Integrin expression in normal, hyperplastic, dysplastic, and malignant oral epithelium

JOURNAL OF PATHOLOGY, VOL. 169 235-243 (1 993)

INTEGRIN EXPRESSION IN NORMAL, HYPERPLASTIC, DYSPLASTIC, AND

MALIGNANT ORAL EPITHELIUM JUDITH JONES*, MASARU SUGIYAMA*t, FIONA M. WATT? AND PAUL M. SPEIGHT*

*Joint Department of Oral Pathology, The London Hospital Medical College and the Institute of Dental Surgery, Eastman Dental Hospital, 256 Gray's Inn Road, London WCIX 8LD, U.K.; TKeratinocyte Laboratory,

Imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London WCZA 3PX, U.K.

Received 4 June 1992 Accepted24 July 1992

SUMMARY We have examined the distribution of a range of integrin subunits in normal and lesional oral mucosa. The a2, a,, u6,

p,, and 8, subunits were highly expressed in normal epithelium, and there was weaker, more variable expression of a, and a,. Expression of all subunits was highest in the basal layer of normal epithelium, but extensive staining above the basal layer was also observed, particularly in the floor of the mouth and the lateral margin of the tongue. In dysplastic lesions and hyperplastic epithelium adjacent to ulcers, suprabasal staining was even more pronounced. Staining patterns in squarnous cell carcinomas showed considerable variation, both within and between individual tumours: in some areas there was staining reminiscent of normal epithelium, but uniform staining throughout tumour islands, and patchy and variable cytoplasmic and pericellular staining were also seen. Thirteen out of 17 carcinomas showed some loss of integrin expression: six out of ten moderately well differentiated tumours and all the poorly differentiated tumours. Focal loss of u6 and D4 was most commonly observed, but loss of u2 and uj also occurred. Since integrins regulate not only keratinocyte adhesion, but also the initiation of terminal differentiation, the changes in integrin expression that we have observed may have significance for the behaviour of individual tumours.

KEY womx-Integrins, squamous cell carcinoma, oral epithelium, ulcers, dysplasia.

INTRODUCTION

Integrins are a family of cell surface receptors that mediate cellkcell and cell-extracellular matrix adhesion.'-3 Each integrin consists of an a and a /j' subunit which are non-covalently associated transmembrane glycoproteins. Integrins play an important role in regulating a wide range of cellular interactions during growth, development, differentiation, and the immune response.'-3

Stratified squamous epithelia express integrins of the /I,, b4, and p5 fa mi lie^.^-'^ Studies with cultured epidermal cells suggest that keratinocyte integrins regulate three distinct processes: cellkell

Addressee for correspondence: Dr Paul M. Speight, Depart- ment of Oral Pathology, Institute of Dental Surgery, Eastman Dental Hospital, 256 Gray's Inn Road, London WClX8LD, U.K.

a d h e ~ i o n , ~ . ' ~ adhesion to extracellular matrix proteins,'-8 and commitment to terminal differen- t i a t i ~ n . ' ~ In normal epidermis, integrin expression is largely confined to the basal layer. However, during wound healing and in psoriatic lesions integrins are also expressed by su rabasal, terminally

Carcinomas are characterized by invasion of malignant cells into the underlying connective tissues and by an ability to metastasize to distant sites. This process involves an alteration of cell-matrix and cellkcell interactions which may be associated with changes in the expression or function of integrins. In addition, aberrant integrin expression or function may act as a signal for loss of control of proliferation and differentiation in neoplasia.*' In support of this idea, altered integrin expression has been reported in a number of malignant epithelial

differentiating keratinocytes. II ,%

0022-341 7/93/020235-09 $09.50 0 1993 by John Wiley & Sons, Ltd.

236 J. JONES ETAL.

tumours, including squamous and basal cell carcinomas of the kin,'^,^* and adenocarcinomas of the ~ o l o n , ~ ~ , ~ ~ b r e a ~ t , ~ ~ - ~ ' and pancreas.29 Loss of integrins has been noted in aggressive lesions and has been associated with a oor prognosis in breast and colorectal tumours.

Little is known about the distribution of integrins in either normal or pathological oral epithelium. The pattern of differentiation and keratinization differs in different regions of the mouth and this might be reflected in site-specific variation in integrin expression. The purpose of our study was to examinep, and p4 integrin expression in normal oral epithelium and to determine whether it is altered in hyperproliferative, dysplastic, and neoplastic lesions.

