Hybridization in Situ IV Collagenases in Human Skin ...

Localization of Messenger RNA for Mr 72,000 and 92,000 Type IV Collagenases in Human Skin Cancers by in Situ Hybridization Charles Pyke, Elisabeth Ralfkiær, Piricko Huhtala, et al. Cancer Res 1992;52:1336-1341. Published online March 1, 1992.

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Downloaded from cancerres.aacrjournals.org on July 9, 2011 Copyright © 1992 American Association for Cancer Research

[CANCER RESEARCH 52, 1336-1341, March 1, 1992]

Localization of Messenger RNA for MT 72,000 and 92,000 Type IV Collagenases in Human Skin Cancers by in Situ Hybridization1 Charles Pyke,2 Elisabeth Ralfkiaer, Piricko Huhtala, Tina Hurskainen, Keld Dane, and Karl Tryggvason Finsen Laboratory [C. P., K. D.] and Department of Pathology [E. R.J, Rigshospitalet, Strandboulevarden 49, DK-2100 Copenhagen 0, Denmark, and Biocenter and Department of Biochemistry, University ofOulu, SF-90570 Oulu, Finland [P. H., T. H., K. T.J

ABSTRACT We have examined the expression of 2 type IV collagen degrading enzymes (M, 72,000 and 92,000 type IV collagenases) in human skin cancer by in situ hybridization. In all cases of infiltrating carcinomas of squamous cell (9 of 9) and basal cell (5 of 5) types, messenger RNA for the M, 72,000 type IV collagenase was present in numerous fihrnhlusts. These were especially abundant in the stroma adjacent to the invasive tumor nodules. Malignant cells were negative for mRNA for the M, 72,000 enzyme in all cases as were all other epithelial as well as endothelial cells. mRNA for the M, 92,000 type IV collagenase was present in all 9 squamous cell and in 3 of the 5 basal cell carcinomas. In all these cases, a subpopulation of tissue macrophages was found to be positive, while malignant cells showed a signal for M, 92,000 type IV collagenase in 6 of the squamous cell carcinomas but in none of the basal cell carcinomas. In all cases, the signal for this mRNA was confined to cells located at the tumoral/stromal interface or in the close vicinity of tumor nodules. No mRNA for any of the 2 collagenases was detected in 3 biopsies of normal skin. In vitro studies have indicated that collagenases are involved in the degradation of the extracellular matrix during cancer invasion. The present findings are consistent with such a role of the A/,72,000 and 92,000 type IV collagenases in squamous and basal cell carcinomas in situ. The findings also demonstrate that degradative enzymes are not necessarily produced by the malignant cells themselves but may be generated by induction or recruitment of nonmalignant stromal cells.

INTRODUCTION Studies on cultured cells and in experimental systems have indicated that several enzymes participate in degradation of the extracellular matrix during cancer invasion as well as during tissue remodeling under nonmalignant conditions. Serine pro teases and metalloproteases appear to be the most important of these enzymes (1-7). Basement membranes are of particular interest in cancer invasion since they frequently are the first matrix hindrance to be traversed in the process of invasion. The basement membranes are ubiquitous thin sheet-like struc tures that separate epithelia, endothelia, and muscle cells from the underlying stroma. Type IV collagen forms the tight struc tural basement membrane network, and mechanisms leading to the breakdown of this molecule have consequently been the focus of extensive research in the field of invasion and metastatic spread of tumor cells. Type IV collagen is resistant to a range of proteolytic enzymes, but it can be specifically cleaved in the helical, pepsin-resistant region by 2 members of the metalloproteinase family, the type IV collagenases. A A/, 72,000 type IV collagenase was originally purified from a metastatic mouse tumor (8, 9), and a related enzyme, MT Received 8/23/91; accepted 12/5/91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported financially by the Danish Biotechnology Pro gramme, the Danish Cancer Society, the Danish Medical Research Council, the Finnish Cancer Society, the Finnish Cancer Institute, the Academy of Finland, and the Harboe Foundation. 2 To whom requests for reprints should be addressed, at: Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, DK-2100 Copenhagen 0, Denmark.

