Expression of hemidesmosomal and extracellular matrix proteins by ...

American Journal ofPathology, Vol. 146, No. 6, June 1995 Copyright © American Societyfor Investigative Pathology

Expression of Hemidesmosomal and Extracellular Matrix Proteins by Normal and Malignant Human Prostate Tissue

Ray B. Nagle,* Junshan Hao,* J. David Knox,* Bruce L. Dalkin,t Virginia Clark,* and Anne E. Cress$ From the Departments of Pathology,* Surgery,t and Radiation Oncology,t University of Arizona Health Sciences Center, Tucson, Arizona

The progression of prostate carcinoma may be influenced by the biochemical nature of the basal lamina surrounding the primary carcinoma ceUs. As a flrst step toward understanding this process, the composition and structure of the basal lamina in normalprostate, prostatic intraepithelial neoplasia, and human carcinoma were determined. In addition, a comparison was made between the attachments of the normal basal ceU to its underlying basal lamina and those made by primary prostate carcinoma. The normal basal ceUs form botb focal adhesions and hemidesmosomal-like structures as observed by transmission electron microscopy. The normal basal ceUs exhibited a polarized distribution of hemidesmosomal associated proteins including BP1 80, BP230, HD1, plectin, laminin- y2 (B2t), collagen VII, and the corresponding integrin laminin receptors v6g31 and a6g34. The expression and distribution pattern of these proteins were retained in theprostate intraepithelial neoplasia lesions. In contrast, the carcinoma ceUs uniformly lacked hemidesmosomal structures, the integrin a6f34, BP180, laminin-y2 (B2t), and coUagen VII but did express BP230 (30%), plectin, HD1 (15%), and the integrin laminin receptors cv3 (31 and c6.(1. These results suggest that, although a detectable basal lamina structure is present in carcinoma, its composition and celular attachments are abnormaL The loss of critical celular attachments may play a role in influencing the progression potential of prostate carcinoma. (Am J Pathol 1995, 146-1498-1507)

Although prostate carcinoma has become the most commonly diagnosed visceral neoplasm in males,1

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little is known of its basic biology. Previously, using sensitive immunohistochemical techniques on frozen sections, we have shown that an ephemeral basal lamina is formed by most groups of malignant cells regardless of the histological grade.2 The use of specific antibodies demonstrated that the basal lamina formed by carcinoma is similar in composition to the basal lamina of normal glands in that its major components are type IV collagen, entactin, and laminin. Additionally, the laminin subchains al (A), a2 (M), (1 (B1), ,32 (S), and yl (B2) have all been shown to be present in the human prostate.3'4 Our previous studies have shown that in the normal prostate gland the basal cells attach to the laminin of the basement membrane primarily through two integrin pairs, a6f31 and a6f34.2 The laminin receptor a6f31 forms focal adhesions and the a6j34 integrin pair, in the epidermis, is associated with hemidesmosomes.2 We and others have shown that carcinomas maintain the a6131 integrin but lose the ability to form the a6134 pair.2'5 In this study these findings have been extended by examining normal and malignant glands of the prostate for the presence of focal adhesions and hemidesmosomes and the proteins related to them. We demonstrate for the first time that hemidesmosomes are formed by the normal prostate glands. In addition, the distribution of the newly described laminin subchain y2 (B2t)6 in normal and malignant prostate is characterized.

Materials and Methods Human Tissue Specimens Thirty-nine specimens of primary prostatic carcinoma containing adjacent nonmalignant glands were obSupported by National Institutes of Health Grant 2PO1 CA 56666OlAl. Accepted for publication February 21, 1995. Address reprint requests to Dr. Ray B. Nagle, Department of Pathology, Rm. 5226, Health Sciences Center, University of Arizona, 1501 N. Campbell Avenue, Tucson, AZ 85724.

