Sonic Hedgehog Pathway Promotes Metastasis and ...cancerres.aacrjournals.org/content/canres/71/22/7061.full.pdf · Molecular and Cellular Pathobiology Sonic Hedgehog Pathway Promotes

Molecular and Cellular Pathobiology

Sonic Hedgehog Pathway Promotes Metastasis andLymphangiogenesis via Activation of Akt, EMT, and MMP-9Pathway in Gastric Cancer

Young A. Yoo1, Myoung Hee Kang2, Hyun Joo Lee3, Baek-hui Kim3, Jong Kuk Park6, Hyun Koo Kim4,Jun Suk Kim5, and Sang Cheul Oh5

AbstractActivation of sonic hedgehog (Shh) signaling has been implicated in progression of a variety of tumors. In this

study, we elucidated a role for Shh in the invasion of gastric tumors and determined themechanism by which Shhis regulated. Immunohistochemical analysis of 178 primary human gastric tumor biopsies indicated that Shhexpression was positively correlated with lymph node metastasis, high lymphatic vessel density, and poorprognosis. Inmouse xenograftmodels of human gastric cancer, enforced expression of Shh significantly enhancedthe incidence of lungmetastasis comparedwith nonexpressing controls. Mechanistic investigations revealed thatphosphoinositide 3-kinase (PI3K)/Akt inhibition blocked Shh-induced epithelial–mesenchyme transition, theactivity of matrix metalloproteinase 9 (MMP-9), and lymphangiogenesis, reducing tumor invasiveness andmetastasis. Taken together, our findings establish that Shh signaling promotes the metastasis of gastric cancerthrough activation of the PI3K/Akt pathway, which leads to mesenchymal transition and MMP-9 activation.These findings offer preclinical validation of Shh as a candidate therapeutic target for treatment of metastaticgastric cancers. Cancer Res; 71(22); 7061–70. �2011 AACR.

Introduction

The sonic hedgehog (Shh) signaling pathway plays a criticalrole in stem cell maintenance and specifying patterns of cellgrowth and differentiation during embryonic development (1).Shh activates its signaling by binding to the receptor, Patched(Ptch), which relieves Ptch-mediated repression of a down-streammembrane protein related to G protein-coupled recep-tors, Smoothened (Smo). Upon activation, Smo promotesnuclear translocation of a family of transcription factors [CiinDrosophila and Glis (Gli1, Gli2, and Gli3) in vertebrates], andsubsequently activates target genes through Glis (2).Recently, the Shh signaling pathway was shown to not only

be involved in induction of angiogenesis in an adult mamma-

lian systembut also in the control of themotility andmigrationof multiple cell types (3). Studies conducted by Hochman andcolleagues have suggested that the components of the Shhsignaling pathway participate directly in cell migration andangiogenesis, whereas inhibition of this pathway blocks Shh-induced cell migration and angiogenesis (4). In addition, Shhand Gli1 expression has also been shown to affect epithelial-to-mesenchymal transition (EMT) in pancreatic cancer cell linesand lymphatic metastasis in esophageal squamous cell carci-noma (5, 6). These observations imply that disregulated Shhsignaling is correlatedwith the severity of the associated tumorand contributes to maintain metastatic behavior. Therefore,understanding these pathways in tumor metastasis will pro-vide great promise for the discovery of novel therapeutics andthe treatment of metastatic disease. Nevertheless, the exactrole and regulatory mechanisms of Shh signaling in tumori-genesis and metastatic progression of gastric cancer are con-troversial and need further classification. To clarify the func-tional role of Shh signaling in gastric cancer progression, weused multiple approaches in testing the gene, both in vitro andin vivo, and validated the outcome results in a clinical setting.In this study, we found that the Shh signaling pathway, actingthrough the phosphoinositide 3-kinase (PI3K)/Akt pathway,may contribute to tumor metastasis by inducing EMT andmediating matrix metalloproteinase 9 (MMP-9) activity.

Materials and Methods

Cell lines and culturesAGS and KATOIII were obtained from the American Type

Culture Collection and MRC-5, SNU-5, SNU-16, SNU-216,

Authors' Affiliations: 1Brain Korea 21 Program for Biomedical Science;2Graduate School of Medicine; Departments of 3Pathology and 4Thoracicand Cardiovascular Surgery; 5Division of Oncology/Hematology, Depart-ment of Internal Medicine, Korea University College of Medicine, KoreaUniversity; and 6Laboratory of Radiation Tumor Physiology, Korea Instituteof Radiological and Medical Sciences, Seoul, Korea

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

Y.A. Yoo and M.H. Kang contributed equally to this work.

