Src Kinase Is a Novel Therapeutic Target in ... › content › canres › ... · Alexey Tyryshkin, Abhisek Bhattacharya, and N. Tony Eissa Abstract Lymphangioleiomyomatosis (LAM)

Molecular and Cellular Pathobiology

Src Kinase Is a Novel Therapeutic Target inLymphangioleiomyomatosis

Alexey Tyryshkin, Abhisek Bhattacharya, and N. Tony Eissa

AbstractLymphangioleiomyomatosis (LAM) is a progressive cystic lung disease affecting some women with tuberous

sclerosis complex (TSC). Sporadic LAM can develop in women without TSC, owing to somatic mutations in theTSC2 gene. Accumulating evidence supports the view of LAM as a low-grade, destructive, metastasizingneoplasm. The mechanisms underlying the metastatic capability of LAM cells remain poorly understood. Theobserved behavior of LAM cells with respect to their infiltrative growth pattern, metastatic potential, and alteredcell differentiation bears similarity to cells undergoing epithelial–mesenchymal transition. Here, we reportincreased levels of active Src kinase in LAM lungs and in TSC2�/� cells, caused by a reduction of autophagy.Furthermore, increased Src kinase activation promoted migration, invasion, and inhibition of E-cadherinexpression in TSC2�/� cells by upregulating the transcription factor Snail. Notably, Src kinase inhibitors reducedmigration and invasion properties of TSC2�/� cells and attenuated lung colonization of intravenously injectedTSC2�/� cells in vivo to a greater extent than control TSC2þ/þ cells. Our results reveal mechanistic basis for thepathogenicity of LAM cells and they rationalize Src kinase as a novel therapeutic target for treatment of LAMand TSC. Cancer Res; 74(7); 1996–2005. �2014 AACR.

IntroductionTuberous sclerosis complex (TSC) is an autosomal domi-

nant disorder caused by mutation in either the TSC1 or TSC2tumor suppressor genes (1). Lymphangioleiomyomatosis(LAM), a pulmonary manifestation of TSC (2), is a progressivecystic lung disease affecting primarily women of childbearingage. LAM affects 30% to 40% of women with TSC (3, 4) and ischaracterized by abnormal and potentially metastatic growthof atypical smooth muscle-like LAM cells within lungs andaxial lymphatics. Clinical and genetic data suggest a linkbetween the loss of TSC2 function and cell invasion andmetastasis. The mTOR is a serine/threonine kinase that pos-itively regulates cell growth, proliferation, and survival (5).TSC2 is a negative regulator of themTOR complex 1 (mTORC1;refs. 6, 7). Therefore, hyperactivation of mTORC1 and inhibi-tion of autophagy are observed in TSC2�/� LAM cells (8).However, many of the clinical and pathologic features of LAMremain unexplained by our current understanding of thefunction of these genes. Activation of mTORC1 is sensitive toinhibition by rapamycin, which has been used in the treatmentof LAM (9, 10). Rapamycin treatment improved pulmonary

functions and reduced the size of angiomyolipoma (AML) inTSC and LAM subjects. Unfortunately, cessation of rapamycintherapy was followed by regrowth of tumors and the decline ofpulmonary functions (9, 10). Accordingly, alternative or com-binational therapies are needed to treat LAM. Identification ofnovel therapeutic targets, other than mTOR, might allow suchtherapy.

Accumulating evidence supports the hypothesis that LAM isa low-grade, destructive,metastasizing neoplasm (11, 12). LAMcells are found in blood, urine, and chylous fluids of LAMsubjects with AML (13). If themetastatic hypothesis for LAM iscorrect, then AML or renal tumors might be the source.Consistent with this notion, the morphology and immunohis-tochemical characteristics of AML and LAM cells are verysimilar. However, not all subjects with LAM have detectableAML, and the uterus has also been proposed as a potentialsource (11, 12).

Collectively, the observed behavior of LAMcells with respectto their infiltrative growth pattern, metastatic potential, andaltered cell differentiation is reminiscent of cells undergoingepithelial–mesenchymal transition (EMT; ref. 14). Src familykinases are nonreceptor tyrosine kinases and key regulators ofcellular proliferation, survival, motility, invasiveness, and EMT(15). Signaling through Src kinase suppresses transcription ofE-cadherin by upregulating the transcriptional repressorsSnail/Slug (16).

