Stem Cell Marker Nestin Is Critical for TGF-b1-Mediated ... · 7/2/2013 · 2013 AACR. Introduction The incidence of pancreatic ductal adenocarcinoma (PDAC) is rising to the ﬁfth

Signal Transduction

Stem Cell Marker Nestin Is Critical for TGF-b1-MediatedTumor Progression in Pancreatic Cancer

Huei-Ting Su1, Ching-Chieh Weng1, Pi-Jung Hsiao2,3, Li-Hua Chen1, Tzu-Lei Kuo1, Yu-Wen Chen1,Kung-Kai Kuo2,4,5, and Kuang-Hung Cheng1,2

AbstractThe stem cell marker nestin is an intermediate filament protein that plays an important role in cell integrity,

migration, and differentiation. Nestin expression occurs in approximately one third of pancreatic ductaladenocarcinoma (PDAC), and its expression strongly correlates with tumor staging and metastasis. Little isknown about the mechanisms by which nestin influences PDAC progression. Here, nestin overexpression inPDAC cells increased cell motility and drove phenotypic changes associated with the epithelial-mesenchymaltransition (EMT) in vitro; conversely, knockdown of endogenous nestin expression reduced the migration rateand reverted cells to a more epithelial phenotype. Mouse xenograft studies showed that knockdown of nestinsignificantly reduced tumor incidence and volume. Nestin protein expression was associated with Smad4 statusin PDAC cells; hence, nestin expression might be regulated by the TGF-b1/Smad4 pathway in PDAC. Weexamined nestin expression after TGF-b1 treatment in human pancreatic cancer PANC-1 and PANC-1shSmad4 cells. The TGF-b1/Smad4 pathway induced nestin protein expression in PDAC cells in a Smad4-dependent manner. Moreover, increased nestin expression caused a positive feedback regulator of the TGF-b1signaling system. In addition, hypoxia was shown to induce nestin expression in PDAC cells, and the hypoxia-induced expression of nestin is mediated by the TGF-b1/Smad4 pathway. Finally, the antimicrotubuleinhibitors, cytochalasin D and withaferin A, exhibited anti-nestin activity; these inhibitors might be potentialantimetastatic drugs. Our findings uncovered a novel role of nestin in regulating TGF-b1-induced EMT. Anti-nestin therapeutics may serve as a potential treatment for PDAC metastasis. Mol Cancer Res; 11(7); 1–12.�2013 AACR.

IntroductionThe incidence of pancreatic ductal adenocarcinoma

(PDAC) is rising to the fifth leading cause of cancer-related mortality. The condition is associated with thepoorest prognosis among all gastrointestinal cancers, witha 5-year survival rate less than 4% (1, 2). A main reason forthis extremely poor prognosis is the cancer's tendency toinvade adjacent tissues and metastasize to regional lymphat a relatively early stage. Patients with PDAC frequently

experience disease recurrence with a high incidence ofhepatic metastases, peritoneal dissemination, and lymphnode as they undergo resection (3). These features under-score the pressing need to develop new therapeutic strat-egies specifically aimed at preventing invasion and metas-tasis in PDAC, to improve the survival rates and clinicaloutcomes.Tumormetastasis is a complex phenomenon involving the

extension of cell processes through the formation and/ordisassembly of focal adhesion complexes and polymerizedactin filaments (4, 5). Epithelial–mesenchymal transition(EMT) is a fundamental cellular process necessary for tumormetastasis (6, 7). EMT can be induced by a wide variety ofstimuli, such as inflammatory cytokines [including inter-leukin (IL)-1, TNF-a, and TGF-b1] or hypoxia (8–10).TGF-b1 has been described as a major EMT-inducingsoluble factor in a range of tumors; this pleiotropic cytokinemediates the transcriptional regulation of downstreamEMT-related genes through the formation of the Smad2/3–Smad4 complex (11–14). During PDAC carcinogenesis,TGF-b-1 signaling is constitutively associated with tumorprogression in advanced PDAC (15, 16).When treated withTGF-b1, many pancreatic carcinoma cell lines show amorphologic alteration in EMT, losing epithelial markers

Authors' Affiliations: 1Institute of Biomedical Science; 2National Sun Yat-SenUniversity–KaohsiungMedicalUniversity JointResearchCenter,Nation-al Sun Yat-Sen University; 3Division of Endocrinology and Metabolism,4Cancer Center, and 5Division ofHepatobiliopancreatic Surgery,Departmentof Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

H.-T. Su and C.-C. Weng contributed equally to this work.

Corresponding Author: Kuang-Hung Cheng, Institute of BiomedicalSciences, National Sun Yat-Sen University, Kaohsiung, Taiwan 80424.Phone: 886-7-5252000, ext. 5817; Fax: 886-7-5250197; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-12-0511

�2013 American Association for Cancer Research.

