Amerncan Journal ofPathology, Vol. 152, No. 1, January 1998

Copyright Amen1can Societyfor Investigative Pathology

Altered Cadherin and Catenin Complexes in theBarrett's Esophagus-Dysplasia-AdenocarcinomaSequence

Correlation with Disease Progression and Dedifferentiation

Tracey Bailey,* Leigh Biddlestone,tNeil Shepherd,t Hugh Barr,t Philip Warner,* andJanusz Jankowski*From the Cranfield Biotechnology Centre,* Cranfield University,Cranfield, the Gloucester Gastroenterology Group,tGloucestershire Royal Hospital, Gloucester, and the Departmentof Oncology,* Queen Elizabeth II Hospital, Birmingham,United Kingdom

The maintenance ofadult tissue architecture is largelydependent on the function ofcadherins. E-cadherin isexpressed in most epithelia, although it may be co-

expressed with P-cadherin in basal layers of stratifiedepithelia. Adhesive function of cadherins relies on

interactions with catenins. Many reports have char-acterized reduced expression of cadherins andcatenins in tumors, including those of the gastroin-testinal tract. This study aimed to characterize expres-sion of E- and P-cadherins, and the catenins, in theprogression of Barrett's esophagus to adenocarci-noma. Immunohistochemical analysis and Westernblotting were performed on paraffin-embedded andfresh-frozen tissue using antisera to the selected cad-herins and catenins. The results of this study haveshown inappropriate expression of cadherins andcatenins in neoplastic Barrett's mucosa. There was a

significant reduction of E-cadherin expression as theBarrett's metaplasia-dysplasia-adenocarcinoma se-

quence progressed (P < 0.01). In contrast, P-cad-herin, expressed in basal layers of squamous esoph-agus, was usually absent from Barrett's and dysplasiabut was expressed in 17 of 24 carcinomas, especiallyat the advancing tumor edge. Reduced expression ofcatenins was also seen, but in some specimens, inmmu-noreactvity was observed in neoplastic nuclei, suggest-ing mediation of a nuclear function such as transcrip-tional regulation. (AmJ Pathol 1998 152:135-144)

The incidence of adenocarcinoma of the esophagus israpidly increasing in the United States and Western Eu-rope. This is in contrast to squamous cell carcinoma ofthe esophagus, the incidence of which is decreasing.1Increased prevalence of adenocarcinoma coincides with

an increasing prevalence of Barrett's esophagus, inwhich squamous mucosa is replaced by columnar epi-thelium. Patients with Barrett's are at increased risk ofacquiring adenocarcinoma of the esophagus,23 whichhas a poor prognosis, and many patients with this tumorwill die as a result of it.4 It is hypothesized that cancer

arises in Barrett's esophagus through a multistep se-

quence of events.5 It is initiated by chronic gastro-esoph-ageal reflux leading to the presence of columnar epithe-lium, which may be of gastric or intestinal type, andprogresses to dysplasia and finally adenocarcinoma. Anincreased understanding of the molecular events thatoccur during the progression of Barrett's esophagus tocancer might allow improved diagnostic and screeningtechniques and ultimately lead to improved managementof the disease. One target for study is the mediation ofcell adhesion.Tumor growth and metastasis involve a series of

events from invasion at the site of the primary tumor togrowth at a distant site. Many, if not all, of these stepscould involve changes in the ability of cells to adhere toeach other or the extracellular matrix.6 Cadherins are celladhesion molecules expressed in solid tissues.7 They areimportant for initiating and maintaining cell-cell contactand may also be involved in signal transduction6 and cellpolarity.9 At a molecular level cadherins may be dividedinto subclasses corresponding to their tissue distributionincluding E-cadherin (epithelial cadherin or uvomoru-lin)10 and P-cadherin (placental cadherin).11 Normal ad-hesive function of cadherins requires the interaction ofthree proteins termed a- ,- and y-catenin (plakoglo-bin).12,13 It has been shown that mutants of these pro-teins prevent the correct functioning of cadherins. E-cadherin is the primary regulator of cell adhesion inepithelia.14 It is first expressed before implantation andthen during development becomes confined to epithelialtissues.15.16 The blocking of E-cadherin-mediated adhe-

Funded by Gloucester Gastroenterology Group and Cranfield Universityas part of the Gloucestershire Royal and Cranfield University Institute ofMedical Sciences.