Corresponding Authors: Young A. Yoo, Division of Oncology/Hematol-ogy, Korea University Guro Hospital, 97 Gurodong-gil, Guro-ku, Seoul152-703, Korea. Phone: 82-2-2626-1998; Fax: 82-2-862-4453;E-mail: [email protected] and Sang Cheul Oh. Phone: 82-2-2626-3060;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-1338

�2011 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 7061

Research. on August 6, 2019. © 2011 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 5, 2011; DOI: 10.1158/0008-5472.CAN-11-1338

http://cancerres.aacrjournals.org/

SNU-638,MKN-28,MKN-45, andMKN-74were purchased fromthe Korea Cell Line Bank. Adult human dermal lymphaticmicrovascular endothelial cells (HMVEC-dLyAd) wereobtained from Lonza and the cells were maintained accordingto manufacturer's instructions.

Authentication and characterization of cell linesEach cell was characterized by cytogenetic analysis. SNU-5,

SNU-16, SNU-216, and SNU-638 were initially characterized byPark and colleagues (7). MKN-28, MKN-45, and MKN-74 werecharacterized byKitano andKitamura (8). Origin ofMRC-5wasconfirmed by nucleoside phosphorylase, glucose-6-phosphatedehydrogenase, and lactate dehydrogenase isoenzyme electro-phoresis. HMVECs stain positive for acetylated LDL and vonWillebrand's (factor VIII) antigen and stain negative forsmooth muscle a-actin.

Clinical samplesThe archival paraffin-embedded tissues were used from

178 patients who had undergone surgical excision for gastriccancer at Korea University Guro Hospital between March2002 and April 2004. Tissue samples were obtained fromKorea Lung Tissue Bank, assigned and supported by theKorea Science & Engineering Foundation in the Ministry ofScience & Technology. Patient clinical characteristicsaccording to the International Union Against Cancer, asrevised in 2002, are shown in Table 1. This protocol wasreviewed and permitted by Institutional Review Board ofGuro Hospital.

Animal studies using a subcutaneous and tail veininjection model

Exponentially growing cells (1 � 106) in 0.1 mL of PBS wereprepared and then injected into the subcutis or tail veins of 6-week-old BALB/c nu/nu mice (Charles River Laboratories).Mice were sacrificed 7 weeks after inoculation of the cells, andmetastatic lesions on the lungs were counted macroscopically.Primary tumor tissues were harvested, weighed, and processedfor further analyses.

Reverse transcriptase PCR analysis, luciferase assay,immunohistochemical staining, cell proliferation assay,wound healing, Matrigel invasion assays, Westernblotting, immunofluorescence staining, and gelatinzymography

The above analyses were conducted as described earlier(9, 10). Antibodies, sources, clones, and dilutions used inimmunofluorescence staining are listed in SupplementaryTable S1. The sequences of primers used for reverse tran-scriptase PCR (RT-PCR) analysis are shown in Supplemen-tary Table S2.

In vitro kinase assay and in vitro tube formation assayNonradioactive Akt kinase assays were carried out

according to the manufacturer's protocol (Cell Signaling).Quantitative analysis of lymphatic vessel density (LVD)was conducted in sections, which were single stained forD2-40.

Statistical analysisThe similar methods were used for statistical analysis of in

vitro and in vivo data described previously (9). The relation-ships between variables were assessed with a Spearman rankcorrelation coefficient. Difference in the incidence of lungmetastasis was analyzed by Fischer exact test. Kaplan–Meiersurvival curves were constructed for patients positive andnegative for Shh. Differences between survival curves weretested for statistical significance by a log-rank test. P values lessthan 0.05 were defined as statistically significant.

Results

Elevated expression of Shh is correlated with advancedclinical stage, lymph node metastasis, and poorprognosis in patients with gastric cancer

To investigate the role of Shh pathway in gastric cancermetastasis, we first conducted RT-PCR for components of theShh pathway in a panel of gastric cells. As expected, thecomparison of various gastric cancer cell lines or tissuesrevealed that all of the components of this pathway wereexpressed in diverse gastric tumor cells to a different extent(Supplementary Fig. S1A and B). Interestingly, we detectedsignificantly higher expression of Shh andGli1 in gastric tumortissues obtained from patients with lymph node metastasiscompared with patients without lymph node metastasis(Fig. 1A). Furthermore, we showed that there was a significantpositive correlation between the levels of Shh and Gli1 tran-scripts (P < 0.01, r ¼ 0.766, Fig. 1A).

