expression of snail in pancreatic cancer promotes metastasis and chemoresistance

Journal of Surgical Research 141, 196–203 (2007)

Expression of Snail in Pancreatic Cancer Promotes Metastasisand Chemoresistance

Tao Yin, M.D.,* Chunyou Wang, M.D.,*,1 Tao Liu, M.D.,* Gang Zhao, M.D.,*Yunhong Zha, M.D.,† and Ming Yang, M.D.*

*Pancreatic Center, Department of General Surgery, †Department of Neurology, Union Hospital, Tongji Medical College, HuazhongUniversity of Science and Technology, Wuhan City, Hubei Province, People’s Republic of China

Submitted for publication June 17, 2006

doi:10.1016/j.jss.2006.09.027

Background. Pancreatic cancer is a dreadful malig-nancy. Because of its tendency to metastasis and itsresistance to chemotherapy, the prognosis remainspoor. Snail is a transcriptional factor that endows ep-ithelial cells with migratory and anti-apoptotic abili-ties. Its expression has been demonstrated in manytumors. We hypothesized that Snail may be expressedin pancreatic cancer, and it may confer invasive andchemoresistant properties.

Materials, methods, and results. We immunohisto-chemically examined Snail expression in pancreaticcancer and found that it was expressed in 20 of 56(36%) samples of pancreatic cancer. The Snail expres-sion had a close correlation with lymph node invasionand distant metastasis. After transfecting Snail cDNAinto pancreatic cancer cell line Panc-1, we found thatSnail triggered overt epithelial to mesenchymal tran-sitions in Panc-1 cells. The tumor invasive ability invitro was evaluated using a transwell invasive cham-ber. Snail dramatically promoted the invasive abilityof Panc-1 cells. Chemosensitivity of Panc-1 cells to5-fluorouracil or gemcitabine after Snail transfectionwas assayed by MTT cell proliferation assay. Overex-pression of Snail enhanced the chemoresistance to5-fluorouracil of gemcitabine at different dosages.Moreover, Snail-transfected Panc-1 cells producedmore spontaneous metastasis than parental untrans-fected cells after orthotopically injected into the pan-creas of nude mice.

Conclusion. Snail is expressed in pancreatic cancer;it confers enhanced invasive ability and chemoresis-

1 To whom correspondence and reprint requests should be ad-dressed at Pancreatic Center, Department of General Surgery,Union Hospital, Tongji Medical College, Huazhong University ofScience and Technology, Jiefang Avenue 1277#, Wuhan City, HubeiProvince, 430022 P.R. China. E-mail: Chunyouwang52@yahoo.
com.cn.
1960022-4804/07 $32.00© 2007 Elsevier Inc. All rights reserved.

tance to pancreatic cancer cells. Snail may be a markerfor predicting the malignancy of pancreatic cancer. Fur-ther therapy target to Snail may be of great benefitto pancreatic cancer patients. © 2007 Elsevier Inc. All rights

reserved.

Key Words: pancreatic cancer; Snail; metastasis;chemoresistance.

INTRODUCTION

Pancreatic cancer is a highly malignant carcinoma,which is a leading cause of cancer-related deaths in thedeveloped world [1, 2]. Despite improvement in diag-nosis and treatment, the prognosis remains poor. Mostpatients diagnosed with pancreatic cancer cannot re-ceive curative surgical resection. Even after surgicalresection, the 5-year survival rate is only about 20%[3]. One major characteristic of pancreatic cancer is itsearly systemic dissemination and its extraordinary lo-cal tumor progression [4, 5]. At the time of diagnosis, alarge percentage of pancreatic cancer patients havefound locally invasion or distant metastasis. Besides,this malignancy is resistant to nearly all of the existingchemotherapeutic agents. To improve the prognosis ofpancreatic cancer, there is an urgent need to elucidatethe molecular mechanisms underlying the malignantbehaviors of pancreatic cancer.

Metastasis is a multistep process in which cancercells must decrease adhesion with their neighbors, ac-quire the ability to migrate, and invade to surroundingtissues. This has often been correlated with loss ofepithelial properties and acquisition of fibroblast-likephenotype. The phenomenon is referred to as epithelialto mesenchymal transitions (EMT) in which polarizedepithelial cells are converted into motile mesenchymal
cells. EMT is crucial in embryo development, wound

197YIN ET AL.: SNAIL IN PANCREATIC CANCER

repair, and tissue remolding, in which it is under tightregulation. While in cancer, the tightly regulated pro-cess is out of control. This is often used by cancer cellsto have unlimited migrating abilities [6, 7]. Moreover,EMT may be a mechanism for cancer cells to survive inthe aberrant environment: fetal rat hepatocytes thatunderwent EMT can resist the apoptotic effects in-duced by TGF-�1 [8]. EMT has been shown to occur inthe progression of many malignancies such as breastcancer, melanoma, and pancreatic cancer [9–11].