Accepted for publication October 10, 1997.

Address reprint requests to Tracey A. Bailey, Cranfield BiotechnologyCentre, Cranfield University, Cranfield, Beds, MK43 OAL, UK.

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136 Bailey et alAJPJanuary 1998, Vol. 152, No. 1

sion by monoclonal antibodies was shown to lend inva-sive properties to cells in culture.17 Subsequently, E-cadherin has been proposed as an important suppressorof invasion18'19 and has been investigated in a variety ofcancers and cancer cell lines. In colorectal cancer, levelsof both protein expression and levels of E-cadherinmRNA have been shown to be significantly different be-tween survivors and nonsurvivors.2021 Studies correlat-ing E-cadherin immunoreactivity have also been carriedout in bladder tumors,22 gastric carcinomas,23'24 thoracicneoplasms,25 and squamous cell carcinoma of theesophagus.26 Expression of E-cadherin has also beenstudied in Barrett's esophagus, dysplasia, and adenocar-cinoma. In gastric-type epithelium, a uniform type ofstaining of high intensity is revealed by use of anti-E-cadherin antibodies with immunohistochemistry. How-ever, reduced or absent staining is noted in 25% ofintestinal-type Barrett's.27 In dysplasia, staining becomesfurther reduced or disorganized.25 Staining tends to bestrong, compared with normal tissues, in well differenti-ated adenocarcinomas but weaker in poorly differenti-ated tumors.27As catenins are required for the function of cadherins,

it would follow that their mutation might facilitate tumori-genesis through loss of cadherin-mediated adhesion. Intwo stomach cell lines, deletion mutations in f-catenindisrupted the interaction between E-cadherin and a-cate-nin and led to loss of cell-cell adhesion.28 In a humanlung cancer cell line that strongly expressed E-cadherinat the cell membrane, deletions were found in the a-cate-nin gene that caused loss of its protein in the cells, whichlacked tight cell-cell adhesion.29 Immunohistochemistryhas been used to show reduced a-catenin and f3-cateninexpression in carcinomas of the esophagus, stomach,and colon,3032 but it is not clear whether these reduc-tions are due to mutations in the genes themselves orsome other mechanism of reduction, such as reducedtranscription. Mutations have so far only been describedin cell lines, and a study of human gastric and breastcarcinomas found no such changes.33The aim of this study was to characterize the expres-

sion of cadherins and catenins in the Barrett's esopha-gus-dysplasia-adenocarcinoma disease sequence. Im-munohistochemistry and Western blotting were used toexamine cellular distribution of these proteins and quan-tify their expression. Changes in immunoreactivity werethen correlated with grade and stage of disease to as-sess the relevance of each protein component in diseaseprogression.

Materials and Methods

Human TissuesSections for immunohistochemistry were cut from 51 par-affin blocks from Barrett's esophagus resection speci-mens from the archives of Gloucestershire Royal Hospitaland Cheltenham General Hospital. In all patients thediagnosis of Barrett's esophagus had been confirmed byendoscopic and histopathological criteria. Thus, all

cases showed histological evidence of Barrett's esopha-gus with the presence of intestinalization. Tissue wasobtained from 28 Barrett's patients: 26 male and 2 fe-male; age range, 39 to 76 years (age at time of biopsy);mean age, 63.4 years. Sections from 10 blocks containedsquamous epithelium, 13 contained regions of gastricmetaplasia, 6 had regions of intestinal metaplasia, 8showed dysplasia, and 24 showed invasive adenocarci-noma. Fifteen lymph nodes containing metastatic esoph-ageal adenocarcinoma were also studied. Fresh tissuefrom esophagectomy and esophago-gastrectomy spec-imens for Western blot analysis was taken during surgeryat Gloucestershire Royal Hospital. Tissue was availablefrom six individuals. Normal gastric mucosa was ob-tained from all six patients, squamous esophageal mu-cosa from five patients, metaplastic tissue from four, andtumor tissue from six. Of the tumor tissues, one was welldifferentiated, two moderately differentiated, and threepoorly differentiated. Esophageal mucosa was taken dis-tant from the tumor site when possible. Barrett's mucosawas selected on the basis of its red velvety appearancein the lower esophagus, and the presence of metaplasiawas confirmed by routine histology of adjacent tissuesections. Tissue specimens were snap-frozen in liquidnitrogen and stored at -70°C before processing.

