Elevated level of SUMOylated IRF-1 in tumor cells interferes with IRF-1-mediated apoptosis

Article (PDF Available)inProceedings of the National Academy of Sciences 104(43):17028-33 · November 2007with14 Reads
DOI: 10.1073/pnas.0609852104 · Source: PubMed
Abstract
SUMOylation of transcription factors often attenuates transcription activity. This regulation of protein activity allows more diversity in the control of gene expression. Interferon regulatory factor-1 (IRF-1) was originally identified as a regulator of IFN-alpha/beta, and its expression is induced by viral infection or IFN stimulation. Accumulating evidence supports the theory that IRF-1 functions as a tumor suppressor and represses the transformed phenotype. Here we report that the level of SUMOylated IRF-1 is elevated in tumors. Site-directed mutagenesis experiments disclose that the SUMOylation sites of IRF-1 are identical to the major ubiquitination sites. Consequently, SUMOylated IRF-1 displays enhanced resistance to degradation. SUMOylation of IRF-1 attenuates its transcription activity, and SUMOylated IRF-1 inhibits apoptosis by repression of its transcriptional activity. These data support a mechanism whereby SUMOylation of IRF-1 inactivates its tumor suppressor function, which facilitates resistance to the immune response.
Elevated level of SUMOylated IRF-1 in tumor cells
interferes with IRF-1-mediated apoptosis
Junsoo Park*
, Kwangsoo Kim
, Eun-Ju Lee*, Yun-Jee Seo
, Si-Nae Lim*, Kyoungsook Park*, Seung Bae Rho
,
Seung-Hoon Lee
§
, and Je-Ho Lee*
*Molecular Therapy Research Center, Sungkyunkwan University, Seoul 135-710, Korea;
Korea Basic Science Institute, Gwangju 500-757, Korea;
Research
Institute, National Cancer Center, Gyeonggi-do 411-769, Korea;
§
Department of Life Science, Yong-In University, Gyeonggi-do 449-719, Korea; and
Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea
Edited by Peter M. Howley, Harvard Medical School, Boston, MA, and approved September 12, 2007 (received for review November 7, 2006)
SUMOylation of transcription factors often attenuates transcrip-
tion activity. This regulation of protein activity allows more diver-
sity in the control of gene expression. Interferon regulatory factor-1
(IRF-1) was originally identified as a regulator of IFN-
/
, and
its expression is induced by viral infection or IFN stimulation.
Accumulating evidence supports the theory that IRF-1 functions as
a tumor suppressor and represses the transformed phenotype.
Here we report that the level of SUMOylated IRF-1 is elevated in
tumors. Site-directed mutagenesis experiments disclose that the
SUMOylation sites of IRF-1 are identical to the major ubiquitination
sites. Consequently, SUMOylated IRF-1 displays enhanced resis-
tance to degradation. SUMOylation of IRF-1 attenuates its tran-
scription activity, and SUMOylated IRF-1 inhibits apoptosis by
repression of its transcriptional activity. These data support a
mechanism whereby SUMOylation of IRF-1 inactivates its tumor
suppressor function, which facilitates resistance to the immune
response.
tumor suppressor Ubc9 SENP1
S
UMOylation is a posttranslation modification, in which the
SUMO moiety (known as SMT3 in yeast) is attached to the
lysine residues of target proteins. A lthough SUMOylation shares
many common features with ubiquitination, its role in cellular
met abolism is diverse (1). Ubiquitination is generally, but not
always, involved in proteasomal protein degradation, whereas
SUMOylation affects numerous processes, including subcellular
localization, modulation of transcriptional activity, and en-
hanced protein stability (1, 2). A striking characteristic is that a
small f raction of the substrate is SUMOylated at any given time,
but SUMOylation alters the long-ter m fate of the modified
protein after rapid de-SUMOylation (3). Over the past few years,
several studies have focused on the role of SUMOylation in
tumorigenesis (4). Recent reports indicate that Ubc9, the single
SUMO E2 ligase catalyzing the conjugation of SUMO to target
protein, is overexpressed in ovarian cancer (5), and the SUMO-
ylation status of reptin modulates the invasive activity of cancer
cells with metastatic potential (6). However, at present, limited
infor mation is available on the relationship between SUMO-
ylated proteins and tumors.
Interferon regulatory factor-1 (IRF-1) was originally identi-
fied as a regulator of IFN
/
. IRF-1 expression is dramatically
up-regulated on v iral infection and stimulation by the IFN
family, including IFN
, IFN
, IFN
, and TNF
(7). Accumu-
lating ev idence supports the theory that IRF-1 functions as a
tumor suppressor (8–11) and represses the transformed pheno-
t ype (10, 12–14). In human tumors, IRF-1 is inactivated to
prevent apoptosis and cell cycle arrest by genetic mechanisms,
such as gene deletion and exon skipping (11, 15–17). IRF-1 is a
substrate of both ubiquitination and SUMOylation (18, 19). In
the present work, we screened the SUMOylated proteins in
tumor cells and found that the level of SUMOylated IRF-1 is
sign ificantly increased. We propose that the elevated level of
SUMOylated IRF-1 in tumor cells interferes with IRF-1-
mediated apoptosis.