Recent results have shown that, in cancer cells in which theSrc pathway is hyperactive, autophagosomes promote degra-dation of the active tyrosine kinase Src, enabling tumor cellsurvival (17). Thereby, decreased autophagy due to an activa-tion of mTORmay play a critical role in accumulation of activeSrc kinase in LAM cells. Hyperactivity of Src has been

Authors' Affiliation: Department of Medicine, Baylor College of Medicine,Houston, Texas

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: N. Tony Eissa, Baylor College of Medicine, OneBaylor Plaza, BCM 285 Suite 535E, Houston, TX 77030. Phone: 713-798-3657; Fax: 713-798-2050; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-1256

�2014 American Association for Cancer Research.

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implicated in the development of several types of humancancers and in their progression to metastases (18). There areno prior studies addressing potential activation of Src in LAM.Here, we report that Src kinase is activated in LAM cells. In thisstudy, we examined the potential underlying mechanisms ofSrc activation in LAM cells and tested Src as a novel thera-peutic target in LAM.

Materials and MethodsReagents and antibodiesThe following antibodies were used for immunoblot anal-

ysis: pSrc(Tyr416), pStat3(Tyr705), Stat3, pErk1/2(Thr202/Tyr204), Erk1/2, S6, pS6(Ser235/236), pFAK(Tyr925), pFAK(Tyr397), mTOR, U0126 (all from Cell Signaling Technology),tuberin, rabbit E-cadherin, MMP9, Snail (all from Santa CruzBiotechnology),mouse E-cadherin (BDBiosciences), Src (Milli-pore), pSrc(Tyr418; LifeSpan Biosciences), and HMB45 (EnzoLife Sciences). Src kinase inhibitors PP2 and SU6656 werepurchased from Calbiochem. Rapamycin, dasatinib, and sar-acatinib were purchased from LC Laboratories.

Cell culture and tissue samplesEker rat embryonic fibroblasts (EEF)4 (TSC2þ/þ) and EEF8

(TSC2�/�) were maintained in Dulbecco's Modified EagleMedium (DMEM)/F12 mixture (1:1) containing 10% heat-inactivated FBS. Lung tissues of normal subjects and subjectswith LAM were obtained from the National Disease ResearchInterchange.

Plasmids, siRNA, and cell transfectionSpecific TSC2 (J-003029-11 and J-003029-12), ATG7

(J-0095596-11 and J-0095596-12), and Src (J-080144-11 andJ-080144-12) siRNAs were purchased from Dharmacon. Cat-ionic lipid–mediated transient transfection of plasmids wasdone using Lipofectamine 2000 (Invitrogen).

Immunofluorescence and histochemistryCellswere grown on glass coverslips,fixed in either cold pure

methanol or 4% paraformaldehyde, permeabilized by0.2%Triton X-100, and blocked in 10% normal goat serum.Primary antibody incubation was done at 4�C overnight in ahumidified chamber followed by a 30-minute incubation atroom temperature with Alexa Fluor 594–labeled secondaryantibodies. Coverslips were mounted by SlowFade gold anti-fade reagent with 40,6-diamidino-2-phenylindol dihydrochlor-ide (DAPI). Tissue sections were deparaffinized, incubatedovernight with primary antibodies at 4�C in a humidifiedchamber, and then washed and incubated with biotinylatedsecondary antibodies for 30 minutes at room temperature.Slides were developed using the Vectastatin Elite ABC Kit(Vector Laboratories) and counterstained with hematoxylin.Images were viewed using a Zeiss Axiovert microscope.

Wound-healing assayCells were plated in a 10-cm plate and allowed to form a

confluent monolayer that was then scratched with a sterilepipette tip (200 mL), washed with medium to remove floated

and detached cells. Wound areas were photographed (magni-fication, �100) at the start and 18 hours after treatment toassess the degree of wound closure. Data are expressed inmm2 � 1,000.

Cell invasion assayCells were studied using Matrigel inserts (BD Biosciences).

Serum-deprived cells (5 � 104 cells) were loaded in the uppercompartment of the chambers and the bottomwells were filledwith chemoattractant (complete media with 10% FBS). Afterincubation for 18 hours, the membranes were processed andthe nonmigrating cells were removed from the upper chamberwith a cotton swab and the inserts were fixed with methanoland stained with 1% Toluidine blue. The invading cells werecounted in six random fields under a microscope.

Real-time PCRRNAs were purified using the RNeasy Mini Kit (Qiagen) and

cDNA synthesis was performed using the cDNA Reverse Tran-scription Kit (Applied Biosystems). mRNA expression wasmeasured using a real-time detection system (Applied Biosys-tems StepOnePlus) in 96-well optical plates using PerfeC-TaqPCR FastMix (Quanta Biosciences). 18S was used as anendogenous control. All analyses were performed in triplicate,and means were used for statistical calculations.