Snail is a transcriptional factor that is required formesoderm and neural crest formation during meta-zoan development. It has been identified as a key reg-ulator for EMT during embryonic development [12, 13].Snail is overexpressed when ectodermal epithelial cellstransit into mesenchymal cells [14, 15]. Moreover,Snail has been implicated in the progression of cancerin which it converts cancer cells with migratory prop-erties by triggering EMT. Ectopic expression of Snail inepithelial cancer cells led to repression of epithelialmarkers, activation of mesenchymal markers, and ac-quisition of migratory ability [16]. Besides, Snail maypromote metastasis through conferring resistance tocell death [17]. Overexpression of Snail has been dem-onstrated in the progression of many malignanciessuch as breast cancer, colon cancer, gastric cancer, andmelanoma [18–21]. However, expression of Snail inpancreatic cancer and its significance has not beenelucidated. We hypothesized that Snail may be expressedin pancreatic cancer and may endow pancreatic cancercells with invasive and chemoresistant abilities.

To test our hypothesis, we immunohistochemicallyexamined the Snail expression in pancreatic cancertissues. Besides, we transfected the Snail-encoding se-quence into pancreatic cancer cell line Panc-1 andtested the changes of biological behavior after trans-fection. We found that Snail enhanced the invasiveability and chemoresistance of Panc-1 cells.

MATERIALS AND METHODS

Tissue Samples and Immunohistochemistry

Fifty-eight paraffin-embedded tissue samples of pancreatic cancerwere obtained from the Pancreatic Center of Union Hospital, TongjiMedical College, Huazhong University of Science and Technologybetween 2000 and 2004. The tumors were staged according to thepathologic tumor-node-metastasis staging system [22]. There were14 cases of well differentiation, 18 cases of moderate differentiation,and 24 cases of poor differentiation. The paraffin-embedded tissuesamples were consecutively cut into 4-�m-thick sections and weremade for hematoxylin and eosin staining and for immunohistochem-ical studies.

The immunohistochemical staining was performed with LAB-SA(Labeled streptavidin biotin) method using a Histostain-SP kit(Zymed, San Francisco, CA). The primary antibody was polyclonalanti-Snail antibody (Santa Cruz Biotechnology, Santa Cruz, CA)(dilution, 1:100). Briefly, after deparaffinization and rehydration, thesections were boiled in 0.01 mol/L citrate buffer (pH 6.0) for 15 min
in a microwave oven. Then they were immersed in 3% hydrogen
peroxide in methanol for 10 min to block the endogenous peroxidaseactivity and incubated with serum blocking solution to block nonspe-cific binding. The sections were incubated with primary antibody at4°C overnight, followed by incubation with biotinylated second anti-body and enzyme conjugate. Staining was developed by adding dia-minobenzidine chromogen. The sections were counterstained withhematoxylin. The results of immunostaining were assessed by twopathologists without knowing the patients’ clinicopathological de-tails. Snail expression in the tumors was graded according to theproportion of positive cells. Positive Snail expression was defined if�5% of cancer cells were stained.

Cell Culture and Transfection of Snail

The pancreatic cancer cell line Panc-1 was obtained from ATCC(Manassas, VA, USA) and was maintained in Dulbecco’s modifiedEagle’s medium (DMEM) supplemented with 10% fetal bovine se-rum, penicillin (100 U/mL), and streptomycin (100 �/mL) at 37°Cwith 5% CO2. The Snail expression plasmid pCAGGS-HA-Snail waskindly provided by Masayuki Ozawa (Kagoshima University, Japan)and was transfected into Panc-1 cells using Lipofectamine 2000(Invitrogen, Carlsbad, CA). The transfection procedure was carriedout according to the protocol. After transfection for 72 h, stabletransfectants were selected by adding 600 �g/mL of G418 (Gibco-BRL, Gaithersburg, MD) for 3 weeks and maintained with 200�g/mL of G418. Successful transfection was confirmed by Westernblot using anti-hemagglutinin (HA) antibody. Two resistant cloneswere selected for further study. They were named Panc-1 Snail1 andPanc-1 Snail2, respectively.

Immunoblotting Analysis

Western blotting was performed according to the procedure de-scribed previously [23]. Briefly, pancreatic cancer cells were culturedand lysed in a lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1%Triton X-100, 0.1% NP40, 0.5% sodium deoxycholate, 1 mM phenyl-methylsulfonyl fluoride, and gabexate mesilate). Equal amounts ofprotein were loaded onto SDS-polyacrylamide gels and transferredonto a nitrocellulose membranes. The membranes were blocked with5% nonfat milk powder in Tris-buffered saline for 1 h and incubatedwith primary antibodies against vimentin (1:600; Sigma, St. Louis,MO), E-cadherin (1: 500; Santa Cruz Biotechnology), and HA (1:800;Lab Vision, Fremont, CA). After washing with Tris-buffered salinecontaining 0.1% Tween 20, the membrane was incubated with sec-ondary, horseradish peroxidase-coupled antibodies (Pierce, Rock-ford, IL) and visualized using enhanced chemiluminescence (Pierce).�-Actin was used as internal controls.