Results
SUMOylation often affects subcellular localization, stability,
and transcriptional activity of target proteins (2, 3), and it is
proposed that specific SUMOylated proteins exist in tumor cells
to enhance tumorigen icity. As shown previously, SUMOylated
proteins migrate slowly because of the attached moiety, and are
thus readily detected by immunoblot analysis. To identify the
SUMOylated proteins in tumors, a series of immunoblots were
performed with cell lysates extracted from MRC-5 (control) and
MCF-7 (tumor sample). Because the SUMO isopeptidase is
capable of readily cleaving the isopeptide bond between SUMO
and the t arget protein, cell lysates were carefully prepared by
direct boiling to inactivate the enzymes (20). Among several
signaling molecules, IRF-1 displayed a shif ted band in tumor
samples in an anti-IRF-1 immunoblot (Fig. 1A Left). The specific
modifications of IRF-1 in tumors were further confirmed by
immunoblotting with other normal and tumor cell lines (data not
shown). Ectopic coexpression of IRF-1 and SUMO-1 induced
SUMOylation of IRF-1 in HEK293 cells (Fig. 1B). The SUMO-
ylated IRF-1 protein comig rates with a 75-kDa band found in
HeL a tumor cells (Fig. 1C). Our findings were further supported
by immunoprecipitation assays with the anti-SUMO-1 antibody.
MCF7 cell lysates were immunopurified with the anti-SUMO-1
antibody, and then the immunoprecipitates were probed with
anti-IRF-1 antibody. Analysis of immunoprecipitates confir med
that IRF-1 is SUMOylated in tumor cells [supporting informa-
tion (SI) Fig. 5]. Ectopic expression of SENP1, an SUMO
isopeptidase, resulted in a dramatic reduction in shifted SUMO-
IRF-1 bands. In addition, longer ex posure of control HeLa cells
led to multiple bands, and the level of modified forms were
sign ificantly decreased upon SENP1 expression, suggesting that
IRF-1 is modified by SUMO-1 at multiple positions in tumor
cells (Fig. 1D). These results were confirmed by findings from
tumor tissues. It is important to note that SUMOylated proteins
can be easily de-SUMOylated until inactivation of the SUMO
isopeptidase by boiling. Because of the large number of steps
required to homogenize tumor tissue, it was dif ficult to obt ain
Author contributions: J.P. designed research; J.P., K.K., Y.-J.S., and S.-N.L. performed
research; E.-J.L., S.B.R., S.-H.L., and J.-H.L. contributed new reagents/analytic tools; J.P.,
E.-J.L., K.P., S.-H.L., and J.-H.L. analyzed data; and J.P. and K.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: IRF-1, interferon regulatory factor-1; ISRE, interferon stimulated response
element; Luc, luciferase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide; NEM, N-ethylmaleimide.
To whom correspondence should be addressed. E-mail: jeholee@gmail.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0609852104/DC1.
© 2007 by The National Academy of Sciences of the USA
17028–17033
PNAS
October 23, 2007
vol. 104
no. 43 www.pnas.orgcgidoi10.1073pnas.0609852104
clear shifted bands as obtained with the tumor cell lines.
However, upon longer exposure, five of five ovarian tumors
c ontained the shif ted SUMOylated forms of IRF-1 (Fig. 1E
Left), whereas nor mal ovarian tissues ex pressed low amounts of
IRF-1 protein (Fig. 1E Left). To confirm that the shifted bands
observed in ovarian tumor tissue lysates were SUMOylated
for ms of IRF-1, tumor cell lysates were immunopurified with
anti-SUMO-1 antibody, followed by immunoblot analysis with
anti-IRF-1 antibody (Fig. 1E Right).
Among components of SUMOylation machinery, Ubc9 is the
sole SUMO E2 conjugation enzyme, and PIAS3 is known to be
the E3 enzy me for IRF-1 SUMOylation (19). Recently, it has
been reported that the levels of Ubc9 and PIAS3 were generally
increased in tumor cells (5, 21). Because IRF-1 is extensively
c onjugated with SUMO-1, we examined the expression levels of
Ubc9 and PI AS3 in tumor cells. The levels of Ubc9 and PIAS3
are elevated in the MCF7 cell line, compared with MRC5 (Fig.
1 A Right). Consistent with recent findings by other investigators,
we also detected the elevated levels of Ubc9 and PIAS3 in
tumors (Fig. 1E).