MolecularCancer

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and gaining mesenchymal ones. This result confirms thatTGF-b is one of the most prominent EMT inductors(13, 17, 18).Tumor cells undergo EMT by losing epithelial char-

acteristics such as cell adhesion, cell–cell contact, andremodeling of the cytoskeleton; simultaneously, theyacquire mesenchymal phenotypes that facilitate invasionand migration to distal organs (19, 20). The cytoskeletonorganizes other constituents of the cell, maintains cellshape, and is responsible for the internal movement of cellorganelles and cell locomotion (21). Three major types offilaments comprise the cytoskeleton: actin filaments,microtubules, and intermediate filaments, which areknown to be crucial modulators of the structural organi-zation of cells and cell migration (22). Nestin is a largeintermediate filament protein of class VI; it displays ashort N-terminus and an unusually long C-terminus,which interacts with other cellular components, micro-tubules, and other intermediate filaments (includingvimentin or desmin) to form heterodimers and mixedpolymers. The long carboxy-terminal portion of nestinprotrudes from the filament body and might function as alink or cross-bridge between the intermediate filamentsand microtubule (23, 24).Nestin was first described as a functional stem cell

marker in embryonic and adult central nervous system(CNS) stem cells and is used extensively to identifyprogenitor cells in several tissues (25). In addition, recentresearch has characterized nestin as a cancer stem cellmarker in various cancers; these include brain tumors,ovarian, head and neck, prostate, and pancreatic cancers(26–30). Nestin has also been implicated in pancreasdevelopment and pancreatic cancer progression (30). Onein vitro study reported that nestin-positive cells derivedfrom isolated pancreatic islets were able to differentiateinto pancreatic endocrine or exocrine cells (31, 32).Moreover, Kawamoto and colleagues suggested that nestinexpression in pancreatic cancer cells might be associatedwith nerve and stromal invasion (30). Matsuda andcolleagues reported that nestin played an important rolein pancreatic cancer cell migration, invasion, and metas-tasis by selectively modulating the expression of actin andcell adhesion molecules (33). Furthermore, in a previousstudy, nestin-expressing cells were reported to be the cellsof origin for pancreatic intraepithelial neoplasia (PanIN)lesions (34). However, before our research, no study hadattempted to determine the underlying mechanisms bywhich nestin promotes metastasis in pancreatic cancer.In this study, we first determined that nestin expression

may correlate with Smad4 status in PDAC. Furthermore, wefound that nestin expression was necessary to mediate EMTinduced by TGF-b1 and to promote PDAC migration andinvasion. More important, nestin promoted TGF-b1/Smadsignaling by increasing the expression of TGF-b1, TbR1,and TbR2, which led to constitutively active TGF-b1/Smadsignaling in nestin-positive PDAC cells. These results pro-vided a molecular-level understanding of nestin action inpancreatic cancer metastasis.

Material and MethodsCell culture, RNA isolation, and cDNA synthesisImmortalized normal human pancreatic ductal cells

(HPDEC) were obtained from Dr. Tsao (Ontario CancerInstitute, Princess Margaret Hospital, University HealthNetwork, Toronto, Ontario, Canada) (35). The HEK293Tand human PDAC cell lines were obtained from the sourcesdescribed previously (36, 37). We administered TGF-b1 (5ng/mL), hypoxia, and inhibitors treatment according topreviously described procedures (37). The RNA isolationand cDNA synthesis from the cell lines were also conductedaccording to previously described procedures (38, 39).

Lentivirus production and shRNA for gene knockdownThe plasmids required for short hairpin RNA (shRNA)

lentivirus production were purchased from the NationalRNAi Core Facility, Academia Sinica (Taipei, Taiwan).The pLKO.1-shRNA vectors used for knockdown ofnestin and smad4 were TRCN0000014729 (Nestin) andTRCN0000010323 (Smad4). The pLKO.1-shEGFP con-trol plasmid was TRCN00000-72190 (EGFP). Lentivirusproduction and infection were conducted according to apreviously described protocol (39).

Western blotting and immunofluorescenceWestern blotting and immunofluorescence analyses were

conducted as previously described (36, 39). Primary anti-bodies used were nestin antibodies (Sigma); smooth muscleactin (SMA, ab5698); N-cadherin (ab12221); TGF-b1(ab66043; Abcam); E-cadherin (sc-8426); vimentin (sc-32322); Smad4 (sc-7966); CD44 (sc-18849; Santa CruzBiotechnology); and mouse anti-b-actin (Sigma). E-cad-herin (#4065), Smad2 (#3122) and p-Smad2 (#3104; CellSignaling).

Quantitative RT-PCR analysisQuantitative real-time PCR (qRT-PCR) amplification

and results of the DCT measurements were conducted asdescribed in previously published methods (39). The primersequences were listed in Supplementary Table S1.

Transient transfections and luciferase reporter assaysEGFP-C1-nestin expression vector and pNesPE-Luc (with

50 flanking 4 kb promoterþ intron II) reporter construct wereprovided as a gift from Dr. Jing [Shanghai Institutes for Bio-logical Sciences (SIBS), Chinese Academy of Sciences, Shang-hai, China]. Transient transfections and luciferase reporterassays were conducted as previously described (39–41).

In vitro migration/invasion assayFor Transwell migration and the wound closure assays,

cells were pretreated with 0.02% (0.2 mg/mL)mitomycin Cand followed previously published protocols (37, 39).

In vivo xenograft mouse studySpecific pathogen-free male C.B17/lcr-SCID mice, 8

weeks of age, were purchased from BioLASCOTaiwan Co.,Ltd.. Mice were treated according to the institutional

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guidelines for the care and use of experimental animals. Thedetailed protocol for establishing subcutaneous injection ofxenograft tumor models in severe combined immunode-ficient (SCID) mice was conducted using our previouslydescribed method (39), and orthotopically grown pancreaticcancer xenografts were conducted according to a previouslypublication (42).

