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The Korean Journal of Pathology2008; 42: 351-7

Background : Slug is a member of the Snail family of transcription factors, and it plays a cru-cial role in the regulation of the epithelial-mesenchymal transition by suppression of severalepithelial proteins and adhesion molecules, including E-cadherin. Methods : The aim of thepresent study was to examine the significance between the expression of Slug in colorectaladenocarcinoma (CRA) specimens and the clinicopathological parameters of CRA, as deter-mined by immunohistochemical analysis, and to determine the correlation between the Slugand E-cadherin expressions in non-neoplastic colorectal mucosa (n=45), primary CRA (n=109) and metastatic CRA (n=17). A semiquantitative scoring system was applied based onthe intensity and extent of the positive immunohistochemical staining. Results : The expres-sions of Slug and E-cadherin were associated with the depth of tumor invasion (pT) (p=0.019,p=0.001, respectively), and these expressions showed a significant inverse correlation (p<0.001) each other. Conclusions : Our results demonstrated a positive role for Slug in thedevelopment of CRA, and Slug is a mediator of tumor invasion in CRA. In addition, an up-regulated Slug expression is significantly correlated with the loss of an E-cadherin expres-sion, which suggests that Slug may play some role in the epithelial-mesenchymal transition(EMT) by down-regulating the E-cadherin expression.

Key Words : Slug transcription factors; E-cadherin; Colon; Adenocarcinoma; Immunohisto-chemistry

Ran Hong1∙∙Dong-Yul Choi1Sung-Chul Lim1,2∙∙Chae-Hong Suh1Keun-Hong Kee1∙∙Mi-Ja Lee1

351

The Differential Expressions of the Epithelial-Mesenchymal Transition

Regulator, Slug and the Cell Adhesion Molecule, E-cadherin

in Colorectal Adenocarcinoma

351 351

Corresponding AuthorSung-Chul Lim, M.D.Department of Pathology, Chosun University Hospital,588 Seoseok-dong, Dong-gu, Gwangju 505-717,KoreaTel: 062-230-6343Fax: 062-226-5860E-mail: sclim@chosun.ac.kr

*This work was supported by the Korea Science andEngineering Foundation (KOSEF) grant funded by theKorea government (MEST) through the ResearchCenter for Resistant Cells (R13-2003-009).

Department of 1Pathology, 2ResearchCenter for Resistant Cells, College ofMedicine, Chosun University, Gwangju,Korea

Received : September 19, 2008Accepted : October 9, 2008

The normal epithelium is a highly regulated tissue structurethat requires a tight interaction between neighboring cells.1

Strong adhesive junctions between epithelial cells, and betweenthe epithelial cells and the extracellular matrix (ECM) inhibitthe movement of individual cells, and enable the cells to formuniform epithelial cell sheets with polarized apical and basal sur-faces.1 The loss of adhesion, increased cell motility and reversiblededifferentiation are marked in the process of wound healing,and especially in the developmental processes of embryogenesisand in gastrulation where the mesoderm is formed.2 The mor-phological and phenotypical changes that occur during theselater processes are referred to as the epithelial-mesenchymal tran-sition (EMT). The EMT is one of the central mechanisms thatinduces invasion and metastasis of tumors, and it is a processwhere by epithelial cells lose their polarity and they are convert-

ed to mesenchymal phenotypes.3,4 The acquisition of a fibrob-lastic phenotype is accompanied by the loss of E-cadherin andthe dissociated cells from the epithelial tissue freely migrate.5

This is an essential event during the gastrulation movementand neural crest formation, but it has also been suggested toplay a fundamental role during the early steps of invasion andmetastasis of carcinoma cells. It has been proposed that the samemolecules that trigger the EMT are involved in tumor progres-sion, invasion, and metastasis.5

E-cadherin is a transmembrane glycoprotein, localized in theadherens junctions that are typically found in epithelial cells,and it plays an important role in the maintenance of the struc-tural integrity of epithelial sheets.6 The loss of an E-cadherinexpression had been reported in several gastrointestinal cancers,including advanced colorectal carcinomas, esophageal adeno-

