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LYMPHOID NEOPLASIA
S1PR1 is an effective target to block STAT3 signaling in activated B cell–like
diffuse large B-cell lymphoma
*Yong Liu,1 *Jiehui Deng,1 Lin Wang,1 Heehyoung Lee,1 Brian Armstrong,2 Anna Scuto,3 Claudia Kowolik,3
Lawrence M. Weiss,4 Stephen Forman,5 and Hua Yu1
Departments of 1Cancer Immunotherapeutics and Tumor Immunology, 2Neurosciences, and 3Molecular Medicine, Beckman Research Institute, City of Hope,
Duarte, CA; and Departments of 4Pathology and 5Hematology/Hematopoietic Cell Transplantation, City of Hope, Duarte, CA
STAT3 plays a crucial role in promoting
progression of human cancers, including
several types of B-cell lymphoma. However, as a transcription factor lacking
its own enzymatic activity, STAT3 remains
difficult to target with small-molecule
drugs in the clinic. Here we demonstrate
that persistent activated STAT3 colocalizes with elevated expression of
S1PR1, a G-protein–coupled receptor for
sphingosine-1-phosphate (S1P), in the tumor cells of the activated B cell–like sub-
type of diffuse large B-cell lymphoma
patient specimens. Inhibition of S1PR1
expression by shRNA in the lymphoma
cells validates that blocking S1PR1 affects expression of STAT3 downstream
genes critically involved in tumor cell
survival, proliferation, tumor invasion,
and/or immunosuppression. Using S1PR1
shRNA, or FTY720, an antagonist of S1P
that is in the clinic for other indications,
we show that inhibiting S1PR1 expression down-regulates STAT3 activity and
causes growth inhibition of the lymphoma tumor cells in vitro and in vivo.
Our results suggest that targeting S1P/
S1PR1 using a clinically relevant and
available drug or other approaches is
potentially an effective new therapeutic
modality for treating the activated B cell–
like subtype of diffuse large B-cell lymphoma, a subset of lymphoma that is less
responsive to current available therapies.
(Blood. 2012;120(7):1458-1465)
Introduction
Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous
disease, including subtypes with diverse origins and gene expression profiles.1-6 One of the most aggressive histologies, activated B
cell–like DLBCL (ABC-DLBCL) remains a challenge for effective
therapy, despite extensive studies of morphology and gene expression patterns.5-10 In vitro, cells of the ABC-DLBCL subtype have
gene expression patterns similar to those of activated peripheral
blood B cells, such as the NF-␬B downstream genes IRF4, CD44,
and cyclin D2.11 At the same time, these tumor cells also secrete
IL-6 and IL-10 through autocrine/paracrine pathways.12 Consistent
with this, the constitutive activation of NF-␬B and Janus kinase
(JAK) is shown to promote tumorigenicity of malignant B-cell
lymphoma, including ABC-DLBCL.13
One promising candidate for ABC-DLBCL targeted therapy is
signal transducer and activator of transcription 3 (STAT3). STAT3
is a transcriptional factor that is constitutively activated in many
types of cancer, contributing to tumor progression via several
mechanisms.14-20 STAT3 is shown to be activated in the ABCDLBCL subtype.20,21 Approximately one-third of ABC-DLBCL
patients have gain-of-function mutations in the adaptor protein
MYD88, which leads to JAK and STAT3 activation, as well as IL-6
and IL-10 secretion.13 IL-6 and IL-10 secretion can further increase
STAT3 activation levels within tumor cells via an autocrine
feedback loop.14 IL-6–activated STAT3 is crucial for survival of
several types of cancer cells, including multiple myeloma, a
plasmacytic B-cell malignancy.14,22 IL-10 is also important for
STAT3-mediated immune suppression in the tumor microenviron-
ment through paracrine mechanisms.15,23 When targeting STAT3
within tumor cells, their proliferation and survival ability decrease
concurrently with down-regulation of STAT3 downstream genes
that are involved in these processes.14,15
Although STAT3 could be a good candidate for B-cell lymphoma therapy, as a transcriptional factor it is hard to target. Recent
discoveries show that STAT3 can also be activated by the
sphingosine-1-phosphate receptor 1 (S1PR1), a G-protein–coupled
receptor.24 G-protein–coupled receptors are more tractable therapeutic targets than transcriptional factors.25,26 S1PR1 is important for
normal B-cell secondary lymphoid organ retention as well as
splenic marginal zone localization.27,28 In tumor cells, S1PR1 can
maintain persistent activation of STAT3, in part through JAK2
kinase.24 However, it remains unknown whether S1PR1 is crucial
for STAT3 activation in ABC-DLBCL. One of the most effective
S1PR1 antagonists is FTY720, an analog of the S1PR1 ligand, S1P,
with an even higher binding affinity than the natural ligand.29
FTY720 has been in the clinic for certain autoimmune diseases.30,31
Our present study demonstrates that S1PR1 and STAT3 are
coactivated in ABC-DLBCL tumor cells. Inhibiting S1PR1 expression using either shRNA, or a small-molecule drug, FTY720, leads
to inhibition of STAT3, induction of tumor cell growth arrest and
apoptosis. Inhibiting S1PR1 in vivo using both human/mouse
xenograft and syngeneic models led to STAT3 inhibition and strong
antitumor effects. Our study provides a novel druggable target for
certain B-cell lymphoma subtypes that are less responsive to
chemotherapy plus rituximab.
