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Therapeutics, Targets, and Chemical Biology
Aberrant SYK Kinase Signaling Is Essential forTumorigenesis
Induced by TSC2 InactivationYe Cui1,Wendy K. Steagall2, Anthony M.
Lamattina1, Gustavo Pacheco-Rodriguez2,Mario Stylianou3, Pranav
Kidambi1, Benjamin Stump1, Fernanda Golzarri1,Ivan O. Rosas1,
Carmen Priolo1, Elizabeth P. Henske1, Joel Moss2, andSouheil
El-Chemaly1
Abstract
Somatic or germline mutations in the tuberous sclerosiscomplex
(TSC) tumor suppressor genes are associated closelywith the
pathogenesis of lymphangioleiomyomatosis, a rareand progressive
neoplastic disease that predominantly affectswomen in their
childbearing years. Serum levels of the lym-phangiogenic growth
factor VEGF-D are elevated significantlyin
lymphangioleiomyomatosis. However, there are gaps inknowledge
regarding VEGF-D dysregulation and its cellularorigin in
lymphangioleiomyomatosis. Here, we show thatincreased expression
and activation of the tyrosine kinase Sykin TSC2-deficient cells
and pulmonary nodules from lymphan-gioleiomyomatosis patients
contributes to tumor growth. Sykkinase inhibitors blocked Syk
signaling and exhibited potent
antiproliferative activities in TSC2-deficient cells and
animmunodeficient mouse xenograft model of
lymphangioleio-myomatosis. In TSC2-deficient cells, Syk signaling
increasedthe expression of monocyte chemoattractant protein
MCP-1,which in peripheral blood mononuclear cells (PBMC)
stimu-lated the production of VEGF-D. In clinical isolates of
PBMCsfrom lymphangioleiomyomatosis patients, VEGF-D expres-sion was
elevated. Furthermore, levels of VEGF-D and MCP-1 in patient sera
correlated positively with each other. Ourresults illuminate the
basis for lymphangioleiomyomatosisgrowth and demonstrate the
therapeutic potential of targetingSyk in this and other settings
driven by TSC genetic mutation.Cancer Res; 77(6); 1492–502. �2017
AACR.
IntroductionTuberous sclerosis complex (TSC), an inheritable
disorder
resulting from mutations in either the TSC1 or TSC2 gene,
isassociated with benign tumors in multiple tissues,
cognitiveimpairment, seizures, and skin abnormalities (1). TSC
defi-ciency causes aberrant activation of the mTORC1 signaling
andthereby results in excessive protein synthesis and
uncontrolledcell proliferation (2). Up to 30% of women with TSC
developlymphangioleiomyomatosis (LAM), a rare and
progressiveneoplastic disease that predominantly affects women in
theirchildbearing years and ranks as the third leading cause of
TSC-related death (3, 4). Other than TSC-associated
lymphangio-leiomyomatosis (TSC-LAM), there is a separate form of
the
disease with a distinct clinical entity called sporadic
lymphan-gioleiomyomatosis (S-LAM), which is caused by somatic
ratherthan germline mutations in the TSC genes (5). Lung
lesionsfrom lymphangioleiomyomatosis patients are characterized
byexcessive growth of smooth muscle–like cells
(lymphangioleio-myomatosis cells) and cyst formation, leading to
gradualairflow obstruction and potentially death from
respiratoryfailure within two decades (4).
It is estimated that up to 3,500 patients worldwide and
1,400patients in the United States have been diagnosed with
lym-phangioleiomyomatosis and that up to a quarter million wom-en
may have lymphangioleiomyomatosis (6). Significantefforts have been
made over the past two decades to identify,develop, and implement
effective therapeutic strategies tocombat this potentially fatal
disease. On the basis of its abilityto inhibit mTORC1 activation of
downstream kinases, siroli-mus (rapamycin) has become an
established treatment forlymphangioleiomyomatosis (7, 8). Rapamycin
has been shownto suppress TSC2-null xenograft tumor growth in
animal mod-els (9, 10). Correspondingly, rapamycin treatment leads
toshrinkage of angiomyolipomas (AML; ref. 11) and
lymphan-gioleiomyomas (12), reduction of chylous effusions (13),
andpreservation of lung function in patients with
lymphangioleio-myomatosis (7). Rapamycin decreases serum levels of
VEGF-D,which is elevated in a majority of
lymphangioleiomyomatosispatients and has been widely used as a
diagnostic biomarker(14, 15). However, the mechanism for its
upregulation is notfully known. Although lymphangioleiomyomatosis
cells aregenerally thought to be the source of VEGF-D (15), we
havepreviously shown that silencing TSC2 in human lung
1Division of Pulmonary and Critical Care Medicine, Brigham and
Women'sHospital, Harvard Medical School, Boston, Massachusetts.
2Cardiovascular andPulmonary Branch, National Heart, Lung, and
Blood Institute, NIH, Bethesda,Maryland. 3Office of Biostatistics
Research, National Heart, Lung, and BloodInstitute, NIH, Bethesda,
Maryland.
Note: Supplementary data for this article are available at
Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
Y. Cui and W.K. Steagall contributed equally to this
article.
J. Moss and S. El-Chemaly contributed equally as senior
authors.
Corresponding Author: Souheil El-Chemaly, Brigham and Women's
Hospital,75 Francis Street, Boston, MA 02115. Phone: 617-732-6869;
Fax: 617-582-6102;E-mail: [email protected]
doi: 10.1158/0008-5472.CAN-16-2755
�2017 American Association for Cancer Research.
CancerResearch
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fibroblasts does not lead to increases in VEGF-D levels
(16),suggesting that TSC2-deficient cells may not be the direct
sourceof excessive VEGF-D in lymphangioleiomyomatosis.