352 Ran Hong∙Dong-Yul Choi∙Sung-Chul Lim, et al.

carcinomas and gastric and pancreatic cancers.7-10 Therefore, itis important to identify the molecular mechanisms that regu-late the expression of E-cadherin. Multiple mechanisms inacti-vate the E-cadherin mediated cell adhesion system in cancer,such as a gene mutation, promoter hypermethylation, chromatinrearrangement, post-translational truncation or modification,and the recently characterized transcriptional repression.11-14

EMT inducers such as Zeb1, SIP1, Slug, E47, and Twist havebeen shown to induce the EMT and metastasis through the repres-sion of E-cadherin.15 Slug is a member of the Snail family oftranscription factors, and Slug is highly expressed in the cellsundergoing the EMT in the developing chick embryo, wherethe function of this protein is critical for the formation of theprimitive streak and the endocardial cushions, decondensingsomites and in closure of the palate.16 Moreover, Slug plays acrucial role in the regulation of EMT by suppressing severalepithelial marker proteins and adhesion molecules, includingE-cadherin.16 Several recent studies have reported that the expres-sion of Slug in breast carcinomas and esophageal carcinomas isaccompanied with shorter survival.16 In the present study, 109colorectal adenocarcinomas were examined for the expression ofSlug and E-cadherin by the use of immunohistochemical analy-sis, and we investigated the association of these expressions withthe clinicopathological factors and also the prognostic impactof the expression of Slug in colorectal adenocarcinoma.

MATERIALS AND METHODS

Patients

Among the patients who underwent curative surgery for col-orectal adenocarcinoma at Chosun University Hospital fromJanuary 1992 to December 2004, the present study was con-ducted on a non-consecutive series of 109 patients. The patientswere selected where their paraffin embedded tissues were rela-tively well preserved, the medical records were complete, andthe patient’s status had been followed-up. Patient survival wasconfirmed through telephone interviews and by a mail inter-view. The patients who underwent preoperative chemoradio-therapy and emergency surgery, and the patients who had evi-dence of hereditary non-polyposis colorectal cancer or familialadenomatous polyposis were excluded from the study. The var-ious clinicopathological parameters of the patients were con-firmed by a review of the patient medical records and patholo-gy files. The relationship of the clinicopathological parameters

for the patients and the immunohistochemical findings with sur-vival was investigated for all of the 109 patients. Informed con-sent was obtained from each subject according to the institu-tional guidelines, and the Ethics Committee of the UniversityHospital approved the research protocols.

Histopathological analysis

Microscopic examination

Each tumor was re-evaluated by retrospective analysis of themedical records and the tissue slide files from the Departmentof Pathology. Age, gender, the tumor size, and histological sub-types, the degree of differentiation, the depth of tumor inva-sion, the status of lymph node metastases and the presence of adistant metastasis were assessed. The stage was defined accord-ing to the TNM staging system of the American Joint Com-mittee on Cancer.17

The examined tissues were fixed in 10% neutral formalin, andthe prepared paraffin embedded tissues were sectioned 4-5 μmin thickness. Hematoxylin and eosin staining was performed,and the sections were examined under a light microscope. Arepresentative area suitable for study purposes was selected, andthe slides were prepared for immunohistochemical analysis.

Immunohistochemical staining

All of the specimens in this study were tested using a goatpolyclonal antibody against Slug (Santa Cruz Biotechnology,Santa Cruz, CA, USA), and a mouse monoclonal antibody againstE-cadherin (Santa Cruz Biotechnology), according to the man-ufacturer’s protocol.