AntibodiesAntibodies used for immunohistochemistry were HECD-1(anti-human E-cadherin M. Takeichi), anti-P-cadherin(Transduction Laboratories, Lexington, KY), anti-a-cate-nin, anti-13-catenin, and anti-y-catenin (K. Herrenknecht).Anti-E-cadherin, anti-a-catenin, anti-f-catenin, and anti-y-catenin (Transduction Laboratories) were used forWestern blotting.

ImmunohistochemistryImmunohistochemistry was performed using an avidin-biotin-peroxidase complex (ABC) method (Dako, HighWycombe, Bucks, UK). Slides were dewaxed and rehy-drated in xylene-ethanol series, endogenous peroxi-dases were blocked with a hydrogen peroxide solution,and a protocol for microwave antigen retrieval was fol-lowed. Slides were then incubated with 20% normal goatserum for 20 minutes to prevent nonspecific binding be-fore incubating with the primary antibody for 1 hour. TheABC kit was used according to the manufacturer's in-structions. Washing between steps was with Tris-buff-ered saline (TBS, 50 mmol/L Tris base, 150 mmol/L so-dium chloride, pH 7.6). A solution of 200 mg of 3,3'-diaminobenzidine tetrahydrochloride was used as achromogen, and slides were then counterstained withMayer's hematoxylin (R. A. Lamb, London, UK). Sectionsof normal colon were simultaneously processed as con-trol tissues in all instances, except with use of anti-P-cadherin antibodies where normal skin sections wereused.

Cadherins, Catenins, and Barrett's Esophagus 137AJPJanuary 1998, Vol. 152, No. 1

Evaluation of Staining

Staining intensity was graded using a method previouslyutilized.26 Cells staining less intensely than those in thepositive control were defined as negative, and the sectionwas then scored semiquantitatively according to the pro-

portion of positive cells: +, more than 90% positive; +/-,between 10% and 90% positive; or -, less than 10%positive. Sections showing more than 90% of cells to bepositive were described as having preserved type ex-pression, and other categories were described as re-

duced type. Cellular localization of stain was also noted.

Statistical AnalysisResults from immunohistochemistry were analysed by x2

test rules for a table comparing number of sections ofeach histological type with each category of stainingintensity. Statistical significance was accepted for P <

0.05. Results were also ranked to give a median stainingresult for each histological type.

Western Blot Analysis

Protein expression levels of E-cadherin, a-catenin,,f-catenin, and y-catenin were quantified using Westernblotting with appropriate antisera. In each instance,banding from tumor or metaplastic tissue lysates wascompared with that gained from normal gastric mucosa

from the same patient as a positive control. The tech-nique was also used to show that the full-length proteinwas being detected (if present).

Approximately 1 cm3 of tissue was placed in 1 ml ofextraction buffer (0.5% Triton X-100, 10 mmol/L Tris/HCI,pH 7.5, 25 mmol/L KCI, 180 mmol/L CaCI2, 120 mmol/LNaCI, 2% 2-mercaptoethanol, and appropriate proteaseinhibitors). Tissue was then homogenized for 1 to 2 min-utes in an alcohol/ice bath, and placed on a shaker for 10minutes while still on ice. Samples were then centrifugedfor 5 minutes at 1000 rpm at less than 0°C. The super-natant was aliquoted and stored at -800C until required.

Before electrophoresis by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis, samples werethawed on ice and then boiled for 5 minutes with an equalvolume of loading buffer (100 mmol/LTris, pH 6.8, 4% w/vSDS, 20% glycerol, 5% 2-mercaptoethanol, 0.2% bromo-phenol blue). After cooling on ice, samples were loadedon a 7.5 or 10% polyacrylamide gel according to degreeof separation required, along with a prestained marker inone well (Kaleidoscope, BioRad Laboratories, HemelHempstead, UK). Electrophoresis was carried out at 200V for at least 1 hour.

After electrophoresis, proteins were transferred topolyvinylidene difluoride membrane (BioRad) using aBioRad mini-trans blot apparatus at 40 mA overnight inCAPS buffer (10 mmol/L 3-cyclohexylamino-1-propane-sulfonic acid, 10% v/v methanol, pH 11.0). Membranesfor immunodetection were incubated with 3% w/v bovinealbumin for 1 hour to prevent nonspecific binding andthen incubated for 1 hour with the primary antibody be-

fore use of the Dako ABC kit as a detection system withdiaminobenzidine as a chromogen.