It should be noted, however, that more than half of
lym-phangioleiomyomatosis patients enrolled in the
MulticenterInternational Lymphangioleiomyomatosis Efficacy and
Safetyof Sirolimus (MILES) clinical trial did not exhibit enhanced
orstabilized lung function during the rapamycin treatment phase(7),
and as such, it is absolutely imperative to evaluate newtherapies
that may expand options for patients with lymphan-gioleiomyomatosis
and other malignancies characterized byactivation of the mTORC1
pathway who are unresponsive orintolerant to rapamycin treatment.
The identification of elevat-ed Src kinase activity in
lymphangioleiomyomatosis cells hasled to an ongoing clinical trial
evaluating the therapeutic effi-cacy of an Src inhibitor
(saracatinib) in lymphangioleiomyo-matosis patients (17). Similar
to Src kinase, spleen tyrosinekinase (Syk) is a non–receptor
tyrosine kinase and a key com-ponent of both innate and adaptive
immunity. Syk also con-tributes to the initiation and metastatic
progression of multipletypes of solid tumors. For instance, Syk
plays a role in breastcancer–invasive cell growth (18) and acts as
an oncogenic driverin small cell lung cancer (19). In addition,
upregulated Sykexpression corresponds with metastasis of both
prostate cancers(20) and squamous cell carcinomas of the head and
neck (21).More intriguingly, studies have shown that Syk
regulatesmTORC1 activation in acute myeloid leukemia and
lymphoma(22, 23). Taken together, the aberrant expression of Syk
invarious malignancies and the association between Syk andmTORC1
prompt us to hypothesize that Syk may be tightlyinvolved in
lymphangioleiomyomatosis pathogenesis.
In this study, we found deregulated Syk expression and
acti-vation in TSC2-deficient cells and in
lymphangioleiomyomatosislung lesions. We also determined the
therapeutic efficacy ofclinically relevant Syk inhibitors in
curbing TSC2-null tumorprogression. Furthermore, we identified a
unique cascade ofSyk-dependent signals between TSC2-deficient cells
and periph-eral blood mononuclear cells (PBMC) that eventually
inducedVEGF-Dexpression.Given these collectivefindings, Syk could
be atherapeutic target for the treatment of
lymphangioleiomyoma-tosis and other diseases associated with
mutations in the TSCgenes.
Materials and MethodsHuman studies
All human subjects gave their written informed consent.Human
samples were acquired under protocols approved by theNational
Heart, Lung, and Blood Institute Institutional ReviewBoard
(Protocol 95-H-0186).
Lung tissues. Pulmonary parenchyma from subjects with
lym-phangioleiomyomatosis (n ¼ 6) was fixed in 10%
neutral-buff-ered formalin and subsequently embedded with
paraffin.
Clinical features of patient cohort. Serum was collected from
27lymphangioleiomyomatosis patients for a total of 200 visitsbefore
(80 visits) and after (120 visits) sirolimus treatment.
Thediagnosis of lymphangioleiomyomatosis was confirmed
byhistopathology or the presence of cystic disease by CT scanplus
TSC, angiomyolipomas, high VEGF-D levels, and/or lym-
phangioleiomyomas. Three of the patients have LAM-TSC; therest
have sporadic lymphangioleiomyomatosis. There are 2Asian, 1 white
Hispanic/Latino, and 24 white non–Hispanic/Latino patients. Age and
visit information are in SupplementaryTable S1.
Luminex analysis of serum samples. Monocyte
chemoattractantprotein (MCP)-1 and VEGF-D were measured in serum
(dilution1:2) using magnetic bead–based multiplex screening assays
fromR&D Systems on a Luminex IS100 instrument (Luminex
Corp.).Data were analyzed using Bio-Plex Manager Pro 6.1
analysissoftware (Bio-Rad). If a measurement exceeded or fell below
thelimit of detection, either the upper or lower limit of detection
wassubstituted.
PBMCs. Blood samples (20 mL) were collected from
healthyvolunteers (n ¼ 16) and subjects with
lymphangioleiomyo-matosis (n ¼ 22). PBMCs were isolated by
Ficoll-Paque Plus(GE Healthcare) gradient centrifugation.
Characteristics ofhealthy volunteers and lymphangioleiomyomatosis
subjectswhose PBMCs were studied are summarized in
SupplementaryTable S2.
Cell cultureCells were incubated at 37�C in a humidified 5% CO2
atmo-
sphere. TSC2-null ELT3 cells, first isolated in 1995, were
derivedfrom the Eker rat uterine leiomyoma as described previously
(24).Cells were generously provided by Dr. Cheryl Walker
(MDAnderson Cancer Center, Houston, TX) and authenticated by
thestudy team by Western blotting for tuberin. ELT3-V cells
(vector)or ELT3-T (TSC2 addback) cells were cultured in DMEM
supple-mented with 10% FBS. TSC2-null ELT3-V cells and
TSC2-reex-pressing ELT3-T cells are denoted as TSC2� and TSC2þ,
respec-tively. Cellswere serum-starved overnight before theywere
treatedwith R406 (1 mmol/L; Selleckchem), rapamycin (20 nmol/L;
LCLaboratories), or DMSO (Sigma-Aldrich) as control vehicle forthe
indicated times. PBMCs were isolated, seeded, and incubatedwith
recombinant human MCP-1 (10 ng/mL, R&D Systems) for24
hours.
RNAiSequences for rat Syk and STAT3 siRNA were designed
using
an online siRNA selection program (25) and were synthesizedby
Sigma-Aldrich. Please refer to Supplementary Table S3 forsiRNA
sequences. TSC2� cells were transfected with siRNA fortargeted gene
suppression or with MISSION siRNA UniversalNegative Control
(Sigma-Aldrich). Cells were transfected with1 nmol/L siRNA using
Lipofectamine RNAiMAX (Thermo Fish-er Scientific).