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tained at the time of radical prostatectomy. Twenty of these cases were used primarily to study integrin expression. An additional twenty cases, including one case from the integrin group, were used to determine the abundance of the hemidesmosome proteins. An additional thirteen specimens containing high grade prostatic intraepithelial neoplasia (PIN)7 were studied for integrin expression. Ten normal autopsy prostates from young men dying from trauma were used as normal controls. All tissue was snap frozen in isopentane cooled in liquid nitrogen. Cryosections 3 to 4 p thick were reacted with monoclonal antibodies for indirect immunofluorescence microscopy and examined in a Zeiss photomicroscope equipped with a 50-mm mercury epi-illuminator. Sections were additionally examined by using a Zeiss laser scanning confocal microscope (LSM 10). Primary antibodies used included polyclonal antisera specific for Engelbreth-Holm-Swarm transplantable mouse tumor laminin, the y2 (B2t) truncated form of laminin, and the 230-kd and 180-kd bullous pemphigoid antigens. Monoclonal antibodies used were specific for the HD1 500-kd hemidesmosomal protein, the al to ca6, cav, 131, and (4 integrins and collagen VII. All antibodies, dilutions used, references, and sources are listed in Table 1. Specific antikeratin primary antisera (also listed in Table 1) were used to specifically stain normal basal cells and differentiate malignant from benign glands. The primary extracellular matrix and hemidesmosomal proteins were detected by secondary antibodies coupled to fluorescein isothiocyanate. Antikeratin antibodies used were the polyclonal 18A antibody21 (reacts with both malignant and benign epithelium and

highlights the basal cells), monoclonal antibody KA4 (reacts with cytokeratins 14, 15, 16, and 19 and detects both malignant and benign epithelium), and KA1 (specifically reacts with cytokeratins 5 and 14 and highlights only the basal cells of normal glands).22 The keratin antibodies were detected by secondary antibodies labeled with Texas Red. An additional twenty specimens were fixed in 4% paraformaldehyde and 1% glutaraldehyde and processed for routine transmission electron microscopy. Photomicrographs were taken with a JEOL 100CX transmission electron microscope. Three cases containing examples of PIN were examined.

Results Two types of attachment structures at the basal cell/ basal lamina junction were observed in the electron micrographs of normal prostate glands. The first structure was characterized by cytoplasmic plaques, subcytoplasmic dense plates, fine filaments spanning the lamina lucida, and striated anchoring fibers extending into the adjacent interstitium (Figure 1A). This structure is therefore similar in morphology to the hemidesmosome of the epidermis.23 A second type of junction was identified that consisted simply of electron-dense condensations in the basal cytoplasm next to the basal lamina. These latter structures correspond to adhesion plaques that lack anchoring filaments, subcytoplasmic dense plates, and anchoring fibrils (Figure 1A). The thin basal lamina of primary prostatic carcinomas was less organized than that of normal glands

Table 1. Antibody Reagents

Specificity Laminin

Clone

y2 (B2t) laminin subchain J20 LH7.2 Collagen VII AlA5 p1 j4 3E1 al Ts2/7 a2 P1E6 ca3 P1B5 ca4 P4G9 P1D6 a5 ca6 GOH3

cv

BP1 80

BP230 HD1 Plectin General keratin Keratins 5,14 Keratins 14,15,16,19 * Unpublished data.

Type Antibody Rabbit polyclonal Rabbit polyclonal Mouse MAb Mouse MAb Mouse MAb Mouse MAb Mouse MAb Mouse MAb Mouse MAb Mouse MAb Rat MAb

Dilution 1:100 1:100 1:25

1:80 1:80 1:100 1:100 1:200 1:100 1:400 1:75 VNR147 Mouse MAb 1:100 233 Mouse MAb 1:4 J17 Rabbit polyclonal 1:80 1OC5 Mouse MAb 1:100 121 Mouse MAb 1:15 21 Rabbit polyclonal 1:50 18A Rabbit polyclonal 1:60 KA1 Mouse MAb 1:1000 KA4 Mouse MAb 1:1000

Source ICN, Costa Mesa, CA* Dr. J. Jones, Northwestern University, Chicago, IL* Dr. E. Birgit Lane, University of Dundee, Dundee, UK8 T Cell Diagnostics, Cambridge, MA9 Telios, San Diego, CA10 Dr. M. E. Hemler, Boston, MA1

Telios12 Telios12 Telios12 Telios12 Accurate, Westbury, NY13 Telios14,15 Dr. K. Owaribe, Nagoya University, Nagoya, Japan16 Dr. J. Jones17 Dr. J. Jones18 Dr. K. Owaribe19 Dr. G. Wiche, University of Vienna, Vienna, Austria20 Dr. R. Nagle21 Dr. R. Nagle22 Dr. R. Nagle22

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C.