To allow equalization of loading of epithelial proteins,an immunodetection was performed using CAM 5.2 (Im-perial Cancer Research Fund, London, UK). This detectsa number of epithelial cytokeratins, and the intensity ofbands on the resulting blots allows an estimate to bemade of the relative amounts of epithelial proteins, sosample loading can be adjusted accordingly.34 Equaliza-tion in such a manner allowed a more accurate compar-ison of protein expression levels by the comparison ofband intensity of each sample by Western blotting.

Results

ImmunohistochemistryMorphological Distribution of E-Cadherin

Median results are indicated in Figure 1 to illustrate stain-ing trends in each histological stage of disease progres-sion. Mucosal epithelial cells in normal colon sections,used as a positive control, stained strongly with HECD-1at the basolateral membranes (Figure 2B). Weak immu-noreactivity was also observed in the cytoplasm. Colonsections processed without primary antibodies stainedonly with the blue of the Mayer's hematoxylin counterstain(Figure 2A). In squamous epithelium of normal esopha-gus, there was only weak staining of the most basal layer,but cell membranes in the parabasal layers of the tissuestained with comparable intensity to the positive control.Membranous staining reduced in intensity toward theluminal edge of the tissue. Cytoplasmic staining was alsoseen in cells of the parabasal layer (Figure 2C).

In gastric-type metaplasia, a polarity of staining wasnoted with immunoreactivity strongest at the basal mem-branes, decreasing in intensity along the lateral mem-branes and disappearing at the apex of cells of thesuperficial layer (Figure 2D). Staining also occurred in thecytoplasm with similar cellular distribution. In intestinal-type metaplasia, immunoreactivity was more reduced,and one case showed very low cytoplasmic immunore-activity, although another case showed quite strongmembranous immunoreactivity.Two patterns were seen in dysplastic tissue, but in all

instances immunoreactivity was reduced. In 50% ofcases, cytoplasmic staining was seen. In the remainingsections studied, there was no immunoreactivity in themajority of the tissue except for isolated regions of weakmembranous or cytoplasmic staining (Figure 2E).

Immunoreactivity in adenocarcinoma was reduced inall instances but was otherwise variable in both intensityand location. Variations of E-cadherin expression in car-cinomas were seen not only between individuals but alsowithin cases. However, there appeared to be some cor-relation between reduced immunoreactivity and tumordifferentiation, with seven of nine poorly differentiatedtumors having strong immunoreactivity in less than 10%of cells in comparison to one of eight well differentiatedtumors and one of seven moderately differentiated tu-

138 Bailey et alAJPJanuary 1998, Vol. 152, No. 1

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Figure 2. E-cadherin immunoreactivity. A: Colonic mucosa as a negative control with no primary antibody, stained with Mayer's hematoxylin only (magnification,X 100). B: Colonic mucosa plus HECD-1 at a 1 in 100 dilution as a positive control, immunoreactivity showing as brown against the blue counterstain of Mayer'shematoxylin (x 100). C: Squamous esophageal mucosa with membranous immunoreactivity plus some cytoplasmic staining in basal layers (X 200). D: Gastric-typecolumnar epithelium (Barrett's) showing polarized immunoreactivity (X 100). E: Dysplastic tissue with reduced immunoreactivity (X200). F: Poorly differentiatedadenocarcinoma with no immunoreactivity (X400).

Cadherins, Catenins, and Barrett's Esophagus 139AJPJanuary 1998, Vol. 152, No. 1

Figure 3. P-cadherin immunoreactivity. A: Epidermis with anti-P-cadherin antibodies at a 1 in 100 dilution as a positive control (X 100). B: Gastric-type Barrett'swith only very weak immunoreactivity in the superficial layer but strong immunoreactivity in parietal cells (X100). C: Immunoreactivity at the leading edge ofa poorly differentiated adenocarcinoma with cells appearing to show some polarity of P-cadherin expression as an example of its up-regulation in this case(X200). D: Center of same tumors as C with almost no immunoreactivity (X200).

mors. Of 15 primary tumors known to be lymph nodepositive, 9 showed reduced, cytoplasmic immunoreactiv-ity to E-cadherin or were negative (Figure 2F).Reduced expression of E-cadherin was also present in

all of 15 lymph nodes with metastatic carcinoma, with 5showing less than 10% of cells staining. Those carcino-mas with more cells staining tended to retain immunore-activity at the cell membranes.When histological stage of disease progression was

compared with category of staining intensity, the x2 testdemonstrated a statistically significant decrease in stain-ing intensity with disease progression (P < 0.01).