Animal studiesAll animal experimental procedures were approved
by the
Institutional Animal Care and Use Committee at Brigham
andWomen's Hospital (Boston, MA). The mouse xenograft tumormodel
was established as described previously (9, 10). TSC2�
cells (2.5� 106; ELT3-V) were subcutaneously injected
bilaterallyinto the flank and shoulder of female immunodeficient
C.B17scid mice (Taconic). When tumor surface area reached 40
mm2,mice were randomly assigned to one of the following
treatmentsthrough intraperitoneal injection: (i) vehicle control
(n¼ 5mice,
Syk Signaling Regulates Growth of TSC2-Deficient Cells
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three divided doses at 3-hour intervals per day); (ii) R788 (n ¼
5mice, 80 mg/kg, three divided doses at 3-hour intervals per
day;Selleckchem); and (iii) rapamycin (n ¼ 4 mice, 3 mg/kg, 3
timesper week). The dose, frequency, and route of drug
administrationwere chosen on the basis of published studies (9,
26). Miceunderwent 12 days of designated treatment and were then
sacri-ficed. Prior to the treatment phase, tumors were measured
with aVernier caliper 3 times per week, while during the
treatmentphase, these measurements were recorded daily. Tumor
surfacearea was calculated as follows: p � (major diameter/2) �
(minordiameter/2). After tumors were excised, final tumor volume
wascalculated as follows: (4/3) � p � (length/2) � (width/2)
�(height/2).
IHC and immunofluorescenceFormalin-fixed, paraffin-embedded
tissue sections were
deparaffinized in xylene and rehydrated through a gradedseries
of ethanol, followed by antigen retrieval with citratebuffer (pH
6.0) for 20minutes at 95�C. Sections were incu-bated with a primary
antibody or an isotype control in ahumidified chamber overnight at
4�C. Please refer to Supple-mentary Table S4 for a complete list of
antibodies. For IHC,sections were incubated with appropriate
secondary antibo-dies using an HRP-DAB Cell & Tissue Staining
Kit (R&DSystems) according to the manufacturer's instructions.
Forimmunofluorescence, sections were incubated in Alexa
Flu-or–conjugated secondary antibodies (Thermo Fisher Scientif-ic)
for 1 hour at room temperature in the dark. Slides were thenmounted
using mounting medium containing 4,6-diamidino-2-phenylindole-2-HCl
(DAPI; Vector Laboratories Inc.). Todetect in vivo cell apoptosis,
terminal deoxynucleotidyl trans-ferase–mediated dUTP nick end
labeling (TUNEL) stainingwas performed using the In Situ Cell Death
Detection Kit,TMR red (Roche Applied Science) according to the
manufac-turer's recommendations.
RNA extraction and real-time PCRReal-time PCR was performed as
described in Supplementary
Material andMethods. Please refer to Supplementary Table S5 fora
list of primer sequences.
Statistical analysisData are presented as mean � SEM. Two-tailed
Student t test
was used to compare two groups. Variables with more than
twofactorswere evaluated by one-wayANOVA, followed by the Tukeypost
hoc test. Mixed effects models were used to incorporate thewithin
and between subject variability as well as the repeatedmeasurements
assessing the effect of rapamycin before and aftertreatment.
Correlation coefficients between two variables werecalculated with
Pearson correlation test. Analyses were performedby using GraphPad
Prism 5.0 (GraphPad Software) and SAS(Information Technology
Services). P < 0.05 was consideredsignificant.
ResultsDeregulated Syk expression and activation are identified
inTSC2-deficient cells
As we hypothesize that Syk is a critical kinase involved
inlymphangioleiomyomatosis pathogenesis, we first sought
todetermine whether Syk expression is TSC2 dependent. We usedthe
TSC2-deficient ELT-3 cell line derived from a uterine leio-myoma in
an Eker rat with a germline mutation in TSC2 gene(27). Real-time
PCR analysis showed that Syk expression wasupregulated almost
4-fold in TSC2� cells (ELT3-V) comparedwith TSC2þ cells (ELT3-T;
Fig. 1A). Western blot analysisverified the elevated Syk expression
in the absence of tuberin(the protein encoded by TSC2 gene) and
revealed that TSC2�
cells exhibited increased phosphorylation of Syk at
Tyr525/526(Fig. 1B), which is pivotal for Syk activation and its
down-stream signal transduction (28). As expected, TSC2
deficiency
Figure 1.
TSC2-deficient cells display deregulatedSyk expression and
activation. A, Real-time PCR analysis of rat Syk mRNA inTSC2� cells
compared with TSC2þ cells.Results were expressed as the foldchange
relative to TSC2� cells. Datarepresent means � SEM of
threeindependent experiments. � , P
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led to mTORC1 hyperactivation characterized by increasedpresence
of phospho-p70S6 kinase, which was almost comp-letely abolished by
rapamycin treatment. Notably, we foundthat treatment with rapamycin
yielded a concomitant reductionof both phospho-Syk and total Syk
expression (Fig. 1B), sug-gesting that Syk is mTORC1 dependent.
Consistent with published data (29), we confirmed thattreatment
with R406 (a Syk inhibitor) effectively blocked Sykphosphorylation
in TSC2-deficient cells, but did not modulatetotal Syk expression
(Fig. 1C). As Syk has been reported to play asignificant role in
mTORC1 activation in hematopoietic malig-nancies (22, 23), we
queried whether Syk inhibition could affectmTOR signaling in
TSC2-deficient cells as well. As anticipated,we found that R406
decreased the levels of phospho-p70S6kinase in TSC2� cells (Fig.