23-2r

MATERIALS AND METHODS

Selection of' t issue

A total of 75 specimens were examined. There were 37 samples of normal oral mucosa from sites that differ in keratinization (Table I); these samples

Table I-Details of the specimens of normal oral mucosa

Site n Type of epithelium

Cheek 16 Para-keratinized Hard palate 10 Ortho- keratinized Lateral tongue 5 Non-keratinized Floor of mouth 6 Non-keratinized

were obtained either from the margins of surgical specimens not associated with malignancy or from sudden-death post-mortems within 40 h of death. Thirty-eight samples of lesional tissue, representing three types of lesion, were also examined. Squamous cell carcinomas were moderately (10 specimens) or poorly differentiated (7) and were located on the lateral or ventral surface of the tongue, the floor of the mouth, buccal sulcus, or lower alveolus. Cases of epithelial dysplasia (1 I ) were subjectively graded as mild or moderate; although it is considered to be a premalignant lesion, no case used in this study was associated with malignant change. The healing ulcers (10) were cases of non-specific ulceration from

the tongue or buccal mucosa and were associated with trauma.

Primary ant ihodies Table I1 gives details of the primary antibodies

used to stain sections. All antibodies were used on all tissues, except for 13C2, which was applied only to normal oral epithelium, and anti-laminin, which was only used on selected cases of carcinomas. A range of dilutions of every antibody was tested.

Staining Six pm frozen sections were cut, fixed in 100 per

cent acetone for 10 min at room temperature, air- dried for 1 min, and then washed in PBS containing 1 mM calcium chloride and magnesium chloride (PBSABC). Sections were incubated with primary antibody diluted in PBSABC for I h at room temperature (except BIIG2, which was applied either for 1 h at room temperature or for 18 h at 4°C) and then washed in PBSABC. Biotin-conjugated secondary antibodies (anti-mouse, anti-rat, or anti- rabbit as appropriate; Sigma Chemical Company, St Louis, U.S.A.) were applied at a concentration of 1 :200 for 30 min and then the sections were washed in PBSABC. Slides were incubated with avidin- biotin complex (Dakopatts, U.K.) for 30 min, washed three times in PBSABC, and developed in a 50 pg/ml solution of diaminobenzidine (Sigma Chemical Company) and 0.03 per cent H,O, in PBS for 10 min. Sections were lightly counterstained with Mayers haematoxylin. As a control, primary antibodies were omitted from the staining sequence.

Within normal stratified squamous epithelium, positive staining was assessed in four cell compartments: basal layer (the layer adjacent to the basement membrane), suprabasal layer (one or two cell layers above the basal layer), prickle cell layer (the bulk of the epithelium, composed of cells with identifiable desmosomes), and the superficial layer (the outermost or keratinized layers).

In moderately differentiated squamous cell carcinomas, the compartments examined were cells lying adjacent to the basement membrane (equivalent to basal cells), cells lying above these (equivalent to suprabasal cells), cells lying in the centre of the tumour islands (equivalent to superficial cells), and those cells lying between the centre of the tumour islands and the suprabasal equivalent (equivalent to prickle cells). In poorly differentiated squamous cell carcinomas, cell layers could not be identified and an overall assessment of the tumour section was made.

INTEGRIN EXPRESSION IN ORAL EPITHELIUM 237

Table 11-Primary antibodies

Specificity Antibody Species Source

a1 TS 217 Mouse M. E. Hemler3” a2 HAS6 Mouse F. M. Watt a3 VM-2 Mouse American Tissue Culture Collection3’ ‘4 B-5G10 Mouse M. E. Hemler3* a4 P4G9 Mouse Telios Pharmaceuticals Inc. a5 BIIG2 Rat C . H. Damsky” ‘6 GoH3 Rat A. Sonnenbergs4

13C2 Mouse M. A. Horton3’ Anti-CD 29 Mouse Janssen Pharmaceutica 3E1 Mouse E. Eng~a11~~

? B4 Involucrin DH 1 Rabbit F. M. Watt3’ Laminin Anti-laminin Rabbit M. J. Warb~rton-’~

RESULTS

Normal epithelium Cheek-Strong positive staining of the epi-

thelium was observed with antibodies to a2, a3, a6, PI, and /I4 (Fig. l a and results not shown). All subunits showed high levels of expression in the basal and immediately suprabasal layers with weaker and patchy expression in the prickle cell layers. The superficial layers were never stained with antibodies to a,, a6, PI, and P4 (see Fig. la), but a2 showed occasional staining. All subunits had a pericellular distribution, but a6 and b4 showed a relative concentration at the basal aspect of the basal cells, giving a strong band-like pattern at the basement membrane zone. Faint cytoplasmic staining was observed in the basal and suprabasal cells for all subunits.