Submitted December 16, 2011; accepted June 17, 2012. Prepublished online
as Blood First Edition paper, June 28, 2012; DOI 10.1182/blood-2011-12-399030.
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.
*Y.L. and J.D. contributed equally to this study.
The online version of this article contains a data supplement.
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© 2012 by The American Society of Hematology
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BLOOD, 16 AUGUST 2012 䡠 VOLUME 120, NUMBER 7
Methods
Reagents and cell lines
FTY720 was synthesized by the Synthetic and Biopolymer Chemistry Core
at City of Hope. The pLKO.1 nontarget shRNA control, human S1PR1
shRNA lentiviral vectors, and anti–␤-actin (AC-15) were purchased from
Sigma-Aldrich; anti–Bcl-xL, anti-poly (ADP-ribose) polymerase (H-250),
anti-phosphorylated Stat1 (p-Stat1; Tyr701), anti-phosphorylated Stat3
(p-Stat3; Tyr705), anti-phosphorylated Stat5 (p-Stat5; Tyr694), and anti-Stat5
were from Cell Signaling Technology; anti-S1PR1 (A6), anti-Stat1 (E23),
and anti-Stat3 (C-20) were from Santa Cruz Biotechnology; and antiSurvivin was from Novus Biologicals. AlexaFluor-488 and AlexaFluor-546
secondary antibodies were purchased from Invitrogen. FITC- and
allophycocyanin-conjugated antibodies to annexin V were from BD Biosciences PharMingen. Human ABC-like DLBCL cell lines Ly3 and Ly10 were
kind gifts from Dr B. Hilda Ye (Albert Einstein College of Medicine, Bronx,
NY) and Dr L. M. Staudt (National Cancer Institute, Bethesda, MD),
respectively. Ly3 cells were cultured in IMDM supplemented with 10%
FBS. Ly10 cells were cultured in IMDM supplemented with 20% FBS.
Murine lymphoma cell line A20 was purchased from ATCC and cultured in
RPMI 1640 medium supplemented with 10% FBS.
Lentivirus transduction
The green fluorescent protein (GFP) tagging lentivirus vector eGFPffluc_epHIV7 was a gift from Dr Michael Jensen (University of Washington, Seattle, WA). The shRNA part of pLKO.1 nontargeting shRNA control
and S1PR1 shRNA lentiviral vectors were subcloned into GFP-tagging
lentiviral vectors. The production of lentivirus was performed as previously
described.21 Ly3 cells were transduced with shRNA expressing lentivirus,
and the infected cells were sorted based on their GFP signals.
Patient specimens and immunohistochemistry and
immunofluorescent staining
Paraffin-embedded patient tissue samples were obtained from the archive
files of the Pathology Core of City of Hope Comprehensive Cancer Center,
with approval from the Institutional Review Board (COH IRB11234). For
immunohistochemical staining, paraffin-embedded sections were deparaffinized and hydrated through xylene and graded ethanol series, followed by
staining with antibodies against p-Stat3 (Cell Signaling) and S1PR1 (Santa
Cruz Biotechnology) and examined under Olympus AX70 automated
upright microscope. For immunofluorescent staining, AlexaFluor-488 and
AlexaFluor-546 (Invitrogen) were used as secondary antibodies; nuclei
were counterstained with Hoechst 33432 (Invitrogen) and examined using
Zeiss LSM510 Meta inverted confocal microscope (Carl Zeiss) with
20⫻/0.5 objectives. The images were captured using Zeiss LSM Image Browser
Version 4.2 software and analyzed using Image-Pro plus Version 6.3 (Media
Cybernetic Inc). The immunohistochemical staining images were taken on
an Aperio ScanScope AT system with 20⫻/0.75 objectives and analyzed
using Aperio ImageScope Version 11.1 software (Aperio Technologies).