Mouse in vivo imagingFemale CB17-SCIDmice, 6 to 8weeks of age, were purchased

from The Jackson Laboratory. Cells were transfected withpGL4.51[luc2/CMV/Neo] vector (Promega), expressing lucif-erase, using Lipofectamine 2000 (Invitrogen). For intravenousinjections, 1� 106 cellswere injected into the retro-orbital vein.Ten minutes before imaging, animals were injected withLuciferin (Promega; 120 mg/kg, i.p.). Bioluminescent signalswere recorded using Xenogen in vivo imaging system (IVIS;Xenogen). Total photon flux at the chest regions was analyzed.All animal studieswere performed in accordancewith protocolapproved by the Institutional Animal Care and Use Committeeof Baylor College of Medicine (Houston, TX).

Statistical analysisAll blots and data are representative of at least three

independent experiments. The Student t test was used, andP values of less than 0.05 were considered to be statisticallysignificant.

ResultsEnhanced Src activation in LAM lungs

We evaluated tissue samples of lungs of normal subjects andof subjects with LAM. LAM lungs showed collections of LAMcells, which were identified by HMB45 antibodies (Fig. 1A;refs. 11, 19). Phosphorylation of the ribosomal protein S6 wasincreased in LAM lung tissues compared with normal lungs(Fig. 1B). These data confirm that mTOR is activated in lungtissues of subjects with LAM, as expected to occur secondary toTSC2 deficiency. One of the consequences of mTOR activationis inhibition of autophagy, which was evident by the accumu-lation of the autophagy substrate p62 (20) in LAM lungs

Src Kinase Is a Target in Lymphangioleiomyomatosis

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(Fig. 1C). Importantly, we found increased phosphorylation ofSrc onTyr416 in LAM lung tissues comparedwith normal lungs(Fig. 1D). These findings were further confirmed by analyzinghuman LAM lungs by immunofluorescence (SupplementaryFig. S1). Moreover, there was strong correlation betweenexpression of phospho-Src andHMB45-positive cells. However,some HMB45-negative cells contained phospho-Src as well,consistent with the notion that not all LAM cells are HMB45-positive (19). Phosphorylation of Tyr416, in the activation loopof the kinase domain, upregulates Src kinase activity (21).These data suggest that Src is activated in lung tissues ofsubjects with LAM. To confirm that Src activation in LAMlungs had functional consequences, we tested activation ofSTAT3, which is a downstreammediator of Src. We found thatLAM lungs had elevated phosphorylated STAT3 (Fig. 1E),suggesting that STAT3 is activated in LAM lungs, consistentwith a prior report (22).

We then wanted to confirm that the increased activities ofSrc and STAT3, observed in LAM lungs, were specific to LAMcells. We isolated cells from LAM lung explants and identifiedLAMcells using antibodies against HMB45.We found that cellspositive for HMB45 had increased phospho-(Y416)-Src andphospho-(Y705)-STAT3, whereas non-LAM cells did not exhib-it such increase (Supplementary Fig. S2). These data confirmthat LAM cells have increased Src and STAT3 activities.

Src and STAT3 are activated in TSC2�/� cellsWe studied EEFs TSC2þ/þ wild-type (EEF4) and TSC2�/�

mutant cells (EEF8). These cells are well characterized as acellular model for LAM and TSC (14, 23). EEF8 cells (TSC2�/�)

did not express Tuberin, had increased activity of mTOR, andsuppressed autophagy (Supplementary Fig. S3). Activation ofmTORwas evident by increased phosphorylation of mTOR andof its substrate p70S6 kinase. Inhibition of autophagy wasshown by reduction of LC3-II and by increased level of autop-hagy substrate p62 protein. Accumulation of p62 in EEF8 cellswas not caused by increase of its mRNA. The above dataconfirmed prior reports that TSC2�/� EEF8 cells have themolecular features of LAM cells. We then investigated whetherTSC2�/� cells have increased Src activity. We found thatphosphorylation of Src was increased in the TSC2�/� cells (Fig.2A and Supplementary Fig. S4). We also found that TSC2�/�

cells had increased phosphorylated STAT3 (Fig. 2B). IncreasedSTAT3 translocation to the nucleus was observed in TSC2�/�

cells (Fig. 2C). Importantly, inhibition of Src by PP2 or Su6656reduced STAT3 phosphorylation (Fig. 2D). We then wanted toconfirm that the increase in Src and STAT3 activities was adirect result of TSC2 deficiency. To this end, siRNA-mediatedknockdown of TSC2 in Hela cells resulted in increased Src andSTAT3 activities (Fig. 2E), essentially recapitulating the phe-notype of TSC2�/� EEF8 cells. That phenotype was also con-firmed by the finding of increased phosphorylation of ribosom-al protein S6 (Supplementary Fig. S5), which indicated that theactivation of mTOR was similar to that observed in EEF8 cells.Moreover, overexpression of Src kinase in wild-type EEF4 cellsled to increased Src activity and increased STAT3 phosphor-ylation (Fig. 2F). These data suggest that TSC2�/� cells haveincreased Src activity, similar to that found in human LAMlungs. They also show that STAT3 activation in TSC2�/� cells isa downstream event of Src activation.