Sections were also stained with antibodies to the a, , a4, a5, and a, subunits. a, and a4 were not detected. a, was expressed in the basal layer of all specimens and some specimens showed positive suprabasal staining. a5 showed only occasional weak cytoplasmic staining of basal cells. Tntraepi- thelial dendritic cells, presumably Langerhans cells, were positive for a4 and Dl in all specimens and for u5 in five specimens.

Hard palate,JEoor of mouth, und lateral tongue- The integrin staining patterns observed at other sites in the oral cavity were essentially similar to those seen in cheek epithelium (Figs Ib, Ic, and results not shown). However, in the floor of mouth and lateral tongue there was a general increase in expression above the basal layers. a2, p3, and B, were frequently and strongly expressed in the prickle

cell layers and in some cases the whole thickness of the epithelium was stained (Fig. lc). In these cases, the prickle cells co-expressed integrin subunits and involucrin (results not shown). a5 was not observed in keratinocytes in any of the specimens, although Langerhans cells and endothelial cells were positively stained.

Hyperplastic and dysplastic epithelium In dysplastic lesions and in hyperplastic cheek

epithelium adjacent to ulcers, increased integrin expression above the basal layer was consistently observed (Fig. 2). In the hyperplastic epithelium, a2, a3, a6, and b1 were frequently expressed throughout all cell layers and the /3, subunit was expressed in the suprabasal and prickle cell layers (Figs 2a, 2b, and results not shown). PI, P4, and a6 (Fig. 2c) were seen above the basal layer in dysplastic epithelium but staining in the prickle cell layers was not as extensive as in hyperplasticepithelium, and staining for u2 and a3 was similar to that observed in normal cheek.

Pericellular staining for a5 was seen in the basal layer in four hyperplasias and two dysplasias. a , and a4 were negative in all specimens.

Squamous cell carcinomas Moderately well diferentiated-The u3, a,,b1, and

8, subunits were expressed in all tumours but the pattern of expression varied between tumours and between different areas of the same tumour. Four different staining patterns were observed (Figs 1 and 3). In some sections, there was a ‘normal’ pattern, with strong pericellular staining of basal and suprabasal cells but weak or negative staining of the terminally differentiated cells at the centre of tumour

238 J . JONES ET AL.

Fig. I-Normal epithelium and squamous cell carcinomas. (a) Cheek epithelium shows strong basal and immediately SUprdbaSdl staining for&. (b) Floor of mouth shows strong stainingforp,polarized to the basement membrane zone. There is weakerpericellular staining immediately above the basal layer. (c) Staining for a, is present throughout the thickness of epithelium from the floor of mouth. (d, e) Poorly differentiated squamous cell carcinomas stained for 8, (d) or a6 (e). Note the loss of polarized expression at the epitheliakonnective tissue interface which is patchy and focal in (d) and more extensive in (e). (f) A moderately differentiated tumour stained for al- The tumour islands (*) are completely negative, although adjacent vessels show normal expression

islands (Fig. 3a). A second pattern showed positive staining throughout the tumour, even within well differentiated tumour islands which were positively stained for involucrin (Fig. 3b and results not

shown). The third pattern was of patchy and variable cytoplasmic and pericellular staining.

The final pattern was significant loss of integrin expression (Table 111). Two tumours showed patchy


Fig. 2-Hyperproliferative and dysplastic epithelium. (a) a2 is expressed in all cells at the advancing front of a healing ulcer. (b) u, expression throughout all cell layers in hyperproliferative epithelium. (c) A case of mild epithelial dysplasia showing increased ab expression with strong and variable expression on the pleomorphic suprabasal cells

Fig. 3-Staining patterns observed in squamous cell carcinomas cell carcinomas. (a, b) Moderately well differentiated; (c) poorly differentiated. (a) B, staining showing a ‘normal’ staining pattern with strong basal and suprabasal staining and loss of expression among kerdtinizing cells in the Centre of the tumour island. (b) aa staining showing strong positivity throughout the tumour. (c) a, staining showing a patchy and variable pattern with loss of expression on some cells

loss of p4 at the basement membrane zone, three showed loss of a6, and one case had patchy loss of both a6 and p4. In most tumours, a, and u3 were expressed throughout tumour islands but one case showed loss of a3 among the basal cells in addition

to loss of B,. One case was completely negative for the a, subunit (Fig. 1 f).

a, and a4 were not detected in any of the tumours. There was occasional weak pericellular staining for a5 in four cases.