Next, we conducted immunohistochemical staining for Shhin 178 gastric cancer specimens. Clinicopathologic analysisrevealed that the expression of Shhwas strongly elevated in thecases with advanced stages (stages II–IV) andwith lymph nodemetastasis compared with early stage (stage I, P ¼ 0.039) andnonmetastatic cases (P ¼ 0.042; Table 1 and SupplementaryFig. S1C). The results also indicate that in advanced stages(stages II–IV) of gastric cancer (n¼ 147), patients with positiveexpression of Shh had a significantlyworse rate of survival thanthe patients with low expression of the gene (P¼ 0.002 by log-tank test; Fig. 1B). On the basis of univariate Cox regressionanalysis, the death risk of patients with increased Shh expres-sion was 3.2 times higher than the risk of patients with areduced expression of the gene. Indeed, the overall survival ratewas significantly lower in patients with gastric cancer at stagesII to IV than those at stage I (P¼ 0.02 by log-tank test; Fig. 1B).Taken together, these results clearly show that elevated expres-sion of Shh is correlated with advanced clinical stage, lymphnode metastasis, and poor prognosis in patients with gastriccancer.

Crucial function of Shh signaling on invasiveness andmetastatic potential of gastric cancer cells

Next, we testedwhether or not the Shh signaling pathway isinvolved in progression to metastatic disease in gastriccancer, especially in late tumorigenesis, including migrationand invasion. Our results showed that N-Shh treatmentsignificantly stimulated the migration and invasiveness ofthese cells (Fig. 1C, and D). In contrast, the stimulatory effects

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Cancer Res; 71(22) November 15, 2011 Cancer Research7062




of N-Shh on cell migration and invasion were completelyabolished by cyclopamine (a Shh signaling inhibitor) or Gli1-specific siRNA treatment (Supplementary Fig. S2 A).The AGS cell line expressing Shh and containing only vector

was subcutaneously injected into athymic mice, then theformation and growth rate of the tumor was monitored every4 days for 24 days. All of the vector only and Shh expressing cellshad similar growth rates during the indicated period, suggest-ing that overexpression of Shh did not significantly affecttumor growth rate (Supplementary Fig. S2B). This result isconsistent with data obtained from in vitro BrdU labeling andfluorescence-activated cell sorting analysis that assesses theproliferation and the cell-cycle distribution of the vector onlyand Shh expressing AGS cells (Supplementary Fig. S2C). On thecontrary, tumors stably expressing Shh showed nearly 5 timesmore colony formation on lung surfaces compared with thetumors containing only vector (Supplementary Fig. S2D). Inaddition, Shh significantly increased the number of mice withmetastasis to the lung, and similar results were also obtained ina tail vein injection model (Fig. 1E).To further determine whether there is a causal correlation

between Shh expression and metastatic function of gastriccancer, Shh shRNA expressing highly metastatic SNU-16 cellswere compared with vector only expressing cells for theprogression of lung metastases. Lung metastasis developed in

9 of the 10 (90%) mice inoculated with nonspecific shRNA onlyexpressing cells, whereas only 1 of the 10 (10%) mice thatinoculated with Shh shRNA expressing cells developed lungmetastases (Fig. 1E). These results strongly suggest that Shhsignaling enhances the metastatic potential of gastric cancercells.

Shh signaling pathway induces lymphangiogenesis andmorphologic changes of gastric cancer cells through theEMT process and MMP-9 activity

In the context of tumor progression, EMT is an early eventin tumor invasion and increased motility and invasion arepositively correlated with EMT (11). Here, we found thattreatment of SNU-216 and MKN-74 cells with N-Shh for 72hours exhibited dramatic changes in cell morphology, from acuboid, epithelial-like shape to a spindle, fibroblastic-likeappearance, consistent with EMT (Supplementary Fig. S3A).Concomitant with the change in phenotype, the cells treatedwith N-Shh showed strong suppression of E-cadherin 72hours after addition, whereas strong expression of Snail,which is known to repress expression of the E-cadherin gene,were detected in cells treated with N-Shh. These alterationswere completely recovered by pretreatment with cyclopa-mine before N-Shh stimulation or by knocked down Gli1(Fig. 2A).

Table 1. Relationship between Shh expression and other clinicopathologic features in gastric cancer

Clinicopathologic feature (numberof cases examined)

Number of cases Shh expression Pa

Positive Negative

Age (174)>60 79 37 (46.8) 42 (53.2) 0.071�60 95 68 (71.6) 25 (28.4)

Gender (173)Male 124 82 (66.1) 42 (33.9) 0.095Female 49 23 (46.9) 26 (53.1)

Lauren's classification (162)Intestinal type 57 41 (72) 16 (28) 0.021Diffuse type 105 61 (58.1) 44 (41.9)

Histologic grade (73)WDb 5 4 (80) 1 (20) 0.018MDc 47 36 (76.6) 11 (23.4)PDd 21 8 (38) 13 (62)

Lymph node status (174)No metastasis 46 19 (41.3) 27 (58.7) 0.042Metastasis 128 88 (68.8) 40 (31.2)

TNM staging (178)I 31 13 (41.9) 18 (58.1) 0.039II–IV 147 92 (62.6) 55 (37.4)

NOTE: The values in parenthesis are expressed in percentage.aStatistical analysis was conducted using Student t test. P < 0.05 was considered statically significant.bWell differentiated.cModerately differentiated.dPoorly differentiated.

