Akt Girdin Signaling in Cancer-Associated Fibroblasts ...cancerres.aacrjournals.org/content/canres/75/5/813.full.pdf · utes at 37 C with a collagenase solution containing 400 mg

Microenvironment and Immunology

Akt–Girdin Signaling in Cancer-AssociatedFibroblasts Contributes to Tumor ProgressionYumiko Yamamura1,2, NaoyaAsai1, Atsushi Enomoto1,Takuya Kato1, Shinji Mii1,Yuji Kondo3,Kaori Ushida1, Kaoru Niimi4, Nobuyuki Tsunoda6, Masato Nagino6, Shu Ichihara5,Koichi Furukawa3, Kengo Maeda2, Toyoaki Murohara2, and Masahide Takahashi1

Abstract

PI3K–Akt signaling is critical for the development, progres-sion, and metastasis of malignant tumors, but its role in thetumor microenvironment has been relatively little studied.Here, we report that the Akt substrate Girdin, an actin-bindingprotein that regulates cell migration, is expressed and activatedby Akt phosphorylation in cancer-associated fibroblasts (CAF)and blood vessels within the tumor microenvironment. Lewislung tumors grafted into mice defective in Akt-mediated Gir-din phosphorylation (SA transgenic mice) exhibited a decreasein both CAF infiltration and tumor growth, compared withwild-type (WT) host control animals. Contrasting with the

findings of other studies, we found that Akt-dependent phos-phorylation of Girdin was not a rate-limiting step in thegrowth of endothelial cells. In addition, Lewis lung tumorsdisplayed limited outgrowth when cotransplanted with CAFderived from tumor-bearing SA transgenic mice, comparedwith CAF derived from tumor-bearing WT mice. Collectively,our results revealed a role for Akt-mediated Girdin phosphor-ylation in CAF during tumor progression, highlighting theneed to inhibit Akt function in both tumor cells and cellsthat comprise the tumor microenvironment. Cancer Res; 75(5);813–23. �2015 AACR.

IntroductionAlterations of key genes and signal transduction pathways are

central to the development and progression of malignanttumors (1). Although these driver mutations events are com-monly associated to the cancer's cell-of-origin, recent studieshave shown that the tumor's microenvironment can also play asignificant role in malignant progression (2–5). Tumor cellsrecruit a diverse array of cell types to their surrounding stroma,including cancer-associated fibroblasts (CAF), endothelialcells, and pericytes that constitute tumor vessels, immune cellssuch as tumor-associated macrophages (TAM), and adipocytes,leading to the formation of highly complex neoplastic tissues(6–10). In turn, the tumor microenvironment facilitates tumorprogression and metastasis by providing a matrix for the

integration of intricate interaction networks suitable to main-tain and nourish tumor cells, as well as suppress the normalimmunologic antitumor defenses (1, 11–13). The supportivefeatures provided by stromal cells populating the microenvi-ronment increase tumor tissue diversity, making it difficult totreat patients with cancer uniformly.

CAF originating from fibroblasts, bone marrow–derivedfibrocytes, and mesenchymal stem cells can localize to thetumor microenvironment and promote tumorigenesis throughmultiple mechanisms (5, 14, 15). CAF secrete numerous onco-genic growth factors, chemokines, and cytokines and degradeextracellular matrix (ECM) proteins, thereby promoting bothtumor proliferation and invasion (5, 11, 16, 17). CAF physi-cally assist in the invasion of surrounding tissue by activelyremodeling the ECM, subsequently providing routes that canbe exploited by tumor cells (14, 18). On the basis of thesefindings, CAF gene-expression assays have been developed asuseful prognostic indicators in recent clinical studies (19–23).Clearly, it is important to dissect the mechanisms mediating thelocalization, proliferation, and activity of CAF, as such insightsmay lead to the development of novel therapies that target thetumor microenvironment (24).

Recently, the mechanisms of CAF development and differ-entiation have attracted much attention (5). These studies haverevealed that mechanical forces regulated by ECM stiffness,TGF-b1, and other soluble factors secreted from tumor cellsact in synergy with TGF-b1 signaling as a critical factor in CAFdevelopment (5, 25, 26). However, relatively limited evidenceaddresses the question of how intracellular signaling pathwaysregulate the dynamic cellular activities in CAF. For instance, theactivation of Janus kinase 1 by proinflammatory cytokinesleads to Rho kinase–dependent signaling, which may induceECM remodeling, and increase CAF contractility and motility

1Department of Pathology, Nagoya University Graduate School ofMedicine, Showa-ku, Nagoya, Japan. 2Department of Cardiology,Nagoya University Graduate School of Medicine, Showa-ku, Nagoya,Japan. 3Department of Biochemistry, Nagoya University GraduateSchool of Medicine, Showa-ku, Nagoya, Japan. 4Department ofObstetrics and Gynecology, Nagoya University Graduate School ofMedicine, Showa-ku, Nagoya, Japan. 5Division of Pathology, Depart-ment of Advanced Diagnosis, Nagoya Medical Center, Nagoya, Aichi,Japan. 6Department of Surgery, Nagoya University Graduate Schoolof Medicine, Showa-ku, Nagoya, Japan.