J. JONES ETAL. 240

Table 111-Loss of integrins in squamous cell carcinomas

Case a2 ‘3 a6 p4

Moderately difkrentiated ** 1 2 3 4 5 6 7 8 9

10

Poorly direrentiated 11 12 13 14 15 16 17

* * * *

*

* *

*

* * * * * *

*Patchy, focal loss. **Total loss.

Poorly diferentiated-The overall pattern of integrin expression was similar to that observed in the moderately differentiated tumours, with variable staining both within and between tumours (Figs Id, le, and 3c). Although there was no evidence of terminal differentiation, as judged by lack of staining for involucrin, the centres of some tumour islands were still integrin-negative.

Loss of specific integrin subunits was more extensive than in the moderately differentiated tumours (Table 111). Two cases showed focal loss of a6 or/?, at the basement membrane zone (e.g. Fig. Id) and five showed loss of both u6 and p,. In two of the latter, the loss was extensive and involved at least 25 per cent of the epithelial-connective tissue interface (Fig. le). Staining for u2 and u3 was reduced in all cases, but four showed extensive loss among cells adjacent to the connective tissue (‘basal cells’) (Fig. 3c). Two cases which showed extensive loss of a6 and p4 were stained for laminin and showed a pattern of loss which was similar in distribution to the integrin loss (results not shown).

The prevalence of integrin loss is summarized in Table 111. Thirteen of the 17 carcinomas showed some loss of integrin expression: six of ten moder-

ately differentiated tumours and all the poorly differentiated tumours. The loss was more extensive in the poorly differentiated tumours, except for the one moderately differentiated case which lacked a?.

DISCUSSION

We found that the major integrin subunits expressed in the stratified squamous epithelium of four different areas of the oral cavity were the same as those expressed in the e p i d e r m i ~ ’ ~ ~ ’ ’ ~ ’ ~ ’ ~ and gingiva.” The u2, a3, a6, D,, and p, subunits were highly expressed, whereas expression of as and a, was weaker and more variable, and a, and u4 could not be detected. The u2, u3, u5, a,, and PI subunits generally had a uniform pericellular distribution, although in some sections a2 and u3 were polarized to either the apical or basal aspect of basal cells. The a6 and /3, subunits were also found on all surfaces of basal cells, but there was a relative concentration of these subunits on the basal surface, consistent with the association of ad4 with h e m i d e s m o s ~ m e s . ~ ~ ~ In cultured epidermal keratinocytes, and a$, mediate adhesion to collagen and laminin, and a3pI is also an epiligrin receptor; a$, is a fibronectin receptor, a$, is a receptor for vitronectin, and the ligand for ad4 remains to be e~tablished.~~’,~,~,’~,~~

In the epidermis, integrin expression is largely confined to the basal layer, but in normal oral mucosa, extensive staining above the basal layer was also observed, particularly in the floor of mouth and lateral border of the tongue, where prickle cells were found to express both integrins and the terminal differentiation marker inv0lucrin.4~ In the epidermis, integrin expression by keratinocytes undergoing terminal differentiation is observed during wound healing and in psoriasis, suggesting a correlation with hyperproliferation, perhaps as a consequence of exposure to inflammatory cyto- kines.” It is thus interesting to note that the rate of cell turnover in non- and para-keratinbed epithelium of the mouth is approximately twice that of the epiderrni~.~’

In the hyperproliferative epithelium at the edge of healing ulcers, enhanced staining of a,, u3, PI, u6, and p4 was observed above the basal layer; suprabasal expression of u6 was more extensive thanp,, suggesting that u6 may form a heterodimer with p,.” In addition, the a5 subunit was expressed in a pericellular distribution in four out of ten specimens, whereas in normal cheek epithelium pericellular staining was not usually seen. Keratinocyte

INTEGRIN EXPRESSION IN ORAL EPITHELIUM 24 1

adhesiveness to fibronectin is upregulated during epidermal wound healing and a&, expression is increased in keratinocytes migrating out of skin ex plant^.^' In contrast, in epidermal suction blister wounds, in which the basement membrane is intact, a, expression is not increased,“ and in corneal wounds the presence or absence of a basement membrane does not significantly affect the a5 staining pattern.47 The present findings indicate an individual variation in us expression.