Figure 1. Sections of normal and LAM lungs were stained with LAM cell marker HMB45 antibody (A) or lysed and analyzed by immunoblot analysis usingantibodies against S6 and phospho-S6 (B), p62 (C), Src kinase and phospho-Src (Y416) kinase (D), or STAT3 and phospho-STAT3 (Y705; E). Blottingwith b-actin antibody was used as a loading control. Scale bar, 100 mm.

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Enhanced activation of the Src kinase signaling pathwayin TSC2�/� cellsThe activation of STAT3 in TSC2�/� cells suggested that

other Src downstream substrates might also be activated inthese cells. One important Src partner is focal adhesion kinase(FAK). We found that levels of FAK were increased in EEF8cells, likely secondary to increase of its mRNA (Fig. 3A and B).Moreover, we found overphosphorylation of FAK on Y397 andY925 sites in EEF8 cells (Fig. 3C and D). FAK-Y397 autopho-sphorylation plays an important role in FAK binding to Srckinase and forming of an active FAK–Src complex (24). Recruit-ment of Src kinase results in phosphorylation of FAK-Y925 andtriggers a Ras-dependant activation of the mitogen-activatedprotein kinase (MAPK) pathway (25). To evaluate the MAPKpathway activation in EEF8 cells, we examined phosphoryla-tion of Erk. We found increased level of phosphorylated Erk inEEF8 cells, compared with EEF4 cells (Fig. 3E). To determinethe effect of Src kinase on Erk phosphorylation, we over-expressed Src kinase in EEF4 cells and we found markedlyincreased level of Erk phosphorylation (Fig. 3F). To confirmthat the increased phosphorylation of Erk in EEF8 was causedby Src kinase, we found that Src-specific siRNA led to adecrease of Erk phosphorylation in EEF8 cells (Fig. 3G). More-over, Src kinase inhibition by any of four different inhibitors(PP2, SU6656, dasatinib, and saracatinib) reduced Erk phos-

phorylation in EEF8 cells (Fig. 3H). Taken together, these dataindicate the Src signaling pathway is activated in TSC2�/�

cells.

Autophagy inhibition results in Src kinase activationLoss of the TSC2 gene leads to mTOR activation and autop-

hagy inhibition (26). Recently, a role for autophagy has beenshown in degradation of active Src (17). We hypothesized thatautophagy inhibition in EEF8 contributes to Src activation inthese cells. To test this hypothesis, we used siRNA to knock-down autophagy related gene 7 (Atg7) in wild-type EEF4 cells.Atg7 knockdown resulted in inhibition of autophagy asshown by reduction of autophagy marker LC3 type II, andincreased active phosphorylated Src (Supplementary Fig. S6Aand S6B). Furthermore, mouse embryonic fibroblasts derivedfrom Atg7�/� mice and Atg5�/� mice had increased active Src(Supplementary Fig. S6C and S6D). Finally, treatment of wild-type EEF4 cells with the autophagy-lysosome pathway inhibitorchloroquine resulted in increased active Src (SupplementaryFig. S6E). Thus, autophagy inhibition caused by several inde-pendent methods led to accumulation of active Src kinase.Moreover, we found that the phospho-Src levels decreased afterinduction of autophagy by starvation in TSC2�/� EEF8 cells(Supplementary Fig. S6F). These data suggested that autophagywas involved in modulation of cellular Src kinase activity.

Figure 2. Activation of Src and STAT3 in TSC2�/� cells (EEF8). A and B, cell lysates of EEF4 and EEF8were subjected to immunoblot analysis using antibodiesagainst Src kinase and phospho-Y416-Src (A) or STAT3 and phospho-Y705-STAT3 (B). C, EEF4 and EEF8 cells were fixed and immunolabeledby phospho-STAT3 antibodies followed by IgG conjugated to Alexa Fluor 594 (red). Cells were stained with DAPI to visualize nuclei (blue). Graph, percentageof pStat3 (nuclear localization)-positive cells (n ¼ 3). D, EEF4 and EEF8 cells were treated with either DMSO or Src kinase inhibitors PP2 (25 mmol/L),or SU6656 (Su; 10 mmol/L) for 4 hours before lysis. Cell lysates were subjected to immunoblot analysis. E, HeLa cells were transfected for 72 hours withcontrol nontarget (NT) siRNA or TSC2-specific siRNA. Cells were lysed and immunoblot analysis was done. Graphs, quantification of phospho-Srcand phospho-STAT3, normalized to total Src or STAT3 expression, respectively, n ¼ 3. F, EEF4 cells were transfected for 24 hours with Src kinase or LacZcontrol plasmids. Cell lysates were analyzed, compared with EEF8, by immunoblotting. Blotting with b-actin antibody was used as a control. Data,mean � SD. �, P < 0.05; ��, P < 0.001. Scale bar, 10 mm.