Figure 1. A: Electron micrograph showing the basal cell attachment to the underlying basal lamina (BL). Note hemidesmosome-like structures with cytoplasmic dense plaques, subcytoplasmic dense plate (arrows), fine anchoring fila ment transversing the lamina lucida, and the striated anchoringfibrils (double arrows). Note also focal adhesions (open arrows) with electron-dense material in the cytoplasm adjacent to the cell membrane but lacking the other structures. Magnification, X38,000. B: Electron micrograph showing the relationship between a primary prostate carcinoma cell and its underlying basal lamina (BL). Note focal adhesions (open arrows) but absence of hemidesmosomes. Magnification, X 12,000.

Protein Expression in Human Prostate

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and focally discontinuous. Focal adhesion plaques present (Figure 1B), although well formed hemidesmosomes were not observed in carcinoma.

Discussion

were

Immunohistochemistry Laminin was seen surrounding all normal glands, PIN lesions, and carcinomas regardless of histological grade. Laminin was also seen surrounding blood vessels as well as smooth muscle cells of the stroma (Figure 2, A and B). The truncated laminin subchain y2 (B2t) was expressed by normal glands, prostatic ducts, and PIN lesions (Figure 3C) but was not detected in the basal lamina surrounding carcinomas (Figure 2, C and D). Similarly, the collagen type VIl anchoring fibrils were produced by normal prostate gland, ducts, and PIN lesions (Figure 3E) but were not seen in any case of carcinoma (Figure 2, E and F). The prostate glands in the ten normal controls expressed the a1l31 laminin receptor, the collagen receptor a2f31, the epiligrin receptor a3f31, the a4f1 fibronectin receptor, the a6f31 laminin receptor, the avf3l vitronectin receptor (data not shown), as well as the hemidesmosome-associated a6f34 heterodimer at the basal cell/basal lamina interface (Figure 2, G and H). The a6f31 and a6f34 receptors are also seen at the basal cell/basal lamina interface in PIN lesions (Figure 3A, Table 3). In the twenty primary carcinomas specifically studied for integrin expression, only one case expressed either a2 or a5 and two different cases expressed a3 (data not shown). Eighteen cases expressed a6 associated with lesser amounts of 131, and the polarity of expression at the basal aspects of the cells was replaced by diffuse expression in the cytoplasmic membranes of the carcinoma cells. In a larger group of thirty-nine cases of carcinoma examined for (34, no expression of P4 was detected, although adjacent nonmalignant glands revealed strong expression. The hemidesmosome-associated proteins BP180, BP230, HD1, and plectin were expressed along the basal aspect of basal cells in normal glands (Figure 4, A-E). Plectin is also associated with the intercellular desmosome and was therefore also expressed by luminal cells of the normal glands (Figure 4E). In the 20 cases of carcinoma, the transmembrane protein BP180 was not expressed (Figure 4A). In contrast, 6 of the 20 carcinoma cases (30%) expressed BP230 in a diffuse cytoplasmic pattern (Table 2). The 500-kd protein, HD1,19 was expressed in 3 cases (15%). Plectin24 was expressed in an intracellular pattern in all 20 cases (Table 2).

The high incidence of prostate carcinoma discovered in aging men coupled with the highly variable rate of progression of the disease constitute a major problem in the clinical management of the individual patient.25 The slow rate of progression seen in most men is assumed to be in part a result of the fact that the invasive tumors secrete and surround themselves with a basal lamina that presumably retards their migration through the stroma.2 Our previous work had shown that the basal lamina of invasive tumors was similar in chemical composition to that formed by normal prostate glands in that it contained type IV collagen, entactin, and laminin, including the subchains al (A), a2 (M), (31 (B1), and 32 (S).2,3 In the current study we have shown that the recently described truncated form of laminin, y2 (B2t),6 was absent in the de novo formed basal lamina of prostate carcinoma although found in the basal lamina surrounding normal prostate glands and ducts. SCC12 human epidermal carcinoma cells under normal culture conditions do not form hemidesmosomes. These cells can be induced to form hemidesmosomes when they are grown on a matrix containing the y2 (B2t) laminin subchain produced by the 804G rat bladder cell line.26 Recent immunological studies and partial cDNA sequencing of y2 (B2t) laminin subchain show that it corresponds to one of the subchains of kalinin, a disulfide-linked heterotrimer that forms part of the anchoring filaments spanning the space between the cytoplasmic membrane and the lamina lucida.6 On the basis of recent sequencing data, kalinin27 is now regarded to be similar or even identical to the molecules BM600/nicein28'29 and epiligrin.30'31 In this study we document by ultrastructure and immunohistochemistry the presence of hemidesmosome structures formed at the interface of the normal glandular basal cell and the underlying basal lamina. These structures are similar in morphology to those described previously in the basal cells of stratified and complex epithelia, including a cytoplasmic plaque associated with intermediate filaments, a sub-basal dense plate with anchoring filaments in the basal lamina, and anchoring fibrils in the immediately adjacent stroma.32 The hemidesmosome proteins BP1 80, BP230,33 HD1 ,19 the associated integrin pair a614,34 the anchoring filamentassociated laminin subchain variant y2 (B2t),6 as well as the anchoring fibril-associated type VII collagen38 were all demonstrated by the specific antibodies to