Mice strains for genetically engineered mouse models ofPDAC, necropsy, histopathology, andimmunohistochemistryThe mouse strains included in this study were PDX-Cre

and LSL-KrasG12D, which were obtained from NationalCancer Institute Mouse Models of Human Cancers Con-sortium (NCI-MMHCC). All experiments were carried outon a more than 96.9% C57BL/6J background. Mice geno-typing, tissue processing, and IHC staining were conductedas previously described (36, 43). The IHC analysis wasconducted as previously described using anti-Smad4 (SantaCruz Biotechnology), anti-vimentin (Santa Cruz Biotech-nology), anti-nestin (Sigma), anti-nestin antibody (clone10C2; Abcam), anti-E-cadherin (Santa Cruz Biotechnolo-gy), anti-CD44 (Santa Cruz Biotechnology), and anti-TGF-b (Santa Cruz Biotechnology). Images were captured using aCarl Zeiss. Axioskop 2 plus microscope (refs. 36, 44).

Human samplesHuman PDAC tumor tissues were collected during

surgical resections at Chung-Ho Memorial Hospital ofKaohsiung Medical University (KMU; Kaohsiung, Tai-wan). The surgically resected tumor samples were imme-diately fixed in 10% formalin and embedded in paraffin

(FFPE) using standard protocols (39). Sections from eachspecimen were confirmed by pathologists and gradedhistologically. Representative FFPE human PDAC blockswere further selected for analysis of nestin. The studyprotocol was approved by the Institutional Review Boardat KMU (39).

Statistical analysisData are presented as mean � SEM. The data measured

on continuous scale were statistically analyzed using Studentt test and categorical data were subjected to x2 test. A P valueof less than 0.05 was considered significant.

ResultsNestin expression in the normal versus malignantpancreasTo study the role of nestin in the pancreas and malignant

formation, we analyzed nestin expression in the pancreasusing IHC and immunofluorescence analyses. Consistentwith some previous reports, our findings revealed thatnestin-positive cells were observed mainly in the cytoplasmof a limited number of vascular endothelial cells, intrapan-creatic nerves, and within the islets of normal (mouse andhuman) pancreatic tissues.Weak or no detectable expressionof nestin was evident in the pancreatic acinar cells (Fig. 1Aand B; Supplementary Fig. S1; refs. 44, 45). We also stainedtissues from a set of mouse and human malignancies. Inthe PanIN lesions of Pdx-Cre LSLKrasG12Dmice, IHC andimmunofluorescence analyses indicated an increase innestin protein expression in the cytoplasm. PanIN lesionsin Pdx-Cre KrasG12Dmice showed strong positive stainingfor nestin, which implied that nestin expression was induced

Figure 1. Nestin expression innormal (human and mouse)pancreas, tumors, and PDAC cells.A, IHC staining for nestin in theendothelial cells and islets ofpancreas fromwild-typemice (a) andPdx-Cre Kras mouse PanIN (b). Notepositive staining for nestin in normalislets and in PanINs duct. c,Immunofluorescent detection ofnestin (green) expression in Pdx-CreKras mouse PanIN. Cell nuclei werecounterstained with 40,6-diamidino-2-phenylindole (DAPI; blue). B, IHCanalysis of nestin expression innormal human pancreas (a) andPanINs (b). c, Immunofluorescenceanalysis of nestin (green) expressionin human PanIN. Images were takenat �100 and �400 magnifications.C, Western blot analysis of nestinand Smad4 expressions in humannormal pancreatic duct and PDACcells.

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from an early stage of pancreatic malignant transformation(Fig. 1A).To verify this finding, we further conducted IHC and

immunofluorescence analyses to evaluate the nestin ex-pression in human PDAC tissues. Nestin expression wasobserved mainly in the cytoplasmic region of malignantductal structures; the same pattern has been described formouse PDAC tissues (Fig. 1A and B). Next, we furtherevaluated nestin expression in 6 human PDAC cell linesby Western blot analysis. Nestin was expressed in variousamounts in the pancreatic cancer cell lines tested, with thehighest expression observed in wild-type Smad4-expres-sing HPDEC, PANC-1, and MiaPaCa-2 cells; relativelylow expression was observed in Smad4-mutated or nullPDAC cells (AsPC-1, BxPC3, and CFPAC-1). As shownin Fig. 1C, nestin was highly expressed in Smad4-positivePDAC cells. Our findings indicated that signaling throughSmad4 may regulate nestin expression in PDAC cells.Because Smad4 is a central signaling molecule of TGF-b–induced signal transduction, we hypothesized that nes-tin expression status might depend on TGF-b1/Smad4signaling.

Nestin promoted tumor cellmigration and induced EMTin PDACWe investigated whether nestin played a direct functional

role in facilitating PDAC cell migration. Three independentPDAC cell lines, namely PANC-1, AsPC-1, and MiaPaCa-2, were selected to examine the expression and role of nestinin regulating PDAC cell migration. We induced the over-expression of nestin in MiaPaCa-2 or AsPC-1 cells bytransfecting the cells with the EGFP-C1-nestin expressionvector to overexpress nestin protein (see Materials andMethods). Nestin knockdown was conducted using nestinlentiviral shRNA in PANC-1 cells, and stable cell linesresistant to puromycin were selected. Cell migration andinvasion were further evaluated by using the wound-scratchmethod and SPL insert Transwell system using a track-etched polycarbonate membrane, pretreated by coating withtype I collagen (2 mg/mL). As shown in Fig. 2A, the PANC-1 nestin shRNA cells displayed significantly lower closurecompared with the control cells (P < 0.05). We thenextended our experiments to examine the overexpression ofnestin in other PDAC cell lines (AsPC-1, MiaPaCa-2, andBxPC3). The results confirmed that nestin promoted PDACcell in vitro migration.Next, in vitro invasion was measured by the number of

tumor cells that penetrated the collagen-coated membraneof the insert and attached themselves to the bottom well.Invasion was further assessed by counting the number ofcell colonies under a microscope after crystal violet stain-ing. Our findings showed that the invasion of PANC-1nestin shRNA cells was attenuated compared with thecontrol (Fig. 2B). Similar results were obtained fromnestin-overexpressing MiaPaCa-2 cells compared withvector control cells; these findings confirmed that over-expression of nestin increased PDAC cell invasive poten-tial in vitro.