1C). To corroborate these findings,we transfected TSC2� cells with
Syk siRNA and found thatknocking down Syk expression dampened
mTORC1 activity,similar to the effects of R406 (Fig. 1D). Taken
together, our datasuggest that the Syk and mTORC1 pathways are
reciprocallyregulated.
Syk inhibition reduces proliferation of TSC2-deficientcells in
vitro
TSC2 deficiency and the constitutive activation of
mTORC1signaling induce uncontrolled cell growth, a distinctive
char-acteristic in lymphangioleiomyomatosis (2). To
determinewhether the abnormal proliferation of TSC2� cells is
Sykdependent, we treated these cells with either R406 or
rapamycinand monitored cell density at different time points. R406
was aseffective as rapamycin in diminishing cell proliferation
(Fig.2A). Correspondingly, TSC2� cells treated with either R406
orrapamycin exhibited a substantial increase in the percentage
ofG1-phase cells accompanied by a reduced fraction of cells in
theS-phase, indicating that R406 and rapamycin induced G1
cell-cycle arrest (Fig. 2B; Supplementary Fig. S1). The
antiprolifera-tive effects of R406 and rapamycin were further
corroborated byWestern blot analysis, which revealed that both
treatments wereassociated with decreased proliferating cell nuclear
antigen
(PCNA) expression in TSC2� cells, although neither
treatmentactivated caspase-3 (Fig. 2C).
Syk inhibition suppresses TSC2-null xenograft tumordevelopment
in vivo
On the basis of the antiproliferative effects of R406 in vitro,
weevaluated the activity of R788, the prodrug of R406, in
animmunodeficient mouse model bearing subcutaneous TSC2-nullELT-3
xenograft tumors (9, 10). As expected, TSC2-null xenografttumors
treated with control grew progressively. In contrast, bothR788 and
rapamycin treatment resulted in a striking and persis-tent decrease
in the tumor area (Fig. 3A). After 12 days oftreatment, R788
significantly reduced xenograft tumor burdento a similar degree as
rapamycin, evidenced by the volume of theexcised tumors (Fig. 3B
and C). In agreement with our observa-tions in TSC2� cells in
vitro, Western blot analysis of tumorhomogenates showed that
expressions of phospho-Syk and phos-pho-p70S6 kinase were similarly
decreased in R788- and rapa-mycin-treated groups compared with
controls, whereas total Sykprotein expression was only reduced in
the rapamycin-treatedgroup (Fig. 3D; Supplementary Fig. S2). In
addition, we foundreduced immunostaining of PCNA in the tumors of
R788- andrapamycin-treated animals (Fig. 3E). Nonetheless,
quantificationof TUNEL staining showed no difference in the
percentage ofpositively labeled cells among all experimental
conditions(Fig. 3F), indicating that tumor shrinkage after
treatment was notcaused by the induction of apoptosis. Thus, these
data demon-strate that TSC2-null xenograft tumor growth is Syk
dependent invivo and provide further rationale to test the efficacy
of Sykinhibitors in future clinical trials.
Syk regulates MCP-1 expression via STAT3 signalingin
TSC2-deficient cells
Loss of functional TSC2 in embryonic fibroblasts has
beendescribed to mediate increased MCP-1 production (30). In
thecurrent study, wemade a similar observation that TSC2
deficiencyled to the upregulation of MCP-1 gene expression
(Supplemen-tary Fig. S3). To evaluate whether Syk signaling is
involved in this
Figure 2.
Inhibition of Syk suppresses growth of TSC2-deficient cells
through the induction of cell-cycle arrest. TSC2� cells were
treated with DMSO (vehicle control),R406 (1 mmol/L), or rapamycin
(20 nmol/L). A, Effects of R406 and rapamycin on cell proliferative
capacity. Cell proliferation was determined at theindicated time
points after treatment. B, Effects of R406 and rapamycin on
cell-cycle distribution. Cells were harvested 24 hours after
treatment. Resultswere expressed as a percentage of cells in G1, S,
and G2–M phases. Data represent mean � SEM of at least three
independent experiments. MW,molecular weight; NS, not significant.
� , P < 0.05; �� , P < 0.01; ��� , P < 0.001 (vs. DMSO),
by one-way ANOVA. C, Cells were collected 24 hours aftertreatment.
Equal amounts of protein from whole-cell lysates were analyzed
byWestern blot using antibodies against PCNA, cleaved caspase-3,
and caspase-3.b-Actin was used as a loading control. All
experiments were repeated at least three times.
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pathologic process, we treated TSC2� and TSC2þ cells with
R406and analyzed the levels of MCP-1 in cell culture supernatants.
Wefound that increased MCP-1 secretion from TSC2� cells wasmarkedly
attenuated by pharmacologic blockade of Syk kinase
activity (Fig. 4A). To further confirm the role of Syk in
theinduction of MCP-1, we silenced Syk using siRNA in TSC2�
cellsand observed a dramatic reduction in MCP-1 production
com-pared with control siRNA (Fig. 4B).
Figure 3.
Syk inhibition impairs TSC2-null xenograft tumor development.
Female CB17-SCID mice were inoculated with TSC2� cells
subcutaneously. Mice were treatedwith control vehicle (n ¼ 5), R788
(n ¼ 5), or rapamycin (n ¼ 4) through intraperitoneal injection
after the tumor surface area reached 40 mm2. A,Tumor surface area
was measured daily with a caliper, and the tumor growth curve was
plotted. Results were expressed as a percentage of baselinetumor
surface area before treatment. B, Mice were sacrificed 12 days
after the initial treatment. Representative gross appearance of
mice bearing xenografttumors and excised tumors is displayed. Scale
bar, 1 cm. C, Volume of the excised tumors. D, Equal amounts of
protein from tumor homogenates wereanalyzed by Western blot
analysis using antibodies against phospho-Syk, Syk, phospho-p70 S6
kinase, and p70 S6 kinase. b-Actin was used as a loadingcontrol. E,
Left, representative images of PCNA (red) immunofluorescence
staining in tumor tissue; right, percentage of PCNA-positive cells.