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I 'I

Figure 2. Double-stained indirect immunofluorescence images comparing the same field on the right and left. The tissue sections contain both primary carcinoma (c) and adjacent nonmalignant glands (n). Primary antisera are as follows: A, antilaminin; B, anticytokeratin K44; C, anti- y2 (B2t) laminin; D, anticytokeratin KA4; E, anti-collagen type VII; F, anticytokeratin 18A; G, anti-a6 integrin; and H, anti-,84 integrin. Magnification, X210.

-m

-.

Protein Expression in Human Prostate 1503 AJPJune 1995, Vol. 146, No. 6

Figure 3. Double-stained indirect immunofluorescence images comparing the same field on the right and left. Th-ese illustrations are taken from PIN lesions stained with primary antibodies as follows: A, anti-a6 integrin; B, anticytokeratin KA4; C, anti-y2 (B2t) laminin; D, anticytokeratin KA4; E, anti-collagen type VII; and F, anticytokeratin 18A. Magnification, X210.

localize to the basal aspects of the basal cells of normal glands (Figure 5). These independent observations confirm that the normal basal cell is attached to its underlying basal lamina in part through stable hemidesmosomes. In addition, the structure contained cytoplasmic plaques lacking sub-basal plates or anchoring fibrils that most likely represent

adhesion plaques anchored to the E8 domain of laminin through the a6131 integrin pair. In carcinoma, the basal cells are lost as defined by antibodies to the basal cell-specific cytokeratins 5 and 14. The carcinoma cells appear to make contact with their surrounding matrix mainly through a6f31 focal adhesions. The transmembrane proteins BP180

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1.'

_

v-"A

Loll

W.

I

I. t #A ., ..^

t#

''

"

;

6 *.t.' .O- '

*

Figure 4. Double-stained indirect immunofluorescence images comparing the same field on the right and left. The tissues sections contain both primary carcinoma (c) and adjacent nonmalignant glands (n). Primary antisera are as follows: A, anti-BP180; B, anticytokeratin KA4; C, antiHD1; D, anticytokeratin 18A; E. antiplectin; and F. anticytokeratin KA4. Magnification, X210.

and f4 integrin are lost as well as the y2 (B2t) laminin component of the anchoring filaments and the type VIl collagen of the anchoring fibrils. Ultrastructural analysis reveals plaque-like condensations as described above but the absence of the anchoring filaments and fibrils characteristic of hemidesmosome structures.

The depolarization or loss of basal expression of

a6f34 has been reported previously in tumors and in normal keratinocytes growing and migrating under wound healing conditions.37'38 The mechanism that controls the a6f34 distribution is unknown, but it has been postulated that changes in the phosphorylated state of the p unit could be impor-


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Table 2. Immunohistochemical Results

Case

Gleason Grade III III IV III V III 11 III III III

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

V

11 III IV III V

Il-Ill III III IV

BP180

BP230

HD1

Plectin

a6

f34

LM

Collagen VIl

y2 (B2t)

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

+

+

+

+

+

0 +

0 0

+ + + + + + +

+

+

+

+

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

+ + + + + + + + +

+ + 0 + + + + + +

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

+

+ + + + + + + + +

0 +

0 0 f+ 0 0

0 0 0 0 0 f+ 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 +

+ + + + + + + + + +

+ + + + + + + + +

+, Positive; 0, negative; ft, focally positive

tant.39 The a6f31 phenotype, which appears to be the major integrin pair maintained by the carcinoma cells, is expressed by migrating populations of cells during normal development.40-43 Our results imply that the carcinoma cells are less stably attached to the basal lamina and therefore may have enhanced migratory potential. An interesting observation is that, in the preinvasive PIN lesions, both the surrounding lamina and the hemidesmosomes are intact, suggesting that the interactions of the basal cells with the basal lamina is in these lesions normal although the proliferating population of cells have assumed many of the cyto-

logical characteristics of invasive carcinoma.44 It has been postulated that prostate carcinomas are derived from a population of amplified basal cells that share certain cytokeratins in common with basal cells.45 Our observation that hemidesmosomeassociated proteins HD1, BP230, and plectin are synthesized by at least some carcinomas supports this hypothesis. It is unclear at this point what causes the simultaneous abnormalities in the de novo synthesized basal lamina and the associated attachment proteins, although our results suggest that the inability to synthesize hemidesmosome proteins collagen VII and y2 (B2t) and/or some other factor critical for

Hemidesmosome

IF

ts

Figure 5. Schematic diagram depicting location of hemidesmosome proteins. Note that structures are not necessarily drawn to scale.