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

CorrespondingAuthor:Masahide Takahashi, Department of Pathology, NagoyaUniversity Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya466-8550, Japan. Phone: 81-52-744-2093; Fax: 81-52-744-2098; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-14-1317

�2015 American Association for Cancer Research.

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(5, 27). However, other intracellular signaling pathways thatdefine cytoskeletal control and migratory responses in CAFremain to be elucidated.

Here, we report that the actin-binding proteinGirdin (girders ofactin filaments; also known as Ga-interacting vesicle-associatedprotein) is expressed and phosphorylated in CAF in both clinicalcancer tissues and tumors in animal models. Girdin is phosphor-ylated by Akt, which functions downstream of PI3K activation toregulate cellmigration andpolarization (28–32). In this study, weused a knock-in mouse line (designated SA mice) engineered toexpress a Girdin mutant that lacks the Akt phosphorylation site atSerine 1416 (S1416A). We found that the growth of allogeneictumors was decreased in SA mice compared with wild-type (WT)counterparts. Notably, the cotransplantation of tumor cells witheither skin fibroblasts or CAF derived from SA mice also atten-uated tumor growth, further indicating the in vivo relevance ofGirdin phosphorylation in formation of the tumor microenvi-ronment. To our knowledge, although clinical relevance is yet tobe clearly defined, these findings demonstrate that the PI3K–Aktpathway is important for the cellular behavior of CAF and suggestthat inhibiting PI3K–Akt signaling not only in tumor cells, butalso CAF, could improve the efficacy of tumor therapy.

Materials and MethodsAntibodies and reagents

Antibodies used in this study were purchased from: polyclonalrabbit anti-human Girdin (Santa Cruz Biotechnology); polyclonalrabbit anti-humanGirdin and polyclonal rabbit anti-human phos-pho (1416-Ser)-Girdin (IBL, Gunma, Japan); polyclonal sheepanti-human Girdin (R&D Systems); monoclonal mouse anti-human a-smooth muscle actin (a-SMA) and monoclonal mouseanti-human vonWillebrand Factor (vWF;Dako);monoclonal anti-mousephospho(473-Ser)-Akt (Cell Signaling Technology);mono-clonal mouse anti-human prolyl 4-hydroxylase b (P4HB; AcrisAntibodies); monoclonal rat anti-mouse CD31 (Dianova); anti-mouse CD68 and CD68-APC (BioLegend); anti-mouse CD16/CD32 and 7-amino-actinomycin D (7-AAD; BD Biosciences).PE-conjugated platelet-derived growth factor receptora (PDGFRa)and PE-conjugated isotype-matched negative control (eBioscience)were used for sorting CAF. Collagenase type II/IV and deoxyribo-nuclease I were purchased from Worthington.

Generation of Girdin SA knock-in miceConventional gene-targeting techniques were used to generate

the SA knock-in mice, in which the Akt phosphorylation site ofGirdin (1416-serine) was mutated to alanine, as described pre-viously (33–35). Resultant mice homozygous for the SA mutantallele in a 129Sv background were mated with C57BL/6 mice togenerate mice with a C57BL/6 genetic background.

Cell cultureLLC (Lewis lung adenocarcinoma) and GFP-tagged LLC cells

(GFP-LLC)were purchased from the ATCC and AntiCancer Japan,respectively. Cells were cultured in DMEM supplemented with10% FBS.

Immunohistochemistry, immunofluorescence,immunoprecipitation, and Western blot analysis

Tissue sections from patients with invasive ductal breast carci-nomawere obtainedwith informedpatient consent at theNagoya

Medical Center (20 cases) and Nagoya University Hospital (86cases), as previously described (36). For IHC studies, formalde-hyde-fixed, paraffin-embedded tumor tissue sections were depar-affinized, and antigens were retrieved by boiling samples in targetretrieval solution at pH 9 (Dako) for 30 minutes. Subsequently,tissue sections were washed with PBS, blocked with blockingreagent (Dako), and incubated overnight at 4�Cwith appropriateprimary antibodies diluted in antibody diluents (Dako). Tissuesections were then washed, treated with 5% hydrogen peroxide/ethanol solution for 15 minutes at room temperature, andincubated at room temperature for 30 minutes with horserad-ish peroxidase–conjugated anti-mouse, anti-rat, or anti-rabbitIgG secondary antibodies (EnVision System; Dako), followedby signal detection with diaminobenzidine (DAB) solution.

For immunofluorescence, tissue sections were incubated over-night at 4�C with appropriate primary antibodies diluted inantibody diluents (Dako). Sections were then washed with PBSand incubated at room temperature for 1 hour in PBS containingAlexa Fluor 488/594–conjugated secondary antibodies (LifeTechnologies). The sections were thenmounted with PermaFluor(ThermoFisher Scientific), andfluorescencewas examinedusing aconfocal laser-scanning microscope (LSM 700, Carl Zeiss). Wefollowed conventional procedures for immunoprecipitation andWestern blot studies, as described previously (28).

Cell proliferation assaysSkin fibroblasts were cultured in triplicate (2� 103 cells/well in

96-well plates) in DMEM containing 20% FBS. Cell proliferationwas monitored by measuring the conversion of a water-solubletetrazolium salt in water-soluble tetrazolium-1 (WST-1) assays(RocheApplied Science). TheWST-1 reagent (10mL)was added to100mLof cell suspension and incubated for 1 hour. Absorbance inwells was measured in a microplate reader set at a wavelength of450 nm with a reference wavelength of 620 nm.

