Changes in Integrin and E-Cadherin Expression in Neoplastic Versus Normal Thyroid Tissue

Changes in Integrin andE-Cadherin Expression inNeoplastic Versus NormalThyroid Tissue

Guido Serini, Livio Trusolino,Enrico Saggiorato, OttavioCremona, Marco De Rossi,Alberto Angeli, Fabio Orlandi,Pier Carlo Marchisio*

Background: The functional organiza-tion of polarized epithelia dependsmostly on adhesion molecules belong-ing to the integrin and cadherinfamilies. These molecules either recog-nize basement membrane components,such as laminins, or form intercellularjunctions via homotypic interactions.Such tissue organization is often dis-rupted upon neoplastic transformation,and the resulting loss of functionalpolarization and cell cohesion mightbe a prerequisite for the invasive andmetastatic behavior of carcinomas.Purpose: We studied modifications ofthyroid adhesive mechanisms atvarious stages of neoplastic progressionin terms of adhesion molecule expres-sion, topography, and functional reg-ulation by tyrosine kinases. Startingfrom this working hypothesis, wesought to identify one or more biologi-cal markers that would be suggestive ofmalignant transformation and poorerprognosis and that could be developedas a reliable indicator(s) in earlydiagnostic steps. Methods: The studywas carried out on both surgicalsamples and the corresponding fine-needle aspiration biopsy smears(numbers of specimens collected: 19adenomas, seven follicular carcinomas,13 papillary carcinomas, and 39 normaltissues). Immunohistochemistry oftissue sections and smears andimmuno-precipitation and westernblot analysis of protein extracts weredone with a battery of monoclonal andpolyclonal antibodies. Northern blot-ting was performed on RNA extractsfrom frozen tissue samples and use ofan integrin subunit b4 complementary

DNA probe. Results: Our findings canbe summarized as follows: 1) In normalthyroid cells, the cooperative role ofintegrin a6b4 and laminin 5/kalinin inhemidesmosome-mediated adhesion ismissing, and recognition of the basallamina occurs via integrin a3b1 andlaminin 1 and/or 2 (this pattern beingmaintained in adenomas but altered incarcinomas regardless of their histo-type or differentiation grade); 2) onlyin carcinomas with clinical and/orhistologic aggressiveness do neoexpres-sion of integrin subunit b4 and loss oflaminin 2/merosin occur, indicating denovo assembly of integrin a6b4; 3)pericellular redistribution and cyto-skeletal disconnection of the E-cad-herin-catenin complex occur; and 4)basal E-cadherin tyrosine phosphory-lation decreases in carcinomas as com-pared with that in normal andadenomatous tissues. Conclusions:The malignant progression of thyroidtumors involves marked rearrange-ments of cell±basement membraneand cell±cell adhesion molecules andchanges in their cytoskeleton linkage.These rearrangements are also easilyand reproducibly detected on fine-needle aspiration biopsy smears. Impli-cations: Immunodetection of adhesionmolecules in sections and/or fine-needle smears may complement thetoolbox of thyroid surgical patholo-gists; it may expand the possibilitiesof achieving a correct early diagnosisof thyroid tumors and of gaining someprognostic information on thyroidtumors. [J Natl Cancer Inst1996;88:442-9]

The induction and maintenance of a

polarized and differentiated epithelial

phenotype depend on expression, mem-

brane sorting, and function of surface

adhesion molecules (1) that provide

domain-dependent mechanical and che-

mical information and are responsible for

defining and maintaining cell topology

and the functional polarity typical of

epithelia. Such tissue organization is

often disrupted upon neoplastic transfor-

mation, and the resulting loss of the

polarized distribution and/or functional

properties of adhesion molecules might

be a prerequisite for the invasive and

metastatic behavior of carcinomas (2,3).

Epithelial cells adhere to the basement

membrane zone mostly via integrins (4-

6); these integrins are transmembrane a/bheterodimeric proteins that mediate re-

cognition of the basal lamina and attach-

ment to laminins (represented in epithelial

basal lamina by laminin 1/EHS1

[a1b1g1], laminin 2/merosin [a2b1g1],

and laminin 5/kalinin [a3b3g2]) (7). The

major integrin restricted to the basal

domain of the basal layer in squamous

epithelia (8-11) and in most columnar

epithelia (12,13) is a6b4, which usually

codistributes with hemidesmosomes (14-

16) and laminin 5/kalinin.

Epithelial cell±cell association is pri-

marily mediated by E-cadherin (17-19), a

structural component of zonula adherens

involved in forming homotypic bonds and

in linking the microfilament network

(20,21) via a chain of cytoskeletal pro-

teins termed `̀ catenins'' (22-24).