The EMT process is necessary for cell migration; there-fore, we attempted to uncover the molecular mechanismsinvolved in regulating nestin-activated PDAC cell migra-tion. We examined the effects of nestin expression in theregulation of EMT. First, we used qRT-PCR analysis toexamine EMT-related markers in PANC-1 nestin shRNAcells and GFP control cells; the examined markers includ-ed mesenchymal markers of vimentin, SMA, N-cadherin,epithelial marker of E-cadherin, and nestin mRNA levels.The results of our qPCR analysis showed that knockdownof nestin increased the expression of E-cadherin mRNAand decreased the levels of vimentin, SMA, and N-cad-herin mRNA (Fig. 2C). Similar results were obtainedusing nestin-overexpressing MiaPaCa-2 cells comparedwith control. Western blotting was conducted to detectthe protein expressions of EMT-related markers inPANC1 nestin shRNA cells and nestin-overexpressingMiaPaca-2 cells versus controls. The Western blot analysisresults were similar to the preceding analysis; protein levelsof vimentin, SMA, and N-cadherin had decreased signif-icantly and E-cadherin protein had increased in PANC-1nestin shRNA knockdown cells compared with the con-trol cells. Similar results were obtained using nestin-over-expressing MiaPaCa-2 and AsPC-1 cells, compared withcontrols (Fig. 2D).

Nestin expression is induced by TGF-b1–Smad4pathwayOur previous findings confirmed that nestin is involved

in the EMT process. The TGF-b1–Smad signal is knownto be a potent inducer of EMT during carcinogenesis.Nestin expression may be associated with Smad4 status inhuman PDAC cells. To verify the interactions betweennestin and TGF-b/Smad signaling, we selected PANC-1(Smad4-positive) and AsPC-1 (Smad4 null) PDAC celllines for further study. Our findings indicated that theTGF-b/Smad pathway induced nestin expression, down-regulated E-cadherin, and increased protein expression ofvimentin, SMA, and N-cadherin (Fig. 3A). Most impor-tant, the TGF-b pathway was found to induce nestinprotein expression, and this induction was mediated bySmad4-dependent activation (Fig. 3B and C). Further-more, administration of SB525334 (20 mmol/L; Sigma-Aldrich) inhibited the TGF-b pathway in PANC-1 cells,and led to a significant reduction in nestin expression,as shown in the Western blot analysis (SupplementaryFig. S2).To confirm these findings, we used the nestin reporter

construct pNesPE-Luc to verify that nestin transcriptionalactivity was increased by the TGF-b1/Smad4 pathway inPANC-1 Smad4-positive cells to confirm that Smad4 wasnecessary for TGF-b–induced nestin expression, and 3TP-TGF-b1 reporter construct was used to determine theactive TGF-b1/Smad pathway (Fig. 3D). Rapid tumorgrowth frequently led to hypoxia, which in turn influ-enced signaling events within the tumor cell and eventu-ally activated an angiogenic program and EMT. Previousstudies have shown that the TGF-b pathway can be

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induced by hypoxia, which can further drive TGF-b–induced EMT by several pathways. To examine wheth-er hypoxia stimulated TGF-b1 expression in PDAC cells,we incubated cells under normoxic (N) or hypoxic (2%O2) conditions to detect TGF-b1 protein expression.Under the hypoxic condition, we found a marked increasein TGF-b1 protein expression and an increased expressionof nestin in PANC-1 cells (Fig. 3E). We also found thathypoxia-inducible factor-1 inhibitor, LW6 (10 mmol/L;Merck Millipore) reduced the expression of nestin inPANC-1 cells by qRT-PCR analysis (Supplementary Fig.S3). We speculated that hypoxia induced an increase inthe expression of TGF-b1 cytokine, leading to the induc-tion of nestin expression under hypoxic conditions. Hyp-oxia-induced TGF-b1 protein expression was abolishedafter knockdown of Smad4 or nestin expression in PANC-1 cells (Fig. 3F).

Nestin is involved in TGF-b1/Smad4 signaling to induceEMT in PDACTo test if nestin was involved in TGF-b–induced cell

migration, we examined the function of nestin in TGF/Smad signaling. Western blot analyses were conducted todetect the phosphorylation levels of Smad2 in PANC-1nestin shRNA clonal line and control. Western blotanalyses were also conducted in nestin-overexpressingMiaPaCa-2 cells compared with vector controls, to deter-mine the activation status of TGF-b1/Smad signaling. Asshown in Fig. 4A, the level of phospho-Smad2 (p-Smad2)was reduced several-fold in PANC-1 nestin shRNA cellscompared with eGFP vector controls, whereas the levels oftotal Smad2 remained unchanged. In contrast, weobserved an increase in the p-Smad2 level in nestin-overexpressing MiaPaCa-2 cells compared with controls(Fig. 4A).