F, Left,representative images of TUNEL (red) staining in tumor
tissue; right, percentage of TUNEL-positive cells. Nuclei were
counterstained with DAPI (blue).Scale bars, 50 mm (E and F). Data,
mean � SEM. MW, molecular weight; NS, not significant. � , P <
0.05; �� , P < 0.01; ��� , P < 0.001 (vs. control), byone-way
ANOVA.
Cui et al.
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Considering that Syk regulates STAT3 and
STAT3-dependentsignaling transduction is prominently associated
with the induc-tion of MCP-1 (31, 32), we next set out to explore
whether theSTAT3 pathway interconnects aberrant Syk activation and
theeventual MCP-1 overexpression. Consistent with previous
find-ings (33), we observed that TSC2� cells displayed
elevatedphosphorylation of STAT3 (Fig. 4C). Treatment with
R406abrogated STAT3 activation but did not affect total
STAT3expression. Furthermore, siRNA-mediated silencing of
STAT3resulted in decreased MCP-1 levels in cell culture
supernatantsfrom TSC2� cells (Fig. 4D and E). These data
collectivelyindicate that Syk regulates MCP-1 production through
STAT3signaling in TSC2-deficient cells.
MCP-1 upregulates VEGF-D expression in PBMCsVEGF-D is elevated
in the sera of approximately 70% of
patients with lymphangioleiomyomatosis (14, 15). The prevail-ing
view holds that lymphangioleiomyomatosis cells are thefundamental
source of increased VEGF-D production (15),although the exact
cellular origin of increased VEGF-D has notbeen completely
established. Interestingly, we found that thelevels of VEGF-D gene
expression were comparable betweenTSC2� and TSC2þ ELT-3 cells (Fig.
5A), suggesting that TSC2deficiency might not directly upregulate
VEGF-D. As MCP-1 isoverexpressed in TSC2� cells, we hypothesized
that increasedamounts of MCP-1 recruit PBMCs, culminating in
excessiveVEGF-D production. Indeed, we found that incubation of
lym-phangioleiomyomatosis patient-derived PBMCs with exoge-nous
MCP-1 resulted in an approximately 2.5-fold increase inVEGF-D gene
expression (Fig. 5B). Considering that PBMCs candifferentiate into
tumor-associated macrophages (TAM), whichconstitute a predominant
source of cellular infiltrates around
solid tumors (34), we next examined the presence of CD68þ
TAMs in TSC2-null xenograft tumors. TSC2-null xenografts
wereassociated with the accumulation of CD68þ macrophagesnear the
tumor edges. Treatment with R788 or rapamycin re-sulted in a
remarkable reduction in CD68þ macrophage density(Fig. 5C). In
addition, we found a significant decrease in ratMCP-1 expression in
tumors treated with R788 or rapamycin(Fig. 5D, left). Consistent
with our in vitro data, we did notobserve changes in rat VEGF-D
expression among all groups(Fig. 5D, middle) but found that R788
and rapamycin led todownregulation of mouse VEGF-D (Fig. 5D, right)
in tumors.Further statistical analysis showed that rat MCP-1 levels
corre-lated with the presence of CD68þ cells (r ¼ 0.76, P ¼
0.0017;Fig. 5E, left), which in turn correlated with mouse VEGF-D
levels(r ¼ 0.9, P < 0.0001; Fig. 5E, middle). A positive
correlationalso existed when rat MCP-1 levels were plotted against
mouseVEGF-D levels (r ¼ 0.76, P ¼ 0.0015; Fig. 5E, right).
Takentogether, these data suggest an association between MCP-1,TAM
infiltration, and VEGF-D production.
Aberrant Syk signaling is evident inlymphangioleiomyomatosis
patients
To determine whether our in vitro and in vivo results
wereconsistent with the findings in
lymphangioleiomyomatosispatients, we first performed IHC staining
for Syk expression andactivation in lymphangioleiomyomatosis
lungs.While unaffectedareas in lymphangioleiomyomatosis lungs
exhibited minimalphospho- and total Syk immunoreactivity, abundant
phospho-and total Syk staining was detected in
lymphangioleiomyoma-tosis nodules (Fig. 6A).
To validate the downstream effects of Syk activation, wemeasured
the levels of MCP-1 and VEGF-D in sera from 27
Figure 4.
Aberrant Syk activation induces MCP-1 overexpression via STAT3
signaling in TSC2-deficient cells. A, TSC2� and TSC2þ cells were
treated with either controlvehicle (DMSO) or R406 (1 mmol/L) for 24
hours. B, TSC2� cells were transfected with control siRNA (Ct) or
Syk siRNA for 48 hours. A and B, Levelsof MCP-1 in cell culture
supernatants were determined by ELISA. Data represent means� SEM of
three independent experiments. ��� , P < 0.001 (vs. TSC2�
cellstreated with DMSO), by one-way ANOVA (A); � , P < 0.05, by
Student t test (B). C,TSC2� and TSC2þ cells were treated with
either control vehicle(DMSO) or R406 (1 mmol/L) for 24 hours. MW,
molecular weight. D, TSC2� cells were transfected with control
siRNA (Ct) or STAT3 siRNA for 48 hours. C and D,Equal amounts of
protein from whole-cell lysates were analyzed by Western blot
analysis using antibodies against phospho-STAT3 and STAT3.
b-Actinwas used as a loading control. E, TSC2� cells were
transfected with control siRNA (Ct) or STAT3 siRNA for 48 hours.