In vivo experiments
Murine lymphoma A20 cells (2 ⫻ 106/mouse) and ABC-DLBCL Ly3 cells
(5 ⫻ 106/mouse) transduced with either nontarget shRNA or S1PR1 shRNA
were implanted subcutaneously into 6- to 8-week-old male BALB/c
(National Cancer Institute) and NOD/SCID IL2R␥-null mice (The Jackson
Laboratory), respectively. A20 tumor-bearing mice were treated by intraperitoneal injection with FTY720 (5 mg/kg body weight) or DMSO vehicle
every day, starting when tumors reached 5 to 7 mm in diameter. Mouse care
and in vivo experimental procedures were performed under pathogen-free
conditions and approved by the Institutional Animal Care and Use Committee of
the Beckman Research Institute at City of Hope Medical Center.
Cell proliferation and apoptosis assay
Cells were seeded in triplicates at a density of 5 ⫻ 105 cells/mL into 96- or
24-well plates. Cell proliferation assays were performed using the CellTiter
TARGETING S1PR1 BLOCKS STAT3 IN HUMAN LYMPHOMA
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96 AQueous One Solution Cell Proliferation Assay kit (Promega) after
treatment at the indicated time. The absorbance at 490 nm was measured
using Microplate reader (Bio-Rad). For apoptosis assays, cells were
harvested and stained with annexin V–FITC and propidium iodide and
assessed for the percentage of double-negative population using an Accuri
C6 flow cytometer. For the lentivirus-transduced Ly3 cells, samples were
stained with annexin V–allophycocyanin and 4⬘, 6-diamidino-2phenylindole. Apoptosis data were analyzed using FlowJo Version 7.6.1
software (TreeStar).
Quantitative real-time PCR and PCR arrays
Total RNA was purified by RNeasy Kit (QIAGEN), and cDNA was
synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). Real-time
PCR was performed in triplicates using the Chromo4 Real-Time Detector
(Bio-Rad). The 18S RNA human housekeeping gene was used as an internal
control to normalize the target gene mRNA levels.
Statistical analysis
The correlation between phospho-STAT3 (red) and S1PR1 (green) was
quantified based on Manders colocalization coefficients M1 (red to green)
and M2 (green to red), using Image-Pro plus Version 6.3 software (Media
Cybernetics, Inc). Value ranges from 0 (no colocalization) to 100% (all
pixels colocalize). Statistical analysis between 2 groups was calculated as
2-tailed P value using unpaired Student t test.
Results
STAT3 activity correlated with S1PR1 expression in patient
ABC-DLBCL tumor cells
STAT3 is constitutively activated in ABC-DLBCL, but not in
GCB-DLBCL lymphoma cells.20 To determine whether S1PR1
expression is crucial for STAT3 activation in ABC-DLBCL tumor
cells, we examined S1PR1 and phospho-STAT3 protein levels in a
cohort of 10 ABC-DLBCL patient samples. The B-cell lymphoma
primary tumor cells showed elevated S1PR1 and STAT3 activity, as
determined by immunofluorescent and immunohistochemistry staining (Figure 1A-B). To assess whether S1PR1 was an important
contributor for STAT3 activation in primary ABC-DLBCL tumor
cells, we quantified S1PR1 expression with phospho-STAT3 (pSTAT3) in the tumor cells, using tissue sections double-stained by
S1PR1 and p-STAT3. The expression levels of S1PR1 and p-STAT3
correlated with each other in the same tumor cells within the tumor
tissues from these patients, with average colocalization percentages
of 87.3 (p-STAT3 to S1PR1) and 79.6 (S1PR1 to p-STAT3),
respectively (Figure 1A right). Immunohistochemical staining was
performed using separate tissue sections, further supporting the
finding that S1PR1 expression was elevated in tumor cells with
positive p-STAT3 in ABC-DLBCL (Figure 1B). Consistent with
this, ABC-DLBCL cell lines Ly3 and Ly10 also showed elevated
STAT3 activity and S1PR1 expression as determined by Western
blot analysis (supplemental Figure 1, available on the Blood
Web site; see the Supplemental Materials link at the top of the
online article).