Differentiated thyroid carcinomas ori-

ginating from follicular cells are classi-

fied as a papillary type or a follicular type.

Their poorly differentiated variants en-

compass the tall-cell carcinoma for papil-

lary types (25) and insular (26) and

Hurthle cell (27) carcinomas for follicular

types.

So far, no conclusive data have been

provided about a simple and reliable

method for correct, early diagnosis of

thyroid tumors. Ideally, the preoperative

differential diagnosis between benign and

malignant thyroid neoplasms, performed

on fine-needle aspiration biopsy smears,

could be improved by two potential

findings: 1) changes in integrin expression

and topography and 2) aberrant synthesis,

assembly, and/or adhesive status of the E-

*Affiliations of authors: G. Serini, L. Trusolino, P.C. Marchisio, Department of Biomedical Sciencesand Human Oncology, University of Torino, Italy,and Department of Biological and TechnologicalResearch, San Raffaele Scientific Institute, Milano,Italy; E. Saggiorato, A. Angeli, F. Orlandi, Depart-ment of Clinical and Biological Sciences, Universityof Torino; O. Cremona, Department of BiomedicalSciences and Human Oncology, University ofTorino, and Department of Medical Sciences,University of Torino, Novara Branch; M. De Rossi,Department of Biological and TechnologicalResearch, San Raffaele Scientific Institute.

Correspondence to: Pier Carlo Marchisio, M.D.,Ph.D., Department of Biological and TechnologicalResearch, San Raffaele Scientific Institute, ViaOlgettina 58, 20132 Milan, Italy.

See `̀ Notes'' section following `̀ References.''

REPORTS Journal of the National Cancer Institute, Vol. 88, No. 7, April 3, 1996442

cadherin±catenin complex. Moreover,

these modifications might provide a better

prognostic definition of thyroid

tumors. Accordingly, the aim of this

study was to understand thyroid adhesive

mechanisms during neoplastic progres-

sion in terms of adhesion molecule ex-

pression and functional regulation by

tyrosine kinases. Starting from this work-

ing hypothesis, we sought to identify one

or more biological markers suggestive of

malignant transformation and poorer

prognosis that would be suitable for

development as reliable indicators in

early diagnostic steps. The main morpho-

logic±functional changes observed in

thyroid cancers are pericellular redistribu-

tion of the integrin a3b1 complex, integrin

a6b4 neoexpression, and E-cadherin dis-

orientation as well as a reduction in its

tyrosine phoshorylation level.

Materials and Methods

Surgical Specimens

After clinical staging and fine-needle aspiration

biopsies, fresh neoplastic thyroid tissue was surgi-

cally removed from patients previously treated with

Lugol's iodine solution. All patients underwent

thyroidectomy at San Luigi Gonzaga Hospital, Or-

bassano (Torino, Italy). The tumor tissue was either

directly embedded in OCT 4583 (Miles Scientific,

Naperville, IL) and snap-frozen for further immu-

nohistochemical studies or directly frozen in liquid

nitrogen for protein and RNA extraction. A

portion of this tissue was used for routine

histopathology. Tumor-free tissue was excised

from each patient and processed in parallel. Frozen

sections (5 mm) were serially cut in a Reichert±Jung

cryomicrotome, transferred onto poly-l-lysine-

coated slides, air-dried, and stored overnight at

room temperature.

Histopathologic Classification

In parallel with immunohistochemistry, serial

sections of each specimen were stained with

hematoxylin±eosin to determine the histotype and

subhistotype. We examined 19 surgical samples of

follicular adenomas, seven of follicular carcinomas,

and 13 of papillary carcinomas. In addition, we

examined a total of 39 normal tissue specimens. Of

the seven follicular carcinomas examined, four were

classified as poorly differentiated malignant neo-

plasms (HuÈrthle cell subtypes) and three as well-

differentiated carcinomas; among the 13 papillary

carcinomas, we observed five poorly differentiated

variants (four tall-cell carcinomas and one sclerotic

tumor), two follicular variants, five well-differen-

tiated cancers, and one dedifferentiated carcinoma

with wide anaplastic fields. The histologic features

of each case, together with sex and age information,

TNM staging (28), follow-up, and additional data

are summarized in Table 1.

Fine-Needle Aspiration Biopsies

The fine-needle aspiration biopsies were per-

formed with a 22-gauge ¾ 1.5-inch needle attached

to a 30-mL plastic syringe (29). After aspiration, the

small fluid specimen was expelled from the needle

and smeared onto a polylysine-coated slide. The

smears were air-dried for 2 hours, pre-fixed in

absolute methanol at º20 C, and stored at º80 C.