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Table 3. Immunohistochemical Results on PIN Cases

Cases (n = 13)

BP180

BP230

HD1

Plectin

a6

,B4

LM

Positive Weak Negative

12 1 0

10 2 1

13 0 0

9* 0 0

13 0 0

13 0 0

13 0 0

Collagen VII 13 0 0

y2 (B2t) 13 0 0

*Four cases were not examined for plectin. hemidesmosome assembly represents a key step in the progression from a premalignant lesion to invasive carcinoma.

Acknowledgments The authors thank Drs. Jonathan C. R. Jones, E. Birgit Lane, Katsushi Owaribe, M. E. Hemler, and Gerhard Wiche for supplying important specific antibodies (listed in Table 1). We also thank Dr. Jonathan C. R. Jones for reviewing the manuscript before submission and Ms. Nancy E. Suttle for assistance in preparing the manuscript.

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33. Klatte DH, Kurpakus MA, Grelling KA, Jones JCR: Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells. J Cell Biol 1989, 109:3377-3390 34. Stepp MA, Spurr-Michaud S, Tisdale A, Elwell J, Gipson IK: a6,34 integrin heterodimer is a component of hemidesmosomes: Proc Natl Acad Sci USA 1990, 87: 8970-8974 35. Jones JCR, Green KJ: Intermediate filament-plasma membrane interactions. Curr Opin Cell Biol 1991, 3:127-132 36. Sonnenberg A, Calafat J, Janssen H, Daams H, van der Raaij-Helmer LMH, Falcioni R, Kennel SJ, Aplin JD, Baker J, Loizidou M, Garrod D: Integrin a6,B4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesions: J Cell Biol 1991, 113:907-917 37. Kurpakus MA, Quaranta V, Jones JCR: Surface relocation of a6i34 integrins and assembly of hemidesmosomes in an in vitro model of wound healing. J Cell Biol 1991, 115:1737-1750 38. Gipson IK, Spurr-Michaud SJ, Tisdale AS: Anchoring fibrils form a complex network in human and rabbit cornea. Invest Ophthalmol Vis Sci 1987, 28:212-220 39. Carey TE, Sair TS, Chern C, Liebert M, Grossman HB, Wolf FT, Van Waes C: Blood group antigens and integrins as biomarkers in head and neck cancer: is aberrant tyrosine phosphorylation the cause of altered a6,34 integrin expression? J Cell Biochem 1993, 17F-

(Suppl):223-232 40. Bronner-Fraser M, Artinger M, Muschler J, Horwitz AF: Developmentally regulated expression of a6 integrin in avian embryos. Development 1992, 197:115-211 41. Damsky C, Sutherland A, Fisher S: Extracellular matrix 5: adhesive interactions in early mammalian embryogenesis, implantation and placentation. FASEB J 1993, 7:1320-1329 42. Tozeren A, Kleinman HK, Wu S, Mercurio AM, Byers SW: Integrin a6,B4 mediates dynamic interactions with laminin: J Cell Sci 1994 (in press) 43. Shaw LM, Mercurio AM: Regulation of cellular interactions with laminin by integrin cytoplasmic domains: the A and B structural variants of the a6f31 integrin differentially modulate the adhesive strength, morphology, and migration of macrophages. Mol Biol Cell 1994, 5:679-690 44. Petein M, Patrick M, Van Velthoven R, Pasteels J-L, Brawer MK, Davis JR, Nagle RB, Kiss R: Morphonuclear relationship between prostatic intraepithelial neoplasia and cancers as assessed by digital cell image analysis. Am J Clin Pathol 1991, 96:628-634 45. Verhagen APM, Ramaekers FCS, Aalders TW, Schaafsma HE, Debruyne FMJ, Schalken JA: Colocalization of basal and luminal cell-type cytokeratins in human prostate cancer. Cancer Res 1992, 52:61826187