Migration and invasion assaysDirectional cell migration of fibroblasts was stimulated in

monolayers using an in vitro scratch-wound assay (28, 30). Briefly,fibroblasts isolated fromWT and SA mice were seeded in 35-mmtissue culture dishes, and confluent monolayers were scratchedwith a 200-mL disposable plastic pipette tip and allowed tomigrate toward the wound. To examine PDGF-dependentmigration of fibroblasts, the medium on confluent cells wasreplacedwith freshmedium containing 0.1% FBSwith orwithout10 ng/mL PDGF-BB, followed by scratching. Cells were fixed at 12hours after scratching, and migratory distances were calculatedusing an invertedmicroscope. To assess the invasion offibroblaststhrough Matrigel, cells were first seeded in 24-well TranswellMatrigel invasion chambers (8.0-mm pore size; BD Biosciences).Fibroblasts (2.5 � 104/mL) suspended in serum-free medium inthe upper chamber were allowed to invade for 22 hours. Theremaining fibroblasts on the upper surface of filters were removedby wiping with cotton swabs, and cells that had invaded throughto the lower surfacewere visualized byGiemsa staining.Datawereobtained from three independent experiments that were per-formed in triplicate.

Tumor transplantation experimentsEight- to 10-week-old WT or SA mice were anesthetized with

sodium pentobarbital, and 1 � 106 LLC cells were implanted s.c.

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Cancer Res; 75(5) March 1, 2015 Cancer Research814



into their backs. Tumor sizesweremeasured at day 5, 8, 11, and14with calipers, and tumor volumes were calculated using theformula X2 � Y � 0.5, where X, the smaller diameter and Y, thelarger diameter. Tumors were embedded in paraffin wax at day 7to day 14 for subsequent histologic analyses. For cotransplanta-tion studies, we used LLC cells (2.5 � 104) alone or intermixedwith skin fibroblasts or CAF (2.5 � 105) from WT or SA mice.These cell populations were implanted s.c. into the backs of 8- to10-week-old male WT or SA mice. A minimum of three to fourrandomly picked sections from each animal (n ¼ 5–6 mice/group) were analyzed to measure areas of tumor vessel andquantify the populations of CAF and TAM. The numbers of WTand SAmice used for each assay are indicated in thefigure legends.All animal procedures were performed in compliance with insti-tutional ethical requirements and were approved by the AnimalCare and Use Committee of Nagoya University Graduate Schoolof Medicine.

Isolation of CAF from LLC tumor tissuesCAF from LLC allografts were purified and sorted as

described previously (37), with some modifications. Briefly,GFP-LLC tumors were excised from mice at day 23 after

implantation. Tumors were minced and digested for 13 min-utes at 37�C with a collagenase solution containing 400 mgBSA, 100 mg collagenase II, 100 mg collagenase IV, 300 mgdeoxyribonuclease I, and 80mL PBS, and then passed through a70-mm cell strainer. Cells were washed, red blood cells werelysed with 1� ammonium–chloride–potassium buffer for 3minutes, and the remaining intact cells were washed again. Cellpellets were then resuspended in PBS with 1% BSA, pretreatedwith FcR-block (anti-CD16/32), and incubated on ice withprimary antibodies (PDGFRa-PE, 1:50; CD68-APC, 1:100).Cells were washed and incubated with 7-AAD to indicate celldeath. Live CAF were then sorted on a FACS Aria II flowcytometer (BD Biosciences) by gating for GFP�, CD68�, andPDGFRaþ cells. CAF were sorted to a purity of >90%, as verifiedby post-sort immunofluorescence studies. Antibody specificitywas confirmed using isotype-matched control antibodies.

Data analysisData are presented as the means � S.D for all quantitative

analyses. Statistical significances for experimental data and theanalysis on clinicopathologic features were evaluated with c2

tests.

Figure 1.Girdin expression and Akt-mediatedphosphorylation in the stroma ofhuman breast cancers. A and B,sections from invasive ductal breastcarcinomas were stained with anti-Girdin (A) or anti–phospho-Girdin(1416-Ser; B) antibodies. Areashighlighted with red boxes aremagnified in the adjacent bottom.Yellow and white arrows indicatevessels and stromal fibroblasts,respectively. The far bottom panelsshow immunofluorescence staining/colocalization of Girdin orphosphorylated Girdin (green) withvWF or a-SMA (red) in endothelialcells and CAF, respectively. C,coimmunostaining analysis showedthat Akt phosphorylation (pAkt) isdetected in both a-SMA–positive andphospho-Girdin–positive stromalfibroblasts (arrows). D, Girdinexpression and its phosphorylation inthe stroma of both tumors andadjacent normal tissues wereevaluated by IHC on tissue sectionsfrom patients with invasive ductalcarcinoma (n ¼ 20). The number ofcells positive for Girdin expression andphosphorylation in each tissue wascounted and quantified. H&E,hematoxylin and eosin stain;� , P < 0.01; �� , P < 0.001.