92,000 type IV collagenase, was identified in neutrophilic granulocytes (10). Both enzymes have been shown to cleave native type IV collagen molecules at a single site into 1 of 4 and 3 of 4 size fragments (11, 12). They also degrade elastin (13) and gelatin. The complete primary structure of the 2 type IV colla genases has been derived from cDNA clones (14-16), and the enzymes are the products of 2 separate genes ( 17, 18). Cells of neoplastic origin and oncogene-transformed cells often secrete both M, 72,000 and 92,000 type IV collagenases in culture (15, 19-27). Cultured cells, however, are not neces sarily representative of the cells in the intact tissues from which they are derived, with respect to production of extracellular proteolytic enzymes (5, 28, 29). In this study, we have therefore used in situ hybridization methods to examine the occurrence and location of mRNA for M, 72,000 and 92,000 type IV collagenases in histolÃ³gica! specimens of human skin cancers. MATERIALS

AND METHODS

Materials. The following materials were obtained from the sources indicated: T7 and T3 polymerase, pBluescriptKS(+) plasmid vector (Stratagene, La Jolla, CA); RNasin and DNase I (Promega, Madison, WI); [35S]UTP (1300 Ci/mmol; Amersham DK, Birker0d, Denmark); dithiothreitol and restriction endonucleases (Boehringer Mannheim, Mannheim, Germany, and New England Biolabs, Beverly, MA); K5 autoradiographic emulsion (Ilford, Cheshire, England); Formamide (Fluka, Buchs, Schwitzerland); and salmon sperm DNA (type III; Sigma Chemical Co., St. Louis, MO). All other materials were as described previously (29). Tissue Preparation. Routinely processed, formalin-fixed and paraffinembedded specimens from skin cancers, operated during 1988-1990, were drawn from the files of the Department of Pathology, Rigshospi talet. The specimens were assessed in accordance with standard criteria and included 9 cases of squamous cell carcinomas and 5 cases of basal cell carcinomas of superficial and nodular-ulcerative types. In addition, 3 biopsies from normal skin were examined. Isolation of M, 92,000 Type IV Collagenase cDNA. A Xgtl 1 human fibrosarcoma cell (HT-1080) cDNA library (HT-1048b; Clontech) was screened using 2 endlabeled oligonucleotides. The 2 synthetic oligonucleotides contained nucleotides 118-138 and 2013-2052 from the cDNA sequence for the human M, 92,000 type IV collagenase secreted by SV40-transformed lung fibroblasts (15). Hybridizations and washes were performed using the sodium pyrophosphate method (30). One positive cDNA clone, HG-1, was isolated and subcloned into the PUC19 plasmid. Sequencing was carried out by the dideoxy method of Sanger et al. (31) using Sequenase (United States Biochemical Corp.) and M13 universal primers or specific oligonucleotide primers derived from the sequence of the M, 92,000 type IV collagenase cDNA. The cDNA clone was shown to contain bases 654-2326 in the 2334 base pair long sequence reported previously (15). Preparation of RNA Probes. Restriction fragments from cDNA clones for the human M, 72,000 type IV collagenase (16) and A/r 92,000 type IV collagenase (Ref. 18 and this study) were subcloned using standard techniques (32), and the following subclones were prepared in pBluescriptKS(+): pCOL7201 [5eal(647)-5acl(1284)] and pCOL7202 [0/-Â«II(1528)-/)raII(1956)] (M, 72,000 enzyme); pCOL9201 \Ava\(101 l)-tfiiiiflll(l 751)] and pCOL9202 [//imflII(1751)-EcoRI(2326); Â£coRI-sitecreated by extension from blunt-end)] (M, 92,000 enzyme).