In Vitro Invasion Assay

The invasive assay was performed as described using transwellcell culture chambers (8 �M pore size polycarbonate membrane;Costar, Cambridge, MA) [24]. The membrane was coated with Ma-trigel (BD Biosciences, Bedford, MA). Cells were resuspended inDMEM with 0.5% fetal bovine serum (FBS) to a concentration of 1 �106/mL. The upper chamber was loaded with 100 �L of cell suspen-sion and the lower chamber was loaded with 600 �L of DMEM with10% FBS. After incubation for 24 h at 37°C with 5% CO2, the filterwas fixed with 4% paraformaldehyde and stained with 4 g/L trypanblue (Sigma). The cells on the upper side of the filter were wiped offwith a cotton swab. The cells invaded to the undersurface of themembrane were counted under a microscope. Eight microscopicfields were randomly selected for cell counting. Each assay was donein triplicate. The cells were counted blinded to whether the cell was
transfected.

198 JOURNAL OF SURGICAL RESEARCH: VOL. 141, NO. 2, AUGUST 2007

Cell Proliferation Assay

The effect of Snail on proliferation was demonstrated using MTTassay. In brief, cells were seeded into 96-well plates at 1 � 104 perwell. After incubation for 24, 48, 72, or 96 h, 20 �L of MTT reagent(5 mg/mL; Sigma) was added to each well. Plates were incubated at37°C for 4 h and then the supernatant was removed and 150 �L ofdimethyl sulfoxide (Sigma) was used to dissolve the resultantformazan crystals. The absorption was read at 490 nm using aspectrophotometer. The MTT assay was performed in triplicate.

Cell Viability Assay

The chemoresistance of Panc-1 cells transfected with Snail wasevaluated with MTT cell proliferation assay. Cells were plated in96-well plates with a density of 10,000 cells/well. Three groups weredivided as follows: blank group (no cells); control group (no drug);and experiment group (5-fluorouracil (5-FU); Xudong Haipu Phar-maceutical Inc., Shanghai, China; or gemcitabine; Eli Lilly and Co.,Lily France SA, France). After incubation for 24 h, the cells wereincubated with medium containing various concentrations of either5-FU or gemcitabine for 72 h. Then 5 mg/mL of MTT (Sigma) was

FIG. 1. Immunohistochemical staining of Snail in pancreaticcancer tissue (original magnification, �200). (A) Positive expressionof Snail in pancreatic cancer (low differentiation). (B) Negative ex-pression of Snail in pancreatic cancer (moderate differentiation).
(Color version of figure is available online.)
added to each well. After incubation for 4 h, the supernatant wasreplaced with 150 �L of dimethyl sulfoxide (Sigma). The absorption(A) was read at 490 nm using a spectrophotometer. The survival rateof cells was calculated as a percentage as follows: Aexperiment/Acontrol �100%. Every concentration had three replicate wells and each groupwas repeated three times.

Nude Mice Experiment

The significance of Snail expression in vivo was studied by inoc-ulation of cells into the nude mice. Panc-1 cells transfected withSnail gene or parental Panc-1 cells (2 � 106 cells/mouse) were ortho-topically injected into the pancreas of nude mice (n � 6 for eachvariant). The mice were killed after 1 month following the operation.The viscera were removed and fixed in 4% paraformaldehyde,paraffin-embedded, and sectioned for Hematoxylin-Eosin staining.The National Research Council’s guide for the care and use of labo-ratory animals was followed in our experiment.

Statistical Analysis

The relationship between Snail expression and clinicopathologicalfactors was examined with the �2 test. Differences between othergroups were evaluated using the Student’s t-test and Fisher’ exacttest. P � 0.05 was considered significant.

RESULTS

Expression of Snail in Pancreatic Cancer

We immunohistochemically examined the Snail ex-pression in pancreatic cancer tissues. The positiveSnail staining was mainly found in the nucleus andcytoplasms of a proportion of cancer cells (Fig. 1). Ac-cording to the criteria described earlier, 20 of 56 pan-creatic cancers (36%) were positive for Snail staining,and 36 pancreatic cancers (64%) were negative. More-

TABLE 1

Relationship Between Snail Expression andClinicopathological Factors

Parameters Categories

Snail expression

PNegative Positive

Tumor extent (pT) T1�2 8 2 0.221T3�4 28 18

Node involvement (pN) N0 19 3 0.005*N1 17 17

Distant metastasis (pM) M0 35 13 0.002*M1 1 7

Histological grading (G) High 10 4 0.809Moderate 11 7Low 15 9

Notes. The slides were scored for Snail expression by determiningthe percentage of cancer cells that had positive staining. The rela-tionship between Snail expression and clinicopathological factorswas calculated by �2 test.