Morphological Distribution for P-Cadherin

Squamous cells in the basal layers of skin sectionsshowed strong membranous staining with the use of anti-

P-cadherin antibodies (Figure 3A). In all cases of squa-mous mucosa of the esophagus, distribution of immuno-reactivity mirrored that seen in the control.

Only one gastric-type and one intestinal-type Barrett'smucosal biopsy showed patches of P-cadherin immuno-reactivity in the superficial layer. Two cases with special-ized gastric glandular cells did not stain in the superficialfoveolar layer but showed strong immunoreactivity in thecytoplasm of specialized glandular cells (Figure 3B). Allremaining sections of gastric- and intestinal-type meta-plasia had no immunoreactivity to P-cadherin.Seven of eight cases with dysplasia were negative for

P-cadherin. However, one case of high-grade dysplasiahad weak staining in a small proportion of cells.

Of 24 cases of invasive adenocarcinoma, 7 showed noimmunoreactivity with anti-P-cadherin. Six showed heter-ogeneous staining, whereas eleven showed low immuno-

140 Bailey et alAJPJanuary 1998, Vol. 152, No. 1

reactivity, with expression predominantly around the tu-mor edges (Figure 3, C and D). All lymph nodescontaining metastatic carcinoma showed reduced immu-noreactivity to P-cadherin in comparison with the positivecontrol, and two were reduced in comparison with theirprimary tumor. Most nodes (80%) stained with equivalentintensity to their primary tumor, with five being completelynegative, one with low-level immunoreactivity at the edgeof the node, and the remainder having weak heteroge-neous staining throughout the nodes.

Morphological Distribution of a-Catenin

Staining of strong intensity with anti-a-catenin was ob-served at the basolateral membranes of colonic mucosalcells, and weak immunoreactivity was apparent in thecytoplasm.

Expression of a-catenin, as detected by immunohisto-chemistry, showed a similar distribution in squamousepithelium to the cadherins.

In gastric-type metaplasia, immunoreactivity wasstrongest at the basolateral membranes, with weak stain-ing in the cytoplasm. In 6 of 13 cases, there was areduction in intensity of staining in comparison with thepositive control. In intestinal-type metaplasia, five of thesix cases showed reduction in immunoreactivity.

In dysplastic tissue and adenocarcinoma, cytoplasmicrather than membranous immunoreactivity to a-cateninwas more prevalent. When compared with immunoreac-tivity for the other catenins, it was noted that a-cateninwas membranous in distribution only when either ,B- ory-catenin localized to the membrane. Although there wasreduced immunoreactivity in some cases (including 65%of adenocarcinomas), none were completely negative.Even poorly differentiated tumors, negative for HECD-1,showed staining with anti-a-catenin antibodies. Twelve offifteen lymph nodes with metastases showed some re-duction in staining intensity, but only 31% were reducedin comparison with the primary cancer. Most staining washeterogeneous, both in intensity and cellular location(membranous or cytoplasmic). No statistical significancewas found for changes in a-catenin immunoreactivity withdisease progression.

Morphological Distribution of 13-Catenin

Strong staining was observed in the basolateral mem-branes of colonic mucosa cells, used as a positive con-trol for ,3-catenin immunoreactivity (Figure 4A). In squa-mous epithelium, immunoreactivity was strongest at themembranes of the cells in the basal layers, decreasing inintensity toward the luminal border (Figure 4B). In gastric-type metaplasia, polarity of ,-catenin staining was ob-served in a manner already described for both E-cad-herin and a-catenin (Figure 4C). Four of the thirteencases showed immunoreactivity comparable to the pos-itive control, whereas the remaining nine had some loss instaining intensity. In intestinal-type metaplasia, however,the proportion of cases showing a reduction increased tofive of the six cases studied.