Sonic Hedgehog in Gastric Cancer Metastasis

www.aacrjournals.org Cancer Res; 71(22) November 15, 2011 7063




Next, we evaluated Shh-mediated cell migration and inva-sion to determine whether it resulted from elevated levels ofMMPs, which are well-documented ECM-degradingenzymes, the activity of which is associated with tumorinvasiveness. RT-PCR analysis showed that N-Shh signifi-cantly increased the mRNA levels of MMP-9 in AGS andMKN-28 cells (Fig. 2B). In contrast, abrogating the Shhpathway by cyclopamine or Gli1 siRNA reduced inductionof MMP-9 by N-Shh. Similar results were obtained in West-ern blot (data not shown) and gelatin zymographic analysisof conditioned medium obtained from AGS and MKN-28cells (Fig. 2B). As expected, overexpression of MMP-9 siRNAreduced the ability of Shh to stimulate migration and

invasion of those cells compared with the cells expressingcontrol siRNA (Supplementary Fig. S3B and Fig. 2B). Impor-tantly, in the tail vein experiments,MMP-9 knockdown in thecells expressing Shh also resulted in significant reduction inthe lung metastasis compared with cells expressing only Shh(Fig. 2C). Collectively, these findings suggest that Shh-medi-ated induction of EMT or MMP-9 contributes to Shh path-way-related migration and invasive ability of gastric cancercells.

The metastatic spread of tumors through lymphatic vesselsto local or regional lymph nodes is an early event in metastaticdisease for a variety of human tumors, and nodal metastasisis considered to be dependent on the tumor-induced

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Figure 1. Shhpathway plays a crucial role in invasiveness andmetastatic potential of gastric cancer cells in vivo and in vitro.A, the gastric cancer samplesweredivided into 2 groups according to the lymph node status (LN�, no metastasis; LNþ, metastasis), and then association between expression of ShhmRNA orGli1mRNAandgastric cancermetastasiswas observed byRT-PCR in the left. The correlation between the levels ofShhmRNAandGli1mRNAwas assessedwith theSpearman rank correlation coefficient (right). B, the5-year survival ratewascalculated in 147patientswithgastric cancer at stages II to IV relative to theexpression of theShh (Shhpositive vs. negative, left) or stages (stage I vs. II–IV, right) by theKaplan–Meiermethod.P¼0.002 andP¼0.02were determined bythe log-rank test, respectively. C, gastric cancer cells were treatedN-Shh (0.5mg/mL) alone or togetherwithKAAD-cyclopamine (cyclopamine, 10mmol/L) andthen assayed for their motility (top) and invasiveness (bottom) by wound healing and Matrigel invasion assay as described in the Materials and Methodssection. D, the cells transfectedwithGli1 siRNAor nonspecific (Con) siRNAwere then evaluated in a performancemigration (top) and invasion assay (bottom).Relative mRNA level was normalized to the corresponding b-actin mRNA expression. Bars represent the SD of 3 independent experiments conducted intriplicate. �, P < 0.05; ��, P < 0.01. E, the metastatic potential of either AGS cells expressing Shh/or control vector (Con) or SNU-16 cells Shh shRNA/ornonspecific control shRNA (con shRNA) was assessed by measuring colonization of lung surfaces following subcutaneous (s.c.) or tail vein injection (i.v.) ofthese cells into athymic mice. Seven weeks later, the mice were sacrificed and the number of mice with metastatic lesions to lung was counted.

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lymphangiogenesis (12–14). These findings suggest that Shhsignaling may influence the metastatic capacity of gastriccancer to lymph nodes by inducing lymphangiogenesis. Ourresults showed that cells treated with N-Shh displayed signif-icantly higher levels of expression of Lyve-1, Podoplanin, andVEGF-D than cells treatedwithoutN-Shh (Fig. 2D). The levels ofexpression of thesemolecules in cells treatedwith cyclopamineor Gli1 siRNA were significantly lower than those in the cells

treated with control vehicle. Next, we evaluated the responsesin terms of migration and tubule formation in HMVECs, bothof which are essential steps for lymphangiogenesis in vivo. Asshown in Fig. 2E, N-Shh significantly increased themotility andtubule-like structure formation of HMVECs compared withcontrol vehicle. In contrast, pretreatment of HMVECs withcyclopamine completely inhibited Shh-induced cell migrationand the generation of tubular networks.