To exclude the possibility of a transcriptional increase of
IRF-1 mRNA in tumor tissues, the IRF-1 mRNA level was
examined by RT-PCR analysis. There was no sign ificant differ-
ence between normal and tumor tissues (Fig. 1E), indicating that
the increased expression of IRF-1 protein in tumor is indepen-
dent of transcriptional control. Protein st ability is often regu-
lated by the att achment of SUMO or ubiquitin (22). Because
up-regulation of IRF-1 protein is accompanied by its SUMO-
ylation, we assumed that the level of IRF-1 protein in tumor
c orrelates with its SUMOylation. To test this hypothesis, we
examined whether SUMOylation is involved in IRF-1 protein
st ability. Ectopic coexpression of SUMO-1 and IRF-1 induced
SUMOylation. Treatment with the proteasomal inhibitor,
MG132, induced a dramatic increase in the IRF-1 protein level,
suggesting that IRF-1 is primarily removed by proteasomal
degradation (Fig. 1B). Next, we examined the role of SUMO-
ylation of IRF-1 protein stability under conditions in which
synthesis was blocked by cycloheximide (18). Coexpression of
SUMO-1 conferred IRF protein more resistance to degradation,
demonstrating that SUMOylation governs protein st ability
(Fig. 2A).
To provide further evidence for the role of SUMOylation on
IRF-1 stabilit y, we determined the SUMOylated sites on IRF-1.
An in vitro SUMOylation assay was performed with IRF-1
splicing variants lacking some combination of exons 7, 8, and 9
(SI Fig. 6). SUMOylation sites were mapped within the C-
ter minal domain (SI Fig. 6), and K275 was identified as the major
in vivo SUMOylation target by site-directed mutagenesis of
lysine residues in the C-terminal domain (Fig. 2B). The IRF-1
double mutant (K275,299R) was not SUMOylated in vitro and in
vivo (Fig. 2B). Because SUMO of ten c ompetes with ubiquitin for
the same lysine residues, we examined whether the ubiquitina-
tion and SUMOylation sites in IRF-1 overlap. A n in vivo
ubiquitination assay disclosed sign ificantly diminished ubiquiti-
nation of IRF-1 in SUMO-deficient mutants (Fig. 2 C and D),
and the coexpression of IRF-1 with SUMO-1 decreased the level
of ubiquitinated IRF-1 (SI Fig. 7). The data suggest that the
major ubiquitination and SUMOylation sites are identical. Fi-
nally, we deter mined the stability of wild-t ype and mutant IRF-1
proteins. The SUMO-deficient mut ant (K275,299R) displayed
higher resistance to degradation, indicating that both these lysine
residues control the stability of IRF-1 (Fig. 2 E and F). Taken
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Fig. 1. SUMOylation of IRF-1 in cell lines and tumors. (A) MRC-5 and MCF-7 cells were lysed with SDS-loading buffer, and equal amounts of cell lysates were
immunoblotted with the indicated antibodies. (B) IRF-1 is SUMOylated after coexpression with SUMO-1. HEK293 cells were transiently transfected with plasmids
encoding IRF-1 and FLAG-SUMO-1. Treatment with the proteasomal inhibitor, MG132, blocked IRF-1 degradation. (C) The size of the shifted band in HeLa cells
was similar to that of SUMOylated IRF-1. The SUMOylated IRF-1 band in HEK293 was used as a positive control. (D) SENP1 cleaved the bond between IRF-1 and
SUMO-1. HeLa cells were transiently transfected with a control vector or the plasmid-encoding SENP1. (E) IRF-1 is highly SUMOylated in ovarian tumors. Western
blot analysis was performed by using the indicated antibodies (Left). The star indicates a nonspecific band. RT-PCR results displayed that the amount of IRF-1
mRNA was not significantlychanged between the normal cells andthe tumor. Ovarian tumor cell lysates were immunopurified with either anti-SUMO-1or control
antibody and then incubated with anti-IRF-1 antibody (Right).
Park et al. PNAS
October 23, 2007
vol. 104
no. 43
17029
IMMUNOLOGY
together, our results suggest that SUMOylation of IRF-1 con-
tributes to the enhanced protein stability in tumors.
To clarify the role of IRF-1 SUMOylation in tumors, we fused
SUMO-1 to the C ter minus of IRF-1 (23, 24) (Fig. 3A). The
chimeric protein was used as a per manently SUMOylated form
of IRF-1, designated SUMO-IRF-1 (SUMOylated IRF-1). Be-
cause SUMOylation often changes the subcellular localization
and transcriptional activity (1), we compared the localization
and transcriptional activity of SUMO-IRF-1 with that of IRF-1
protein. IRF-1 is involved in the transcriptional induction of cell
c ycle inhibitor, p21 (WAF/CIP), by positively regulating the p21
promoter (25). Although the SUMO moiety did not affect the
subcellular localization of IRF-1 (data not shown), p21 induction
was absent in SUMO-IRF-1-ex pressing cells, manifesting that
transcriptional activity of IRF-1 is lost in the SUMOylated for m
(Fig. 3B).
Using the reporter assay, we examined whether SUMO mod-
ification of IRF-1 affects IRF-1-mediated transcriptional activ-
it y. As expected, SUMO-IRF-1 repressed IRF-1-induced tran-
scriptional activation from both interferon stimulated response
element (ISRE) and p21 promoters in a dose-dependent manner
(Fig. 3 C and E), in contrast to the SUMO-IRF-1 mutant lacking
the DNA-binding domain (SUMO-IRF-1 N) (Fig. 3D). More-
over, SUMO-IRF-1 did not repress p53-induced transcriptional
activation, indicating that the antagon istic function is specific for
IRF-1 (SI Fig. 8). Furthermore, a SUMOylation-deficient mu-
t ant (K275,299R) of IRF-1 is more active than wild-type IRF-1
in ter ms of activating transcription ISRE-luciferase (Luc) and
p21-Luc reporters (Fig. 3F and SI Fig. 8). Our results suggest that
SUMOylation of IRF-1 suppresses its transcriptional activity.