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Base pair numbers correspond to sequences as listed in EM BO database [acquisition numbers: J03210 (M, 72,000 type IV collagenase), and J05070 (M, 92,000 type IV collagenase)]. Pure plasmid preparations were prepared by banding in CsCl gradients. For pCOL7201, the correct identity of the inserted fragment was assured by sequencing by the dideoxy method of Sanger et al. (31). The plasmids were linearized for transcription using restriction endonucleases, and 5 UKof the linearized plasmids were extracted with phenol and with chloroformnsoamyl alcohol (25:1), precipitated with ethanol, and redissolved in water. Each transcription reaction contained 1 /Â¿glinearized DNA template, and transcriptions were performed essentially as recommended by the man ufacturer of the polymerases. The RNA was hydrolyzed in 0.1 M sodium carbonate buffer, pH 10.2, containing 10 mM DTT3 to an average size of 100 bases. Probe preparations always contained more than 2 x IO6 cpm/fil, and the amount of trichloroacetic acid-precipitable material was usually above 90%. For pCOL7201 and pCOL9201, RNA probes transcribed from opposite strands of the same plasmid template, yield ing sense and antisense transcripts, were adjusted to the same radioac tivity concentration. Probes were stored at â€”¿20Â°C until used. In Situ Hybridization. A modification of a previously described method (29) was used. In brief, paraffin sections were cut, placed on gelatinized slides, heated to 60Â°Cfor 30 min, deparaffinized in xylene, and rehydrated through graded alcohols to PBS (0.01 M sodium phos phate buffer, pH 7.4, containing 0.14 M NaCl). The slides were then washed twice in PBS; incubated with 5 Â¿ig/mlproteinase K in 50 mM Tris/HCI (pH 8.0), with 5 mM EDTA for 7.5 min; washed in PBS (2 min) dehydrated in graded ethanols; and air-dried before the RNA probe (80 pg/Ml) was applied. The hybridization solution consisted of deionized formamide (50%), dextran sulfate (10%), tRNA (1 /ig//*I), Ficoll 400 (0.02%, w/v), polyvinylpyrrolidone (0.02%, w/v), bovine serum albumin fraction V (0.02%, w/v), 10 HIMDTT, 0.3 M NaCl, 0.5 mM EDTA, 10 mM Tris-HCl, and 10 mM NaPO4 (pH 6.8). Sections were covered by alcohol-washed, autoclaved coverslips and hybridized at 47Â°Covernight (16-18 h) in a chamber humidified with 10 ml of a mixture similar to the hybridization solution, except for the omission of probe, dextran sulfate, DTT, and tRNA (washing mixture). After hybridization, slides were washed in washing mixture for 2 x l h at 50Â°C,followed by NTE with 10 mM DTT at 37Â°Cfor 15 min. Following treatment with RNase A (20 Mg/ml) in NTE at 37Â°Cfor 30 min, the sections were washed in NTE at 37Â°C(2 x 30 min), and in 2 liters of 15 mM sodium chloride, 1.5 mM sodium citrate (pH 7.0), with 1 mM DTT for 30 min at room temperature with stirring. Sections were then dehydrated and air-dried. Finally, autoradiographic emulsion was ap plied according to the manufacturer's recommendations, and sections were stored in black airtight boxes at 4Â°Cuntil they were developed after 1-2 weeks of exposure. Immunohistochemistry. Tissue macrophages were identified by Peroxidase-Anti-Peroxidase immunostaining using monoclonal anti-mac rophage antibody KP1 reactive with macrophages in routine sections (code no. M814; DAKO A/S).

ing hair follicles and glands) in 3 biopsies of normal skin contained no detectable mRNA for the M, 72,000 type IV collagenase (data not shown). M, 92,000 Type IV Collagenase.Messsenger RNA for the M, 92,000 type IV collagenase was detected in all 9 squamous cell carcinomas and in 3 of the basal cell carcinomas. In 6 of the squamous cell carcinomas, malignant cells located at the tumoral/stromal interface showed a weak to moderate hybridization signal for the mRNA of this enzyme (Fig. 3). The positive cells were in these cases easily identified as cancer cells by their large nuclei and eosinophilic cytoplasm (Fig. 4). In contrast, none of the basal cell carcinomas contained cells of epithelial origin positive for mRNA for the M, 92,000 type IV collagenase. In all the squamous cell carcinomas and in the 3 positive basal cell carcinomas, relatively few but strongly positive stromal cells were located in the connective tissue surrounding the malignant epithelium (Fig. 3). These large cells were iden tified as a subpopulation of tissue macrophages by immuno staining of adjacent sections with a monoclonal anti-macro phage antibody (CD68; data not shown). In the remaining 2 basal cell carcinomas, no mRNA for the M, 92,000 type IV collagenase could be seen. In 1 case of squamous cell carcinoma in which giant cells were present, an intense hybridization signal was seen in and around these multinucleated macrophages (data not shown). Neutrophilic granulocytes still contained in vessels as well as tissue neutrophils accumulating in inflammatory regions showed no signal in any of the specimens (Fig. 4). No M, 92,000 type IV collagenase mRNA was detected in any cells in 3 biopsies from normal skin. As was the case for the M, 72,000 type IV collagenase, this absence of signal included all hair follicles and glands present in the specimens (data not shown). Control Experiments. Positive control experiments were per formed for the A/r 72,000 and 92,000 type IV collagenases by application of 2 different antisense probes representing 2 nonoverlapping parts of each of the 2 cDNAs (see "Materials and Methods"). These probes were adjusted to the same specific