P � 0.05 was considered to be significant.pT: T1�2: Tumor is limited to the pancreas; T3�4: Tumor extends out

of the pancreas.* Significant value.

over, Snail expression had a close correlation with


lymph node invasion and distant metastasis (Table 1).Seven of eight cases with distant metastasis (87.5%)were positive for Snail expression. Seventeen caseswith lymphatic invasion (50%) showed positive Snailexpression. We did not find any correlation betweenSnail expression and histological grade.

Snail-Induced Overt Phenotypic Changes of Panc-1 Cells

To study the effect of Snail expression on pancreaticcancer, we transfected the expression plasmid of Snailinto Panc-1 cells, and stable clones were selected withG418. The Snail expression was confirmed by Westernblot using antibody-detecting HA epitope tagged withSnail cDNA (Fig. 2 A). The two clones (Panc-1 Snail1,Panc-1 Snail2) we selected lost their epithelial charac-teristics and presented a scattering, spindle-shapedmorphology (Fig. 3).

Snail has been reported to trigger EMT of manyepithelial cells. Ectopic expression of Snail in epithelialcells led to repression of epithelial markers and acti-vation of mesenchymal markers [25–29]. Loss of epi-thelial markers and gain of mesenchymal markers isa recognized criterion to judge occurring of EMT.E-cadherin is not only an epithelial marker but also animportant adhesive molecule for maintenance of theepithelial phenotype through the formation of adher-ent junctions [30]. Repression of E-cadherin has beencorrelated with loss of epithelial phenotype [31]. Snailis a strong repressor of E-cadherin; it triggers EMT bydirect transcriptional repression of E-cadherin expres-

FIG. 2. Western blot analysis of changes in E-cadherin, vimentinexpression after ectopic expression of Snail in Panc-1 cells. (A) Snailexpression was confirmed using anti-HA antibody. No HA was de-tected in Panc-1 cells, whereas HA was expressed in Snail-trans-fected clones. (B) Effects of transfection of Snail gene on E-cadherin,vimentin expression. After Snail gene transfection, epithelial markerE-cadherin was repressed, and mesenchymal marker vimentin wasenhanced.

sion [29]. In our experiments, Snail-transfected clones

showed marked E-cadherin repression concomitant withmorphological changes. Besides, the expression of vi-mentin (a mesenchymal marker) was greatly enhanced(Fig. 2B). We concluded that Snail triggered EMT inpancreatic cancer cells.

Snail Enhanced the Invasive Ability of Panc-1 Cells

Snail has been reported to play an important role forthe acquisition of invasive properties of epithelial cells.We have found that expression of Snail in pancreaticcancer is correlated with metastasis and lymph nodeinvasion. Snail may enhance the invasive ability ofPanc-1 cells. To test this, we investigated the invasiveability of Panc-1 cells after transfection of Snail usingtranswell invasive assay. Cells were seeded on the topof the porous membrane. FBS was used as a chemoat-tractant in the lower compartment. The number ofcells invaded to the lower side of the membrane in-

FIG. 3. Morphology of Panc-1 cells after Snail transfection (mag-nification, �200). Panc-1 cells display a typical epithelial cell mor-phology (A). After transfection of Snail gene, Panc-1 cells acquire a
scattering, spindle-shaped morphology (B).


creased dramatically after Snail transfection (Fig. 4).Snail afforded a significant increase in cell invasion ascompared to parental Panc-1 cells.

Snail Led to Impaired Proliferation of Panc-1 Cells

To investigate the effect of Snail on Panc-1 cellsgrowth, we assessed cell proliferation after transfec-tion using MTT assay. The Snail-transfected Panc-1cells can still proliferate with time going on, but theproliferation is retarded compared to untransfectedcells (Fig. 5).

Snail Enhanced the Chemoresistance of Panc-1 Cells

Epithelial to mesenchymal transition is a processthat facilitates cancer cell migration and invasion. Be-sides, it may also link to aberrant survival and resis-tance to apoptosis of metastatic cancer cells [32]. Snail

FIG. 4. Snail enhanced the invasive ability of Panc-1 cells. Afterthe transwell invasive chamber was incubated for 24 h, the filter wasfixed and stained. The number of cells on the undersurface of thefilter was counted using a microscope. Eight random fields wereselected for counting in three independent experiments. The numberwas represented with mean � SD. The invasive ability of Panc-1 cellsafter transfected with Snail gene increased dramatically (*P � 0.01).