Because IRF-1 belongs to the tumor suppressor family, its
ex pression induces cell death in some cell types (10, 26, 27). To
address whether the tumor suppressor function of IRF-1 de-
pends on its SUMOylation (28), we examined whether the
SUMOylated protein inhibits IRF-1-mediated cell death.
HEK293 cells were transiently transfected with plasmids encod-
ing IRF-1 together with SUMO-1. HEK293 cells expressing high
levels of IRF-1 protein and low levels of SUMO-1 induced cell
blebbing, a typical phenotype of IRF-1-mediated apoptosis
(indicated by arrow heads), whereas cells expressing high levels
of SUMO-1 led to inhibition of IRF-1-mediated apoptosis (Fig.
4A). These findings suggest that SUMOylation of IRF-1 atten-
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Fig. 2. IRF-1 is stabilized by SUMOylation. (A) SUMOylated IRF-1 displayed more resistance to protein degradation. HEK293 cells were transiently transfected
with IRF-1 in the presence or absence of SUMO-1 and treated with 20
M cycloheximide for the indicated periods. The stability of IRF-1 alone (filled circles) and
IRF-1 with the coexpression of SUMO-1 (open circles) was evaluated under conditions in which synthesis was blocked. (B) Generation of SUMO-deficient IRF-1
mutants. Cells were transfected with IRF-1, IRF-1 K254R, IRF-1 K275R, IRF-1 K299R, and IRF-1 K275,299R with SUMO-1 (Upper). The arrow specifies SUMOylated
IRF-1. These constructs were used for the in vitro SUMOylation assay (Lower). The star indicates a nonspecific band. (C) SUMO and ubiquitin target the same lysine
residues of IRF-1. HEK293 cells were transiently transfected with IRF-1 or IRF-1 (K275,299R) and treated with 10
M MG132 for 12 h. Cell lysates were subjected
to immunoblotting with antiubiquitin (Left) and anti-IRF-1 antibodies (Right). (D) Cells were transfected with plasmids encoding Xpress-tagged IRF-1 or
Xpress-tagged IRF-1 mutant (K275,299R). The proteins were immunoprecipitated with anti-Xpress antibody and then immunoblotted with antiubiquitin
antibodies (Right). Whole-cell lysates (WCL) were subjected to immunoblotting with antiubiquitin (Left) and anti-IRF-1 antibodies (Right). (E) The IRF-1 mutant
(K275,299R) displayed greater resistance to protein degradation. The stability of wild-type IRF-1 (filled circles) and IRF-1 mutant (K275,299R) (open circles) was
evaluated. (F) Schematic model showing antagonistic effects of SUMO and ubiquitin on IRF-1 protein stability.
17030
www.pnas.orgcgidoi10.1073pnas.0609852104 Park et al.
uates its tumor suppressive function. Next, we evaluated the
apoptotic activity of IRF-1 and IRF-1 mut ant (K275,299R).
A lthough SUMO-IRF-1 does not change the cell proliferation,
the SUMOylation-deficient mutant (K275,299R) of IRF-1 in-
creased the apoptotic activity compared with wild-type IRF-1
(Fig. 4B), indicating that SUMOylation of IRF-1 inhibits its
apoptotic activit y.
Earlier reports show that treatment with IFN
and TNF
triggers IRF-1 expression, which in turn mediates cell death (29).
We examined whether SUMO-IRF-1 interferes with IRF-1-
induced cell death. We established HEK293 cell lines stably
ex pressing SUMO-IRF-1, and we treated the combination of 100
un its/ml IFN
and 10 ng/ml TNF
. Treatment of IFN
and
TNF
induced IRF-1 expression in both control cells and
SUMO-IRF-1-ex pressing cells and did not change the level of
SUMO-IRF-1 (data not shown). Forty-eight hours after cyto-
k ine treatment, cell proliferation was measured by both 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay and flow-cytometric analysis. A lthough IFN
and TNF
stimulated dramatic apoptosis in HEK293 control cells, SUMO-
IRF-1-ex pressing cells failed to induce apoptosis by cytokine
treatment (Fig. 4C). Flow-cytometry analysis using propidium
iodide staining revealed a t ypical apoptotic pattern. The sub-G
1
population of cells with fragmented DNA was 41.24% for
c ontrol cells and 7.5% for SUMO-IRF-1-expressing cells after
c ytokine treatment (Fig. 4D). Taken together, our results are
c onsistent with our hypothesis that SUMOylation inhibits IRF-
1-mediated apoptosis.