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Figure 2. Nestin increased PDAC cell migration and induced EMT in PDAC. A, representative image of nestin induced increases in cells migration ofthe indicated cell lines bywound-healing assays. Original magnification�400. B, transmembrane invasion assays for GFP control and nestin-overexpressingMiaPaCa-2 cells, or for PANC-1 nestin shRNA cells and eGFP shRNA control. The y-axis represents the fold change in a number of stable poolcells invaded andmigrated, comparedwith control cells. Bars represent SD. �,P < 0.01 versus control, x2 test. C, qPCR analysis of the relativemRNA levels ofvarious EMT markers in a GFP control versus various nestin-overexpressing MiaPaCa-2 cells, or in PANC-1 shRNA control and PANC-1 shNestin cells(mean�SD;n¼3;P<0.01). D,Western blot analysis comparing variousEMT-relatedmarker expressions either inGFPcontrols versusnestin-overexpressingBxPC-3, AsPC-1 (b) and MiaPaCa-2 cells, or in an shRNA eGFP versus PANC-1 nestin shRNA stable transfected cells (a). Total lysates were analyzedfor protein levels of nestin, E-cadherin, N-cadherin, vimentin, SMA, and b-actin.






To explore the mechanism by which nestin expressionpromotes the elevation of p-Smad2 levels, we determined theexpression level of TGF-b cytokine byWestern blot analysisand qRT-PCR analysis. Our findings indicated that knock-down of nestin expression in PANC-1 cells resulted inreduced TGF-b1expression at the mRNA and the corre-sponding protein levels (Fig. 4B andD). Consistent with thisfinding, we also detected an increase in TGF-b1 proteinexpression in nestin-overexpressing MiaPaCa-2 (Smad4-proficient) and AsPC-1 (Smad4-deficient) cells by Westernblot analysis (Fig. 4B and Supplementary Fig. S4). Further-more, we investigated whether TGF-b activity was alteredafter knockdown or overexpression of nestin. Luciferasereporter assays with various TGF-b/Smad signaling respon-sive reporter constructs were used to confirm that nestinmodulated the activation of the TGF-b/Smad signalingpathway in PANC-1 cells (Fig. 4C). Our findings indicatedthat nestin knockdown resulted in the downregulation of theTGF-b1/Smad pathway. A similar result was observed in anincrease of the TGF-b1/Smad signaling luciferase reporteractivity in nestin-overexpressing MiaPaCa-2 cells (Fig. 4C).We also examined the effects of nestin on the levels of TbRreceptors mRNA expression. qRT-PCR analysis revealedthat depletion of nestin results in a decrease of TbR1a and

TbR2a mRNA levels in PANC-1 nestin shRNA cells com-paredwith control cells, whereas SMAD7mRNA levels werenot increased (Fig. 4D). A similar result confirmed anincrease of the TbR1a and TbR2a mRNA levels in nes-tin-overexpressing MiaPaCa-2 cells, clearly showing thatnestin is involved in regulation of TGF-b1/Smad4 signalingin PDAC cells (Fig. 4D).Previous studies have indicated that TGF-b interacts with

the Wnt pathway to modulate EMT at various stages ofdevelopment.Ourfindings showed a significant reduction intranscription activity of the Wnt transcriptional reporterSuper TOPFlash andWnt downstream gene CD44 reporterin PANC-1 nestin shRNA cells, compared with controls(Fig. 5A), which may explain why we next examined theconsequence of knockdown of nestin expression on theregulation of EMT markers expression after TGF-b1 treat-ment. Western blotting revealed that the protein level of themesenchymal marker vimentin was significantly diminishedand the epithelial marker of E-cadherin expression was highin PANC-1 nestin shRNA cells, irrespective of whether theywere treated with TGF-b1 overnight (Fig. 5B). This mayexplain why we observed that PANC-1 nestin shRNA cellsgrow in piled-up structure of irregular round cells (Fig. 5C).In contrast, TGF-b1 treatment of mock-transfected

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Figure 3. TGF-b1–Smad4 signaling regulated nestin expression. A, immunoblot analysis of nestin, TGF-b pathway, and EMT-related proteins expressionin indicated cell lines. B, Western blot analysis of nestin and vimentin expression in PANC-1 cells after TGF-b1 treatment. C, representative Western blotanalysis depicting the elevation of nestin expression in Smad4-positive PANC-1 cells treated with or without TGF-b1. D, luciferase activity driven bythe nestin promoter was assayed in PANC-1 cells after TGF-b1 treatment, compared with PANC-1 smad4 shRNA cells treated with or withoutTGF-b1. The TGF-b1/Smad–responsive reporter 3TP-luciferase (3TP-Luc) was used to confirm TGF-b1 treatment. E, total lysate proteins isolated fromMiaPaCa-2 and PANC-1 cells under normoxia and hypoxia treatments were subjected to Western blotting for nestin, vimentin, TGF-b1, and b-actin.F, immunoblot analysis of TGF-b1 from PANC-1 nestin shRNA and Smad4 shRNA cells compared with control eGFP shRNA cells.

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wild-type PANC-1 cells led to an expression profile sugges-tive of EMT (Fig. 5B). Similar results were obtained whenwe used immunofluorescence staining to detect the expres-sion and cellular localization of nestin and to profile theEMT markers expression in nestin shRNA and controlshRNA PANC-1 cells after treatment with or withoutTGF-b1 (Fig. 5C and D).