Levels of MCP-1 in cell culturesupernatants were determined by
ELISA. Data represent means � SEM of three independent experiments.
� , P < 0.05, by Student t test.
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lymphangioleiomyomatosis patients before and after treatmentwith
sirolimus (Supplementary Table S1). Patients had anaverage
follow-up of 3.8 years before sirolimus therapy (cov-ering 80
visits) and 3.4 years after sirolimus therapy (120visits). We found
that when we corrected for factors previouslyshown to influence the
rate of decline (35), rapamycin reduced
the rate of change in the forced expiratory volume in 1
second(FEV1; P < 0.001) and in diffusion capacity in
carbonmonoxide(P < 0.001; Supplementary Table S1). MCP-1 levels
weresignificantly different before and after sirolimus treatment
(P¼ 0.010), as were VEGF-D levels (P < 0.001). Notably, we
foundthat MCP-1 was a significant predictor of VEGF-D regardless
of
Figure 5.
MCP-1 stimulates VEGF-D expression in PBMCs. A, Real-time PCR
analysis of rat VEGF-D mRNA in TSC2� cells compared with TSC2þ
cells. Results wereexpressed as the fold change relative to TSC2�
cells. B, Real-time PCR analysis of human VEGF-D mRNA in human
PBMCs. PBMCs from fourlymphangioleiomyomatosis subjects were
isolated, seeded, and stimulated with recombinant human MCP-1 (10
ng/mL) for 24 hours. Results wereexpressed as the fold change
relative to PBMCs treated with control vehicle. A and B, Data
represent means � SEM of three independent experiments.NS, not
significant. �, P < 0.05, by Student t test. C, Left,
representative images of CD68 (green) immunofluorescence staining
on TSC2-null tumoredges. Nuclei were counterstained with DAPI
(blue). Scale bar, 50 mm. Right, percentage of CD68þ cells. D,
Real-time PCR analysis of rat MCP-1 (left),rat VEGF-D (middle), and
mouse VEGF-D (right) mRNA in tumor homogenates. C and D, Data, mean
� SEM. NS, not significant. �� , P < 0.01; ��� , P <
0.001(vs. control), by one-way ANOVA. E, Correlation between the
percentage of CD68þ cells, rat MCP-1 mRNA expression, and mouse
VEGF-D mRNAexpression in tumors. Correlation coefficients between
two variables were calculated with Pearson correlation test.
Cui et al.
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sirolimus treatment (P < 0.05; correlation r ¼ 0.346; Fig.
6B;Supplementary Table S1).
On the basis of the strong positive correlation between
thedensity of TAMs and mouse VEGF-D expressions in xenografttumors,
we investigated whether PBMCs could be a potentialsource of VEGF-D
in lymphangioleiomyomatosis. We collectedPBMCs from women with
lymphangioleiomyomatosis andhealthy controls (Supplementary Table
S2), and we detected a6-fold increase in VEGF-D mRNA expression in
the PBMCs oflymphangioleiomyomatosis subjects compared with those
ofhealthy controls (Fig. 6C). Similar to our findings in vitro and
inxenograft tumors, these data collectively suggest that Syk
activa-tion led to increased MCP-1 production, which triggered
over-expression of VEGF-D from PBMCs. Our results also
demonstrat-ed that PBMCs could potentially contribute to increased
VEGF-Dlevels in lymphangioleiomyomatosis.
DiscussionAfter its original discovery in 1988, Syk was
initially investi-
gated almost exclusively in hematopoietic cells because of its
keyfunctions in immunoreceptor signaling. Subsequent studies
showthat Syk can also be detected in nonhematopoietic cells, such
asbronchial epithelial cells (36) and vascular smooth muscle
cells(37). In this study,wedemonstrate for thefirst time that there
is anaberrant increase in the expression and activation of Syk in
TSC2-
deficient cells as well as in pulmonary nodules from
lymphan-gioleiomyomatosis patients. Furthermore, treatment with
rapa-mycin reduces Syk expression and activity, indicating that
Syksignaling is mTORC1 dependent. We also provide
compellingevidence that Syk inhibitors (R406 and R788) effectively
blockSyk signaling and elicit potent antiproliferative activities
in TSC2-deficient cells in vitro and in an immunodeficientmouse
xenografttumor model, indicating a pivotal role of Syk signaling in
TSC2-deficient tumor growth and suggesting the therapeutic
potentialof targeting Syk in lymphangioleiomyomatosis. The
beneficialeffects of R788 (fostamatinib disodium), a prodrug of the
bio-logically active compound R406, have been demonstrated
inexperimentalmodels of lymphoma (38) and rheumatoid arthritis(39).
The overwhelming success in the preclinical phase has laidthe
foundation for several clinical trials evaluating R7880s effectsand
safety profile in autoimmune diseases (40, 41). Collectively,these
properties render Syk inhibition an attractive alternative
inlymphangioleiomyomatosis treatment and an ideal candidate
forpersonalized therapy.
Interestingly, protein expression analysis shows that
Sykinhibitors R406 and R788 reduced the level of phospho-p70S6
kinase, a hallmark of mTORC1 activation, in TSC2-deficient cells in
vitro and in xenograft tumors. Corresponding-ly, siRNA-mediated
knockdown of Syk diminished phospho-p70S6 kinase in TSC2-deficient
cells, indicating that the mod-ulation of mTORC1 signaling by Syk
inhibitors is less likely due
Figure 6.