radioactivity and applied to adjacent sections of 6 (M, 72,000 type IV collagenase) and 5 (M, 92,000 type IV collagenase) of the tumors. In all cases, the 2 probes showed identical hybridi zation patterns (data not shown). As a negative control, sense RNA probes, transcribed from each of the 2 cDNA species (see "Materials and Methods"), were applied to adjacent sections of all specimens. In these sections, no signal was seen (Fig. 3). To evaluate the possibility of cross-hybridization to mRNAs RESULTS for other members of the matrix metalloproteinase family, we performed a computerscan (Fastscan, PC/GENE; IntelliMr 72,000 Type IV Collagenase. In all specimens of squamous Genetics Inc., Mountain View, CA) for regions of homology (9 of 9) and basal (5 of 5) cell carcinomas, mRNA for the M, between each of the probes used and the published cDNA 72,000 type IV collagenase was found in fibroblasts. No differ sequences for the following metalloproteinases: interstitial col ences in hybridization patterns or intensity of signal were found lagenase (33), PUMP-1 (34), stromelysin (35), stromelysin-2 between the 2 cancer types. Numerous fibroblasts in all layers of the skin were positive (Fig. 1), but the hybridization signal (34), stromelysin-3 (36), and neutrophil collagenase (37), as well as either the M, 72,000 or the 92,000 type IV collagenase. was in most cases especially prominent in the stroma surround ing the invasive tumor nodules (Fig. 2). In these places it was The region of highest homology was found between the probe not possible to rule out that other stromal cell types were also pCOL7201 (used for detection of the M, 72,000 type IV colla positive, due to the large number of cells present. Malignant genase) and the M, 92,000 type IV collagenase (70%), but using cells were negative in all cases as were all other epithelial as the hybridization protocol described in this study, we observed well as endothelial cells. Fibroblasts and all other cells (includ- no cross-hybridization to M, 92,000 type IV collagenase mRNA containing cells using this probe. For the other metalloprotei 3 The abbreviations used are: DTT, dithiothreitol; PBS, phosphate-buffered nases, no regions with homologies to any of the probes that saline; NTE, 0.5 M NaCl, 1 mM EDTA, 10 mM Tris-HCl (pH 7.2); u-PA, urokinase-type plasminogen activator. were higher than 59% were encountered. We therefore conclude 1337 Downloaded from cancerres.aacrjournals.org on July 9, 2011 Copyright © 1992 American Association for Cancer Research

TYPE IV COLLAGENASES

IN SKIN CANCER

that cross-hybridization to mRNAs for other known members of the metalloproteinase family is highly unlikely. DISCUSSION