TSC2 deficiency or overexpression of Src promotes EMTTo evaluate EMT in TSC2�/� cells, we examined the level

and cellular distribution of E-cadherin. We found that theexpression and cellular localization of E-cadherin in EEF8 cellswere notably altered (Fig. 4A and B). In wild-type cells (EEF4),E-cadherin was readily detectable and localized predominant-ly at the plasma membrane, in which it is known to play acritical role in adherens junction formation. In contrast, inTSC2�/� cells (EEF8), there was much lower expression of E-cadherin and it did not colocalize with plasma membrane.Instead, most of E-cadherin signals were found in punctatecytosolic structures. One possible explanation for the reduc-tion in E-cadherin in TSC2�/� cells could be due to an increaseof its transcriptional repressor Snail. We found markedincrease in the expression of Snail mRNA and protein in EEF8cells (Fig. 4C). Snail activity, measured by its nuclear translo-cation, was also more pronounced in EEF8 cells (Fig. 4D).Importantly, we observed increased level of Snail in humanLAM lungs (Fig. 4E). Furthermore, the increase in Snail expres-sion was limited to LAM cells identified by positive staining forHMB45 (Supplementary Fig. S2C). Matrix metallopeptidase 9(MMP9), an important marker of EMT, was markedlyincreased in EEF8 cells (Fig. 4F). To confirm a role for theobserved increased Src in TSC2�/� cells in promotion of EMT,we transfected wild-type EEF4 cells with Src and then evalu-ated several EMT markers. Src overexpression resulted inreduction of E-cadherin and increased levels of Snail and

MMP9 (Fig. 4G), essentially recapitulating the phenotypeobserved in TSC2�/� cells. These dramatic changes in theexpression and localization of E-cadherin could account forthe decrease in cell adhesion, increased motility, invasiveness,and metastatic potential of TSC2�/� cells.

Src inhibition reduces EMT markers in TSC2�/� cellsTo validate Src as a potential therapeutic target in LAM, we

treated TSC2�/� cells (EEF8) with Src inhibitors dasatinib orsaracatinib (27, 28). Both inhibitors reduced levels of Snail,whereas rapamycin had no effect (Fig. 5A and B). Src inhi-bition also reduced levels of MMP9, as determined by immu-noblot analysis and further confirmed by gelatin zymogram(Fig. 5C and D). Overall, dasatinib and saracatinib seemed tohave equivalent effects on Src activation (phosphorylation)and on EMT markers. In additional experiments, siRNA-mediated knockdown of Src resulted in decrease of expres-sion of Snail and Mmp9 (Fig. 5E and F). These data areconsistent with prior reports of increased immunoreactivityfor MMPs in lung biopsy specimens from subjects with LAMand TSC2-deficient LAM-like cells (29–31) and suggest thatinhibition or genetic knockdown of Src could reduce EMT inTSC2�/� cells.

Src inhibition attenuates migration activity of EEF cellsUsing wound-healing assay, we found that inhibition of Src

kinase by dasatinib or saracatinib led to reduction ofmigration

Figure 3. Src kinase mediates phosphorylation of FAK and activation of the MAPK pathway in TSC2�/� cells. A–E, EEF4 and EEF8 cells were analyzedby real-time PCR (RT-PCR) for FAKmRNA (A) or by immunoblot analysis using antibodies against FAK (B), phospho-FAK(Y397) (C), phospho-FAK(Y925) (D),or Erk and phospho-Erk (Thr202/Tyr204; E). B, C, and E, graphs indicate quantification of the immunoblots, n ¼ 6, 3, or 4, respectively. Protein expressionwas normalized to b-actin expression and then to the EEF4 group. Phospho-Erk was normalized to total Erk and then to the EEF4 group. F, EEF4cells were transfected for 24 hours with a plasmid encoding Src kinase or LacZ as a control and analyzed by immunoblot analysis. Graph, quantification ofthe immunoblots. Phospho-Erk was normalized to total Erk expression, n ¼ 3. G, EEF8 cells were transfected with Src-specific siRNA or control (NT)siRNA for 72hours and analyzedby immunoblot analysis.Graph, thequantification of the immunoblots,n¼4.H, EEF8 cellswere treated for 4 hourswith eitherDMSO or Src inhibitors PP2 (25 mmol/L), SU6656 (10 mmol/L), dasatinib (0.5 mmol/L), or saracatinib (1 mmol/L) and then analyzed by immunoblotanalysis. Blotting with actin antibody was used as a control. Data, mean � SD; n �3. �, P < 0.05; ��, P < 0.001.

