aberrant syk kinase signaling is essential for tumorigenesis ......with recombinant human mcp-1 (10...

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.

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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

www.aacrjournals.org Cancer Res; 77(6) March 15, 2017 1493




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

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.






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.

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Cancer Res; 77(6) March 15, 2017 Cancer Research1496




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.






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.





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.






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.





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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Cui et al.




2017;77:1492-1502. Published OnlineFirst February 15, 2017.Cancer Res Ye Cui, Wendy K. Steagall, Anthony M. Lamattina, et al. Induced by TSC2 InactivationAberrant SYK Kinase Signaling Is Essential for Tumorigenesis

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aberrant syk kinase signaling is essential for tumorigenesis ......with recombinant human mcp-1 (10...

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