Specifically targeting S1PR1 using shRNA decreased
STAT3-mediated cell proliferation and survival
To investigate whether blocking S1PR1 can decrease STAT3
activation and in turn restrain ABC-DLBCL cell proliferation and
survival, we transduced Ly3 ABC-DLBCL tumor cells with both
control and S1PR1 shRNA-expressing lentiviruses. Specific knockdown of S1PR1 gene expression led to tumor cell growth inhibition
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LIU et al
BLOOD, 16 AUGUST 2012 䡠 VOLUME 120, NUMBER 7
Figure 1. Coexpression of S1PR1 and phospho-STAT3 in ABC-DLBCL patient tumor cells. (A) Left panel: immunofluorescent staining of S1PR1 (green) and
phospho-STAT3 (red) in ABC-DLBCL patient tumor tissue sections. Shown are 3 independent ABC-DLBCL patient tissue sections, representing 10 tested patient samples.
Scale bar represents 50 ␮m. Right panel: quantification of 10 tested patient tissue sections for percentages of overlapping red (p-STAT3) and green (S1PR1) channels, shown
as Manders colocalization coefficients M1 (p-STAT3 to S1PR1) and M2 (S1PR1 to p-STAT3), respectively. For each patient tumor section, 10 random fields were chosen to
calculate M1 and M2, valued and normalized as mean, which is represented by a dot. One tumor tissue section from each of the 10 patients was analyzed.
(B) Immunohistochemistry staining of S1PR1 and phospho-STAT3 of the same region, using consecutive ABC-DLBCL patient tissue sections. Scale bar represents 50 ␮m.
and apoptosis (Figure 2A). S1PR1 inhibition reduced STAT3
activity as well as expression of its downstream proliferation/
survival-related genes, including proliferation gene MCL-1 and
antiapoptotic genes BCL-XL and SURVIVIN, at both mRNA and
protein levels (Figure 2B-C). Furthermore, blocking S1PR1/STAT3
signaling not only lowered expression of genes related to proliferation and survival but also those related to extracellular matrix
degradation, such as MMP-9 (Figure 2B).
Inhibiting S1PR1 reduced primary ABC-DLBCL tumor growth
and invasion
Because blocking S1PR1/STAT3 signaling decreased ABCDLBCL tumor cell proliferation and apoptosis in culture, we
wanted to validate the effects of S1PR1/STAT3 inhibition on tumor
progression in vivo. We implanted Ly3 tumor cells, with or without
S1PR1 shRNA expression, into NOD/SCID IL2R␥-null mice,
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BLOOD, 16 AUGUST 2012 䡠 VOLUME 120, NUMBER 7
TARGETING S1PR1 BLOCKS STAT3 IN HUMAN LYMPHOMA
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Figure 2. Effects on specific inhibition of S1PR1 in Ly3 ABC-DLBCL cell line. (A) Cell proliferation and apoptosis were assessed 24 or 48 hours after culturing Ly3 tumor
cells with or without S1PR1 shRNA knockdown. Data represent 3 independent experiments. ***P ⬍ .001. **P ⬍ .01. *P ⬍ .05. (B) Real-time PCR showing differences in
mRNA expression levels of STAT3 downstream genes in control and S1PR1 shRNA lentivirus-transduced Ly3 tumor cells. ***P ⬍ .001. **P ⬍ .01. *P ⬍ .05. Each sample was
examined by triplicates. (C) Western blot analysis of protein expression levels of STAT3 downstream genes in both control and S1PR1 shRNA-expressing Ly3 tumor cells. Data
represent 3 independent experiments.
which lack a mouse immune system. Results from this set of
experiments showed that specifically blocking S1PR1 expression
in human ABC-DLBCL tumor cells decreased primary tumor
growth in xenograft models (Figure 3A). Furthermore, one of the
biggest obstacles to B-cell lymphoma treatment is invasion/
extranodal infiltration of the tumor cells.32 S1PR1 knockdown
reduced approximately 75% of the number of lung metastatic
nodules in the xenograft tumor model (Figure 3B). Consistent with
our in vitro results, blocking S1PR1/STAT3 signaling also downregulated expression of genes related to proliferation and survival
both at mRNA and protein levels in growing tumors (Figure 3C-D).