Girdin Phosphorylation and Cancer-Associated Fibroblasts

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ResultsExpression and Akt-mediated phosphorylation of Girdin inCAF infiltrating in human breast cancers

We and others have previously shown that Girdin is expressedand phosphorylated by Akt at Serine-1416 in some, but not all,cases of breast cancer, colon cancer, and glioblastoma (30, 36, 38,39). These studies also demonstrate that Girdin is expressed inmost cancer cell lines and in freshly isolated tumor-initiating cellsfrom patients with glioblastoma, in which it controls migrationand stem cell differentiation. However, our comprehensivescreening of numerous invasive ductal breast carcinoma tissuesrevealed that Girdin expression and phosphorylation are notlimited to tumor cells. IHC and immunofluorescence studiesconducted in the present study demonstrates that Girdin is alsoexpressed and phosphorylated in cells that constitute cancerstroma, including vWF-positive endothelial cells and a-SMA–positive CAF, and is accompanied by Akt phosphorylation (Fig.1 A–C). Dual immunolabeling indicates that most of the Girdin-positive cells are CAF, endothelial cells, and pericytes, which

constitute tumor vessels (Fig. 1A and B); however, Girdin expres-sion did not extend to CD68-positive macrophages (data notshown). In addition, we also show that Girdin expression andphosphorylation in CAF was significantly higher in the tumortissues compared with the adjacent normal tissues (Fig. 1D).Girdin phosphorylation was found in approximately 63% ofGirdin-positive CAF in tumor tissues, whereas it was less than40% in adjacent normal tissues (Fig. 1D, right). These data implythat Girdin expression in the tumor microenvironment is impor-tant for the progression of human cancers.

Akt-mediated Girdin phosphorylation is crucial for CAFinfiltration into tumor tissues transplanted in mice

To address the role of Akt-mediated Girdin phosphorylationin development of the tumor microenvironment, we usedphospho-null Girdin knock-in mice (SA mice), previouslygenerated in our laboratory, in which the Akt phosphorylationsite (serine 1416) has been mutated to alanine (Fig. 2A;refs. 33–35). Western blot analysis of lysates prepared from

Figure 2.Growth retardation of LLC tumors andlimited intratumoral CAF infiltrationin mice deficient for Girdinphosphorylation. A, generation of SAknock-in–mutant mice defective inAkt-mediated Girdin phosphorylation,as described previously (33). B,Western blot analysis of brain lysatesisolated from WT, Girdin-deficient(KO, knockout; refs. 40, 50), and SAknock-in mice at the first postnatal(P0) day using anti-Girdin and anti–phospho-Girdin (p-Girdin) antibodies .Equivalency of protein loading isshown by Coomassie Brilliant Blue(CBB) staining of the membrane;Mr, molecular marker. C, LLC tumorvolumes implanted into WT (n ¼ 12)and SA (n¼ 10)mice. The diameters ofimplanted tumors were measured andtheir volumes were estimated on thedays indicated. � , statisticallysignificant difference (� , P < 0.005)was observed at day 14 afterimplantation. D, vasculature formationin LLC tumors. Endothelial cells intumor sections were immunolabeledwith anti-CD31 antibody andvisualized by fluorescencemicroscopy(green fluorescence, left; WT, n ¼ 4;SA, n ¼ 4) or with DAB (brown, right;WT, n ¼ 5; SA, n ¼ 6). Quantificationand representative staining imagesare shown; N.S., not significant. E andF, CAF and TAM infiltration in LLCtumors implanted intoWT (n¼ 4) andSA (n ¼ 4) mice was visualized bya-SMA and CD68 staining,respectively. The numbers of a-SMA–(E) and CD68- (F) positive cells inintratumoral regions of LLC tumorswere quantified; � , P < 0.05;N.S., not significant.

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WT and SAmouse brains indicated that Girdin expression levelswere comparable, whereas Girdin phosphorylation was nearlyabolished in SA mice (Fig. 2B). Syngeneic C57BL/6 murine LLCcells were transplanted into WT and SA mice and grew to formtumors. Engraftment ratio of the implanted tumors was 100%in both WT (12/12) and SA (10/10) groups. The growth ofLLC tumor allografts was significantly retarded in SA micecompared with WT mice (Fig. 2C), suggesting that SA miceare defective in developing a supportive tumor microenviron-ment or inducing the host–tumor interactions that enhanceLLC cell proliferation.

To examine which stromal cell subpopulation(s) depend onGirdin phosphorylation to promote tumor growth, we used IHCto quantify the area of tumor vessels and the CAF and TAMpopulations present in LLC tumors excised fromWT and SAmice(Fig. 2D–F).We have previously shown that Girdin is expressed innascent immature endothelial cells and that its phosphorylationis important for angiogenesis in the retinal vascular plexus(34, 40). Thus, it was surprising that CD31 immunostainingrevealed no significant differences in tumor vessel formationbetween WT and SA mice (Fig. 2D). However, the number ofhost-derived a-SMA–positive cells not associated with the vascu-lature was significantly decreased in SAmice. These cells appeared

to colocalize with the CAF invading the tumor tissue (Fig. 2E).Wealso found that the numbers of TAM were comparable betweenWT and SA mice, despite their known roles in tumor progression(Fig. 2F; ref. 4). This finding suggests that the Akt–Girdin pathwayspecifically contributes to CAF infiltration, but not to the infil-tration of vessel cells or TAM.