We examined aspiration biopsy smears from 10

adenomas, five follicular carcinomas, and five

papillary carcinomas. Written informed consent

was obtained from each subject with the approval

of the San Luigi Gonzaga Hospital review board.

Antibodies

The primary monoclonal antibodies (MAbs)

used in this study (with the investigators who

provided them) were as follows: MAR4 to integrin

subunit b1 and MARE (30) to integrin subunit a6

(from S. MeÂnard, Istituto Nazionale Tumori,

Milano, Italy), GOH3 to integrin subunit a6 (from

A. Sonnenberg, The Netherlands Cancer Institute,

Amsterdam), F2 to integrin subunit a3 (from L.

Zardi, Istituto Scientifico per lo Studio a la Cura

dei Tumori, Genova, Italy), AA3 and S3-41 to

integrin subunit b4 (31) (from V. Quaranta, Scripps

Research Institute, La Jolla, CA), IA9 to integrin

subunit b5 (from M. Hemler and R. Pasqualini,

Dana-Farber Cancer Institute, Boston, MA), GB3

to laminin BM600/nicein (32) (from P. Verrando,

Laboratoire de Recherches Dermatologiques,

Faculte de MeÂdecine, Nice, France), and 5H2 to

laminin 2/merosin (33) (from E. Engvall, Wenner

Gren Institute, Stockholm, Sweden). Other MAbs

were commercially obtained: SAM-1 to integrin

subunit a5 and Gi9 to integrin subunit a2 (Immu-

notech, Marseille, France), LAM-89 to laminin 1

and PT-66 to phosphotyrosine-containing proteins

(Sigma Chemical Co., St. Louis, MO), HECD-1 to

human E-cadherin (Takara Shuzo Co., Kyoto,

Japan), and MAbs to a-catenin and b-catenin

Table 1. Clinicopathologic features of patients with thyroid carcinomas

TNM{

Case* Age, y/sex Histotype{ Subhistotype At presentation At recurrence Follow-up} b4 expression

FC1 39/female DFC Well-differentiated carcinoma T2aN0M0 NED (32) +FC2 75/female PDC HuÈrthle cell carcinoma T4aN1bM0 M+ (PUL) AWD (32) ºFC3 27/female DFC Well-differentiated carcinoma T3aN0M0 NED (31) +FC4 51/female DFC Well-differentiated carcinoma T2aN0M0 NED (30) ºFC5 31/male PDC Hurthle cell carcinoma T2aN0M0 NED (29) +FC6 33/female PDC Hurthle cell carcinoma T1aN0M0 NED (15) +PC7 46/female DPC Follicular variant T4bN0M0 NED (27) ºPC8 30/female PDC Tall-cell carcinoma T2aN1aM0 NED (26) +PC9 45/female PDC Tall-cell carcinoma T4bN1bM0 M+ (OTH)|| AWD (26) +PC10 41/female DPC Follicular variant T4aN1bM0 NED (25) ºPC11 70/male DPC Well-differentiated carcinoma T2bN0M0 NED (24) ºPC12 27/female PDC Sclerotic variant T4aN1bM0 NED (24) +PC13 31/female PDC Tall-cell carcinoma T4bN0M0 NED (23) +PC14 16/female DPC Well-differentiated carcinoma T2aN0M0 NED (40) ºPC15 61/female PDC Tall-cell carcinoma T2aN0M0 NED (21) +PC16 60/male DPC Well-differentiated carcinoma T1N0M0 NED (13) ºPC17 37/female DPC Well-differentiated carcinoma T2aN0M0 NED (5) ºPC18 73/female DDC Wide anaplastic fields T4aN0M+ (OTH)} DOD (5) ºFC19 34/female PDC Hurthle cell carcinoma T2N0M0 NED (2) +PC20 65/female DPC Well-differentiated carcinoma T4N1M0 NED (3) +

*FC = follicular carcinoma; PC = papillary carcinoma.{DFC = differentiated follicular carcinoma; PDC = poorly differentiated carcinoma; DPC = differentiated papillary carcinoma; DDC = dedifferentiated carcinoma.{According to Hermanek and Sobin (28). M+ = presence of metastases; PUL = pulmonary metastases; OTH = metastases in other organs.}NED = no evidence of disease; AWD = alive with disease; DOD = dead of disease. Months after surgical resection are indicated in parentheses.||Mediastinum.}Trachea.

443Journal of the National Cancer Institute, Vol. 88, No. 7, April 3, 1996 REPORTS

(Affiniti, Nottingham, U.K.). Rabbit polyclonal

antiserum R5710 to integrin subunit b4 was

provided by V. Quaranta; rabbit antiserum R1542

to integrin subunit b1 was a gift of L. Languino

(La Jolla Cancer Research Foundation, CA). For

immunoperoxidase staining and immunofluores-

cence, MAbs were used at a final immunoglobulin

concentration of 10-40 mg/mL. Immunoprecipita-

tions were performed with 4 mg MAbs per sample.