FIG. 5. Snail led to impaired proliferation of Panc-1 cells. Theproliferation of cells was demonstrated by MTT assay. There arethree groups: Panc-1, Panc-1 Snail1, and Panc-1 Snail2. The exper-iment was performed in triplicate and Student’s t-test was used forstatistical analysis. The absorbance was used to indicate the relativecell number. The results are expressed by means � SD. As we can seein the figure, Snail-transfected Panc-1 cells showed a retarded
growth compared to parental untransfected cells (*P � 0.01).
cannot only trigger EMT, it can also confer resistanceto cell death and provide a selective advantage for cellsto migrate to distant territories [33]. Moreover, aber-rant expression of Snail in breast cancer cells protectsagainst apoptosis induced by genotoxic stress [34]. Sowe hypothesized that expression of Snail in pancreaticcancer may promote chemoresistance.

To test this, we investigated the sensitivity of Panc-1cells transfected with Snail to chemotherapeutic agent:5-FU and gemcitabine. The cell viability was deter-mined by MTT assay. After using 5-FU for 72 h, wefound that ectopic Snail expression conferred a signif-icant protection to Panc-1 cells at different dosages.Especially at dosages of 25 �g/mL, which approximatesthe peak plasma levels of 5-FU achieved during che-motherapy [35], the two clones we selected (Panc-1Snail1, Panc-1 Snail2) showed an improved survivalcompared to those untransfected Panc-1 cells (P �0.005). The study was repeated using gemcitabine.Snail afforded a similar protection to Panc-1 cells (Fig. 6).

Nude Mice Experiments

To determine the significance of Snail expression invivo, Panc-1 cells and Panc-1 cells transfected with

FIG. 6. Snail afforded overt chemoresistance to 5-FU and gem-citabine. Various dosages of 5-FU and gemcitabine were applied toPanc-1 and Snail-transfected Panc-1 cells for 72 h. The cell viabilitywas assayed by MTT assay. We found that Panc-1 cells transfectedwith Snail gene showed an overt resistance to 5-FU or gemcitabine atdifferent dosages. (A) Snail afforded a significant protection to 5-FUat 10–200 �g/mL (*P � 0.01). (B) Snail afforded a significant resis-tance to gemcitabine at 10–200 �g/mL (*P � 0.01).

Snail were orthotopically injected into the pancreas of


nude mice. As we can see in Table 2, Panc-1 cellsdisplayed weak ability to metastasize in vivo. Of the sixmice injected with Panc-1 cells, two mice had lymphnode invasion, and no mice was found to have hepaticmetastasis. Panc-1 cells transfected with Snail showedenhanced metastatic potential; most mice (4/6) devel-oped spontaneous hepatic metastasis, and all micewere found to have lymph node invasion (Fig. 7). Theseresults demonstrated that Snail enhanced the invasiveand metastatic ability of Panc-1 cells in vivo.

DISCUSSION

In our experiment, we demonstrated that Snail isexpressed in pancreatic cancer. Twenty of 56 (36%)cases of pancreatic cancer were positive for Snail ex-pression. Snail may be used by a part of cancer cells toits own purpose. In our study, we found that Snail hada close correlation with lymph node invasion (P �0.005) and distant metastasis (P � 0.002). Patientswith lymphatic invasion and distant metastasis had ahigh Snail-positive expression rate. It suggested thatSnail may be involved in the progression of pancreaticcancer. Snail may serve as a marker for predicting themalignancy of pancreatic cancer after operation.

Snail has been demonstrated to be expressed in aseries of malignancies such as breast cancer, gastriccancer, and hepatic cancer [18, 20, 36]. In hepatic can-cer, Snail expression independently correlated with tu-mor invasiveness [36]. In breast cancer, Snail has beenshown to be present in all of the infiltrating ductalcarcinomas presenting lymph node metastases. Theseresults are inconsistent with ours. In breast cancer,Snail expression inversely correlates with the grade ofdifferentiation of the tumors [20]. In our experiment,we did not find any correlation between Snail expres-sion and differentiation. This may be attributed to thelimited number of our cases, because we found theSnail expression increased with the increase of dedif-ferentiated cases.

To test the significance of Snail expression in pan-creatic cancer, we transfected the Snail cDNA intopancreatic cancer cells Panc-1. After transfection, the

TABLE 2

Effect of Snail Expression on Pancreatic Tumors inNude Mice

Cell line

Hepatic metastasis* Lymph node invasion*

Negative Positive Negative Positive

Panc-1 6 0 4 2Panc-1-Snail 2 4 0 6

* P � 0.05 versus Panc-1 and Panc-1-Snail.

invasive ability of Panc-1 cells increased dramatically.