Recent studies have shown that CAF degrade or remodel theECM surrounding tumors to generate tracks and passages, alongwhich tumor cells can invade and proliferate (14, 18). Therefore,we studied CAF invasion in the peritumoral region of tumors andfound that peritumoral CAF infiltration was significantlyimpaired in SAmice compared with that observed inWT counter-parts (Fig. 3A). This finding was confirmed by staining for P4HB,anotherCAFmarker,which showed that P4HB-positive cells had adecreased presence in the peritumoral regions in SA mice (Fig.3B). Additional immunostaining and quantification of a-SMAand P4HB-positive cells within the peritumoral region confirmedthat SA mice are likely defective exclusively in the recruitment ofCAF, independent of the other host-derived cells that surroundtumors (Fig. 3C). Collectively, these findings suggest that Akt-mediated Girdin phosphorylation is important for the specificrecruitment of CAF, but not vessel cells or TAM, to peritumoralregions.

Figure 3.Limited CAF infiltration in theperitumoral region of LLC tumorsin mice deficient for Girdinphosphorylation. A and B, CAFinfiltration in the peritumoral region ofLLC tumors implanted intoWT (n¼ 5)and SA (n¼ 6) mice was visualized bya-SMA (A) and P4HB (B) staining. Theareas highlighted with boxes aremagnified in the adjacent panels.Yellow arrows, representativeinfiltrated CAF. Right, quantification ofa-SMA– or P4HB-positive cells inperitumoral regions of tumors. Dottedlines, interfaces between tumors andsurrounding stroma; � , P < 0.05. C, thenumbers of a-SMA– (left) and P4HB-(right) positive cells per field werequantified. Frequencies of a-SMA–and P4HB-positive cells in peritumoralregions of LLC tumors were expressedas a percentage of total cells, asdetermined by DAPI staining. Dottedlines, interfaces between tumors andsurrounding stroma; � , P < 0.05;�� , P < 0.005.





Roles ofGirdin phosphorylation infibroblast proliferation andmobilization

To identify the cellular processes and activities of fibroblastsregulated by Akt-mediated Girdin phosphorylation, we firstexamined the properties of cultured primary skin-derived fibro-blasts isolated from WT and SA mice (Fig. 4). Basal Girdinphosphorylation was detected in primary fibroblast lysatesfrom WT mice but not SA mice, as shown by Western blotanalysis (Fig. 4A, left). WST-1 proliferation assays also dem-onstrate that, compared with WT fibroblasts, SA fibroblastsgrow at a slower rate, particularly during the later phase ofsubconfluent culture (after 5 days; Fig. 4A, right). In addition,we found that serum- and PDGF-dependent directional migra-tions in culture dishes are significantly impaired in SA fibro-blasts (Fig. 4B). Considering that Girdin associates with theactin cytoskeleton (28), it is possible that Girdin incorporatesinto the machinery that regulates a-SMA–mediated contractileactivity in fibroblasts, in which its phosphorylation by Aktpromotes cell migration.

Interestingly, but for reasons presently unclear, WT and SAfibroblasts shownodifference in invasiveness through the ECM inresponse to multiple growth factors in sera, as determined byMatrigel invasion assays (Fig. 4C). This finding contradicts anearlier study showing that Girdin is crucial for cancer cell invasion

(30). A potential explanation for these discordant results is thatGirdin phosphorylation may not exert a potent effect on fibro-blast ECM invasion, a property that may diverge from that ofcancer cell invasion.

Significance of Girdin phosphorylation in fibroblasts inpromoting tumor growth

To test whether Girdin phosphorylation affects protumorigenicproperties offibroblasts, we next grafted LLC cells intermixedwithWT or SA skin fibroblasts (henceforth referred to as LLC/WT andLLC/SA, respectively) into WT C57BL/6 mice and monitoredtumor growth (Fig. 5). As a negative control experiment, onlyLLC cells were injected into WT mice (Fig. 5A, left). Engraftmentratio of the implanted tumors was comparable among the threegroups (LLC/WT, 21/21; LLC/SA, 12/12; LLC only, 16/17). Con-sistent with the findings of previous studies reporting tumor-supportive functions of fibroblasts (11, 41), we found that LLC/WT tumors grew at a faster rate than those derived from LLC cellsalone (Fig. 5A, right). Significantly, LLC/SA tumors failed to growas well as those coimplanted with WT fibroblasts, implicating atumor-supportive role of Girdin phosphorylation in fibroblasts.