For immunoblotting, 2 mg/mL MAbs or 10 mg/mL

polyclonal antibodies were used. For control

purposes, irrelevant antibodies were routinely used.

Indirect Immunoperoxidase Technique

Experiments were performed as previously

described (34). Briefly, cryostat sections were

fixed for 10 minutes in a chloroform-acetone

mixture (1:1) at 4 8C, air-dried, and incubated for

10 minutes in phosphate-buffered saline (PBS)

supplemented with 1% serum from the same species

as that for the secondary antibody. Serial sections

were overlaid with 50 mL of different antibodies in

Tris-buffered saline (TBS) in 0.4% bovine serum

albumin (BSA) and incubated at room temperature

for 30 minutes in a moist chamber. After a thorough

wash in PBS, the sections were incubated with the

appropriate biotinylated secondary antibody and

processed for the ABC method (avidin±biotin±

peroxidase complex) using the Vectastain ABC Kit

(Vector Laboratories, Inc., Burlingame, CA). After

the sections were washed three more times, 100 mL

of substrate was added for 5-10 minutes and was

prepared as follows: 5 mg of 3-amino-9-ethylcarba-

zole (Sigma Chemical Co.) was dissolved in 1 mL

of N,N-dimethylformamide (Merck, Darmstad, Fed-

eral Republic of Germany) supplemented with 9 mL

of 100 mM sodium acetate (pH 52) and 100 mL of

12% H2O2. All samples were counterstained with

Mayer's hemalum solution, mounted in Kaiser's

glycerol gelatin (Merck), and examined with a Zeiss

Axiophot photomicroscape (Zeiss, Jena, Federal

Republic of Germany) equipped with 16¾ and 63¾planapochromatic lenses.

Indirect Immunofluorescence Microscopy

Smears were fixed in a chloroform±acetone

mixture (1:1) for 10 minutes at 4 8C and air-dried.

After a 15-minute saturation with TBS-BSA (0.4%

at 37 8C), the primary antibodies (MAbs AA3 and

S3-41 to b4 and MAR6 to a6) were layered onto

slides and incubated in a moist chamber for 30

minutes. After rinsing in TBS containing 0.4%

BSA, the slides were incubated with the appropriate

rhodamine-tagged secondary antibody (Dakopatts,

Copenhagen, Denmark) for 30 minutes at 37 8C.

Coverslips were mounted in Mowiol 4-88 (Hoechst

AG, Frankfurt/Main, Federal Republic of Germany).

Routine observations were carried out in a Zeiss

Axiophot photomicroscope equipped for epitluores-

cence. Fluorescence images were recorded on

Kodak T-Max 400 films exposed at 1000 ISO and

developed in T-Max Developer for 10 minutes at

20 8C.

Detergent Solubilization

Surgical samples were directly snap-frozen in

liquid nitrogen, pulverized in a B-Braun Mikro-

Dismembrator (B-Bran, Melsungen, Federal Repub-

lic of Germany), and lysed in extraction buffer (TBS

[pH 8], 1% Triton X-100, 0.5% Nonidet P-40, and 2

mM CaCl2) containing a mixture of phosphatase

and protease inhibitors (2 mM sodium orthovana-

date, 50 mM sodium fluoride, 1 mM phenylmethyl

sulfonyl fluoride, 2 mg/mL leupeptin, and 2 mg/mL

pepstatin A) by several passages in a Dounce

homogenizer (PBI International, Milan, Italy) on

ice. Tissue lysates and insoluble material were

collected and centrifuged for 10 minutes at 20 000

rpm at 4 8C. Supernatants were saved; in some

experiments, detergent-insoluble pellets were resus-

pended in a buffer containing 50 mM Tris-HCl (pH

8.8) and 1% sodium dodecyl sulfate (SDS), soni-

cated, boiled for 5 minutes, and recentrifuged. SDS

extracts were diluted 10-fold in extraction buffer,

and supernatants were adjusted to 0.1% SDS before

immunoprecipitation.