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In this study, we have used /// situ hybridization methods to examine the distribution of mRNA for the M, 72,000 and 92,000 type IV collagenases in histological samples of human squamous (n = 9) and basal cell (n = 5) carcinomas. In both skin cancers, mRNA for the M, 72,000 type IV collagenase was detected in all cases. It was confined to fibroblasts, while no signals were seen in the malignant cells. By contrast, mRNA for the M, 92,000 type IV collagenase was present in the malignant cells in 6 of 9 squamous cell carcinomas. Labeling of basal carcinoma cells was not seen, but strong staining was observed in tissue macrophages surrounding the malignant epithelium in all the squamous carcinomas and in 3 of the 5 basal cell carcinomas. Control samples, consisting of 3 biopsies from normal skin, were also investigated. The results indicate that the mRNAs for the 2 proteins are confined to diseased conditions. To test whether the antisense hybridization seen could be due to the presence of mRNAs different from, but with a strong homology to, parts of the mRNAs of the 2 type IV collagenases, hybridizations were carried out for each mRNA using 2 differ ent transcripts covering 2 nonoverlapping parts of the cDNA. In all cases, hybridization signals were seen over cells with a similar localization in adjacent sections, confirming the speci-

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Fig. 1. In situ hybridization of a squamous cell carcinoma with a '"S labeled antisense RNA probe for A/, 72,000 type IV collagenase. b, high magnification of the area shown at the open arrow in a. Intense signal is observed over numerous spindle-shaped fibroblasts (straight arrows in b). Note the absence of signal above nonfibroblastic cells (curved arrows). Bars, 100 firn (a) and 25 urn (b).

Fig. 2. In silu hybridization of a basal cell carcinoma with an antisense probe for M, 72,000 type IV collagenase mRNA. Micrograph is a darkfield image to highlight the numerous positive fibroblasts (white areas) surrounding tumor nodules that show no hybridization signal (dark rounded areas marked "t"). Bar, 170 firn.

Fig. 4. High magnification of areas at open arrows in Fig. 3 (a and b are from Fig. 3, a and b, respectively). Cancer cells at the tumoral/stromal interface show hybridization signal for M, 92,000 type IV collagenase (straight arrows). Note that 2 neutrophilic granulocytes in the surrounding stroma are negative (curved arrows). Bar, 17 ^m.

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Fig. 3. In situ hybridization of asquamouscell carcinoma with an antisense mRNA probe for M, 92,000 type IV collagenase (a). Both macrophages (curved arrows) and malignant cells are positive. Note that only the tumor cells bordering the tumor/stroma interface show signal (straight arrows). An adjacent section from the same specimen was hybridized with a sense RNA probe (b) where no silver grain accumulation can be seen above any tissue structures. Enlargements of the areas at open arrows in a and b are seen in Fig. 4. a'and b' are darkfield images of a and b, respectively. Bar, 4(1/.in.

ficity of the staining for the 2 mRNAs. To further evaluate the confined to conditions in which degradation and remodeling of possibility of cross-hybridization, a computer scan for homolthe extracellular matrix is taking place. An immunohistochemical study of common and desmoplasogies between the probes used and regions of cDNAs for known related members of the metalloproteinase family was per tic basal cell carcinomas of the skin showed that only the latter formed. This scan did not suggest that cross-hybridization type of tumors contained M, 72,000 type IV collagenase imshould occur. In addition we used, as a negative control, sense munoreactivity (detected by polyclonal antibodies), and in these cases the staining was confined to tumor cells (39). Imnumo transcript probes adjusted to the same radioactivity. Previous reports have indicated that M, 72,000 type IV reactivity was absent in 15 of 15 cases of the common variety. collagenase is synthesized by cultured normal human fibroblasts This is in apparent disagreement with the present study in (26, 27). In one in vitro study, human skin fibroblasts secreted which 5 of 5 basal cell carcinomas of the common types (super ficial and nodular ulcerative) contained abundant mRNA for type IV collagenase activity when proliferating in vitro, whereas the secretion ceased when the cells reached confluency (38). this collagenase in fibroblasts. There are 2 possible explanations Culturing of skin fibroblasts inflicts on these cells a phenotype for this apparent disagreement: (a) the collagenase IV mRNA characteristic of active fibroblasts, e.g., they exhibit a migratory found in the fibroblasts is not translated; and (b) collagenase behavior, at least until they reach confluency. These in vitro IV is present, but the immunohistochemical method was not observations are in keeping with the findings in the present sensitive enough to detect it. A final clarification of this issue study, which show that fibroblasts in all malignant specimens will await further studies, which also should include the con comitant use of in situ hybridization and immunohistoinvestigated contained mRNA for this type IV collagenase, whereas none of the normal skin biopsies was positive. It is chemistry. conceivable that synthesis of this enzyme by skin fibroblasts is In two recent immunohistochemical studies of human breast 1339