Nestin knockdown reduces in vivo tumorigenicity andinhibited TGF-b1/Smad4–mediated EMT in PANC-1xenograft tumorsNestin has been associated with mitotic spindle organi-

zation and mitosis, and cytokinesis plays a key role in cellproliferation (46). We thus investigated the function ofnestin in PDAC tumorigenesis in vivo. We subcutaneouslyinjected the left and right flanks of SCID mice with stablePANC-1 transfected with nestin shRNA or EGFP shRNAcontrol cells, to confirm the in vivo effect of nestin onpancreatic carcinogenesis. After 2 months, mice that hadbeen injected with the EGFP control or nestin shRNA-transfected cells showed the expected tumor formation. Weconclude that PANC-1 nestin shRNA cells display reducedtumorigenic properties in vivo, compared with the control

groups. Tumor sizes and weights were also 3- to 5-foldsmaller than in controls (Fig. 6A). Hematoxylin and eosin(H&E) staining and IHC assays were conducted to examinethe xenografts in the SCIDmice generated from the injectedPANC-1 cells (nestin shRNA vs. EGFP shRNA control; Fig.6B).Histopathologic analyses of representative tumor sections

showed that the wild-type PANC-1 tumors generally com-prised spindle-shaped cells with a slender cytoplasm, oftenwith elongated cytoplasmic processes. In contrast, PANC-1nestin shRNA tumor cells appeared less dense, with a moreflattened morphology resembling cobblestones. Analysis ofnestin expression by immunohistochemistry showed areduced cytoplasmic expression in PANC-1 nestin shRNAtumors. In these tumors, E-cadherin staining was modestlyincreased but the expression of vimentin decreased, whereasit was high in the wild-type controls (Fig. 6B). Moreover,CD44 staining resulted in decreased protein staining of themembrane and cytosol when nestin was knocked down. Wealso observed that nestin shRNA PANC-1 tumors displayeddecreased TGF-b1 expression and an inhibition of TGF-b1/Smad signaling, with greater Smad4 staining observed in thecytoplasm, compared with the control groups (Fig. 6B).

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Figure 4. Nestin activates TGF-b1 signaling and induces an upregulation of TGF-b1 and its receptors expression in PDAC cells. A, Western blot analysis oftotal Smad2 and p-Smad2 protein levels in protein lysates isolated from the eGFP control and nestin shRNA-transfected PANC-1 cells (a), or in nestin-overexpressed MiaPaCa-2 cells and GFP control (b). B, Western blot analysis of TGF-b1 in nestin shRNA transfected PANC-1 cells (a) and nestinoverexpressed MiaPaCa-2 cells (b) and their corresponding controls. C, activation of TGF-b1 signaling pathways was measured by different TGF-b1responsive reporter constructs (3TP-Luc; SBE4-LucandPAI-1-Luc) in nestin shRNA-transfectedPANC-1 cells (a) andnestin-overexpressedMiaPaCa-2 cells(b) and their corresponding controls. Mean�SEM (n¼ 3). �,P < 0.05. D, qRT-PCR analysis of TGF-b1, TbR1a, TbR2, and Smad7mRNA levels from total RNAextracted rom the eGFP control and nestin shRNA-transfected PANC-1 cells (a), or in nestin-overexpressed MiaPaCa-2 cells and GFP control (b). Resultsshow the mean of 3 experiments � SEM and are normalized to the GAPDH gene.






Identification of antinestin drugs with in vitro andin vivo antimetastatic activityWe selected several potential intermediate filament–tar-

geted inhibitors to test if these compounds might displayantinestin effects or anti-invasive (antimetastatic) propertiesin PDAC. We selected 4 antitumor drugs, namely cisplatin(a DNA-damaging agent), paclitaxel (antimicrotubule com-pound), cytochalasin D (antiactin agent), and withaferin A(an antimetastatic agent; refs. 47, 48). For the cytotoxicologyexperiments, we first determined the IC50 values of eachcompound and applied a dose 5-fold below the IC50 value, toeliminate a possible cytotoxic effect on proliferation and toinvestigate the drug's antinestin activity in vitro. The cyto-toxic drug cisplatin was used as a positive control forapoptotic analysis. To investigate the effect of the inhibitorson cellular nestin, cells were treated with 0.5 mmol/Lcisplatin, paclitaxel, withaferin A, and cytochalasin D forovernight, and then cell extracts were analyzed by Westernblotting. The results showed that both withaferin A andcytochalasin D treatment were able to inhibit the nestinprotein level (Fig. 7A). This conclusion was also confirmedby immunofluorescence analysis (Fig. 7B).Finally, we investigated whether withaferin A and cyto-

chalasin D antinestin inhibitors displayed antimetastaticability in vitro using in vitro Transwell migration assays.

The invasion of PANC-1 cells treated with 0.5 mmol/L ofcytochalasin D and withaferin A had decreased signifi-cantly compared with control or cisplatin groups (P <0.01). We used quantitative analysis to determine thenumber of PANC-1 cells treated with cytochalasin D orwithaferin A that migrated to the lower side of the bottomwells using 0.5% crystal violet staining. The results indi-cated a 3-fold reduction in cell migration activity com-pared with the cisplatin or control groups (Fig. 7C). Forin vivo metastasis analysis, we first used a commonly usedTranswell cell migration assay to enrich high metastaticPANC-1 cells and repeated 6 times for enrichment ofnestinhigh subpopulation of PANC-1 cells (SupplementaryFig. S5A). After the final round of enrichment, migratedcells were observed with high-level expression of nestin ascompared with the parental PANC-1 cell line by Westernblot analysis (Supplementary Fig. S5B). PANC-1nestinhigh cells were then orthotopic injected in to thepancreas of SCID mice for in vivo metastasis analysis todetermine the in vivo effects of cytochalasin D and with-aferin A inhibitors on our orthotopic PDAC xenograftmodels. Our results confirmed the cytochalasin D orwithaferin A treatment suppresses PDAC tumor develop-ment and metastasis formation in vivo (SupplementaryFig. S5C).