Aberrant increase of Syk activation andexpression is identified
inlymphangioleiomyomatosis (LAM). A,Representative IHC staining
oflymphangioleiomyomatosis lungs. Fromleft to right,
immunoreactivity forphospho-Syk and total Syk visualizedwith
diaminobenzidine (DAB) is observedin lymphangioleiomyomatosis
lungnodules. Absence of immunoreactivitywith nonimmune IgG in
thelymphangioleiomyomatosis lung (n ¼ 6lymphangioleiomyomatosis
patients).Scale bars, 500 mm (top) and 50 mm(bottom). B, Serum was
collected from27 lymphangioleiomyomatosis patientsfor a total of
200 visits before (80 visits,y-axis on the left) and after (120
visits,y-axis on the right) sirolimus treatment.MCP-1 and VEGF-D
were measured inserum using magnetic bead–basedmultiplex screening
assays. Correlationcoefficients between levels of MCP-1 andVEGF-D
were calculated with Pearsoncorrelation test. C, Real-time PCR
analysisof human VEGF-DmRNA in human PBMCsfrom healthy volunteers
as control (n¼ 16)and lymphangioleiomyomatosis patients(n ¼ 22).
Results were expressed as thefold change relative to healthy
controlsubjects. Data, means � SEM. �� , P < 0.01,by Student t
test.
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to an off-target effect attributed to nearly all tyrosine
kinaseinhibitors. Our findings suggest that the Syk pathway is
notonly a downstream target of mTORC1 signaling, but alsoengages to
regulate mTORC1 activity in a reciprocal manner,which is consistent
with previous studies describing the indis-pensable role of Syk in
mTORC1 signaling in lymphoma andacute myeloid leukemia (22, 23).
This feedback loop, with Sykboth upstream and downstream of the
mTORC1 pathway,makes it challenging to separate the effects of Syk
on TSC2-null cells independent of its effects on mTORC1.
Nevertheless,it is important to acknowledge that Syk kinase
activity is theprincipal but not the only target for Syk
inhibitors. An in vitropharmacologic profiling reveals that R406
inhibits crucialkinases in VEGFR signaling, including VEGFR1 and
VEGFR2(42). Interestingly, it was reported that an inhibitor of
VEGFRsignaling (axitinib) restrained TSC2-null tumor developmentin
a mouse model of lymphangioleiomyomatosis (43), indi-cating that
signaling pathways besides mTORC1 could alsocontribute to
lymphangioleiomyomatosis pathogenesis. Inlight of the
multifactorial aspects of lymphangioleiomyoma-tosis, we see
tremendous potential in utilizing a multitargetcompound such as an
Syk inhibitor for lymphangioleiomyo-matosis treatment.
Lymphangioleiomyomatosis has been labeled as "a low
grade,destructive, metastasizing neoplasm" (44). Similar to
cancer,lymphangioleiomyomatosis generates a proinflammatory
envi-ronment (45). In fact,TSC2deficiencyhas beendirectly
associatedwith elevated levels ofMCP-1 (30), a potent chemotactic
factor formonocytes, although the exact underlying mechanisms of
thisphenomenon remain to be elucidated. Here, we employed
acombination of pharmacologic and genetic manipulations todefine
the signaling pathways and discovered that Syk mediatesincreased
production of MCP-1 in TSC2-deficient cells through
aSTAT3-dependent mechanism. Our findings further extend pre-vious
reports that Syk directly activates STAT3 (31) and STAT3mediates
MCP-1 production (32).
Serum VEGF-D concentration is elevated in most
lymphan-gioleiomyomatosis patients and has been confirmed to be
areliable diagnostic and prognostic biomarker of
lymphangio-leiomyomatosis (14, 15). Previous investigations have
also
shown that VEGF-D contributes to lymphangioleiomyomatosiscell
proliferation in vitro (46) and correlates with
lymphaticinvolvement (14). Despite intense research efforts and
majoradvances in the understanding of VEGF-D biology, the origin
ofexcessive VEGF-D in lymphangioleiomyomatosis patients'
seraremains enigmatic. Traditionally, lymphangioleiomyomatosiscells
are considered as the major source for elevated VEGF-Dlevels based
on the evidence that VEGF-D immunostaining ispresent in
lymphangioleiomyomatosis cells (15). Notably,
lym-phangioleiomyomatosis cells also express VEGFR3 (46), a
recep-tor for VEGF-D, and therefore, it is difficult to ascertain
whetherVEGF-D originates within lymphangioleiomyomatosis cells
orwhether the observed VEGF-D immunostaining may be in partdue to
ligand–receptor complex internalization. Here, we pro-vide evidence
that endogenous VEGF-D is not directly modu-lated by TSC2
deficiency and mTORC1 activation in vitro and inmouse xenograft
tumors. These data suggest the possibility thatcellular sources of
VEGF-D other than lymphangioleiomyoma-tosis cells may exist in the
lymphangioleiomyomatosis micro-environment and be sensitive to the
effect of rapamycin.Remarkably, we also found that VEGF-D
expression is elevatedin PBMCs from lymphangioleiomyomatosis
patients and couldbe further enhanced by exogenous MCP-1
stimulation. Thesefindings suggest that PBMCs could be the missing
link betweenaberrant MCP-1 production from TSC-2 deficient cells
andincreased VEGF-D concentration in peripheral blood. The
directrole of PBMCs in VEGF-D production was further
substantiatedin the mouse xenograft tumor model. Quantification of
TAMimmunostaining and real-time PCR analysis of tumor homo-genates
suggest that TAM infiltration correlates with gene expres-sions of
rat MCP-1 and mouse VEGF-D. Furthermore, we foundthat the gene
expression of rat MCP-1 also correlates with that ofmouse VEGF-D.