Expression of integrin subunits above the basal layer was also increased in dysplastic epithelium, but was slightly less marked than in hyperplastic epithelium. However, these were mild dysplasias and none progressed to frank malignancy. It will therefore be necessary to examine more cases in order to determine whether or not the patterns of integrin staining in dysplasias have any prognostic significance.

Integrin expression in squamous cell carcinomas showed considerable variation, not only between tumours, but also within different areas in the same tumour. There was no simple correlation with differentiation, as assessed by morphology or involucrin expression. In some tumours, areas that were well differentiated lacked integrins and thus mirrored the expression of integrins in normal oral mucosa, whereas in others highly differentiated cells were integrin-positive, resembling the situation in hyperplastic epithelium.

The variable integrin expression that we observed fits well with observations on other epithelial tumours. Altered and variable integrin staining patterns have been reported in squamous cell carcinomas of the skin’7 and head and n e ~ k . ~ ’ , ~ ~ Decreased or increased integrin staining in breast carcinomas2* and moderately or poorly differentiated colorectal adenocarcinomas has also been r e p ~ r t e d . ~ ~ , ~ ~

There are some indications that the variation in integrin staining patterns has prognostic value. In squamous cell carcinomas of the head and neck, increased staining with a monoclonal antibody to thep, integrin subunit has been associated with poor p r o g n o ~ i s . ~ ~ ~ ~ ~ However, in several other cases, poor prognosis is associated with a loss of integrins. There was a significant association between loss of the u2 subunit in colon carcinomas and the for- mation of lymph node or distant me tas t a se~ ,~~ and progressive loss of the u2, a3, and PI subunits has been associated with loss of tumour differentiation in colorectal adenocarcinomas.2’ In invasive and infiltrating ductal carcinomas of the breast, decrease or loss of a2, a3, and/or 8, has been ~bserved”~~’,~*

and there is also a report of loss of ah at the basal membranes of such tumours.2h Both in oral squamous cell carcinomas and in breast carcinomas, loss of staining for a6 at the basement membrane zone is correlated with loss of laminin.*‘

In our study some loss of staining for the a2, a3, a6, and b4 subunits was observed in squamous cell carcinomas of the oral cavity. Two out of ten moderately differentiated tumours and four out of seven poorly differentiated tumours showed areas of loss of the u2 and/or the u3 subunits. In one moderately differentiated tumour, no a2 was detected; this tumour was particularly aggressive and presented in stage IV. Although overall expression of u6 was increased, with most tumours showing strong positive staining throughout the tumour islands, there was focal loss of a6 and P4 from cells at the epithelial-connective tissue interface. Loss of a6 and 0, was more widespread in poorly differentiated lesions and may thus be associated with poor prognosis.

In conclusion, it is apparent that the expression of Dl and P4 integrins in oral mucosa is similar to that described in skin, but expression is present more superficially. Within the oral cavity, integrin expression varies only slightly between different sites. In situations where epithelium is rapidly pro- liferating, such as at the margin of a healing ulcer, integrin expression is increased in intensity and distribution. Variable expression of integrins was noted in squamous cell carcinomas, with total absence of a2 in one tumour and frequent loss of u6 and 8, at the basement membrane zone. The results support previous work which has shown a relationship between integrin loss and poorly differentiated lesions and is in agreement with the suggestion that alterations in cell adhesion receptors may contrib- ute to the uncontrolled pattern of growth typical of malignant epithelial cells.” Further work will address this issue and will investigate the role of integrins in modulating the behaviour of malignant cells in vitro.

ACKNOWLEDGEMENTS

We are very grateful to everyone who provided antibodies for this study. M.S. is on leave from the 1 st Department of Oral and Maxillofacial Surgery, Osaka University. J.J. was the recipient of an MRC Advanced Course Studentship.

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Integrin expression in normal, hyperplastic, dysplastic, and malignant oral epithelium