Immunoprecipitation

Experiments were carried out as previously

described (35). Tissue extracts were adsorbed onto

protein A±Sepharose CL-4B (Pharmacia LKB Bio-

technology AB, Uppsala, Sweden) previously incu-

bated with normal mouse serum (Sigma Chemical

Co.). Precleared lysates were incubated with MAb

HECD-1 to E-cadherin, and immuno-complexes

were collected by protein A-Sepharose CL-4B

coupled with rabbit anti-mouse immunoglobulin G

(Pierce Chemical Co., Rockford, IL). After seven

washes with extraction buffer, the final pellet was

boiled in Laemmli buffer (36) in the presence of 4%

b-mercaptoethanol, and proteins were processed for

SDS±polyacrylamide gel electrophoresis (PAGE)

(8% polyacrylamide gels). For quantitative recovery

of E-cadherin±catenin complexes, protein concen-

trations were normalized (BCA Protein Assay

Reagent Kit; Pierce Chemical Co.), MAb HELD-1

was titrated in normal thyroid lysates, and saturating

amounts of MAb were used in each immunopreci-

pitation experiment.

Western Blot Analysis

Frozen surgical samples were pulverized using a

B-Braun Mikro-Dismembrator (37). For semiquan-

ritative recovery of epithelium-specific components

from the crude extract, each sample was divided into

200-mg pieces and processed in parallel for western

blot analysis and for routine histology to evaluate

the ratio of stromal contamination according to

histologic examination. The pulverized tissues

were solubilized in boiling Laemmli buffer and

sonicated; 300 mg of presumptive epithelial proteins

was loaded onto each lane. Alternatively, immuno-

complexes, recovered from immunoprecipitation

experiments after elution in boiling Laemmli buffer,

were directly loaded onto gel lanes. Materials were

fractionated by SDS±PAGE (8% polyacrylamide

gels) under nonreducing conditions, and proteins

were electrophoretically transferred to nitrocellulose

filters (Hybond; Amersham Life Science, Inc.,

Arlington Heights, IL) and analyzed as described

previously (38). Filters were probed with rabbit

antiserum R1542 to integrin subunit b1, R5710 to

integrin subunit b4, and MAbs to E-cadherin, a-

catenin, b-catenin, and phosphotyrosine-containing

proteins. Specific binding was detected by the

Enhanced Chemiluminescence System (Amersham

Life Science, Inc.).

Northern Blot Analysis

Total RNA was isolated from pulverized, snap-

frozen tissues by the acid guanidium method (39)

using a Dounce homogenizer on ice, and northern

blots were performed with 10 mg total RNA per

lane. Ethidium bromide at a concentration of 0.2 mg/

mL was added before electrophoresis to 1 lo agarose

gels containing formaldehyde to verify the integrity

of the RNA by short-wavelength UV detection and

to monitor the equivalence of loading before and

after transfer to GeneScreen Plus filters (Du Pont

NEN, Boston, MA) (i.e., the integrity and relative

amounts of ribosomal RNAs were assessed). For

additional RNA quantitation, filters were stained

with 0.04% methylene blue in 0.5 M sodium acetate.

The integrin subunit b4 complementary DNA

(cDNA) clone (1.5-kilobase [kb] EcoRI fragment)

was labeled with random priming (Megaprime DNA

labeling system; Amersham Life Science, Inc.) and

[32P]deoxycytidine triphosphate (3000 Ci/mmol;

Amersham Life Science, Inc.). Membranes were

pretreated and hybridized in 50% formamide

(Merck) and 10% dextran sulfate (Sigma Chemical

Co.) at 42 C. Blots were washed twice with 2¾sodium chloride±sodium citrate (SSC) at room

temperature for 10 minutes, then twice with 2¾SSC±1% SDS at 60 8C for 30 minutes, and finally

twice with 0.l¾ SSC at room temperature for 30

minutes followed by exposure to autoradiography

for 24 hours at º80 8C with intensifying screens.

Results

Expression and Regulation ofIntegrins and Laminins in Normal andNeoplastic Thyroid Tissues

In normal thyroid tissue, integrin

chains a3 and b1 were exclusively ex-

posed at the basal domain of follicular

thyroid cells (Fig. 1a, panel A), whereas

a2 and a5 could not be detected (not

shown). The a6b4 complex was found to

be absent in thyroid cells (Fig. 1e, panel

A).

The basement membrane of normal

thyroid tissue was composed of laminins

1 and 2 but not laminin 5 (not shown). The

parallel lack of expression of both integrin

a6b4 and laminin 5 at the basal aspect of

thyroid cells strongly argues for the

absence of hemidesmosomes in thyroid

follicular cells. In fact, transmission

electron microscopy as well as immuno-

staining with human sera reacting with

hemidesmosome-specific bullous pem-

phigoid antigens revealed that thyroid

cells do not assemble hemidesmosomes

(not shown). The integrin subunits b3, b5,

and av did not show any obvious im-

munoreactivity.