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Figure 2. Effect of Shh signaling pathway on EMT, MMP-9 activity, and lymphangiogenesis. A, the cells were treated with N-Shh (0.5 mg/mL) alone,cyclopamine (10 mmol/L), nonspecific Con siRNA, or Gli1 siRNA for 72 hours. Western blotting with anti–E-cadherin or anti-Snail was carried out. B, left, thecells treatedwithN-Shh (0.5 mg/mL) alone or togetherwith cyclopamine (10mmol/L) for 24 hourswere collected andRNAextractwas subjected to quantitativereal-time PCR using specific primers for MMP-9 (top). The conditioned culture media of the same set of samples were subjected to zymography assay forMMP-2andMMP-9 (bottom).Bars represent theSDof 3 independent experiments conducted in triplicate. †,<0.05, ††,<0.01, comparedwith cells treatedwithcontrol vehicle. �, P < 0.05, ��, P < 0.01, compared with cells treated with N-Shh. Right, AGS cells were transfected with MMP-9 siRNA or nonspecific(Con) siRNA for 24 hours, followed by stimulation with N-Shh (0.5 mg/mL). Right, themigration and invasive ability were evaluated by wound healing (top) andtheMatrigel invasion assay (bottom) after 24 hours. The data are expressed as the means of 3 independent experiments� SD. �, P < 0.05; ��, P < 0.01. C, themetastatic potential of cells expressing Shh, control vector (Con), MMP-9 shRNA, or nonspecific control shRNA (con shRNA) was assessed by measuringcolonization of lung surfaces following tail vein injection (i.v.) of these cells into athymic mice. Seven weeks later, the mice were sacrificed and the number ofmice withmetastatic lesions to lung was counted. D, AGS cells were treated with N-Shh (0.5 mg/mL) alone or a combination of either cyclopamine (10 mmol/L)orGli1 siRNAandRNAextractwas subjected toRT-PCRusing the indicatedprimers.RelativemRNA levelwas normalized to the correspondingb-actinmRNAexpression. E, HMVECs were treated with N-Shh (0.5 mg/mL) alone or together with cyclopamine (10 mmol/L), and then the migration ability was evaluatedby wound healing assay after 24 hours (top panel). Cyclopamine-pretreated HMVECs were seeded on the top of growth factor-reducedMatrigel and treatedwith or without N-Shh (0.5 mg/mL) for 9 hours (bottom panel).






PI3K/Akt signaling pathway is required for Shh-mediated EMT, MMP-9 activity, and lymphangiogenesisin gastric cancer cells

Recently, numerous studies have shown that PI3K/Aktpathway is an essential component of Shh signaling andinhibition of PI3K/Akt pathway with the PI3K/AKT inhibitor,LY294002, results in impairment of Shh morphogenic activity(15–17). Western blot analysis showed that the activation ofPI3K-dependent Akt phosphorylation in AGS, MKN-28, andSNU-216 cells was induced as early as 30 minutes after N-Shhstimulation (Supplementary Fig. S4A and Fig. 3A). However,blockade of Shh signaling by cyclopamine completely reversedthe enhancement of Akt phosphorylation (Fig. 3A). In vitro Aktkinase assay showed that N-Shh treatment enhanced GSK-3a/b phosphorylation, whereas the cyclopamine blocked N-Shh–activated an increase in the phosphorylated GSK-3a/b (Sup-plementary Fig. S4B). Notably, nonphosphorylatable Aktmutant (DN-Akt) significantly reduced the ability of N-Shh tostimulate activity of the Gli-luciferase reporter (Fig. 3A). Sim-ilar results were also obtained in real-time RT-PCR analysis for

Gli1 target gene (Ptch andGli1; data not shown). In addition, inspite of the presence of added N-Shh, treatment of these cellswith LY294002 or DN-Akt suppressed Shh-mediated Akt acti-vation, as well as cell migration and invasion (SupplementaryFig. S4C and Fig. 3B). Similar inhibition was observedwith Akt1siRNA (Supplementary Fig. S4D).