and colon cancer, the A/r 72,000 type IV collagenase was reported to be predominantly present in the neoplastic cells (40, 41). The apparent discrepancy with the results in the present study most likely reflects that this collagenase is pro duced by different cell types in different types of cancer. To our knowledge, no previous reports are available on the histolÃ³gica! localization of the M, 92,000 type IV collagenase or its mRNA. The present finding of mRNA for the M, 92,000 enzyme in tissue macrophages surrounding the malignant epi thelium agrees with the in vitro observation that a A/r 92,000 type IV collagenase/gelatinase is produced by isolated human macrophages and neutrophils (10, 15). The failure to detect mRNA for this enzyme in neutrophils in our study may reflect a lower copy-number of the mRNA for the M, 92 type IV collagenase. Alternatively, these short-lived tissue neutrophils may already contain all the pro-type IV collagenase needed, when they enter the tissues from the circulation. In vitro studies suggest that the collagenolytic activity is needed for macro phages to traverse the endothelial wall during extravasation and cell migration (42). It is also possible that the macrophages may participate in cancer-induced tissue degradation. The finding that mRNA for the M, 92,000 type IV collagen ase was found in all 9 squamous carcinomas (and in 6 of these cases malignant cells at the tumoral/stromal interface were positive), but in only 3 of the basal cell carcinomas (and in none of these cases in the malignant cells), suggests that differences exist between the 2 cancers in their mechanism of invasion. Overall, this is in agreement with the observation that the behavior and infiltration pattern of these cancers is markedly different, the squamous cell carcinomas usually being the most aggressive. Involvement of cells in the tumor stroma in the generation of extracellular proteolytic activity in basal cell skin carcinomas has previously been suggested by an immunofluorescence study of interstitial collagenase (43). In that study, no specific staining of the carcinoma cells was seen, whereas the surrounding con nective tissue showed staining for the interstitial collagenase. The collagenase appeared primarily to be located extracellularly, but the degree of resolution did not permit definitive cellular localization. More recently, additional evidence for the involvement of stromal cells in generation of proteolytic activity has in other types of cancer been suggested by histolÃ³gica! studies of the metalloproteinase stromelysin-3 and of the urokinase pathway of plasminogen activation. By in situ hybridization of breast cancer tissue, stromelysin-3 mRNA was thus exclusively de tected in stromal cells and not in the cancer cells themselves (36). In colon adenocarcinoma, u-PA immunoreactivity and mRNA were found in fibroblast-like cells in the tumor stroma and not in the cancer cells. These u-PA-producing cells sur rounded invasive foci of cancer cells. In contrast, mRNA for the cell surface receptor for u-PA was confined to the cancer cells and a subset of tissue macrophages and neutrophils at these foci (28, 29). Receptor binding of the zymogen pro-u-PA strongly enhances plasmili generation (44), and it is very likely that the histolÃ³gica! findings reflect that u-PA (generated by the fibroblast-like stromal cells) exerts its proteolytic activity after being bound to the receptor on the cancer cells. Moreover, mRNA for the u-PA inhibitor PAI-1 is confined to endothelial cells in the tumor stroma in colon cancer, and the inhibitor probably serves to protect the tumor stroma against degradation (45). Together with the findings in the present study, this indicates that the regulation of extracellular matrix degradation

IN SKIN CANCER

during cancer invasion is the result of a concerted action, not only of several proteolytic enzyme systems but also of several cell types, including both the malignant cells and nonmalignant cells in the tumor stroma. The cancer cells appear to serve as both players and conductors in this process. Hormones, cytokines, and growth factors are likely to be implicated in the induction of the enzymes, receptors, and inhibitors, and studies of such inducing factors will be important topics for investiga tions in the future. ACKNOWLEDGMENTS The excellent technical assistance of Britt Nagel, Lone L0vgren, and Jette Mandelbaum is gratefully acknowledged.

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