of its
specific function in T cell lymphomagenesis.
Whether E2F2 might also play a role in tumor maintenance
remains to be investigated. Further experiments will be necessary
to decipher the exact mechanism by which E2F2 exerts its tumor
suppressor action in vivo. In summary, these studies reveal speci-
ficity among E2F activators in MYC-induced lymphomagenesis,
highlighting an apoptotic role for E2f2 in this process.
Materials and Methods
Generation and Maintenance of Mice. The E2f1?/?, E2f2?/?, and
Teto-Cre mice were generous gifts from Michael Greenberg (Chil-
dren’s Hospital, Boston, MA), Stuart Orkin (Harvard Medical
School, Boston, MA), and Andreas Nagy (Samuel Lunenfeld
Research Institute, Toronto, ON, Canada), respectively. The gen-
eration of the conditional E2f3 knockout mice (E2f3LoxP/LoxP) has
been described (20). Genotyping of mice was performed by PCR
from genomic DNA isolated from mouse tails. All tumor studies
were performed with mice bred into FVB (fifth generation). Mice
monitored for tumor formation over a period of 1 year, as de-
Statistical Analysis. Kaplan–Meier curves were generated, and MS
times with 95% confidence intervals (32) were calculated. Propor-
tional hazards assumptions were confirmed, and the log-rank test
was found to be appropriate to compare the survival curves in all
cases. Bonferroni adjustments for multiple comparisons were used.
Although this is a conservative method of adjustment, the P values
found in these data were on the extreme ends, and a less-
conservative method would have lead to the same conclusions. In
the case where the proportional hazard assumption was not met,
were compared between groups before and after the cross-over of
the survival curves.
This work was funded by National Institutes of Health Grants R01
CA85619 and P01 CA097189 (both to G.L.) and by a translational award
by the Leukemia and Lymphoma Society of America (to G.L.). R.O. is
supported by a T32 CA106196 fellowship in Cancer Genetics, and G.L.
is the recipient of the Pew Charitable Trusts Scholar Award and the
Leukemia and Lymphoma Society Scholar Award.
1. Arvanitis C, Felsher DW (2005) Cancer Lett 226:95–99.
2. Jonkers J, Berns A (2004) Cancer Cell 6:535–538.
3. Pelengaris S, Khan M, Evan G (2002) Nat Rev Cancer 2:764–776.
4. Sherr CJ, McCormick F (2002) Cancer Cell 2:103–112.
5. Adhikary S, Eilers M (2005) Nat Rev Mol Cell Biol 6:635–645.
6. Evan GI, Vousden KH (2001) Nature 411:342–348.
7. Sears R, Nevins JR (2002) J Biol Chem 277:11617–11620.
8. Attwooll C, Denchi EL, Helin K (2004) EMBO J 23:4709–4716.
9. Cobrinik D (2005) Oncogene 24:2796–2809.
10. Dimova DK, Dyson NJ (2005) Oncogene 24:2810–2826.
11. Trimarchi JM, Lees JA (2002) Nat Rev Mol Cell Biol 3:11–20.
12. Field SJ, Tsai FY, Kuo F, Zubiaga AM, Kaelin WG, Livingston DM, Orkin SH, Greenberg
ME (1996) Cell 85:549–561.
13. Yamasaki L, Jacks T, Bronson R, Goillot E, Harlow E, Dyson NJ (1996) Cell 85:537–548.
14. Murga M, Fernandez-Capetillo O, Field SJ, Moreno B, Borlado LR, Fujiwara Y, Balomenos
D, Vicario A, Carrera AC, Orkin SH, et al. (2001) Immunity 15:959–970.
15. Zhu JW, Field SJ, Gore L, Thompson M, Yang H, Fujiwara Y, Cardiff RD, Greenberg M,
Orkin SH, DeGregori J (2001) Mol Cell Biol 21:8547–8564.
16. Gaubatz S, Lindeman GJ, Ishida S, Jakoi L, Nevins JR, Livingston DM, Rempel RE (2000)
Mol Cell 6:729–735.
17. Giangrande PH, Zhu W, Rempel RE, Laakso N, Nevins JR (2004) EMBO J 23:1336–1347.
18. Gartel AL, Radhakrishnan SK (2005) Cancer Res 65:3980–3985.
19. Felsher DW, Bishop JM (1999) Mol Cell 4:199–207.
20. Wu L, Timmers C, Maiti B, Saavedra HI, Sang L, Chong GT, Nuckolls F, Giangrande P,
Wright FA, Field SJ, et al. (2001) Nature 414:457–462.
21. Cantrell DA (2002) Nat Rev Immunol 2:20–27.
22. Eischen CM, Weber JD, Roussel MF, Sherr CJ, Cleveland JL (1999) Genes Dev 13:2658–2669.
23. de Stanchina E, McCurrach ME, Zindy F, Shieh SY, Ferbeyre G, Samuelson AV, Prives C,
Roussel MF, Sherr CJ, Lowe SW (1998) Genes Dev 12:2434–2442.
24. Lissy NA, Davis, Irwin M, Kaelin WG, Dowdy SF (2000) Nature 407:642–645.
25. Sherr CJ, Roberts JM (1999) Genes Dev 13:1501–1512.
26. Kang-Decker N, Tong C, Boussouar F, Baker DJ, Xu W, Leontovich AA, Taylor WR,
Brindle PK, van Deursen JM (2004) Cancer Cell 5:177–189.
27. Keller UB, Old JB, Dorsey FC, Nilsson JA, Nilsson L, MacLean KH, Chung L, Yung C,
Spruck C, Boyd K, et al. (2007) EMBO J 26:2562–2574.
28. Leone G, Sears R, Huang E, Rempel R, Nuckolls F, Park CH, Giangrande P, Wu L,
Saavedra HI, Field, SJ, et al. (2001) Mol Cell 8:105–113.
29. Baudino TA, Maclean KH, Brennan J, Parganas E, Yang C, Aslanian A, Lees JA, Sherr CJ,
Roussel MF, Cleveland JL (2003) Mol Cell 11:905–914.
30. Wikonkal N, Remenyik E, Knezevic D, Zhang W, Liu M, Zhao H, Berton TR, Johnson DG,
Brash DE (2003) Nat Cell Biol 5:655–660.
31. Wloga EH, Criniti V, Yamasaki L, Bronson RT (2004) Nat Cell Biol 6:565–567.
32. Brookmeyer R, Crowley J (1982) Biometrics 38:29–41.
33. Li FX, Zhu JW, Hogan CJ, DeGregori J (2003) Mol Cell Biol 23:3607–3622.
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