To exclude the contribution ofGirdin phosphorylation in othercell types derived from host mice, LLC/WT and LLC/SA cellmixtures were implanted into SA mice (Fig. 5B, left), so as to

Figure 4.Proliferation and migration of skinfibroblasts isolated from WT and SAmice. A, skin fibroblasts isolated fromWT and SA mice were expanded inculture dishes. Total Girdin infibroblast lysates wasimmunoprecipitated (IP) with an anti-Girdin antibody, followed byimmunoblot analysis (IB) by using theindicated antibodies. Twoindependent lots of fibroblasts in eachgroup (WT1, WT2, SA1, and SA2) weretested. Right, proliferation rates ofWTand SA fibroblasts on culture, asmeasured in WST1 assays (n ¼ 4 foreach group); �, P < 0.05. B, directionalcell migration of WT and SAfibroblasts was monitored inmonolayers by using an in vitroscratch-wound assay. Cells wereseeded on tissue culture dishes.Confluent cells were scratched with200 mL disposable plastic pipette tipsand were allowed to migrate towardthe wound for 12 hours in the presenceof FBS or PDGF (10 ng/mL). Dottedlines, the leading front of migratingcells. The graphs show quantificationof migration distances covered byWT(n¼ 3–6) and SA (n¼ 3–6) fibroblasts;��,P<0.05. C, in vitroMatrigel invasionassays. The numbers of WT (n ¼ 4)andSA (n¼4)fibroblasts that invadedthrough Matrigel for 22 hours werequantified; N.S., not significant.

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disregard the role of Girdin phosphorylation in host-derivedvessel and immune cells in development of the tumor stroma.Again, we found that LLC/SA exhibited a retarded growth ratecompared with LLC/WT counterparts (Fig. 5B, right). IHC ontumor sections revealed that infiltration of a-SMA–positive CAFin the peritumoral region was significantly impaired in LLC/SAtumors comparedwith LLC/WT counterparts (Fig. 5C). These datasuggest Girdin phosphorylation in fibroblasts not only enhancestheir proliferative and migratory responses, but also facilitatestumor growth in vivo. Taking together, these data suggest thatGirdin phosphorylation promotes tumor growth by severalpotential direct and/or indirect mechanisms, such as regulatingECM remodeling or growth factor/cytokine networks.

CAF defective of Girdin phosphorylation have limited effectson tumor growth in vivo

To recapitulate the in vivo features of tumor growth and furthervalidate the function of Girdin phosphorylation in CAF, CAF

isolated from WT and SA mice harboring LLC tumors weresubsequently used in our tumor transplantation model (Fig.6). To this end, LLC cells engineered to express GFP (termedGFP-LLC)were allografted intoWTand SAmice. Threeweeks afterimplantation, tumors were harvested and digested with collage-nase to form single-cell suspensions, which were then studied byFACS analysis. Cells were sorted into tumor cell, CAF, and TAMsubpopulation based on expression of GFP, PDGFRa, and CD68expression, respectively (Fig. 6A and B). The purity of sorted CAFwas monitored by immunofluorescence staining, indicating thatcontamination with GFP-LLC cells was less than 10% (data notshown). The role ofGirdin phosphorylation in the tumor growth–promoting ability imparted by CAFwas assessed by coimplantingCAF isolated from LLC tumors grafted in WT and SA mice(henceforth referred to as LLC/WT-CAF and LLC/SA-CAF,respectively; Fig. 6C). The engraftment ratio of the implantedtumors was comparable between the two groups (LLC/WT-CAF,6/6; LLC/SA-CAF, 6/6). Similar to previous experiments, the

Figure 5.Importance of Girdin phosphorylationin fibroblasts in promoting LLC tumorgrowth in vivo. A and B, LLC cells(2.5 � 104) intermixed with WT or SAskin fibroblasts (2.5 � 105) wereimplanted into eitherWT (A) or SA (B)mice, and the volume of each tumorwas measured at the indicated daysafter implantation (right). The samplenumber for each group is indicated inthe graph legends; � , P < 0.05;�� , P < 0.005. C, sections from theallograft tumors developed in theexperiments shown in A, whichcomprised LLC cells and either WT orSAfibroblasts, were stained fora-SMAto visualize CAF. The graph on theright shows the number of a-SMA–positive CAF in each field of theperipheral region of tumors in eachgroup (WT; n ¼ 6, SA; n ¼ 5);�� , P < 0.005.





growth and infiltration of a-SMA–positive CAF in peritumoraltissues were attenuated in LLC/SA-CAF tumors compared withLLC/WT-CAF counterparts (Fig. 6C and D). These data indicatethat Girdin phosphorylation in CAF participates in the forma-tion of the tumor microenvironment, which facilitates tumorgrowth.

As observed with skin fibroblasts (Fig. 4), these findings mightsupport a role for the Girdin phosphorylation–mediated regula-tion of CAF migration in response to certain growth factors andcytokines (Fig. 6E); however, this was not conclusively deter-mined in this study. SA-CAF exhibited defective migratoryresponses following stimulation with TGF-b1 and PDGF, but notwith stromal cell–derived factor 1 (SDF-1), when analyzed by invitro migration assays (although PDGF data were not statisticallysignificant). Our observation that Girdin functions downstream

of TGF-b1 is consistent with a recent study describing the roleof Girdin in liver fibrosis (42). However, we also found thatGirdin phosphorylation seems not to be involved in a-SMAexpression regulated downstreamof TGFb signaling (Supplemen-tary Fig. S1).