The above distribution pattern was

totally retained in follicular adenomas

REPORTS Journal of the National Cancer Institute, Vol. 88, No. 7, April 3, 1996444

(Fig. 1b and f, panel A) but was subverted

in both papillary and follicular carcino-

mas (Fig. 1c, d, g, and h, panel A).

To support immunohistochemical data

and to check the molecular mass of the

integrin subunit b1, we performed im-

munoblot analysis on some normal tissue

and tumor samples. A b1 polyclonal

antiserum identified a 110-kd band pre-

sent at similar intensity levels (Fig. 2,

panel A).

Surprisingly, we observed neoexpres-

sion of the integrin a6b4 heterodimer in 11

of 20 malignant tumors (Fig. 1g and h,

panel A; Table 1). More precisely, two of

three well-differentiated follicular car-

cinomas and nine of 13 poorly differen-

tiated and/or clinically aggressive cancers

were found to be b4 and a6 positive.

Expression of the b4 chain in these

specific tumors was confirmed both by

immunofluorescence in fine-needle as-

piration biopsy smears (Fig. 2, panel B)

and by western blot analysis (Fig. 2, panel

C). Northern blot analysis revealed a

specific 5.5-kb b4 transcript with variable

band intensity in those carcinomas that

were found to express b4 protein in

immunohistochemistry and western blot

experiments (Fig. 2, panel D). Thus, the

data indicate that, in some thyroid can-

cers, a transcriptionally regulated expres-

sion of b4 occurs, which leads to a6b4

membrane exposure.

As with normal thyroid tissue, we did

not observe any b3 or b5 immunoreactiv-

ity in thyroid cancers (not shown).

Among basal lamina components, la-

minin 1 retained the normal pattern within

tumors, whereas laminin 5 remained

undetectable in carcinomas (not shown).

Interestingly, laminin 2 was detected at

high levels in integrin subunit b4-negative

tumors, but it was totally absent in

integrin subunit b4-positive cancers (not

shown). The inverse relationship between

integrin a6b4 and laminin 2 immunoreac-

tivity was strikingly replicable: All carci-

Fig. 1. Adhesion molecule expression and topography in normal and neoplastic thyroid tissues. Panel A: In normal (a) and adenomatous (b) thyroid tissues, theintegrin subunit a3 is exposed at the basal domain of thyroid cells, whereas it is aspecifically redistributed along the cell margins in follicular (c) and papillary (d)carcinomas. The b4 integrin chain is not present in normal (e) and adenomatous (f) follicular cells, but it is strongly expressed in poorly differentiated and/orclinically aggressive follicular (g, case FC1) and papillary (h, case PC13) carcinomas. The integrin subunit a6 expression entirely matches that of b4 (not shown). Bardenotes 16 mm. Panel B: In normal thyroid tissue (a) and in adenoma tissue (not shown), E-cadherin is selectively enriched at cell±cell contacts. In malignant tumors(b), the molecule is conserved but pericellularly redistributed. The expression of b-catenin entirely matches that of E-cadherin (not shown). Bar denotes 16 mm and,for both insets, 4 mm.

445Journal of the National Cancer Institute, Vol. 88, No. 7, April 3, 1996 REPORTS

nomas showing integrin subunit b4 neo-

synthesis did not express laminin 2.

Expression and Functional Statusof the E-cadherin±Catenin Complexin Normal and Neoplastic ThyroidTissues

E-cadherin was detected in thyroid

follicular epithelium at the boundaries

between adjacent cells (Fig. la, panel B).

This strictly lateral topography was main-

tained in adenomas; however, it was

critically subverted in malignant tumors,

where E-cadherin was diffusely distribu-

ted over the entire membrane in all

neoplastic cells (Fig. lb, panel B). The

distribution of b-catenin was identical to

that of E-cadherin in normal, adenoma-

tous, and cancerous thyroid tissues (not

shown).

E-cadherin protein levels following

SDS extraction were identical in normal,

adenomatous, and cancerous thyroid tis-

sues. Quantitative recovery of a- and b-

catenins after non-ionic detergent extrac-

tion was also identical in all samples (Fig.

3, panel A).

The loss of lateral exposure and the

pericellular distribution of E-cadherin

indicate that the association of E-cadherin

with the cytoskeleton could be impaired

in malignant thyroid tumors. To verify

that this is actually the case, we measured

the relative fractions of total E-cadherin

that either were or were not resistant to

extraction by non-ionic detergents (Fig. 3,

panel B). In fact, in normal thyroid tissues

and in thyroid adenomas, E-cadherin was

only partially extractable in nonionic

detergents, with approximately 50% of

the total pool being recovered from the

cytoskeleton-associated fraction; conver-

sely, in malignant tumors, most E-cad-

herin could be extracted by non-ionic

detergents, and the insoluble fraction

represented only a minor portion. These

results support morphologic data and

indicate that the complex is disconnected

from the microfilament network and

undergoes pericellular relocalization

only in carcinomas.