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Figure 5. Nestin regulates Wnt signaling and is crucial for TGF-b1/Smad4–induced EMT. A, reporter assays were conducted using TOP/FOP reporters (Wntsignaling responsive reporter system) andCD44-luc reporter in the eGFP control andPANC-1-shNestin RNA cells. Data shown represent themean�SD for 3independent experiments (�, P < 0.05). B, analysis of EMT markers by Western blot analysis using vimentin and E-cadherin (E-cad) antibodies inPANC-1 shNestin knockdown and control cells, in the presence or absence of TGF-b (5 ng/mL) treatment overnight. C, morphologic and cytochemicalproperties of PANC-1 shNestin knockdown (iii and iv) and control cells (i and ii). PANC-1 shRNAcontrols cells showmoreepithelial cobblestoneappearance (i).PANC-1 nestin shRNA knockdown cells exhibit a sheet-like appearance, and also tend to pile up to form irregular-shaped colonies (iii). E-cadherin(green) staining is increased at the cellular junctions in PANC-1 shNestin knockdown cells (16) compared with control cells (ii). Images were takenat�100 (i and iii) and�1,000 magnifications (ii and iv). D, immunofluorescent staining was conducted for E-cadherin (green) and vimentin (red) expression inPANC-1 nestin shRNA and control cells that treated with or without TGF-b1 for 24 hours. Nuclei were visualized with 40,6-diamidino-2-phenylindole(DAPI; blue). Images were taken at �400 magnifications.

Su et al.





DiscussionNestin, a class VI intermediate filament, similarly to other

eukaryotic intermediate filaments, connects the 3 compo-nents of the cytoskeleton and coordinates changes in the celldynamics (49, 50). Nestin reportedly interacts with vimen-tin or desmin to form heterodimers or polymers; thesestructures provide cellular mechanostructural support,maintain cellular membranes, and restrict organelles to alimited area (51).Nestin has also been found to contribute tothe disassembly of vimentin during mitosis, to regulate theproapoptotic cyclin-dependent kinase 5, and to modulatemitosis-associated cytoplasmic reorganization after becom-ing phosphorylated on Thr316 by Cdc2 kinase (52). Onerecent study indicated that nestin may modulate actin andcell adhesion molecules to control pancreatic cancer cellmigration, invasion, and metastasis (33). However, thesignal mechanisms regulating nestin expression duringmigration remain unknown.Cell migration is initiated or induced by activation of

receptors that trigger remodeling of the cytoskeletonand reordering of subcellular organelles. EMT is increas-ingly recognized as a mechanism, whereby cells in pri-mary noninvasive tumors acquire properties essential formigration and invasion (6, 53). The EMT process isregulated by the secretion of several factors in the tumormicroenvironment, such as IL-6 or TGF-b. The TGF-b1signaling is modulated by a number of biochemicalinteractions, including the association of Smad2/3 withvarious transcriptional cofactors to modulate Smads com-plex stability and possibly also to activate certain non-

Smad pathways (8, 13). Studies using mutant TGF-bRIconstructs that are defective in binding Smads but whichretain signaling by mitogen-activated protein kinases haveshown that Smad pathway is likely to be involved in theEMT process (54).Our previous study on animal models of PDAC sug-

gested that TGF-b1 signaling is activated in PanINs andadvanced cancers; this finding was consistent with obser-vations in human PDAC samples. On the basis of thefindings from our previous research and the current study,we speculate that the excessive TGF-b1 cytokine observedaround PanIN lesions results in increased nestin expres-sion. Subsequently, under this late-stage tumor burden,hypoxia greatly increases TGF-b1 stimulation of nestinexpression, which in turn facilitates tumor cell invasionand metastasis to distant sites. Our engineered mousemodels suggested that active TGF-b1 is required fortumorigenicity of certain subsets of PDAC characterizedby expression of wild-type Smad4 and to acquire a highlyinvasive and undifferentiated phenotype and more met-astatic behavior (36). Moreover, the increased TGF-b1observed in the blood of patients with PDAC is correlatedwith poor prognosis and survival, suggesting that TGF-b1is an important marker for cancer progression (55).In summary, our findings highlight the need for further

functional studies on the role of nestin in TGF-b1–inducedEMT and tumor metastasis. We confirmed that nestinoverexpression induces the EMTof PDAC cells.We showedthat TGF-b1 induced the expression of nestin in PDAC cellsand that this induction was predominantly mediated by the

Figure 6. Effect of nestinknockdown on in vivo tumorigenesis.A, nestin knockdown significantlydecreased in vivo tumorigenesis inPANC-1 cells. Tumor weights andvolumes were measured afterautopsy. Mean � SEM (n ¼ 6).�, P < 0.01; x2 test. B, H&E andIHC staining analyses of the in vivoeffects of nestin knockdown onexpression patterns of EMT-relatedmarkers (E-cadherin, vimentin, andCD44) and TGF-b1 signalingmolecules (Smad4 and TGF-b1).H&E images taken at �100magnification (small inserts are�400). The photographs showrepresentative sections of nestin,Smad4, TGF-b1, E-cadherin,vimentin, and CD44 expressionlevels in PANC-1 nestin shRNA andcontrol groups, respectively. Imageswere taken at �400 magnifications.