Briefly, the 4 μm thick sections that were obtained after for-malin fixation and paraffin embedding were deparaffinized inxylene and they were then rehydrated with distilled water througha graded series of ethanol solutions. The sections were then placedin a glass jar with 10 mM citrate buffer (pH 6.0) and they wereirradiated in a microwave oven for 15 min. The sections wereallowed to cool in the jar at room temperature for 20 min. Theslides were then rinsed with Tris buffered saline (TBS). A block-ing reagent was added for 10 min after quenching the endoge-nous peroxidase activity in 0.3% hydrogen peroxide for 10 min.The slides were then washed as described above and the slideswere subsequently subjected to the primary antibody reactions.Antibodies against Slug (dilution 1:100) and E-cadherin (dilu-tion 1:100) were applied to the tissue sections and the sectionswere allowed to incubate in a moist chamber overnight at 4℃and then for 50 min at room temperature, respectively. After

washing with TBS, a biotinylated link antibody was appliedfor 10 min, and this step was followed by incubation with strep-tavidin peroxidase for an additional 10 min. After removal ofthe excess complex, the localization of antibodies was visualizedby incubating the sections for 15 min in an Ultra Vision PlusDetection System (Lab Vision, Fremont, CA, USA). Counter-staining was performed with the use of Mayer’s hematoxylin.

Analysis and interpretation of staining

For the immunohistochemical staining for Slug, the extentof the nuclear staining was classified according to the percent-age of the nuclear expression of the cells among the tumors as 0(0%), 1 (1-25%), 2 (26-50%), 3 (51-75%), and 4 (76-100%).The intensity of nuclear staining was scored as 0 (negative), 1(weak positive), 2 (medium), and 3 (strong).18 We applied a semi-quantitative scoring system based on the staining intensity andthe extent of the Slug expression in the cells. The sum of theintensity and the extent score were calculated, and we reclassi-fied the sum as score 0 (0), 1 (1-2), 2 (3-5), and 3 (6-7).19 Weused the reclassified score as the final score for the Slug expres-sion. Tumors with a final score of 3 were considered to have ahigh expression. For the evaluation of E-cadherin, the percent-age of tumor cells with a membranous E-cadherin expressionwas scored as 0 (0-5%), 1 (1-25%), 2 (26-50%), 3 (51-75%),and 4 (76-100%). The intensity of the membraneous stainingwas scored as 0 (negative), 1 (weak positive), 2 (medium), and 3(strong). The sum of the intensity and extent scores was used asthe final score (0-7) for E-cadherin. Those tumors with a finalstaining score of 6 or higher were considered to have a highexpression.19 The results were assessed by two pathologists whowere unaware of any of the details regarding the patients’ back-grounds.

Statistical analysis

For statistically analyzing the Slug expression in colorectalcancer, its association with the various clinicopathological fac-tors and the survival rate or survival length, we used the χ2 testand Fisher’s exact test for creating a 2×2 table to compare thecategorical data, and multivariate analysis was also done withusing Cox regression analysis. The life table and statistic signif-icance were computed using the Kaplan-Meier method and thelog-rank test. The statistical significant level was set at p-value

expression was evaluated as 0 (score 0), 1 (score 1-2), 2 (score 3-5), and 3 (score 6 and 7), the Slug expression was significantlyincreased with the increased depth of tumor invasion (p=0.019).When the Slug expression was next evaluated as low (score 0-5)and high (score 6 and 7), the result was the same (p=0.03). Muci-nous adenocaricnomas and poorly differentiated adenocarcino-mas showed a significantly high expression of Slug as comparedwith the well to moderately differentiated adenocarcinomas (p<0.001). However, there was no significant correlation betweenthe Slug expression and the presence of a lymph node metasta-sis, distant metastasis and the clinical staging. Among the 109patients, 25 patients died during the follow-up period. The Slugexpression was not associated with the survival time. Lymphnode metastasis negatively influenced survival, which was sta-tistically significant. Multivariate analysis using Cox regressionanalysis determined that lymph node metastasis was an inde-pendent prognostic factor that influenced patient survival. The

other clinicopathological parameters did not correlate with sur-vival with any significant differences.