Discussion
Here we demonstrate that the level of SUMOylated IRF-1 is
elevated in tumor cell lines and tumor tissues. Site-directed
mut agenesis experiments demonstrate that SUMOylation and
ubiquitination sites overlap, and SUMOylated IRF-1 displays
enhanced resist ance to degradation. The SUMOylated protein
represses IRF-1-mediated transcriptional activation and apopto-
sis. The SUMOylation sites of IRF-1 were determined as t wo
lysines, K275 and K299. Our in vivo findings reveal that lysine at
position 275 appears to be the major SUMOylation site, and this
residue lies within the SUMOylation consensus sequence
(CKEE/
KXE). Coex pression of IRF-1 and SUMO-1 in
HEK293 cells resulted in a single SUMOylated band. SUMO-
ylation of IRF-1 under these c onditions was completely sup-
pressed upon the introduction of a K275R mutation. However,
IRF-1 appears to be SUMOylated at multiple positions in tumors
because several tumor-specific SUMOylated bands were de-
tected and de-SUMOylated by SENP1. Mutant analyses con-
fir med that two lysines, K275 and K299, are involved in SUMO-
ylation of IRF-1. However, it is possible that other lysines
provide ac ceptor sites for additional SUMOylation.
The level of SUMOylated IRF-1 is relatively high compared
with other SUMOylated molecules (3). However, our immuno-
blot data with tumor cell lysates indicate that the remaining
IRF-1 is unmodified in tumors. Our experience with other
SUMOylated proteins suggests that the SUMOylation level of a
given protein is dependent on the sample preparation protocol,
thus special care is required to obtain int act SUMOylated
proteins. Immunoblotting with tumor samples revealed that a
f raction of IRF-1 is SUMOylated in tumors. However, it is
possible that IRF-1 can be partially de-SUMOylated during
sample preparation, and completely SUMOylated IRF-1 is not
detected in tumors because of technical problems. Another
ex planation for unmodified IRF-1 in tumors is that SUMO-
0
1
2
3
4
5
6
7
8
Normalized
Luciferase activity (fold)
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Fig. 3. Inhibition of IRF-1-dependent transcription by SUMOylated IRF-1. (A) Schematic diagram of SUMO-IRF-1 and SUMO-IRF-1 N. K275 and the major
SUMOylation site of IRF-1 at K299. (B) SUMO-IRF-1 does not induce p21 transcription. HEK293 cells were transiently transfected with IRF-1 and SUMO-IRF1. (C)
Transcription of ISRE fused to a Luc gene (ISRE-Luc) was inhibited by SUMO-IRF-1 in the presence of IRF-1 in HEK293 cells. HEK293 cells were cotransfected with
300 ng of ISRE-Luc, 300 ng of an IRF-1 expression plasmid (pcDNA3/IRF-1), and increasing concentrations of plasmid-encoding SUMO-IRF-1 (pcDNA3/SUMO-IRF-1)
(10, 100, and 300 ng). IRF-2 was used as a positive control. (D) SUMO-IRF-1 N does not repress transcriptional activation by IRF-1. (E) The human p21 promoter
was inhibited by SUMO-IRF-1 in the presence of IRF-1. (F) The SUMOylation-deficient mutant (K275,299R) of IRF-1 increased transcriptional activity. HEK293T cells
were transfected with 200 ng of ISRE-Luc and increasing concentrations of plasmid encoding IRF-1 or IRF-1 mutant (5, 10, and 25 ng) (Upper). Cell lysates were
probed with anti-IRF-1 antibodies (Lower) to verify the level of IRF-1 proteins.
Park et al. PNAS
October 23, 2007
vol. 104
no. 43
17031
IMMUNOLOGY
ylation alters the long-term fate of the modified protein af ter
rapid de-SUMOylation (3). Moreover, because it is quite diffi-
cult to SUMOylate a single protein of interest in vivo, SUMO-
fused IRF-1 (SUMO-IRF-1) was prepared to demonstrate the
role of SUMOylated IRF-1 on IRF-1-mediated transcriptional
activit y. SUMO-IRF-1 lost the transcriptional activity (Fig. 3 B
and C). This approach facilitates the elucidation of the role of
SUMOylated IRF-1 (23, 24). However, the SUMOylation pro-
cess is reversible and dynamic, and the SUMO fusion protein
does not behave exactly the same as a native SUMOylated
protein. To further clarify the function of SUMOylated IRF-1,
the SUMOylation-deficient mutant (K25,299R) also was used to
support the functional inactivation of IRF-1 tumor suppressor by
SUMOylation. Further investigation is required for a better
underst anding of SUMOylated IRF-1.
SUMOylation of IRF-1 can enhance the tumor cell evasion of
the immune system. It is known that IRF-1 is inactivated in some
tumors to prevent apoptosis and cell cycle arrest by genetic
mechan isms, such as gene deletion and exon skipping (11,
15–17). We demonstrate that SUMOylated IRF-1 inhibits
c ytokine-mediated apoptosis by c ompeting with unmodified
IRF-1. Thus, SUMOylation of IRF-1 attenuates the tumor
suppressor function that facilitates resistance to the immune
response, such as that during cytok ine treatment. Further in-
vestigation is required to elucidate the role of SUMOylated
IRF-1 in tumorigenesis, cancer prog ression, or drug resistance in
clin ical situations.