We subsequently examined whether or not Shh-inducedEMT, MMP-9 activity, or lymphangiogenesis is mediatedthrough the PI3K/Akt pathway in gastric cancer cells. Immu-nofluorescence staining andWestern blotting showed that theloss of E-cadherin and gain of Snail by N-Shh was completelyreversed by LY294002 or DN-Akt (Fig. 3C and SupplementaryFig. S5A). RT-PCR analysis and gelatin zymography alsoshowed that MMP-9 activity and the expression of lymphan-giogenic factor (Lyve-1, Podoplanin, and VEGF-D) werecompletely abolished by exposure of the cells to LY294002 orDN-Akt, even in the presence of N-Shh (Fig. 3D and E). AftertreatingHMVECswithN-Shh andLY294002, we also found thatLY294002 abrogated the Shh-induced cellularmotility and tubeformation of HMVECs (Fig. 3E). Together, these results showed

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Figure 3. Shh promotes tumormetastasis and lymphangiogenesisvia PI3K/Akt pathway followed byupregulation of EMT and MMP-9 inthe in vitro model. A, the cells weretreated with N-Shh (0.5 mg/mL)alone or together with cyclopamine(10 mmol/L), then Western blottingwith anti–p-Akt, anti-Akt, or anti–b-actin was carried out (left). AGScells were cotransfectedwith 0.5 mgof Con vector or 0.5 mg of DN-Aktand 0.5 mg of Gli-Luc reporterplasmid or 0.1 mg of pRL-TK vector.Twenty-four hours aftertransfection, cells were treated withN-Shh (0.5 mg/mL) or controlvehicle. After incubating for 24hours, pGli1-Luc activity wasmeasured by luciferase assay(right). The amount of luciferaseactivity was normalized to Renillaluciferase activity. B, the cells werepretreated with LY294002(20 mmol/L) for 30 minutes (left) ortransfected with either controlvector (Con), CA-Akt, or DN-Akt(right), followed by stimulation withN-Shh (0.5 mg/mL). The migrationand invasive ability of those cellswere evaluated by wound healing(top) and the Matrigel invasionassay (bottom) after 24 hours.

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that PI3K/Akt signaling pathway is necessary for Shh-mediatedEMT,MMP-9 activity, and lymphangiogenesis in gastric cancercells.

Shh signaling pathway promotes metastatic potential ofgastric cancer cells by activation of the PI3K/Aktpathway in vivoThe effects of Akt activation on Shh-mediated metastatic

capacity of human gastric cancers were further investigatedusing a mouse xenograft model. The metastatic potential ofAGS cells expressing vector only, Shh, DN-Akt, or Shh/DN-Aktwas evaluated by measuring colonization of lung surfaces bytumor cells after subcutaneous or tail vein injection. The tumorgrowth did not reveal significant differences among all 4 tumorxenografts (Supplementary Fig. S5B). In both the tail vein andsubcutaneous experiments, the Shh cells expressing DN-Aktalso showed a significant reduction in the number of lungmetastatic colonies or mice with lung metastasis comparedwith cells expressing only Shh (Fig. 4A). In addition, expressionof those EMT- andmesenchymal-related proteins was evident-

ly decreased in Shh/DN-Akt tumor cells compared with Shhtumor cells (Fig. 4B). As expected, the levels of expression ofMMP-9, Lyve-1, Podoplanin, and VEGF-D mRNA were alsosignificantly decreased in the Shh/DN-Akt tumor cells than inthe Shh tumor cells (Fig. 4C and D). Collectively, these resultsrevealed that the Shh signaling pathway promotes metastaticpotential of gastric cancer cells by sequential activation of thePI3K/Akt pathway, followed by upregulation of the EMTpathway and MMP-9.

Correlation between Shh expression and Akt activity,EMT, or lymphangiogenesis in gastric tumor specimens

Clinicopathologic analysis revealed that phospho-Akt,E-cadherin, or MMP-9 expression levels in tumors with lymphnodemetastaseswas higher than that in tumorswithout lymphnode metastases (E-cadherin: P ¼ 0.024; MMP-9: P ¼ 0.019;Phospho-Akt: P ¼ 0.056; Supplementary Table S3). Therefore,we examined whether or not expression of phospho-Akt,E-cadherin, or MMP-9 was associated with expression of Shhin tumors with lymph node metastases. Phospho-Akt and Shh

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Figure 3. (Continued) C, protein was extracted from SNU-216 cells and resolved by SDS-PAGE and then Western blotting with anti–E-cadherin or anti-Snailantibodies was carried out. b-Actin was used as a control. D, the levels of expression (top) and enzymatic activity of MMP-9 (bottom) in AGS cells weredetermined byRT-PCRandgelatin zymography, respectively. E, AGScellswere pretreatedwith LY294002 (20mmol/L) for 30minutes, followed by stimulationwith N-Shh (0.5 mg/mL), and then RNA extract was subjected to RT-PCR using specific primers for the indicated primers (left). Relative mRNA level wasnormalized to the corresponding b-actinmRNA expression. LY294002-pretreated HMVECs were cultured on Matrigel-coated 24-well plates with or withoutN-Shh stimulation and then assayed for their motility and tube formation bywound hearing (middle) and in vitro tube formation assay (right), respectively. In allcases, the data are expressed as the means of 3 independent experiments �SD. �, P < 0.05; ��, P < 0.01.