Expression of E-cadherin

For all of the patients, 67 cases (61.5%) showed a weakexpression or no expression of E-cadherin, and 42 cases (38.5%)showed a high level of expression (Fig. 2). The E-cadherinexpression had a significant correlation with tumor differentia-tion; the expression level was low in mucinous adenocarcino-mas and poorly differentiated adenocarinomas as comparedwith that of the well to moderately differentiated tumors(Table 3). A significant inverse relationship was observed oncomparison of the Slug and membranous E-cadherin expres-sions (p

Expressions of Slug and E-cadherin in Colorectal Adenocarcinoma 355

Fig. 2. Immunohistochemical staining for Slug and E-cadherin in colorectal adenocarcinomas. (A) Cancer cells are intensely stained forSlug. (B) The cancer cells in the consecutive slices of (A) are faintly stained for E-cadherin. (C) Cancer cells are negative for Slugexpression. (D) Slug-negative tumor cells demonstrate strong immunoreactivity for E-cadherin in the consecutive slices of (C).

C D

A B

Variables np-

valuep-

value

Slug expression

0 1 2 3

Slug expression

Low1 High2

T stage 1 2 1 0 0 1 0.019* 1 1 0.030*2 23 1 4 11 7 16 73 79 4 7 19 49 30 494 5 0 0 2 3 2 3

N stage 0 73 5 7 21 40 0.678 33 40 1.0001 36 1 4 11 20 16 20

M stage 0 106 6 11 31 58 0.511 48 58 1.01 3 0 0 1 2 1 2

Clinical stage Lowa 75 5 7 22 41 0.730 34 41 1.0Highb 34 1 4 10 19 15 19

Differen- W/M 87 6 10 32 39 0.001* 48 39

DISCUSSION

During the EMT, epithelial cells actively downregulate thecell-cell adhesion system, they lose their polarity and they acquirea mesenchymal phenotype with reduced intercellular interac-tion and an increased migratory capacity. De-differentiation,the loss of adhesive constraints and enhanced motility and inva-sion are all hallmarks of increased malignancy, and the EMTprovides a mechanism for carcinoma cells to acquire a moreaggressive phenotype.20 The EMT is controlled by a number ofmaster transcription regulators, including Snail, Twist, ZEB,and Slug.3,21 One of the key factors that regulates the EMT pro-gram is Slug, a member of the Snail family of zinc-finger tran-scription factors. In the present study, the Slug expression wassignificantly correlated with the depth of tumor invasion (pT)(p=0.019) and the differentiation of tumors (p=0.001), but therewas no association with the other clinicopathological factors,including lymph node metastasis, distant metastasis, and theclinical stage. These results were different from the previouslyreported findings by Shiori et al.16 These investigators showed acorrelation of the Slug expression with distant metastasis andthe Duke stage.16 According to our findings, we suggest thatthe Slug expression is important for tumor invasion and dedif-ferentiation as a component of the EMT. However, further eval-

uation of metastasis is required with employing a large numberof metastatic colorectal carcinoma specimens. Snail family tran-scription factors, including Snail (SNAI1) and Slug, have recent-ly been reported to repress the E-cadherin expression, whichmediates cell to cell adhesion and increases cancer invasion invarious malignancies.22 For example, previous studies have deter-mined that Slug is an in vivo repressor of E-cadherin, which isassociated with a poor prognosis for patients with breast carci-noma and esophageal squamous cell carcinoma.23-25 In colorec-tal carcinoma, it has been reported that there is a frequent lossof E-cadherin, and this is is primarily due to promoter hyper-methylation.26 However, the remaining mechanisms that con-trol the loss of E-cadherin in colorectal carcinoma are not yetwell understood. Smits et al.27 have reported that Snail binds E-box motifs in gene promoters, and there are a number of E-box-containing genes that are important in colon cancer biology. Inaddition, E-cadherin is a tumor suppressor that has been impli-cated in the initiation and the promotion phases of colon car-cinogenesis.27 In the present study, 67 cases (61.5%) showed noexpression or a weak expression of E-cadherin. The expressionof E-cadherin was significantly decreased with an increaseddepth of tumor invasion (pT), and this was in accordance withthe previous study by Shiori et al.16 Furthermore, the E-cadherinexpression has a strong inverse correlation with Slug (p

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Expressions of Slug and E-cadherin in Colorectal Adenocarcinoma 357