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ability of both EEF8 and EEF4 cells (Supplementary Fig. S7). Incontrast, mTOR inhibitor rapamycin had no significant effecton cell migration. It should be noted that the migration assayresults reflect, in part, reduction of cell proliferation by Srcinhibitors. We found that EEF8 cell proliferation was increasedcompared with control cells and that Src inhibitors signifi-cantly decreased the proliferation of EEF8 cells (Supplemen-tary Fig. S8). These data suggest that Src inhibition is likely toreduce migration ability of TSC2�/� cells.

Src inhibition reduces invasiveness of TSC2�/� cellsThe invasive properties of EEF4 and EEF8 cells were studied

using Matrigel inserts. After incubation for 18 hours, themembranes were processed and the invading cells werecounted in six random fields. Invasive cells were counted asthe number of migrating cells per field. TSC2�/� cells (EEF8)were much more invasive than control cells (Fig. 6A and B).This behavior is consistent with the notion that TSC2�/� cellshave increased invasive and migratory properties, likely sec-ondary to EMT in these cells. The effect of Src inhibition on theinvasive properties of EEF cells was evaluated. We found thatSrc inhibition by dasatinib or saracatinibmarkedly reduced theinvasiveness properties of EEF8 TSC2�/� cells. In contrast,mTOR inhibitor rapamycin had no significant effect. Further-

more, there was no effect of Src inhibitors on the invasivenessin the EEF4 cells, probably because of low invasiveness of thesecells (Fig. 6C and D). These data suggest that Src inhibition islikely to reduce invasiveness of TSC2�/� cells and that effect isspecific for such cells.

Src inhibition reduces lung colonization of TSC2�/� cellsin vivo

We evaluated the effect of Src inhibition on the metastaticpotential for TSC2�/� cells in vivo. We engineered luciferase-expressing (EEF-Luc) cells to allow in vivo imaging followingtheir injection into mice. EEF8-Luc cells were pretreatedwith vehicle (dimethyl sulfoxide, DMSO), rapamycin (1 mg/mL), saracatinib (1 mmol/L), or both rapamaycin and sar-acatinib. 1 � 106 cells were then intravenously injected intofemale CB17 SCID mice. Six hours following injection ofcells, and 10 minutes before imaging, animals were injectedintraperitoneally with 120 mg/kg, luciferin. Bioluminescentsignals were recorded using the Xenogen in vivo imagingsystem (Fig. 7A and B). Total photon flux at the chest regionwas analyzed. At 24 hours time point after cell injection,mice were sacrificed and their lungs were dissected andimaged in Petri dish (Fig. 7C and D). Saracatinib significantlyreduced the number of EEF8-Luc cells that was detected in

Figure 4. TSC2 deficiency oroverexpression of Src promotesEMT. EEF4 and EEF8 cells orhuman lungs (E) wereanalyzed by immunoblots,immunofluorescence microscopy,or RT-PCR (graphs in C and F) toevaluate E-cadherin (A and B),Snail (C–E), or MMP9 (F). B and D,cells were stained with DAPI tovisualize nuclei (blue). G,EEF4 cells were transfected for16 hours with Src or LacZ controlvector and analyzed byimmunoblot analysis. b-Actinantibody was used as a loadingcontrol. Graphs in G indicatethe quantification of theimmunoblots. Data, mean � SD;n � 3. �, P < 0.05; ��, P < 0.001.Scale bar, 10 mm.





the lungs at 6 and at 24 hours after injection. Rapamycintreatment had no significant effect. Furthermore, we con-ducted in vivo experiments with injection of luciferase-expressing EEF4 cells treated with DMSO or saracatinib.Saracatinib reduced EEF4 cells lung colonization after 24hours but not after 6 hours of the cell injection (Fig. 7E–H).Furthermore, although the decrease in lung colonizationwas significant at 24 hours for both TSC2�/� and TSC2þ/þ

cells, the extent of reduction was not the same. There wasmore reduction in TSC2�/� cells (71.3%) than in TSC2þ/þ

cells (58.7%); compare Fig. 7D and H. Thus, the conse-quences of Src inhibition were more pronounced inTSC�/� cells compared with control cells. These resultssuggest that Src inhibition can reduce the metastatic poten-tial for TSC2�/� cells.