Blocking S1PR1/STAT3 signaling using S1PR1 inhibitor FTY720
induced ABC-DLBCL tumor cell growth inhibition and
apoptosis
Because directly blocking S1PR1 by shRNA can decrease ABCDBCL tumor growth, next we assessed whether using the S1PR1
antagonist FTY720 had antitumor therapeutic effects. Treatment of
FTY720 reduced tumor cell growth in 24 hours as determined by
MTS assay of metabolic rate. The growth inhibition reached more
than 95%, after treatment of Ly3 ABC-DLBCL tumor cells with
5␮M FTY720 for 48 hours (Figure 4A left panel). Ly10 ABCDLBCL tumor cells required relatively higher concentrations
(10␮M) of FTY720 treatment to show profound growth inhibition
(Figure 4A right panel). FTY720 treatment at 5-10␮M range also
reduced cell viability, as determined by annexin V and propidium
iodide double-negative staining (Figure 4B). Because IL-6 and IL-10
are important STAT3 activators and are secreted by ABC-DLBCL
tumor cells, we further tested whether blocking S1PR1 by FTY720
could inhibit production of these cytokines. Results showed that
IL-6 was reduced by FTY720 in Ly3 tumor cells (Ly10 tumor cells
do not produce detectable IL-6). IL-10 secretion was also reduced
by FTY720 treatment in both Ly3 and Ly10 tumor cells (supplemental Figure 2).
We next determined whether blocking S1PR1 using FTY720
could decrease STAT3 activity. We treated Ly3 and Ly10 tumor
cells with FTY720 at the same concentrations used for proliferation
and apoptosis assays to determine S1PR1 and phospho-STAT3
expression levels. FTY720 treatment lowered S1PR1 levels 6 hours
after treatment at a minimum concentration of 5␮M. Phospho-STAT3
was also decreased, accompanied by S1PR1 down-regulation
(Figure 4C). Adding a JAK inhibitor, AZD1480, at 0.5␮M to
FTY720 treatment, had some additive effect on STAT3 inhibition
(supplemental Figure 3). Decreasing of p-STAT3 by FTY720 also
reduced the expression of STAT3 downstream genes critical for
proliferation and survival, including MCL-1 and BCL-XL (supplemental Figure 4A). To test the specificity and potential effects of
FTY720 on the activity of other STAT family proteins, we assessed
pSTAT1 and pSTAT5 levels in Ly3 and Ly10 tumor cells, before
and after FTY720 treatment. However, the levels of pSTAT1 and
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BLOOD, 16 AUGUST 2012 䡠 VOLUME 120, NUMBER 7
LIU et al
Figure 3. Specific silencing of S1PR1 in Ly3 ABC-DLBCL tumor cells inhibited tumor growth and invasion in vivo. (A) Ly3 tumor growth was inhibited by specific S1PR1
silencing; N ⫽ 8. ***P ⬍ .001. Data are representative results from one of 2 independent experiments. (B) Inhibiting S1PR1 by shRNA in ABC-DLBCL tumor cells was
accompanied by a reduction in lung invasion in vivo. Lung nodules were numerated on day 20; N ⫽ 8. *P ⬍ .05. (C) Knocking down S1PR1 reduced expression of STAT3 target
genes in vivo. Western blot analysis to detect expression of STAT3 downstream genes at protein level, using whole tumor lysates prepared from tumors grown from control and
S1PR1 shRNA expressing Ly3 ABC-DLBCL tumor cells. Eight tumors were pooled to prepare the lysates. (D) Real-time PCR to analyze RNA expression levels of Stat3
downstream genes involved in proliferation, survival, and immune response in tumors grown from control and S1PR1 shRNA-transduced tumor cells. Eight tumors were pooled
to prepare RNA, and real-time PCR was performed as triplicates for each sample. ***P ⬍ .001; **P ⬍ .01.
pSTAT5 were not readily detectable in these tumor cells, as
determined by Western blotting (supplemental Figure 4B).