There was a dramatic reduction in immunoreactivity toj3-catenin in dysplasia. In 75% of cases, the reductionwas in excess of 90% in comparison with the positivecontrol. Where staining did occur, it was generally cyto-plasmic.

In adenocarcinoma, moderately and poorly differenti-ated tumors showed weak immunoreactivity (Figure 4D),but well differentiated cancers stained more strongly andretained polarity at the membranes of the cells. Reducedimmunoreactivity was seen in 12 of 15 lymph nodes withmetastatic carcinoma. In comparison to primary tumors77% of invaded lymph nodes stained with equivalent orlesser intensity.The reduction of 3-catenin immunoreactivity with dis-

ease progression was shown to be statistically significant(P < 0.01).

Morphological Distribution of y-Catenin (Plakoglobin)

Sections of colonic mucosa, used as positive controls,showed immunoreactivity to plakoglobin to be strongestat the basolateral membranes.

In squamous esophageal mucosa, immunoreactivitywas most marked around the periphery of cells of thebasal layers, although many cells also showed cytoplas-mic staining. Unlike the pattern observed for othercatenins and the cadherins, there was less reduction ofimmunoreactivity to plakoglobin toward the luminal edgeof the tissue.

Eight of thirteen cases of gastric-type metaplasiashowed strong immunoreactivity around the basolateralmembranes, with weak cytoplasmic staining; three casesshowed this distribution with some reduction in intensityof stain; and two cases were in the most reduced cate-gory for staining (less than 10% of cells with stainingcomparable to the positive control). In intestinal-typemetaplasia, the distribution of staining was similar to gas-tric type with three cases showing reduced immunoreac-tivity.

All dysplastic tissues showed reduced immunoreactiv-ity to plakoglobin, but the reduction was not as dramaticas that for f3-catenin, with only one-third of cases showinga reduction of greater than 90% compared with the pos-itive control.

Immunoreactivity to plakoglobin in adenocarcinomawas highly variable. Some sections showed strong mem-branous and cytoplasmic staining, although at the otherend of the spectrum, some were completely negative forstain. Similar variations were seen in lymph nodes con-taining metastatic carcinoma. Median results are indi-cated in Figure 1. Reduction of plakoglobin with diseaseprogression was not statistically significant.

Nuclear Localization of p- and y-Catenin

One case of dysplasia and one case of poorly differenti-ated adenocarcinoma showed a significant proportion ofcells staining strongly in the nucleus for 13-catenin (Figure5A). This was also seen with y-catenin in two cases withdysplasia (Figure 5B), two adenocarcinomas (Figure 5C),and one lymph node with metastases.

Cadherins, Catenins, and Barrett's Esophagus 141AJPJanuary 1998, Vol. 152, No. 1

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Figure 5. Nuclear immunoreactivity of catenins. A: Nuclear immunoreactivity of 3-catenin in poorly differentiated adenocarcinoma (x200). B: Nuclearimmunoreactivity of 'y-catenin in dysplasia (X200). C: Nuclear immunoreactivity of -y-catenin in an invasive adenocarcinoma (X400).

I

142 Bailey et alAJPJanuary 1998, Vol. 152, No. 1

Table 1. Number of Samples Showing Decreased Protein Expression Levels as Quantified by Western Blot Analysis.

Number of samples with reduced Number of samples with reducedProtein quantified levels in metaplasia levels in adenocarcinoma

E-Cadherin 0/3 (0%) 2/6 (33%)a-Catenin 2/4 (50%) 1/4 (25%)13-Catenin 2/3 (66%) 3/5 (60%)y-Catenin 2/4 (50%) 2/4 (50%)The intensity of banding for tumors and metaplasia was compared with that gained from lysates of normal gastric mucosa from the same patient as

a positive control. Although the same tissue set was used in each instance, the small amount of lysate gained meant it was unfortunately not possibleto use the full set with each antiserum.

Western BlottingDecreased expression was indicated in at least one tu-mor with each antiserum. In metaplastic tissue, de-creased expression was indicated in at least one samplewith each of the catenin antiseva, but not E-cadherin. Thenumber of samples showing decreased expression ineach instance is shown in Table 1. A blot for E-cadherinis shown (Figure 6).