DiscussionThis study has three major novel findings. First, Src is

activated in LAM cells. Second, Src activation contributes tothe pathogenesis of LAM by promoting EMT in TSC2�/� LAMcells. Third, Src inhibition can attenuate the oncogenic andmetastatic potential of LAM cells. A model based on ourfindings is depicted in Supplementary Fig. S9. In LAM cells,the loss of the TSC2 gene results in hyperactivation ofmTORbyRheb. Activation of mTOR increases protein synthesis andproliferation of LAM cells and inhibits autophagy. Autophago-somes are involved in the elimination of active Src kinase fromcells. Autophagy inhibition causes accumulation of phospho-Src(Y416) kinase. Activation of the Src pathway upregulatesEMT genes, including Snail and MMP9 and leads to suppres-sion of E-cadherin.

Hyperactivity of Src has been implicated in the developmentof numerous human cancers and progression to metastases

(18). LAM is currently viewed as a low-grade, destructive,metastasizing neoplasm (12, 11). Recent evidence suggeststhat LAM cells have features similar to cells undergoing EMT.One of the critical steps driving EMT is the repression of E-cadherin, resulting in loss of cell–cell adhesion. E-cadherin is acritical regulator of epithelial junction formation. Dysfunctionof the E-cadherin–mediated cell adhesion system plays animportant role in tumor progression of the relatively benigntumor to invasive, metastatic carcinoma.

Recent studies have shown that, in cancer cells in which theSrc pathway is hyperactive, autophagosomes promote degra-dation of the active tyrosine Src kinase (17). Autophagy isinhibited in LAM cells due to the mTOR activation (7, 26). Inthis study, we show the critical role of autophagy in accumu-lation of active Src kinase in TSC2�/� cells as well as in othermodels of autophagy-deficient cells. Our results indicate thatSrc kinase activation promotes migration and invasion ofTSC2�/� cells, likely secondary to upregulation of Snail tran-scription factor, which supresses E-cadherin expression. Sim-ilar role of Src in promoting cell migration and invasion viaactivation of EMT marker MMP9 has been previouslydescribed in breast cancer (32). Increased immunoreactivityfor MMPs in lung biopsy specimens from subjects with LAMand TSC2-deficient LAM-like cells was also described (29–31).Such activation of MMP9 plays the critical role for the prote-olysis and remodeling of the extracellular matrix that allowscancer cells to invade into the surrounding stroma and pro-motes metastasis.

Src family kinase inhibitor PP2 was found to enhance the E-cadherin–mediated cell adhesion system, which resulted in thesuppression of metastasis of cancer cells (18). Dasatinib andsaracatinib are the most clinically studied Src inhibitors (27,28). Preclinical studies in solid tumor cell lines have shown thatboth dasatinib and saracatinib consistently inhibit cell

Figure5. Src inhibition reduces EMTmarkers in TSC2�/� cells. EEF8cells were treated for 24 hours withvehicle (DMSO), dasatinib (Dasa;0.5 mmol/L), saracatinib (Sara;1 mmol/L), or rapamycin (Rapa;1mg/mL). A, Snail expression wasanalyzed by RT-PCR (top) or byimmunoblot analysis (bottom),n ¼ 4. B and C, cell lysates wereanalyzed by immunoblot analysisusing antibodies against Src,phospho(Y416)-Src or Snail (B), orMMP9 (C). Graphs, quantificationof the immunoblots, n ¼ 3. D, celllysates were analyzed byzymogram of gelatin. Graph,quantification of the zymogram,n ¼ 6. E, EEF8 cells weretransfected for 72 hours with Src-specific siRNA or NT siRNA andanalyzed by immunoblot analysis.Graph, quantification of theimmunoblots, n ¼ 4. F, cell lysatesof E were analyzed for MMP9mRNA using RT-PCR (n ¼ 4). Data,mean� SD. �,P < 0.05; ��,P < 0.01.

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proliferation. Our findings suggest that the selective inhibitionof Src kinase could potentially restore cell adhesion and reducemetastatic tendencies in LAM. Here, we demonstrate thatdasatinib and saracatinib significantly decrease migration andinvasion ability of TSC2�/� cells.