Snail-expressing cells implanted into nude mice metas-tasized widely compared to parental untransfectedcells. This corroborated the role of Snail in pancreaticcancer progression. Snail plays an important role inpancreatic cancer invasion and metastasis.

We found that Snail induced over morphological andphenotypic changes. The transfected cells lost theirepithelial morphology and acquired a spindle, scatter-ing morphology. With the change of cell morphology,the epithelial marker E-cadherin was repressed, andthe mesenchymal marker vimentin was increased.E-cadherin is an important mediator of epithelial ad-hesion and signaling transduction. Loss of E-cadherinexpression has been described in invasion and metas-tasis of many cancers, including pancreatic cancer[37–39]. However the mechanisms underlying changesof E-cadherin expression in pancreatic cancer are notfully known [40]. Snail is recognized to be a strongtranscriptional repressor of E-cadherin; it can repressE-cadherin expression by binding to three E-boxes ofE-cadherin promoter [41]. In fact, inverse correlationbetween Snail and E-cadherin has been observed inmany types of cancer including melanoma, breast can-cer, and hepatic cancer [21, 27, 36]. In our study,E-cadherin was repressed after ectopic expression ofSnail in Panc-1 cells. Repression of E-cadherin in pan-creatic cancer may be one result of Snail expression.

FIG. 7. Snail-enhanced metastasis of Panc-1 cells in vivo. Toevaluate the effect of Snail on Panc-1 cells growth in vivo, we ortho-topically injected the Snail-transfected or untransfected Panc-1 cellsinto the pancreas of nude mice. After 1 month, the mice were sacri-ficed, and the viscera were examined for tumor formation. Panc-1cells had limited invasive and metastatic ability, whereas Snail-transfected Panc-1 cells formed more aggressive tumors. Lymphaticnode invasion (Aa, arrowhead), spontaneous hepatic metastasis (Ab,arrowhead) more frequently occurred. The metastasis was verifiedby hematoxylin-eosin staining (Ba, lymphatic invasion; Bb, hepatic
metastasis). (Color version of figure is available online.)


Snail may accelerate pancreatic cancer progressionpartially through E-cadherin repression.

Vimentin is a Type 3 intermediate filament normallyexpressed in mesenchymal cells, whereas a series stud-ies indicates that vimentin is expressed in the mi-gratory epithelial cells. Vimentin expression may beinvolved in the epithelial cell migration [42]. Transfec-tion of the antisense vimentin cDNA into invasive ep-ithelial cells reduced both their ability to express vi-mentin and their invasive ability [42, 43]. Vimentinexpression may present a migratory phenotype of epi-thelial cells. In our study, after ectopic expression ofSnail in Panc-1 cells, vimentin expression was in-creased and the invasive ability was dramatically pro-moted. We concluded that Snail may confer a migra-tory phenotype to pancreatic cancer cells.

Loss of epithelial marker, gain of mesenchymalmarker is widely used as a criterion for judging EMToccurrence. Loss of epithelial marker E-cadherin iscentral to EMT [30]. On the other hand, vimentin ex-pression is one of the results of epithelial to mesenchy-mal transition [44]. We concluded that Snail inducedEMT of Panc-1 cells. EMT, which is considered to be apivotal process in the metastasis of cancer [6], mayfacilitate pancreatic cancer progression through Snailexpression.

To elucidate the effects of Snail on pancreatic cancercell growth, MTT proliferation assay was performed tocompare the proliferation of Panc-1 and Snail-trans-fected Panc-1 cells. The results indicated that expres-sion of Snail in cancer cells resulted in reduction ofproliferation. This is inconsistent with previous reportthat Snail favors cell shape versus cell division [17].Although increased cell proliferation is crucial for tu-mor formation and growth, this may not be so fortumor malignization and acquisition of an invasivephenotype [45].

Snail is not only an inducer of EMT and cell move-ment, it is considered to be a factor in favor of cellsurviving [45]. When stably expressed in epithelialcells, Snail confers resistance to cell death induced bywithdrawal of survival factors and by pro-apoptoticsignals [17]. Aberrant expression of Snail may promoteresistance to programmed cell death elicited by DNAdamage [34]. Pancreatic cancer is a malignancy thatresists nearly all of the present chemotherapy. Wehypothesize that Snail may confer the chemoresistanceto pancreatic cancer cells. In our experiment, we com-pared the viability of different cells treated with 5-FUand gemcitabine, which are widely used in pancreaticcancer therapy. As a result, Snail-transfected Panc-1cells displayed obvious survival compared to untrans-fected cells. Snail expression may confer chemoresis-tance to pancreatic cancer cells.