DiscussionThis study has demonstrated that reduced expression ofE-cadherin, and reduced expression or aberrant localiza-tion of catenins necessary for its function, occur in theneoplastic progression of Barrett's esophagus. P-cad-herin up-regulation was observed in adenocarcinoma,and in some instances, p- and y-catenins were seen tolocalize to the nucleus.

Both immunohistochemistry (IHC) and Western blot-ting have been used to quantitatively show a decreasedexpression of E-cadherin in the progression of Barrett'sesophagus to adenocarcinoma. Results from IHC showthe reduction is statistically significant (p < 0.01), and allsections examined containing dysplasia or invasive ad-enocarcinoma showed some reduction in immunoreac-tivity with use of HECD-1 with both IHC and Westernblotting. The most poorly differentiated tumors showedthe greatest reduction in immunoreactivity. In addition todecreased staining intensity in IHC, a shift from membra-nous to cytoplasmic expression was also seen. These

137

Figure 6. Westem blot for E-cadherin. Lane 1, marker; lane 2, positivecontrol showing band at 120 kd; lane 3, normal gastric mucosa, patient 1;lane 4, Barrett's mucosa, patient 1; lane 5, tumor, patient 1; lane 6, esophagealmucosa, patient 2; lane 7, normal gastric mucosa, patient 2; lane 8, tumor,patient 2; lane 9, normal gastric mucosa, patient 3; lane 10, tumor, patient 3.Samples were checked for equal epithelial loading by Westem blot usingCAM 5.2. Note less intense banding at 120 kd for tumor samples in lane 5compared with normal gastric mucosa and the absence of a band in lane 10despite loading having been checked by CAM 5.2, indicating complete lossof expression in that sample. The lower molecular weight bands just visibleare thought to have occurred as a result of protein degradation duringprocessing.

results are comparable to similar studies previously pub-lished. 25,27,35

In normal squamous epithelium, the most basal layer ofthe tissue where stem cells occur stains only weakly forE-cadherin. Cytoplasmic expression of E-cadherin oc-curs in the parabasal layers of the tissue where the cellsare actively motile and in earlier stages of differentiation,and then more mature cells show strong membranousimmunoreactivity. In dysplasia and some tumors, cyto-plasmic staining is associated with the lack of functionalcadherins, allowing cell movement and increased prolif-eration and hence facilitating tumor progression.36 Theoccurrence of cytoplasmic staining in abnormal tissuemay be indicative of an event that prevents the insertionof the E-cadherin protein across the cell membrane.A small proportion of carcinomas retained predomi-

nantly membranous staining, but these were moderatelyor well differentiated tumors. Of 15 primary carcinomasknown to have lymph node involvement, 9 showed cyto-plasmic staining or were E-cadherin negative (60%). Thisfigure increased to 86% for poorly differentiated tumorswith lymph nodes showing metastatic adenocarcinoma.

All lymph nodes examined showed a reduction of E-cadherin immunoreactivity in metastatic adenocarcinomacompared with the positive control, and one-third of suchnodes were almost negative. Interestingly, membranousstaining was retained in 67% of invaded nodes, albeit ofreduced intensity. A loss of E-cadherin may well be ex-pected in the metastatic process, as invasion and releasefrom the primary site is a key event that would require lossof tight cell-cell adhesion. In squamous cell carcinomasof the head and neck, E-cadherin expression was foundto be inversely correlated with both loss of tumor differ-entiation and lymph node metastasis.37 In the samestudy, seven of eight infiltrated lymph nodes were E-cadherin negative despite some of the primary tumorsstill expressing the molecule.

P-cadherin is expressed specifically in the basal andparabasal layers of esophageal squamous mucosa,whereas strong E-cadherin immunoreactivity is found inthe parabasal and superficial layers. This overlappingexpression is consistent with that found in many otherepithelial tissues.38 P-cadherin expression was largelyabsent from Barrett's tissue and dysplasia but was seento occur in 75% of carcinomas, notably at the invasiveedge. Class switching from E- to P-cadherin can occur inassociation with separation of tissue layers during em-bryogenesis,38 and it could be speculated that this phe-nomenon might facilitate clonal evolution and movement

Cadherins, Catenins, and Barrett's Esophagus 143AJPJanuary 1998, Vol. 152, No. 1

of transformed epithelial sheets during tumorigenesis. Inbreast cancer, P-cadherin was detected in 20% of infil-trating carcinomas,39 with a significant correlation withreduced E-cadherin expression. It is not yet clear whyco-expression of E- and P-cadherin occurs in somecases but not others. It may, however, be significant thatthe genes for E- and P-cadherin are tandemly arrangedon human chromosome 16 with only 32 kb separatingthem,40 and it might be that their proximity allows amaster cadherin control region regulating cell-type-spe-cific cadherin transcription.