Several recent studies have reported the relationship bet ween
SUMOylation and human diseases (23, 30). However, little is
k nown about SUMOylation in cancer. Screening of signaling
proteins that are preferentially SUMOylated in tumors led us to
identif y a tumor suppressor protein that is extensively SUMO-
ylated. Other signaling molecules preferentially SUMOylated in
tumors possibly exist. Further studies are required to identify
other SUMOylated proteins in tumors and define their roles in
tumorigenesis.
Materials and Methods
Cell Culture and Cell Proliferation Assay. MRC-5, HDF, MCF-7, and
HeL a cells were grown in DMEM supplemented with 10% FBS.
Transfection of HeLa and HEK293 cells was perfor med by using
Fugene 6 (Roche Diagnostics, Indianapolis, IN). Cell g rowth was
measured by using the MTT assay according to the method
described in ref. 31. Cells were seeded onto 24-well plate and
then treated with specific c ytokines for the indicated time
periods. Human IFN
and TNF
were purchased from Sigma–
A ldrich (St. Louis, MO).
Plasmid Construction. To generate SUMO-IRF-1 chimeric pro-
tein, amplified SUMO-1 cDNA was cleaved with XhoI and XbaI
r
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Fig. 4. (A) Coexpression with SUMO-1 attenuates IRF-1-induced cell death. HEK293 cells were transiently transfected with plasmids encoding IRF-1 and
GFP-SUMO-1. At 36 h after transfection, cells were fixed and immunostained with anti-IRF-1 antibody. Whereas IRF-1-expressing cells displayed apoptosis,
coexpression of SUMO-1 blocked cell death. The level of SUMO expression in blebbing cells is low. (B) The SUMOylation-deficient mutant (K275,299R) of IRF-1
has increased apoptotic activity. HEK293 cells were transiently transfected with wild-type IRF-1, IRF-1 mutant (K275,299R), or SUMO-IRF-1. Thirty-six hours after
transfection, cell viability was assessed by MTT assay. (C) SUMO-IRF-1 interferes with cytokine-induced apoptosis. HEK293/vector and HEK293/SUMO-IRF-1 cells
were treated with a combination of 100 units/ml IFN
and 10 ng/ml TNF
, and cell viability was assessed by MTT assays after treatment with cytokines for 48 h.
(D) Flow-cytometric analysis of cytokine-treated cells. SUMO-IRF-1-expressing and control cells were stained with propidium iodide and analyzed by flow
cytometry. Images were obtained for the control treated with cytokine, which displayed a high percentage of subG1 population (Upper) and cell morphology
(Lower). HEK293/vector versus HEK293/SUMO-IRF-1.
*
, P 0.01;
**
, P 0.05;
***
, P 0.001.
17032
www.pnas.orgcgidoi10.1073pnas.0609852104 Park et al.
and subcloned into pcDNA3. IRF-1 cDNA was subcloned into
the Ec oRI and XhoI sites of pcDNA3/SUMO-1 and designated
as pcDNA3/SUMO-IRF-1. Point mut ations of IRF-1 were gen-
erated by using the QuikChange site-directed mutagenesis k it
(Strat agene, La Jolla, CA), and each mutant was completely
sequenced to verify the mutation of the intended sequences. The
reporter plasmid, ISRE-Luc, and p21-Luc were kindly provided
by J. Choe (Korea Advanced Institute of Science and Technol-
ogy, Daejeon, Korea).
Immunoblotting and Immunoprecipitation. For immunoblotting,
cells were harvested and resuspended in lysis buf fer [150 mM
NaCl, 50 mM Hepes (pH 8.0), 0.5% Nonidet P-40] contain ing a
protease inhibitor mixture (Roche Diagnostics). Immunoblot
detection was performed with a 1:1,000 or 1:2,000 dilution of
primary antibody and an ECL system (Amersham Biosciences,
Chicago, IL). To detect the SUMOylated protein, cells were
washed with PBS and directly lysed with SDS-loading buffer [100
mM TrisHCl (pH 6.8), 20% glycerol, 4% SDS, 0.001% bromo-
phenol blue] supplemented w ith 20
M N-ethylmaleimide
(NEM; Sigma–Aldrich), which is an inhibitor of desumoylation
enz ymes. Finally, cell lysates were boiled for 5 min, centrifuged
for 10 min at 15,700 g, and analyzed by SDS/PAGE. To
c onfirm the SUMOylated IRF-1 protein, cells were washed with
c old PBS supplemented with 20
M NEM and lysed by boiling
for 5 min in 150 mM TrisHCl (pH 6.7) buffer containing 5%
SDS, 30% glycerol, and 20
M NEM. Total lysates were diluted
20-fold with lysis buffer containing NEM and a complete
protease inhibitor mixture, incubated with anti-SUMO-1 anti-
body or anti-IgG control antibody, and then immunoprecipitated
with protein A-agarose (Peptron, Daejeon, South Korea). Im-
mune complexes were washed three times with lysis buffer
c ontaining NEM and then subjected to SDS/PAGE analysis,
followed by immunoblotting with anti-IRF-1 antibody. Antibod-
ies for IRF-1, ubiquitin, and SUMO-1 were purchased f rom
Sant a Cruz Biotechnology (Santa Cruz, CA) and Cell Signaling
Technology (Danvers, MA). Ovarian tumor and normal ovary
tissues were obtained from Samsung Medical Center tumor
tissue bank with the approval of the Institutional Review Board.