LAMcells exhibit increased activation of themTORpathway(33). Recent clinical trials in subjects with TSC or LAM usingmTOR inhibitor rapamycin showed that there was a reductionin the size of AMLs and, in some cases, improvement in lungfunction (9, 34). However, cessation of therapy led to the

Figure 6. Src inhibition attenuates invasiveness of TSC2�/� cells. The invasive properties of EEF8 (A) and EEF4 (C) cells were studied using Matrigel inserts.Serum-deprived cells (5 � 104 cells) were loaded in the upper compartment of the chambers. DMSO, rapamycin (1 mg/mL), dasatinib (0.5 mmol/L), orsaracatinib (1 mmol/L) was added for 18 hours. Cells on the surface of theMatrigel were visualized by staining with 1% Toluidine blue. Representative imagesare shown. The invading cells were counted in six random fields (B and D). Data, mean � SD; n � 3. ��, P < 0.001. Scale bar, 100 mm.





regrowth of tumors and diminished pulmonary functions (9,10, 35). In our study, rapamycin had no effect on the migrationand invasion activity of TSC2�/� cells. These data are ofparticular clinical relevance because circulating LAM cellswere found in the blood and plural fluid of women with LAMand these cells might be the source for invasion of LAM cellsinto the lungs (13). Thus, failure of rapamycin to affect cellmigration or invasion of TSC2�/� cells may explain the tran-sient nature of rapamycin efficacy in LAM. Our study suggeststhat the selective inhibition of Src kinase could potentiallyrestore cell adhesion and reduce metastatic tendenciesof TSC2�/� cells in LAM. Saracatinib treatment notablydecreased lung colonization of TSC2�/� cells in vivo. Rapa-mycin either alone or in combination with saracatinib did notprovide additional benefits.

Taken together, our findings highlight a role of Src kinase inthe pathogenesis of LAM. Our data demonstrate that activatedSrc kinase promotes EMT in TSC2�/� cells and increases theirmetastatic potential. Src kinase inhibitors dasatinib and sar-acatinib notably decrease migration and invasion ability ofTSC2�/� cells. These results will be valuable for understanding

the nature of EMT in LAMcells and provide a novel therapeutictarget to prevent LAM cell dissemination. The efficacy ofdasatinib and saracatinib, used as a single agent or in com-bination with mTOR inhibitors might improve treatment out-comes in LAM. The use of multiple drug therapy has theadvantage of reducing the dose of each drug and, thus, canminimize side effect. Overall, our study establishes Src as anovel therapeutic target in LAM and provides encouragementfor further preclinical and clinical studies of the use of Srcinhibitors. Several Src inhibitors are already being tested inclinical trials, which can enhance translation of our findings tothe clinic.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A. Tyryshkin, N.T. EissaDevelopment of methodology: A. Bhattacharya, N.T. EissaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. BhattacharyaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Tyryshkin, N.T. Eissa

Figure 7. Effect of Src inhibition onlung colonization of EEF cellsin vivo. EEF8-Luciferase cells weretreated for 18 hours with DMSO,rapamycin (1 mg/mL), saracatinib(1 mmol/L), or by both rapamycinand saracatinib (A–D). EEF4-Luciferase cells were treated for18 hours with DMSO or saracatinib(1 mmol/L; E–H). Cells were theninjected intravenously into femaleCB17 SCID mice and after 6 hours,lung colonization was measuredusing bioluminescence.Representative images are shown(A and E). Total photon flux persecond present in the chest regionafter injection of EEF cells isexpressed as a percentage ofDMSO-treated EEF cells (B and F).Lungs were dissected 24 hoursafter cell injection andbioluminescence was imaged inPetri dish (C and G). Total photonflux per second present in thedissected lungs after injection ofEEF cells is expressed as apercentage of DMSO-treated EEFcells (D and H). Data, mean � SD;n � 3. �, P < 0.05; ��, P < 0.001compared with DMSO-treatedcells.

Tyryshkin et al.




Writing, review, and/or revision of the manuscript: A. Tyryshkin,A. Bhattacharya, N.T. EissaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): N.T. EissaStudy supervision: N.T. Eissa

AcknowledgmentsThe authors thank Dr. Raymond Yeung of the University of Washington

(Seattle, WA) for kindly providing Eker rat embryos fibroblasts TSC2�/� cellsand wild-type controls. The vector encoding cSrc cDNA was a gift fromDr. Wouter Moolenaar.

Grant SupportThis study was supported by funds from National Heart, Lung, and Blood

Institute (R01 HL69033) and from the NIH Common Fund, through the Office ofStrategic Coordination/Office of the NIH Director, and the National Center forAdvancing Translational Sciences (UH2 TR000961).

The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked advertise-ment in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 2, 2013; revised January 9, 2014; accepted January 14, 2014;published online April 1, 2014.

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Src Kinase Is a Novel Therapeutic Target in ... › content › canres › ... · Alexey Tyryshkin, Abhisek Bhattacharya, and N. Tony Eissa Abstract Lymphangioleiomyomatosis (LAM)