Finally, we examined the tyrosinepho-

sphorylation status of the E-cadherin±

catenin complex in detergent extracts

from normal and neoplastic thyroid tis-

sues (Fig. 3, panel C). In E-cadherin

immunoprecipitates, three bands of 130,

100, and 88 kd, respectively, comigrating

Fig. 2. Panel A: western blot (WB) analysis of b1 integrin chain expression in representative cases fromnormal (NT) and neoplastic thyroid (FA, follicular adenoma; FC, follicular carcinoma; PC, papillarycarcinoma). Panel B: indirect immunofluorescence observation of the integrin subunit b4 in fine-needleaspiration biopsy smears. b4 is undetectable in smears from nodular goiters (a) and adenomas (b), but it ishighly immunoreactive in some papillary (c, case PC13) and follicular (d, case FC1) carcinomas. Bardenotes 2 mm. Panel C: western blot (WB) analysis of b4 integrin chain expression in normal and neoplasticthyroid tissues. Panel D: northern blot (NB) analysis of b4 transcripts in neoplastic thyroid samples. A 5.5-kilobase (kb) transcript corresponding to b4 messenger RNA is detected in the same samples expressing b4

protein but not in b4 protein-negative extracts. kDa = kilodaltons.

REPORTS Journal of the National Cancer Institute, Vol. 88, No. 7, April 3, 1996446

with E-cadherin, a-catenin, and b-catenin,

were identified by western blot analysis

using a phosphotyrosine-specific anti-

body. No tyrosine phosphorylation of g-

catenin±plakoglobin was observed. One

striking observation was that the level of

tyrosine phosphorylation of E-cadherin

was markedly decreased and almost

abolished in thyroid carcinomas; this

finding was consistently observed in all

malignant neoplasms, and it was always

associated with unmodified E-cadherin

protein expression.

Discussion

The epithelial phenotype is character-

ized by the following two discrete mem-

brane domains involved in tissue

polarization and cell cohesion: the basal

domain and the lateral domain. The basal

domain is responsible for recognition of

matrix ligands and attachment to the

basement membrane zone by means of

specific receptors. One major basal recep-

tor is the integrin a6b4 heterodimer that is

also responsible for hemidesmosome as-

sembly (14-16). The lateral domain

mediates epithelial cell alignment by

means of junction-associated adhesion

molecules, e.g., cadherins (40,41) and

integrins of the b1 subfamily (4-6).

Spatial disorganization of, functional

impairment of, or quantitative alterations

in the levels of several adhesion mole-

cules have been widely reported in

epithelial tumors [for references, see

(2,3,42)].

The aim of our study was to investigate

the adhesive mechanisms involved in

Fig. 3. Panel A: western blot analysis of the E-cadherin-catenin complex innormal and neoplastic thyroid tissues. The complex is expressed at comparablelevels, with a conserved stoichiometry, in extracts from both benign andmalignant specimens. Panel B: detergent resistance of E-cadherin in normal andneoplastic thyroid tissues. In normal thyroid tissues and adenomas, E-cadherin islargely resistant to Triton X-100 extraction; in contrast, in carcinomas, it isreadily extracted by non-ionic detergents. Panel C: tyrosine-phosphorylationstatus of the E-cadherin-catenin complex in normal and neoplastic thyroid

tissues. In normal thyroid tissues and adenomas, three bands, comigrating withE-cadherin, a-catenin, and b-catenin, can be observed. In contrast, no tyrosinephosphorylation of E-cadherin is detectable in follicular and papillary carcino-mas. IP = immunoprecipitation; WB = western blot; NT = normal thyroid tissue;FA = follicular adenoma; FC = follicular carcinoma; PC = papillary carcinoma;Sol. = Triton X-100 soluble; Ins. = Triton X-100 insoluble. Molecular massmarkers are indicated on the left in kilodaltons.

447Journal of the National Cancer Institute, Vol. 88, No. 7, April 3, 1996 REPORTS

establishing the polarized phenotype of

thyroid cells and to observe the topo-

graphic and functional modifications of

two classes of relevant adhesion mole-

cules at various stages of neoplastic

progression. In our working hypothesis,

alterations of cellular adhesiveness occur-

ring upon malignant transformation

would result in a rearrangement of adhe-

sion molecule expression and function

that could be readily detected in pre-

operative fine-needle aspiration biopsy

smears as well as in surgical samples.