Reduced-type staining with anti-a-catenin was ob-served in many dysplastic and carcinoma tissues. How-ever, reduction with disease progression was not statis-tically significant. In addition, some tumors with reducedE-cadherin staining retained a-catenin immunoreactivity,although it was usually cytoplasmic in distribution, pre-sumably due to a lack of association with E-cadherin atthe cell membrane. In squamous cell carcinoma of theesophagus, reduction of a-catenin is strongly associatedwith tumor differentiation, infiltrative growth, and metas-tasis (P < 0.01),3° but this would not appear to be thecase in adenocarcinomas arising from Barrett's mucosa,indicating molecular differences between the two esoph-ageal tumor types.

Pulse-chase experiments have shown that a-catenindoes not associate with E-cadherin directly after E-cad-herin arrives at the cell membrane but is added to thecomplex after ,B- and y-catenin.1 It may therefore be thatloss of function of 6- and/or y-catenin is occurring indisease progression in this instance, resulting in the cy-toplasmic localization of a-catenin but not preventing itsexpression. When immunoreactivity to each of thecatenins was compared in each section, there was noinstance in which a-catenin was localized to the mem-brane without the membranous expression of at least oneof the other catenins. This demonstrates the possibility ofmutations in )3- or y-catenin or changes in their phosphor-ylation being more important in the Barrett's esophagus-dysplasia-adenocarcinoma sequence than alterations ina-catenin.

All dysplastic cases showed reduced immunoreactiv-ity to ,B-catenin, as did 75% of adenocarcinomas and85% of metastatic lymph nodes. These reductions weremore pronounced than changes seen in correspondingtissue for E-cadherin, suggesting that this is one of theearliest changes in the cadherin-catenin complex to oc-cur in the progression of Barrett's esophagus to adeno-carcinoma. ,B-Catenin expression levels are not only im-portant due to interactions with cadherins but may alsobe important to disease progression with respect to in-teractions with the adenomatous polyposis coli (APC)gene product42'43 and proposed roles in signal transduc-tion.31'44 In bladder cancer, 34-catenin expression hasbeen found to be of prognostic value, and preservedexpression in cancer correlates to better survival rates.45

Nuclear localization of both B- and y-catenin was seenin some sections of dysplasia, adenocarcinomas, andlymph node metastases by IHC. It has previously beenshown that /3- and y-catenin can interact with the Lef andTcf family of transcription factors in complexes translo-

cated to the nucleus.4647 This suggests that cateninsmay become directly involved in regulation of gene ex-pression. In this study, nuclear localization of cateninsoccurred in cases in which E-cadherin was reduced andcytoplasmic, suggesting that the absence of E-cadherinat the membrane is one factor freeing these catenins toparticipate in functions other than control of cell adhe-sion. Alternatively, lack of control via aberrant phosphor-ylation of catenins in neoplastic cells may occur.48

The identity of target genes controlled by catenin/tran-scription factor complexes is currently of much interest.The type of tissue in which nuclear localization is seensuggests that this may be associated with increasedproliferation, facilitation of tumorigenesis, and a poorlydifferentiated phenotype, so target genes may includethose involved in epithelial-mesenchymal transition.

Conclusions to be drawn from the results of this studyare that alterations in cadherins and catenins are stronglyassociated with progression of Barrett's esophagus toadenocarcinoma. The precise role in disease progres-sion of each of the proteins studied is not yet clear, butour results suggest their biological modes of action to benuclear translocation of catenins mediating epithelial-mesenchymal transition, decreased and inappropriateE-cadherin expression influencing dedifferentiation, andP-cadherin expression at the leading edge of some tu-mors facilitating neoplastic progression.

AcknowledgmentsWe thank M. Takeichi and K. Herrenknecht for supply ofantibodies, the staff of Histopathology at GloucestershireRoyal Hospital for collection of tissue, and C. Hackwoodfor advice on immunohistochemical techniques.

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