Considering that TAMs predominantly differ-entiate from
PBMC-derived monocytes and produce an abun-dant amount of VEGF-D
(47), our findings support a 2-stepmechanism: (i) in TSC2-deficient
cells (primary lymphangio-leiomyomatosis lesions), activation of
mTORC1 and Syk sig-naling induces MCP-1 overproduction via a
STAT3-dependentmanner; and (ii) MCP-1 recruits and activates PBMCs,
leading toincreased VEGF-D expression (Fig. 7). Taken together, our
study
Figure 7.
Schematic illustration depicts the rolesof Syk signaling in
VEGF-Doverexpression and TSC2-null tumorgrowth. In TSC2-deficient
cells,activation of mTORC1 and Syk signalinginduces MCP-1
overproduction via aSTAT3-dependent manner.Subsequently, MCP-1
recruits andactivates PBMCs, leading to increasedVEGF-D expression.
In contrast,treatment with Syk inhibitors impairsproliferation of
TSC2-deficient cells anddecreases the production ofMCP-1. As
aresult, VEGF-D expression is reduced inPBMCs.
Cui et al.
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extends a recent report that proposes alternative sources
ofVEGF-D in lymphangioleiomyomatosis (46), and we demon-strate for
the first time that PBMCs indeed participate in
VEGF-Dproduction.
Consistent with results from the MILES trial (7), this
studydemonstrates that rapamycin treatment effectively preserved
lungfunction and decreased levels of serum VEGF-D in
lymphangio-leiomyomatosis patients. By distinction, we also found
thatrapamycin led to a reduction in serum MCP-1 levels,
whichpositively correlated with the decline in serum VEGF-D
concen-trations. Of particular relevance to the current findings,
elevatedlevels of MCP-1 were previously detected in the
bronchoalveolarlavage fluid from lymphangioleiomyomatosis patients
(48). Thecumulative findings of the current and the prior study
provide anadditional argument for the crucial role of MCP-1 in
lymphan-gioleiomyomatosis pathogenesis. Moreover, the changes
ofMCP-1 in lymphangioleiomyomatosis suggest that it could also be
afeasible target for therapeuticmodulation and could be utilized
topredict the course of the disease. Future studies are required
todetermine the sensitivity and specificity of MCP-1 as a
lymphan-gioleiomyomatosis biomarker.
In conclusion, we delineate a novel cascade of
Syk-dependentsignals and demonstrate that Syk inhibition is a
potential therapyfor patients with lymphangioleiomyomatosis and
perhaps othermanifestations of TSC who are intolerant, allergic, or
unrespon-sive to conventional mTOR inhibitors. Syk inhibitors may
also beparticularly useful for lymphangioleiomyomatosis
patientsawaiting lung transplant, as most transplant programs
requirepatients to stop takingmTOR inhibitors due to concerns
regardingimpaired wound healing (49). Furthermore, we provide
mecha-nistic insight of how cells of the immune system are linked
tothe aberrant regulation of VEGF-D in lymphangioleiomyomato-sis.
In view of the favorable safety profile of Syk
inhibitors,evaluating their therapeutic efficacy in patients with
lymphangio-leiomyomatosis, other manifestations of TSC, and
malignanciescharacterized by activation of the mTORC1 pathway seems
wellwarranted.
Disclosure of Potential Conflicts of InterestNo potential
conflicts of interest were disclosed.
Authors' ContributionsConception and design: Y. Cui, W.K.
Steagall, G. Pacheco-Rodriguez,E.P. Henske, J. Moss, S.
El-ChemalyDevelopment of methodology: Y. Cui, M. Stylianou, C.
Priolo, E.P. Henske,J. Moss, S. El-ChemalyAcquisition of data
(provided animals, acquired and managed patients,provided
facilities, etc.): Y. Cui, W.K. Steagall, A.M. Lamattina, G.
Pacheco-Rodriguez, B. Stump, F. Golzarri, C. Priolo, J. Moss, S.
El-ChemalyAnalysis and interpretation of data (e.g., statistical
analysis, biostati-stics, computational analysis): Y. Cui, W.K.
Steagall, A.M. Lamattina,G. Pacheco-Rodriguez, M. Stylianou, P.
Kidambi, B. Stump, F. Golzarri,I.O. Rosas, J. Moss, S.
El-ChemalyWriting, review, and/or revision of the manuscript: Y.
Cui, W.K. Steagall,A.M. Lamattina, G. Pacheco-Rodriguez, M.
Stylianou, B. Stump, I.O. Rosas,C. Priolo, E.P. Henske, J. Moss, S.
El-ChemalyStudy supervision: J. Moss, S. El-Chemaly
AcknowledgmentsThe authors are grateful to
lymphangioleiomyomatosis patients and their
families for their participation in this research. We would also
like to thankLeigh Samsel, Venina Dominical, and J. Philip McCoy of
the Flow CytometryCore at NIH/NHLBI for their excellent technical
assistance.
Grant SupportThis workwas supported in part by theDepartment
ofDefense (TS130031 to
S. El-Chemaly), NIH grant R01 HL130275 to S. El-Chemaly, the
Anne LevineLAMResearch Fund to S. El-Chemaly, BRImicrogrant
(Y.Cui), The Lucy J. EnglesTSC/LAM Research Program (EP. Henske)
and the Division of IntramuralResearch NIH/NHLBI.
The costs of publication of this article were defrayed in part
by thepayment of page charges. This article must therefore be
hereby markedadvertisement in accordance with 18 U.S.C. Section
1734 solely to indicatethis fact.
Received October 9, 2016; revisedDecember 8, 2016; accepted
December 12,2016; published OnlineFirst February 15, 2017.
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