Immunofluorescence and Confocal Microscopy. Cells were grown on
sterilized glass coverslips, fixed with 4% paraformaldehyde, and
blocked with 0.1% BSA in PBS. Cells were incubated with 1:500
diluted primary antibody in PBS and reacted with 1:5,000 diluted
A lexa 488- or A lexa 568-conjugated secondary antibody (Vector
L aboratories, Burlingame, CA). Finally, slides were washed
three times with PBS and mounted in mounting media (Vector
L aboratories). Images were captured with a c onfocal microsc ope
(Bio-Rad, Hercules, CA).
In Vitro
SUMOylation Reaction. SAE1/SAE2, Ubc9, and SUMO-1
were purchased from LAE Biotech (Rockville, MD), and the in
vitro conjugation reaction was performed with in vitro-translated
IRF-1 according to the manufacturer’s instructions.
We thank J. Choe (Korea Advanced Institute of Science and Technology,
Daejeon, Korea) and J. U. Jung (Harvard University, Cambridge, MA)
for helpful discussions on the manuscript. This work was supported by an
SRC grant from the Korea Science and Engineering Foundation.
1. Seeler JS, Dejean A (2003) Nat Rev Mol Cell Biol 4:690699.
2. Verger A, Perdomo J, Crossley M (2003) EMBO Rep 4:137–142.
3. Hay RT (2005) Mol Cell 18:1–12.
4. Alarc on-Vargas D, Ronai Z (2002) Cancer Biol Ther 1:237–242.
5. Mo YY, Yu Y, Theodosiou E, Rachel Ee PL, Beck WT (2005) Oncogene
24:2677–2683.
6. Kim JH, Choi HJ, Kim B, Kim MH, Lee JM, Kim IS, Lee MH, Choi SJ, Kim
KI, Kim SI, et al. (2006) Nat Cell Biol 8:631–639.
7. Taniguchi T, Ogasawara K, Takaoka A, Tanak a N (2001) Annu Rev Immunol
19:623–655.
8. Moriyama Y, Nishiguchi S, Tamori A, Koh N, Yano Y, Kubo S, Hirohashi K,
Otani S (2001) Clin Cancer Res 7:1293–1298.
9. Lengyel P (1993) Proc Natl Acad Sci USA 90:5893–5895.
10. Tanaka N, Ishihara M, Kitagawa M, Harada H, Kimura T, Matsuyama T,
Lamphier MS, Aizawa S, Mak TW, Taniguchi T (1994) Cell 77:829839.
11. Taniguchi T, Lamphier MS, Tanaka N (1997) Biochim Biophys Acta 1333:
M9–M17.
12. Harada H, Kit agawa M, Tanaka N, Yamamoto H, Harada K, Ishihara M,
Taniguchi T (1993) Science 259:971–974.
13. Tanaka N, Ishihara M, Taniguchi T (1994) Cancer Lett 83:191–196.
14. Pizzoferrato E, Liu Y, Gambotto A, Armstrong MJ, Stang MT, Gooding WE,
Alber SM, Shand SH, Watkins SC, Storkus WJ, Yim JH (2004) Cancer Res
64:8381–8388.
15. Willman CL, Sever CE, Pallavicini MG, Harada H, Tanaka N, Slovak ML,
Yamamoto H, Harada K, Meeker TC, List AF, et al. (1993) Science 259:968–971.
16. Harada H, Kondo T, Ogawa S, Tamura T, Kitagawa M, Tanak a N, Lamphier
MS, Hirai H, Taniguchi T (1994) Oncogene 9:3313–3320.
17. Lee EJ, Jo M, Park J, Zhang W, Lee JH (2006) Biochem Biophys Res Commun
347:882–888.
18. Nakagawa K, Yokosawa H (2000) Eur J Biochem 267:1680–1686.
19. Nakagawa K, Yokosawa H (2002) FEBS Lett 530:204–208.
20. Park J, Seo T, Kim H, Choe J (2005) Mol Cell Biol 25:8202–8214.
21. Wang L, Banerjee S (2004) Oncol Rep 11:1319–1324.
22. Ulrich HD (2005) Trends Cell Biol 15:525–532.
23. Steffan JS, Agrawal N, Pallos J, Rockabrand E, Trotman LC, Slepko N, Illes
K, Lukacsov ich T, Zhu YZ, Cattaneo E, et al. (2004) Science 304:100–104.
24. Ross S, Best JL, Zon LI, Gill G (2002) Mol Cell 10:831–842.
25. Tanaka N, Ishihara M, Lamphier MS, Nozawa H, Matsuyama T, Mak TW,
Aizawa S, Tokino T, Oren M, Taniguchi T (1996) Nature 382:816818.