In summary, we showed that Snail is expressed in
pancreatic cancer. Expression of Snail not only confers
the invasive phenotype to pancreatic cancer cells, butalso promotes chemoresistance. Snail may be a newmarker for predicting the malignancy of pancreaticcancer. Besides, it may be a perfect molecular targetfor therapeutic intervention. Further study on biologi-cal function of Snail may offer great benefit to pancre-atic cancer patients.

ACKNOWLEDGMENTS

We thank Professor Masayuki Ozawa (Department of Biochemis-try and Molecular Biology, Graduate School of Medical and DentalSciences, Kagoshima University, Japan) for providing the Snail-expressing vector. We thank Dr. Quansheng Wang (Union Hospital,Tongji Medical College, Huazhong University of Science and Tech-nology) for providing the Snail antibody.

REFERENCES1. Gunzburg WH, Salmons B. Novel clinical strategies for the

treatment of pancreatic carcinoma. Trends Mol Med 2001;7:30.2. Ohuchida K, Mizumoto K, Murakami M, et al. Radiation to

stromal fibroblasts increases invasiveness of pancreatic cancercells through tumor-stromal interactions. Cancer Res 2004;64:3215.

3. Richter A, Niedergethmann M, Sturm JW, et al. Long-termresults of partial pancreaticoduodenectomy for ductal adenocar-cinoma of the pancreatic head: 25-year experience. WorldJ Surg 2003;27:324.

4. Keleg S, Buchler P, Ludwig R, et al. Invasion and metastasis inpancreatic cancer. Mol Cancer 2003;2:14.

5. DiMagno EP, Reber HA, Tempero MA. AGA technical review onthe epidemiology, diagnosis, and treatment of pancreatic ductaladenocarcinoma. American Gastroenterological Association.Gastroenterology 1999;117:1464.

6. Gotzmann J, Mikula M, Eger A, et al. Molecular aspects ofepithelial cell plasticity: Implications for local tumor invasionand metastasis. Mutat Res 2004;566:9.

7. Thiery JP. Epithelial-mesenchymal transitions in tumor pro-gression. Nat Rev Cancer 2002;2:442.

8. Valdes F, Alvarez AM, Locascio A, et al. The epithelial mesen-chymal transition confers resistance to the apoptotic effects oftransforming growth factor Beta in fetal rat hepatocytes. MolCancer Res 2002;1:68.

9. Shekhar MPV, Pauley R, Heppner G. Host microenvironmentin breast cancer development: Extracellular matrix–stromalcell contribution to neoplastic phenotype of epithelial cells inthe breast. Breast Cancer Res 2003;5:130.

10. Poser I, Dominguez D, de Herreros AG, et al. Loss of E-cadherinexpression in melanoma cells involves up-regulation of thetranscriptional repressor Snail. J Biol Chem 2001;276:24661.

11. Nakajima S, Doi R, Toyoda E, et al. N-cadherin expression andepithelial-mesenchymal transition in pancreatic carcinoma.Clin Cancer Res 2004;10:4125.

12. del Barrio MG, Nieto MA. Overexpression of Snail family mem-bers highlights their ability to promote chick neural crest for-mation. Development 2002;129:1583.

13. Carver EA, Jiang R, Lan Y, et al. The mouse Snail gene encodesa key regulator of the epithelial-mesenchymal transition. MolCell Biol 2001;21:8184.

14. Manzanares M, Locascio A, Nieto MA. The increasing complex-ity of the Snail gene superfamily in metazoan evolution. Trend
Genet 2001;17:178.


15. Duband JL, Monier F, Delannet M, et al. Epithelium-mesenchyme transition during neural crest development. ActaAnat (Basel) 1995;154:63.

16. Cano A, Perez-Moreno MA, Rodrigo I, et al. The transcriptionfactor Snail controls epithelial-mesenchymal transitions by re-pressing E-cadherin expression. Nat Cell Biol 2000;2:76.

17. Vega S, Morales AV, Ocana OH, et al. Snail blocks the cellcycle and confers resistance to cell death. Genes Dev 2004;18:1131.

18. Blanco MJ, Moreno-Bueno G, Sarrio D, et al. Correlation ofSnail expression with histological grade and lymph node statusin breast carcinomas. Oncogene 2002;21:3241.

19. Roy HK, Smyrk TC, Koetsier J, et al. The transcriptional re-pressor SNAIL is overexpressed in human colon cancer. Dig DisSci 2005;50:42.

20. Rosivatz E, Becker I, Specht K, et al. Differential expression ofthe epithelial-mesenchymal transition regulators Snail, SIP1,and twist in gastric cancer. Am J Pathol 2002;161:1881.

21. Poser I, Dominguez D, de Herreros AG, et al. Loss of E-cadherinexpression in melanoma cells involves up-regulation of thetranscriptional repressor Snail. J Biol Chem 2001;276:24661.