FTY720 inhibited Stat3 activity and tumor growth in murine
A20 lymphoma in vivo
Because the tumor microenvironment, especially the immunologic
microenvironment, plays an important role during tumor progression and therapy, we next used murine A20 B-cell lymphoma cells,
which were derived from a BALB/c mouse, to validate whether
S1PR1 could be a good therapeutic target in both tumor cells as
well as tumor stromal immune cells. Consistent with the data from
human ABC-DLBCL cell lines, treatment of the mouse B-cell
lymphoma A20 cell line with FTY70 also reduced cell proliferation
and survival (Figure 5A), abrogated S1pr1 expression, and Stat3
activity (Figure 5B). To determine whether FTY720 could inhibit
tumor growth through targeting S1PR1/STAT3 signaling, A20
B-cell lymphoma cells were implanted into syngeneic BALB/c
mice and treated with FTY720. Subcutaneous A20 tumors in mice
treated by intraperitoneal injection with FTY720 daily at 5 mg/kg
led to a reduction of greater than 50% of tumor growth in vivo
(Figure 5C left panel). Consistent with in vitro treatment results,
FTY720 inhibited Stat3 activity in tumors in vivo, which was
coupled with a reduction in S1pr1 expression (Figure 5C right
panel). Notably, total Stat3 levels were also decreased in the whole
tumor after FTY720 treatment (Figure 5C right panel), which is
probably the result of the ability of phospho-Stat3 to autoregulate
Stat3 expression.
Discussion
Although STAT3 is a promising target for cancer therapy, effectively inhibiting the activity of a transcriptional factor remains a
challenge because of their intracellular localization and lack of
enzymatic activity. Recently, a G-protein–coupled receptor, S1PR1,
is discovered to be important for persistent activation of STAT3 in
certain mouse tumors and human tumor cells.24 S1PR1 is shown to
physically interact with JAK2, leading to increased STAT3 phosphorylation. Activated STAT3 can in turn up-regulate S1PR1
expression at the transcriptional level, thereby forming a feedforward loop.24 STAT3 activation is also contributed by IL-6 and
other factors, leading to further S1PR1 (over)-expression. Compared with STAT3, S1PR1, as a GRCR, has a more advantageous
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BLOOD, 16 AUGUST 2012 䡠 VOLUME 120, NUMBER 7
TARGETING S1PR1 BLOCKS STAT3 IN HUMAN LYMPHOMA
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Figure 4. S1PR1 antagonist FTY720 induced apoptosis and growth inhibition of ABC-DLBCL tumor cells through abrogating S1PR1/STAT3 signaling. (A) FTY720
treatment inhibited ABC-DLBCL tumor cell proliferation. ABC-DLBCL cell lines, Ly3 and Ly10, were treated with FTY720 at different concentrations as indicated for 24 or
48 hours. The relative cell numbers of different treatments were determined by MTS assay. (B) Treating ABC-DLBCL tumor cells with FTY720 induced apoptosis. The
percentage of viable cells was determined by flow cytometry to detect annexin V and propidium iodide (PI) double-negative cells. (C) FTY720 treatment inhibited STAT3 activity
in ABC-DLBCL tumor cells. The levels of S1PR1 and phospho-STAT3 in Ly3 and Ly10 ABC-DLBCL cells after FTY720 treatment (6 hours) were determined by Western blot
analysis. All data are representative of results from 3 independent experiments.
cellular localization as well as the capacity for enzymatic targeting.
However, the importance of S1PR1 in cancer biology is just
beginning to be appreciated, and its potential importance as a target
for human cancer therapy remains to be further assessed. Our study
shows that STAT3 activation and S1PR1 expression colocalize in
patient ABC-DLBCL tumor cells.
S1PR1 is crucial for lymphocyte secondary lymphoid organ
retention for both T lymphocytes and B lymphocytes.28,33 S1PR1 is
also highly expressed in endothelial cells and pericytes, and the
expression of S1PR1 within these cells is important for tumor
angiogenesis and metastasis.34,35 On the other hand, blocking
S1PR1 can retain lymphoma cells in lymphoid organs because of
an egress function defect.29,36 This could potentially limit the
lymphoma cells’ invasive potential. Our results demonstrate the
effectiveness of the S1PR1 antagonist FTY720, which has already
been used clinically for treatment of multiple sclerosis.30,31 This
drug is also shown to be effective in many types of cancer in animal
models.37-39 We show that this clinically available agent can inhibit
ABC-DLBCL cell proliferation and survival in vitro through
decreasing STAT3 activity.