26. Horiuchi M, Yamada T, Hayashida W, Dzau VJ (1997) J Biol Chem 272:11952–
11958.
27. Kirchhof f S, Hauser H (1999) Oncogene 18:3725–3736.
28. Kirchhof f S, Schaper F, Hauser H (1993) Nucleic Acids Res 21:2881–2889.
29. Suk K, Chang I, Kim YH, Kim S, Kim JY, Kim H, Lee MS (2001) J Biol Chem
276:13153–13159.
30. Li M, Guo D, Isales CM, Eizirik DL, Atkinson M, She JX, Wang CY (2005)
J Mol Med 83:504–513.
31. Park K, Kim K, Rho SB, Choi K, K im D, Oh SH, Park J, Lee SH, Lee JH (2005)
Cancer Res 65:749–757.
Park et al. PNAS
October 23, 2007
vol. 104
no. 43
17033
IMMUNOLOGY
    • "Our data illustrate a new approach to correlating the expression patterns of NDRG2, RNF4 and SUMO machinery in lung cancer (Figure 5). There is also potential cross-regulation between ubiquitin and SUMO [32]. As shown in Figure 3B, the NDRG2-K333R mutant cannot be modified with SUMO-1. "
    [Show abstract] [Hide abstract] ABSTRACT: N-Myc downstream-regulated gene 2 (NDRG2) protein is a tumor suppressor that inhibits cancer growth, metastasis and invasion. The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin through its selective recognition and ubiquitination of SUMO-modified proteins. We evaluated NDRG2 SUMOylation in lung adenocarcinoma cells and its underlying molecular mechanism. The results showed that NDRG2 is covalently modified by SUMO1 at K333, which suppressed anchorage independent adenocarcinoma cell proliferation and tumor growth. In human lung adenocarcinomas cells, RNF4 targeted NDRG2 to proteasomal degradation by stimulating its SUMOylation. Endogenous RNF4 expression was increased in human lung adenocarcinomas cells, and there was a concomitant upregulation of SUMO. These findings indicate that SUMOylation of NDRG2 is necessary for its tumor suppressor function in lung adenocarcinoma and that RNF4 increases the efficiency of this process.
    Article · Nov 2014
    • "Increasing evidence supports the theory that IRF-1 functions as a tumor suppressor and represses the transformed phenotype. In human tumors, IRF-1 is deactivated to prevent apoptosis and cell cycle arrest by genetic mechanisms [9]. "
    [Show abstract] [Hide abstract] ABSTRACT: Many traditional Chinese medicine (TCM) formulae have been used in cancer therapy. The JIN formula is an ancient herbal formula recorded in the classic TCM book Jin Kui Yao Lue (Golden Chamber). The JIN formula significantly delayed the growth of subcutaneous human H460 xenografted tumors in vivo compared with the growth of mock controls. Gene array analysis of signal transduction in cancer showed that the JIN formula acted on multiple targets such as the mitogen-activated protein kinase, hedgehog, and Wnt signaling pathways. The coformula treatment of JIN and diamminedichloroplatinum (DDP) affected the stress/heat shock pathway. Proteomic analysis showed 36 and 84 differentially expressed proteins between the mock and DDP groups and between the mock and JIN groups, respectively. GoMiner analysis revealed that the differentially expressed proteins between the JIN and mock groups were enriched during cellular metabolic processes, and so forth. The ones between the DDP and mock groups were enriched during protein-DNA complex assembly, and so forth. Most downregulated proteins in the JIN group were heat shock proteins (HSPs) such as HSP90AA1 and HSPA1B, which could be used as markers to monitor responses to the JIN formula therapy. The mechanism of action of the JIN formula on HSP proteins warrants further investigation.
    Full-text · Article · Aug 2013
    • "A mechanism associated with the human papilloma virus (HPV) 16-encoded E7 oncoprotein has also been reported (26). In addition, previous studies indicate that SUMOylated IRF-1 inhibits apoptosis by repression of transcriptional activity (27). Finally, numerous reports reveal low expression levels of IRF-1 mRNA in specific forms of cancer, including breast cancer and hepatocellular carinoma (28,29). "
    [Show abstract] [Hide abstract] ABSTRACT: The present review focuses on recent advances in the understanding of the molecular mechnisms by which interferon regulatory factor (IRF)-1 inhibits oncogenesis. IRF-1 is associated with regulation of interferon α and β transcription. In addition, numerous clinical studies have indicated that IRF-1 gene deletion or rearrangement correlates with development of specific forms of human cancer. IRF-1 has been revealed to exhibit marked functional diversity in the regulation of oncogenesis. IRF-1 activates a set of target genes associated with regulation of the cell cycle, apoptosis and the immune response. The role of IRF-1 in the regulation of various types of human tumor has important implications for understanding the susceptibility and progression of cancer. In addition, an improved understanding of the role of IRF-1 in the pathological processes that lead to human malignant diseases may aid development of novel therapeutic strategies.
    Full-text · Article · Feb 2013
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