had a weak correlation with regard to the level of expression(r ¼ 0.126), but the correlations were not statistically signif-icant (P¼ 0.183). Of greater importance, the level of expressionof Shh also showed a strong correlation with that of E-cadherin

orMMP-9 (E-cadherin: P < 0.001, r¼�0.447; MMP-9: P < 0.001,r ¼ 0.515; Table 2 and Supplementary Fig. S6A). Finally, tofurther evaluate the role of Shh signaling with respect to lymphnode metastasis and lymphangiogenesis in human gastrictumor specimens, we explored the correlation of expressionof Shh with lymphangiogenesis in 174 gastric cancer speci-mens. To quantitatively analyze the degree of tumor-associ-ated lymphangiogenesis, tumor LVDwas determined by quan-tification of D2-40 (a specific antibody for podoplanin-positivevessels; Supplementary Fig. S6B). Increased LVD was signifi-cantly associated with lymph node metastasis (P < 0.001) andtumor-node-metastasis (TNM) staging (P¼ 0.032, Supplemen-tary Table S4). Importantly, higher rates of LVD were observedin patients in the Shh-positive groups, whereas low rates ofLVD were found in patients in the Shh-negative groups (P <0.001; Table 3). Furthermore, Shh expressions were also sig-nificantly correlated with the presence of lymphatic vesselinvasion (LVI, P¼ 0.014). We also found that the phospho-Akt-positive group had a higher density of tumor lymphatic vesselscompared with the phospho-Akt-negative group (P ¼ 0.051).Taken together, these data suggested that the Shh signalingpathway regulates lymphangiogenesis via amechanism involv-ing the PI3K/Akt pathway.

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Figure 4. Shh promotes tumor metastasis and lymphangiogenesis via PI3K/Akt pathway followed by upregulation of EMT andMMP-9 in the in vivomodel. A,AGS cells expressing vector only (Con), Shh, DN-Akt, or Shh/DN-Akt were injected s.c. or i.v. into athymic mice. Mice were sacrificed 7 weeks after theinoculation of the cells, and tumor colonies on the lung surfaces were counted macroscopically (left). The chart of right panel represents the incidenceof lung metastasis in mice that received intravenous tail injections of each cell. The expressions of EMT marker (N-cadherin, vimentin, and Snail protein; B),MMP-9mRNA (C), and lymphangiogenic marker (Lyve-1, Podoplanin, and VEGF-D; D) in subcutaneous tumors were evaluated viaWestern blot and RT-PCRanalysis, respectively. In all cases, the data are expressed as the means of 3 independent experiments �SD. �, P < 0.05, ��, P < 0.01.

Table 2. Relationship between expression ofShh, E-cadherin, and MMP-9 in lymph nodemetastatic gastric cancer

Numberof cases

Shh P

Positive Negative

E-cadherinPositive 28 10 (35.7) 18 (64.3) <0.001Negative 100 73 (73) 27 (27)

MMP-9Positive 97 68 (70.1) 29 (29.9) <0.001Negative 31 15 (48.4) 16 (51.6)

NOTE: The values in parenthesis are expressed inpercentage.

Yoo et al.





Discussion

Abundant clinical and pathologic evidence indicates thatShh signaling is involved in tumorigenesis (18–20). Althoughsome clinical studies have suggested that Shh expression intumors is correlated with aggressiveness of gastric cancer(19, 21), none of these studies clearly indicate the exact roleof Shh signaling in themetastatic progression of gastric cancerand its underlying mechanism. In this study, we investigatedthe functional role of Shh signaling in gastric cancer progres-sion using multiple experiments, both in vitro and in vivo. Theresults of metastatic examination in vitro and in vivo showedthat Shh functions as an inducer of gastric cancer metastasisand is correlated with clinical stage, lymph node metastasis,and prognosis in patients with gastric cancer.The molecular mechanism by which Shh signaling reg-