Further preclinical studies on the feasibility of using FTY720 to
treat B-cell lymphoma patients are necessary. It will be especially
desirable to study the effect of blocking S1PR1/STAT3 signaling in
both tumor cells and in the tumor immunologic environment using
humanized mouse models with human B-cell lymphoma cells
implanted. In addition to FTY720, a promising drug to target this
pathway, is an antibody against S1P ligand, which has been shown
to effectively inhibit primary tumor growth in various mouse
models and has recently entered clinical trials for other indications.35,40 Another possibility is the use of a novel in vivo siRNA
delivery technology, CpG oligonucelotide-conjugated siRNA, to
specifically target Toll-like receptor 9-positive cells.41,42 Toll-like
receptor 9-positive cells include normal macrophages and B cells
and various types of malignant myeloid and B cells, including
ABC-DLBCL. Using CpG-S1PR1 siRNA, one could target not
only S1PR1/STAT3 signaling in tumor cells but concurrently
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BLOOD, 16 AUGUST 2012 䡠 VOLUME 120, NUMBER 7
LIU et al
Figure 5. Targeting S1pr1/Stat3 signaling inhibited
murine B-cell lymphoma tumor growth in a syngeneic mouse model. (A) Inhibition of S1pr1 induced
apoptosis and growth inhibition of the murine B-cell
lymphoma A20 cell line in vitro. A20 B-cell lymphoma
cells were treated with FTY720 at indicated concentrations. Tumor cell proliferation and apoptosis were examined 24 or 48 hours after treatment. The results represent
3 independent experiments. (B) S1pr1 and phosphoStat3 levels in A20 tumor cells were determined by
Western blot analysis after treating with FTY720 for
6 hours. (C) A20 tumor growth and Stat3 activity were
decreased by FTY720 treatment in vivo. Left panel:
tumor growth curve showing that FTY720 inhibited
A20 tumor growth in vivo; N ⫽ 5. **P ⬍ .01. Data are
representative of 2 independent experiments. Right panel:
Western blotting analysis of S1pr1 and phospho-Stat3
protein levels in A20 tumors after FTY720 treatment
in vivo. Five tumors were pooled to make protein lysates.
Data are representative of 2 independent experiments.
enhance the antitumor immune response by activating tumorassociated dendritic cells, macrophages, and B cells. Taken together, our results suggest that S1PR1 can be a viable target, via
inhibiting STAT3 activity, for treating ABC-DLCL.
Acknowledgments
The authors thank staff members at Pathology Core, Light Microscopy Core, Animal Facility Core, Flow Cytometry Core at Beckman Research Institute City of Hope Comprehensive Cancer
Center for their excellent technical support as well as Sandra
Thomas at Department of Hematology at City of Hope for editing
the paper.
This work was supported by Tim Nesviq Fund at City of Hope
Comprehensive Cancer Center, the National Institutes of Health
(grants P50CA107399 and R01CA146092), the V Foundation
Translational Research Grant, and HEADstrong Foundation, in
memory of Nicholas E. Colleluori.
Authorship
Contribution: H.Y. conceptualized the project and helped to design
the experiments and manuscript writing; Y.L. and J.D. performed
the majority of the experiments, analyzed the data, and prepared the
manuscript; L.W. contributed to the immunofluorescent staining;
H.L. also helped the design of experiments and data interpretation;
B.A. contributed to imaging scanning for the statistical analysis of
immunofluorescent staining data; A.S. contributed to the study of
human ABC-DLBCL cell lines; C.K. performed the lentivirus
transduction experiments; and L.M.W. and S.F. assessed patient
specimens and/or provided clinical relevance.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Hua Yu, Department of Cancer Immunotherapeutics and Tumor Immunology, Beckman Research Institute,
1500 E Duarte Rd, Duarte, CA 91010; e-mail: [email protected].
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2012 120: 1458-1465
doi:10.1182/blood-2011-12-399030 originally published
online June 28, 2012
S1PR1 is an effective target to block STAT3 signaling in activated B cell
−like diffuse large B-cell lymphoma
Yong Liu, Jiehui Deng, Lin Wang, Heehyoung Lee, Brian Armstrong, Anna Scuto, Claudia Kowolik,
Lawrence M. Weiss, Stephen Forman and Hua Yu
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