The fact that the thyroid carcinomas

that switch on b4 expression and switch

off laminin 2 synthesis are those that are

associated with the most aggressive

behavior could be of major importance

both from a biological viewpoint and

from a clinical viewpoint. The role of

integrin subunit b4 is not yet fully under-

stood. The a6b4 heterodimer has been

shown to mediate stable adhesion to

laminin 1, laminin 2, and laminin 5 (43-

45). In contrast, integrin subunit a3-

transfected cells can adhere to laminin 2,

but they fail to attach to and spread on

laminin 1 (46). Therefore, the fact that

normal thyroid tissue expresses one in-

tegrin receptor (a3b1) and two laminin

isoforms (laminins 1 and 2) and yet only

one ligand is recognized by the integrin

heterodimer seems paradoxical. We sug-

gest that, under normal conditions in vivo,

recognition between follicular cells and

the basal lamina is mediated by the

interaction of integrin a3b1 and laminin

2 (and possibly by unknown integrins or

nonintegrin receptors). When synthesis of

laminin 2, which has been shown to be

related to a highly differentiated pheno-

type (33,47), is switched off in a subset of

aggressive thyroid tumors, the de novo

expression of a6b4 might confer a selec-

tive advantage for invasion by providing a

complementary and more versatile lami-

nin receptor capable of triggering recog-

nition events and attachment to laminin 1

that precede infiltration.

Detection of a6b4 exposure can be

achieved easily in smears from fine-

needle aspiration biopsies. If validated

by a large-scale clinical investigation that

is now in progress (Orlandi F, Saggiorato

E, Serini G, Trusolino L, Marchisio PC,

Angeli A: manuscript in preparation), this

finding might be relevant for the pre-

surgical detection of aggressive carcino-

mas and for the differential diagnosis

between follicular adenomas and carci-

nomas.

E-cadherin±b-catenin localization in

normal and adenomatous thyroid tissues

is restricted to the lateral domain of

juxtaposed thyroid cells. In contrast, in

carcinomas, the complex undergoes peri-

cellular diffusion. This subverted topo-

graphy is accompanied by a different

pattern of detergent extractability be-

tween noncancerous and cancerous le-

sions that is consistent with disconnection

of the E-cadherin±catenin complex from

the actin microfilaments. This surface

rearrangement is not associated with any

changed synthesis of E-cadherin and a-

and b-catenins. To our knowledge, this is

the first report in which loss of E-

cadherin±catenin lateral polarization and

removal of the complex from cytoskele-

ton-associated adhesion sites, without

reduced expression of the molecules

involved, have been described in the

progression from benign to malignant

lesions.

Tyrosine phosphorylation of the E-

cadherin±catenin complex plays a key

role in perturbation of the cadherin cell

adhesion system (3,48,49). In normal

adult tissues, specific proto-oncogenic

tyrosine kinases of the src family are

enriched at zonula adherens, where the

level of tyrosine phosphorylation is high

(50). Since a positive association exists in

our data between the three parameters of

decreased tyrosine phosphorylation, loss

of detergent insolubility, and pericellular

redistribution of E-cadherin, we suggest

that tyrosine phosphorylation of the E-

cadherin cytodomain may play a role in

controlling not only the structure of the

whole molecule and, hence, intercellular

adhesion, but also its stability within the

membrane plane. In addition, dissociation

of the E-cadherin±catenin complex from

functional regions could drive the com-

plex away from kinases (51) and phos-

phatases (52) specifically acting on E-

cadherin and responsible for the phos-

phorylation status of the molecule under

normal conditions.

In summary, the addition of a panel of

MAbs to adhesion molecules in the tool-

box of thyroid surgical pathologists, if

validated in larger studies, may expand

the options to reach a reasonably correct

early diagnosis and even gain some

prognostic information from routine nee-

dle biopsies.

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Notes1EHS±laminin is derived from Engelbreth-Holm-

Swarm sarcoma.

G. Serini and L. Trusolino contributed equally tothe experimental work described in this report.

Supported by Target Project `̀ Applicazioni Clin-iche della Ricerca Oncologica'' Consiglio Nazionaledelle Ricerche (Rome), by Associazione Italiana perla Ricerca sul Cancro (Milano), and by theMinistero per l'UniversitaÁ a la Ricerca Scientificaa Tecnologica (Rome).

We gratefully acknowledge the skillful technicalassistance of Germana Cecchini. We are indebted toAndrea Graziani and Daniela Gramaglia for provid-ing the monoclonal antibody to phosphotyrosine.

Manuscript received August 8, 1995; revisedNovember 3, 1995; accepted January 25, 1996.

449Journal of the National Cancer Institute, Vol. 88, No. 7, April 3, 1996 REPORTS