22. Sobin LH, Wittekind CH. TNM classification of malignant tu-mors, 6th ed. New York: John Wiley & Sons, 2002.

23. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: Alaboratory manual. 2rd ed. New York: Cold Spring HarborLaboratory Press, 1989.

24. Tsutsumi S, Yanagawa T, Shimura T, et al. Autocrine motilityfactor signaling enhances pancreatic cancer metastasis. ClinCancer Res 2004;10:7775.

25. Guaita S, Puig I, Franci C, et al. Snail induction of epithelial tomesenchymal transition in tumor cells is accompanied byMUC1 repression and ZEB1 expression. J Biol Chem 2002;277:39209.

26. Yokoyama K, Kamata N, Fujimoto R, et al. Increased invasionand matrix metalloproteinase-2 expression by Snail-inducedmesenchymal transition in squamous cell carcinomas. Int JOncol 2003;22:891.

27. Ohkubo T, Ozawa M. The transcription factor Snail down-regulates the tight junction components independently ofE-cadherin downregulation. J Cell Sci 2004;117:1675.

28. Bolos V, Peinado H, Perez-Moreno MA, et al. The transcriptionfactor Slug represses E-cadherin expression and induces epi-thelial to mesenchymal transitions: A comparison with Snailand E47 repressors. J Cell Sci 2003;116:499.

29. Cano A, Perez-Moreno MA, Rodrigo I, et al. The transcriptionfactor Snail controls epithelial-mesenchymal transitions by re-pressing E-cadherin expression. Nat Cell Biol 2000;2:76.

30. Bates RC, Mercurio AM. Tumor necrosis factor-alpha stimu-lates the epithelial-to-mesenchymal transition of human co-
lonic organoids. Mol Biol Cell 2003;14:1790.
31. Behrens J, Mareel MM, Van Roy FM, et al. Dissecting tumorcell invasion: Epithelial cells acquire invasive properties afterthe loss of uvomorulin-mediated cell-cell adhesion. J Cell Biol1989;108:2435.

32. Bates RC, Goldsmith JD, Bachelder RE, et al. Flt-1-dependentsurvival characterizes the epithelial-mesenchymal transition ofcolonic organoids. Curr Biol 2003;13:1721.

33. Vega S, Morales AV, Ocana OH, et al. Snail blocks the cell cycleand confers resistance to cell death. Genes Dev 2004;18:1131.

34. Kajita M, McClinic KN, Wade PA. Aberrant expression of thetranscription factors Snail and slug alters the response to geno-toxic stress. Mol Cell Biol 2004;24:7559.

35. Wey JS, Gray MJ, Fan F, et al. Overexpression of neuropilin-1promotes constitutive MAPK signalling and chemoresistance inpancreatic cancer cells. Br J Cancer 2005;93:233.

36. Sugimachi K, Tanaka S, Kameyama T, et al. Transcriptionalrepressor Snail and progression of human hepatocellular carci-noma. Clin Cancer Res 2003;9:2657.

37. Pignatelli M, Ansari TW, Gunter P, et al. Loss of membranousE-cadherin expression in pancreatic cancer: Correlation withlymph node metastasis, high grade, and advanced stage.J Pathol 1994;174:243.

38. Yoshida R, Kimura N, Harada Y, et al. The loss of E-cadherin,alpha- and beta-catenin expression is associated with metasta-sis and poor prognosis in invasive breast cancer. Int J Oncol2001;18:513.

39. Cai J, Ikeguchi M, Tsujitani S, et al. Significant correlationbetween micrometastasis in the lymph nodes and reduced ex-pression of E-cadherin in early gastric cancer. Gastric Cancer2001;4:66.

40. Joo YE, Rew JS, Park CS, et al. Expression of E-cadherin,alpha- and beta-catenins in patients with pancreatic adenocar-cinoma. Pancreatology 2002;2:129.

41. Batlle E, Sancho E, Franci C, et al. The transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumor cells. Nat Cell Biol 2000;2:84.

42. Gilles C, Polette M, Zahm JM, et al. Vimentin contributes tohuman mammary epithelial cell migration J. Cell Sci 1999;112:4615.

43. Singh S, Sadacharan S, Su S, et al. Overexpression of vimentin:Role in the invasive phenotype in an androgen-independentmodel of prostate cancer. Cancer Res 2003;63:2306.

44. Ellenrieder V, Hendler SF, Boeck W, et al. Transforminggrowth factor beta1 treatment leads to an epithelial-mesen-chymal transdifferentiation of pancreatic cancer cells requiringextracellular signal-regulated kinase 2 activation. Cancer Res2001;61:4222.

45. Barrallo-Gimeno A, Nieto MA. The Snail genes as inducers ofcell movement and survival: Implications in development and
cancer. Development 2005;132:3151.

expression of snail in pancreatic cancer promotes metastasis and chemoresistance

Documents