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Smad4-dependent pathway. Overexpression of nestin wasshown to increase the Smad2 phosphorylation level, leadingus to hypothesize that nestin might function as a positiveregulator of TGF-b1 signaling. This notion was supportedby evidence that the overexpression of nestin induced TGF-b1 and the expression of its receptors at the RNA and proteinlevels through the Smad4-dependent pathway. Thus, nestin-positive cells apparently use an autocrine positive feedbackloop to maintain the constitute activation of TGF-b signal-ing in PDAC.This process might play an important role in support-

ing the distant metastasis of PDAC in vivo. Three mainfactors would explain this phenomenon, namely (i) theoverexpression of nestin maintains PDAC cells in amesenchymal-like morphology with a high metastaticability; (ii) nestin increases the cellular production ofTGF-b, which acts on surrounding cells to release furthermatrix metalloproteinases and secrete VEGF; it alsofacilitates the degradation of extracellular matrix and

promotes tumor progression; and (iii) excess productionand/or activation of TGF-b1 by nestin-positive PDACcells may mediate the suppression of immune responsesby suppressing IL-2 production, inhibiting CTL activa-tion, and inhibiting Toll-like receptor expression (56,57); these factors help nestin-positive tumor cells escapehost immune surveillance and gain a further advantage ingrowth.This study reports novel findings of a positive cross-

regulatory loop between nestin-TGF-b1/Smad and EMT(Fig. 7D). This nestin–TGF-b1 relationship creates apositive feed-forward loop that controls PDAC tumormetastasis. Overall, our findings provide preliminary evi-dence and support the hypothesis that nestin might play arole in autonomous tumor metastasis promotion by auto-logous activation of TGF-b1/Smad signaling in PDAC.This hypothesis could explain how nestin-mediated signalpathways contribute to the cancer stem cell phenotype,and would imply that nestin is critical to maintain the

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Figure 7. Antimicrotubule inhibitors withaferin A and cytochalasin D targeted nestin protein expression and inhibited cell invasion in PDAC cells. A, PANC-1cells and MiaPaCa-2 cells under treatment with different inhibitors for 24 hours. Cell lysates were analyzed for nestin expression by Western blottingwith a specific antibody. B, immunofluorescence analysis was conducted onPANC-1 cells to detect alterations in nestin proteins after 24-hour treatment withdimethyl sulfoxide (DMSO), cisplatin (Cis), withaferin A (WFA), and cytochalasin D (CD). Nuclei were visualized with 40,6-diamidino-2-phenylindole(DAPI; blue). Imageswere taken at�400magnifications. C, in vitro Transwell invasion assays in PANC-1 andMiaPaCa-2 cells treatedwith cisplatin, withaferinA, or cytochalasin D, and their respective controls. Bar graphs show the relative invasive ability of PANC-1 and MiaPaCa-2 cells treated with DMSO,cisplatin,withaferinA, or cytochalasinD. Thequantitative resultswere normalized in relation toDMSOcontrol groups (mean�SD;n¼3;P<0.01). D, proposedmolecular mechanism of nestin mediated TGF-b-1–induced EMT and plays positive feedback regulator of TGF-b1/Smad signaling after hypoxicstimulus. Increased nestin expression led to enhanced TGF-b1 or its receptors expression to further support an autocrine TGF-b1 signaling cascade. Pac,paclitaxel.

Su et al.





progression of tumor metastasis. Although our study doesnot describe the entire molecular scenario for distantmetastasis in PDAC, it nevertheless makes a major con-tribution to the theory of a metastatic PDAC network andoutlines a strategy here for the treatment of invasion andmetastasis by PDAC.

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

Authors' ContributionsConception and design: H.-T. Su, C.-C. Weng, L.-H. Chen, K.-H. ChengDevelopment of methodology: C.-C. Weng, L.-H. Chen, K.-H. ChengAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): C.-C. Weng, P.-J. Hsiao, L.-H. Chen, T.-L. Kuo, K.-K. Kuo, K.-H.ChengAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): H.-T. Su, C.-C. Weng, L.-H. Chen, K.-H. ChengWriting, review, and/or revision of the manuscript: H.-T. Su, L.-H. Chen, K.-H.Cheng

Administrative, technical, or material support (i.e., reporting or organizing data,constructing databases): P.-J. Hsiao, L.-H. Chen, K.-H. ChengStudy supervision: L.-H. Chen, Y.-W. Chen, K.-K. Kuo, K.-H. Cheng

AcknowledgmentsThe authors thank Drs. Naihe Jing (nestin expression and promoter reporter

constructs), Mark Perella (CD44-Luc reporter), Xi He (TOP/FOP reporter), and SamThiagalingam (3TP-Luc, SBE4-Luc and PAI-luc reporters) for providing cell lines andreagents.

Grant SupportThis work was supported by the grant NSC 98-2320-B-110-003-MY3 (to K.-H.

Cheng) from the National Science Council, Taiwan ROC, grant DOH100-TD-C-111-002 (to K.-K. Kuo) from the Department of Health, Executive Yuan, ROC, andgrant of National Sun Yat-sen University–Kaohsiung Medical University JointResearch Center (to P.-J. Hsiao and K.-H. Cheng).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received August 28, 2012; revised March 1, 2013; accepted March 4, 2013;published OnlineFirst April 3, 2013.

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Published OnlineFirst April 3, 2013.Mol Cancer Res Huei-Ting Su, Ching-Chieh Weng, Pi-Jung Hsiao, et al. Tumor Progression in Pancreatic Cancer

1-MediatedβStem Cell Marker Nestin Is Critical for TGF-

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