ulates cell motility and invasiveness is an intriguing ques-tion. Studies using constitutively activated and dominantnegative Akt mutants have shown that PI3K regulation ofShh signaling is mediated through Akt signaling (15). There-fore, this activated Akt is suggested to be involved in Shhsignal transduction. Furthermore, Akt activation is also bestunderstood in the context of a major cascade stimulatingEMT-related pathway, cell migration, and invasion in vari-ous human cancers. Our results showed that during migra-tion, Akt was strongly phosphorylated at Ser473 by Shh, andthe phosphorylation of this serine residue has previouslybeen found to be involved in the motility and invasiveness oftumor cells (22, 23). Furthermore, according to our finding,Akt modulates Shh signaling through the enhancement ofGli-dependent transcriptional activity. This agrees with thefinding that PI3K/Akt regulates Gli2 transcriptional activityby PKA and GSK-3b phosphorylation in LIGHT cells (15).Although the role of PI3K/Akt in modulating the Shh path-way varies between cell types, Shh-induced modification ofPKA and GSK-3b through AKT might be critical for Shh-mediated EMT and metastasis in gastric cancer. However,

the precise mechanisms controlling Shh/Gli-mediated EMTand metastasis through PI3K/Akt signaling need to beelucidated in detail. In addition, the mechanism wherebyShh signaling induces PI3K/Akt activities is also yet to bedefined. Interestingly, elevation of Akt is a response to theloss of thePTEN, which occurs frequently in metastaticgastric cancer (24–26). Therefore, downregulation of PTENmediated by Shh might contribute to enhance the activity ofAkt and thereby further promote tumor progression.Although PI3K/Akt activities are essential for ligand-depen-dent Shh signaling in metastatic function of gastric cancercell, activation may not be sufficient to drive Gli activation.Recent studies have suggested that oncogenic K-RAS isdirectly involved in activation of the Shh pathway(20, 27, 28). Activating RAS mutations have been frequentlydetected in malignant cells in many gastric cancer patients.Therefore, oncogenic RAS, through the RAF/MEK/MAPKpathway, may play an important role in Shh-mediatedmetastatic potential.

Lymph node metastasis is a common occurrence in cancer,and lymphatic vessels have been reported as an importantroute contributing to metastasis of solid tumors (12–14). Ourdata showed that most patients without lymph node metas-tasis were in the Shh-negative group, whereas patients withlymph node involvement were Shh-positive group. Despite itsclinical relevance, surprisingly little is known about themechanisms of Shh leading to lymphatic metastasis in gastriccancer. Several studies have indicated that one possiblemolecular mechanism promoting metastasis to regionallymph nodes is tumor-induced lymphangiogenesis. Our find-ings indicate Shh signaling, via a mechanism involving thePI3K/Akt pathway, promotes tumor lymphangiogenesis and,consequently, lymph node metastasis, which is in good agree-ment with recent findings (5). However, it is not clear whetheror not Shh signaling plays a role in the formation of thelymphatic vessels and whether or not Shh-mediated lymphan-giogenesis is accompanied by enhanced lymphatic metastasis.This problem may be solved with the establishment of a newmodel of lymphangiogenesis being able to capture the entirenetwork of lymphatics and spread of cancer cells from the siteof inoculation.

Our observations improve our understanding of the mech-anism by which Shh signaling activation occurs as it relates tothe metastatic behavior of gastric cancer. Investigating theexpression of Shh and its related proteins in gastric cancer cellsmay be helpful for determining the therapeutic strategy andantagonists of this signaling may be useful in the clinicalsetting to suppress the spread of tumors through the lymphaticsystem.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Dr. Jae Hun Cheong for providing Gli1 promoter luciferaseconstructs.

Table 3. Relationship between expression ofShh and LVD or LVI in lymph node metastaticgastric cancer

Numberof cases

Shh

Positive Negative P

LVDPositive 98 63 (64.3) 35 (33.7) <0.001Negative 76 42 (55.2) 34 (44.8)

LVIPositive 75 50 (66.7) 26 (33.3) 0.014Negative 99 56 (56.6) 43 (43.4)

NOTE: The values in parenthesis are expressed in percent-age.Abbreviation: LVI, lymphatic vessel invasion.






Grant Support

This work was supported by the Korea Science and Engineering Foundation(KOSEF) grant funded by the Korea government (MEST; no. 2010-0000637), theBrain Korea 21 Project of the Ministry of Education and Human ResourcesDevelopment, Republic of Korea, a Korea University Grant, and a grant of theKoreaHealthcare technology R&DProject, Ministry for Health,Welfare& FamilyAffairs, Republic of Korea (A084953).

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 indicate thisfact.

Received April 21, 2011; revised September 7, 2011; accepted September 22,2011; published OnlineFirst October 5, 2011.

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2011;71:7061-7070. Published OnlineFirst October 5, 2011.Cancer Res Young A. Yoo, Myoung Hee Kang, Hyun Joo Lee, et al. Pathway in Gastric CancerLymphangiogenesis via Activation of Akt, EMT, and MMP-9 Sonic Hedgehog Pathway Promotes Metastasis and

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