Survival of coimplanted CAF in the tumor transplantationmodel

Considering controversy over the fate of coimplanted CAFin the mouse tumor transplantation model and giving implica-tions for further research, we assessed the survival of coim-planted CAF within the developed tumors. To this end, GFP-LLC cells were allografted into the Rosa26–tdTomato reportermice and isolated tdTomato-positive CAF from the developedtumors. The isolated CAF were then coimplanted with GFP-LLC

Figure 6.Importance of Girdin phosphorylationinCAF in promoting LLC tumor growthin vivo. A and B, schematic illustrationof the procedure used for isolatingCAF from tumor-bearing WT and SAmice. GFP-LLC cells were transplantedinto WT and SA mice, and thedeveloped tumors were digested withcollagenase. Cells in suspension werelabeled with the indicated antibodiesand analyzed by flow cytometry.Shown in B is an example ofexpression profiles observedfollowing sequential gating based onCD68, PDGFRa, and GFP expressions.Apoptotic or dead cells were excludedby staining with 7-AAD, a marker ofcell death and apoptosis. C, LLC cells(2.5 � 104) intermixed with CAF(2.5� 105) derived fromWT (n¼ 6) orSA (n ¼ 6) mice were implanted intoWT mice, and the volume of eachtumor was measured at the indicateddays after implantation (bottom);��,P<0.005. D, sections fromallografttumors were stained for a-SMA tovisualize CAF. The graph on the rightshows the number of a-SMA–positiveCAF observed in each field, examiningperipheral regions of tumors fromeach group (WT; n ¼ 8, SA; n ¼ 8);� , P < 0.05. E, the migratory responseof WT- and SA-CAF to recombinantTGF-b1, PDGF, and SDF-1 wasexamined by in vitro scratch-woundassays, and the migration distancescovered byWT (n¼ 6) and SA (n¼ 6)CAF were quantified; � , P < 0.05.

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cells into WT mice (Fig. 7A). Immunofluorescent analysesshowed that the tdTomato-positive CAF populating the intra-tumoral region significantly decreased from days 14 to 21 aftertransplantation. Notably, the ratios of tdTomato-positive CAFto total CAF (both implanted and host-derived) were 56% and16% at day 14 and 21, respectively (Fig. 7B and C). These dataindicate that the implanted CAF did not survive in the tumortissues for an extended period, and leads to the speculation thatthe implanted tdTomato-positive CAF are involved in early, butnot later, stages of tumor progression in tumor transplantationmodels.

Relation between Girdin phosphorylation in CAF andclinicopathologic features in invasive breast cancer

Finally, we performed immunohistochemical analysis by usingtissue sections from 86 patients with invasive ductal breast car-cinoma. However, the data failed to show any significant corre-lation between Girdin phosphorylation in CAF and tumor stagesand grades, histologic features, metastatic rate, or tumor recur-rence, although Girdin phosphorylation tended to be associatedwithHER2-positive cases (Supplementary Fig. S2; SupplementaryTables S1 and S2). No significant correlation may be partiallyattributed to the difference between transplanted animal modelsand progressive human cancers. Because a variety of microenvi-ronmental factors are involved in the progression of humancancer, synergistic or additive effects of Girdin phosphorylationwith those intrinsic factors could influence the clinical outcome ofthe patients.

DiscussionHere, we report that the Akt substrate Girdin is expressed and

phosphorylated in CAF, and exerts tumor growth–promotingeffects with in vivo animal tumor models. Our findings suggesta novel mechanism, whereby Akt signaling is central to bothtumor cells and cells that constitute the tumormicroenvironment.We demonstrate that the Akt-mediated phosphorylation of Gir-din is important for CAF infiltration and tumor cell growth inmice. Given that Girdin is an actin-binding protein and itsphosphorylation regulates actin remodeling (28, 30), it isplausible that Girdin integrates into thick actin cables predom-inantly composed of a-SMA to regulate the contractility of CAF,which could promote ECM remodeling required for tumorgrowth. Although the clinical relevance of our findings are notprovided in this present study and should await further clinicalanalysis, our study provide a foundation for the potentialtargeting of the PI3K–Akt signaling pathway within the tumormicroenvironment, with the goal of developing novel therapiesagainst human malignancies.

One limitation of this study is that it does not address thepotential upstream regulator(s) that may induce Girdin phos-phorylation in CAF. Although speculative, it is interesting toconsider that Girdin phosphorylation in CAF may result fromsignals derived fromproliferating tumor cells. Recent studies haveshown that tumor cells secrete many soluble growth factors,cytokines, and chemokines, including TGF-b1, hepatocyte growthfactor, and SDF-1, which act upon their cognate receptors

Figure 7.The fate of coimplanted CAF duringtumor progression inmice. A, GFP-LLCcells were implanted into the Rosa26-tdTomato reporter mice to developtumors. tdTomato-positive CAF weresorted by flow cytometer, and TAMwere eliminated with CD11bmicrobeads. The isolated CAF werethen coimplanted with GFP-LLC cellsinto WT mice, and the developedtumors were resected for furtheranalysis. B and C, the sectionsprepared from the developed tumorsat the indicated days afterimplantation, which harbor tdTomato-positive CAF (red), were stained fora-SMA (blue). Regions indicated bythe white boxes were magnified in thebottom. The merged images show thenumber of cells double-positive forboth tdTomato and a-SMA (yellowarrows) decreased after implantation(B, day 14; C, day 21 afterimplantation), indicating that theimplanted CAF did not survive at thelate stage of tumor development.Arrowheads, newly infiltrated a-SMA–positive CAF that are negative fortdTomato. The ratios of tdTomato-positive CAF to total infiltrating CAF(both tdTomato-positive implantedand tdTomato-negative host derived)were 56% and 16% at days 14 and 21,respectively.





expressed on CAF to modulate their ability to promote an aggres-sive tumor phenotype (3, 5, 8, 43).Migration assays performed inthis study implicate PDGF and TGF-b1 (42, 44) as candidategrowth factors that may govern the function of Girdin, as fibro-blasts andCAF isolated fromSAmicewere defective in PDGF- andTGF-b1–induced migration (Figs. 4B and 6E). Consistent withthis hypothesis, cumulative evidence has suggested that tumorcells overexpress and secrete PDGF and TGF-b1, and that CAFexpress the cognate receptors for these factors (22, 25, 26, 45, 46).These effects, combined with the known autocrine effects of thesefactors on tumor cells, suggest that CAF may play an integral partin a signaling network regulated by synergy or extensive crosstalkwith tumor cells to provide for the supportive nature of the tumormicroenvironment.

Another intriguing issue in this study is the role of Girdinphosphorylation in tumor vessel formation. We have previouslyshown that Girdin is expressed in nascent small capillaries andcultured endothelial cells, in which it is phosphorylated inresponse to vascular endothelial growth factor stimulationthrough the PI3K–Akt signaling pathway (34, 40). However, ourquantitative analysis of tumor vessel development in allograftedLLC tumors revealed no significant differences in vessel area orbranching morphogenesis between tumors grown in WT and SAhost mice (Fig. 2D). One potential explanation for this discrep-ancy might be that Akt-mediated Girdin phosphorylation isdispensable for the recruitment of endothelial cells in tumortissues, but pivotal for the maintenance of vascular integrity orpermeability. A definitive conclusion regarding this point requiresfurther investigation in tumor tissues and sophisticated imagingtechniques.

A challenging view of tumor stroma is that the distribution ofCAF is not homogeneous and the composition of the tumorstroma is rich in diversity, even within the tumor derived fromthe same tissue (47). The tumor stroma is inconspicuous in somecases, whereas in others it is desmoplastic. Our histologic experi-ments on human breast cancer tissues also indicate the hetero-geneity of Girdin expression and its phosphorylation in both CAFand tumor cells (Fig. 1D). In LLC implanted tumors, a-SMA–positive CAF distributed into both the intratumoral and peritu-moral regions. Although the infiltration of CAF in both regionswere prone to regulation by Girdin phosphorylation (Figs. 2Eand 3), the significance of their distribution patterns are not yetdetermined. Given that clinical investigation and biologic eval-uation of several PI3K and Akt inhibitors are underway (48, 49), itwould be necessary to investigate the effects of these inhibitors onthe distribution of Girdin expression and its phosphorylation inCAF and their relevance to clinical outcomes.

In the present study, the clinical importance of Girdin-depen-dent Akt signaling has not been determined. Our study did not

show any correlation between Girdin phosphorylation in CAFand tumor stages, malignancy grades, and pathologic features ofinvasive ductal breast carcinomas, although Girdin phosphory-lation tended to be associated with HER2-positive cases (Supple-mentary Table S2). Because a variety of microenvironmentalfactors are involved in the progression of human cancer, syner-gistic or additive effects of Girdin phosphorylation with thosefactors may be important for clinical outcome of the patients. Inaddition, the gene-expression profiles of CAF in individual can-cers need to be considered (19–23). Therefore, future studiesshould concentrate on determining the importance of Akt–Girdinsignaling in the tumor microenvironment for the evaluation ofclinical outcomes of the patients with various stages of malignanttumors on an individualized basis.

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

Authors' ContributionsConception anddesign:Y. Yamamura, A. Enomoto, T.Murohara,M. TakahashiDevelopment of methodology: Y. Yamamura, N. Asai, T. KatoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Yamamura, N. Asai, S. Mii, Y. Kondo, N. Tsunoda,M. Nagino, S. IchiharaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Yamamura, K. Niimi, T. Murohara, M. TakahashiWriting, review, and/or revision of the manuscript: Y. Yamamura, A. Eno-moto, K. Maeda, M. TakahashiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Mii, K. Ushida, M. Nagino, S. Ichihara,T. MuroharaStudy supervision: K. Furukawa, K. Maeda, T. Murohara, M. Takahashi

AcknowledgmentsThe authors thank Katsuhiro Kato for helpful discussions, Mayu Isotani-

Sakakibara for Western blot analysis, and Minoru Tanaka for FACS and sortinganalysis.

Grant SupportThisworkwas supported by grant-in-aid for Scientific Researchon Innovative

Areas (22117005), Scientific Research (A) (23249020) and Scientific Research(S) (26221304; M. Takahashi), grant-in-aid for Scientific Research (C)(24390095; N. Asai), and grant-in-aid for Young Scientists (A) (20432255;A. Enomoto) from the Ministry of Education, Culture, Sports, Science, andTechnology of Japan.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 30, 2014; revised November 26, 2014; accepted December 3,2014; published online March 2, 2015.

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2015;75:813-823. Cancer Res Yumiko Yamamura, Naoya Asai, Atsushi Enomoto, et al. Contributes to Tumor Progression

Girdin Signaling in Cancer-Associated Fibroblasts−Akt

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