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Overexpression of Laminin 2 Chain Monomer in Invading Gastric Carcinoma Cells¹

Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813 [N. K., K. Mo., H. T., H. M., K. Mi.]; Department of Pathology, Yokohama City University School of Medicine, Yokohama 236-0004 [Y. N.]; and Clinical Research Institute, Kanagawa Cancer Center, Yokohama 241-0815 [S. Y.], Japan

	ABSTRACT

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Laminin (LN)-5, a heterotrimer of 3, 3, and 2 chains, has been suggested to be involved in tumor cell invasion. The present immunohistochemical study investigated the distribution of the LN 2 chain in 48 different human gastric adenocarcinomas. The immunohistochemical analysis showed two distinct patterns of LN 2 chain expression: (a) extracellular deposition; and (b) cytoplasmic accumulation. The extracellular deposition of the LN 2 chain was typically observed at neoplastic basement membranes of well-differentiated adenocarcinomas. The immunoreactivity was continuous along tumor basement membranes in these tumors but was irregular and diffuse in poorly differentiated carcinomas. These tumor cells coexpressed the LN 3 and 3 chains, suggesting that the LN 2 chain was deposited as the LN-5 complex. In contrast, tumor cells at the invading fronts showed strong cytoplasmic staining for the LN 2 chain without any detectable signal for the LN 3 or 3 chain in both well- and poorly differentiated carcinomas. On the other hand, in vitro analysis by two-dimensional SDS-PAGE demonstrated that human gastric carcinoma cells secrete a high level of LN 2 chain monomer in addition to the LN-5 complex into culture medium. These results indicate that the LN 2 chain can be secreted as a single subunit and might be involved in tumor cell invasion.

	INTRODUCTION

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LN-5,³ which consists of LN 3, 3, and 2 chains, is a LN isoform that is present in the basement membranes of the skin and various epithelial tissues. This protein was originally found in two different sources. Three groups reported this protein as an extracellular matrix protein secreted by cultured human keratinocytes referred to as kalinin, epiligrin, and nicein (1, 2, 3) . In the skin, LN-5 stabilizes the epidermal/dermal junction through binding with integrin ₆₄, which forms the hemidesmosome structure (1 , 4) . Mutation or deletion in the LN-5 genes (LAMA3, LAMB3, and LAMC2) is associated with epidermolysis bullosa, a lethal skin blistering disease (5, 6, 7) . On the other hand, we identified this protein as a large cell-scattering protein (ladsin) secreted by human gastric carcinoma cells (8) . LN-5 promotes the adhesion, migration, and scattering of various types of cultured cells much more strongly than other known extracellular matrix proteins (8, 9, 10) . These activities are mediated mainly by integrin ₃₁ (2 , 9 , 11 , 12) . In culture, LN-5 is produced by human gastric carcinoma cell lines and squamous cell carcinoma lines as well as by normal keratinocytes (8 , 13) . Expression of LN-5 is simulated by growth factors and a tumor promoter in vitro (13 , 14) . These properties of LN-5 suggest its possible roles in tumor invasion and metastasis.

There are some reports showing the expression of LN-5 or its subunits in human cancer tissues. Pyke et al. (15 , 16) analyzed the expression of the LN 2 chain by in situ hybridization and immunohistochemistry and showed that LN-5 is highly expressed in invading colon carcinoma cells. LN-5 is also up-regulated at the site of wound healing of the skin (17) . In most cases of pancreatic adenocarcinomas, tumor cells show cytoplasmic immunoreactivity for the LN 2 chain, but the degree of 2 expression is inversely correlated with the invasive and metastatic potential of the tumor (18) . In prostate carcinomas, the LN 2 chain is not detected, although its mRNA is expressed (19) . Decreased expression of LN-5 has been reported in transformed keratinocytes (17) and breast carcinoma cells (20) . In gastric carcinomas, LN-5 is continuously deposited at the interface between tumor cells and stroma (21) . A recent study has shown discontinuous deposition of LN-5 in colon carcinomas and enhanced intracellular accumulation of the LN 32 heterodimer in the dissociating tumor cells (22) . There are apparent discrepancies among these studies that might arise from the differences in the tumor types and the antibodies used. Thus, the role of LN-5 in tumor malignancy remains unclear.

We recently prepared a monoclonal antibody capable of detecting the LN 2 chain with high sensitivity in both immunoblotting and immunohistochemistry (23) . Using this antibody, we examined the distribution of the LN 2 chain in human gastric carcinoma tissues from 48 patients. Immunohistochemical analyses with antibodies against the LN 3 and 3 chains were also carried out for some tumor tissues to show the coexistence of these LN-5 subunits.

	MATERIALS AND METHODS

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Antibodies.
Mouse monoclonal antibody D4B5 was prepared using the human recombinant LN 2 chain (amino acid residues 382–608) as the antigen (23) . Rabbit polyclonal antibody was raised against the human recombinant LN 3 chain (residues 109–331). Mouse monoclonal antibody against the LN 3 chain (clone 29E) was prepared using purified LN-5 as the antigen. These antibodies could be used for immunohistochemical staining of both formalin-fixed paraffin sections and paraformaldehyde-fixed frozen sections. Mouse monoclonal antibody against the LN 3 chain (kalinin B1) was purchased from Transduction Laboratories (Lexington, KY) and used for immunoblotting.

Tumor Specimens and Sample Preparation.
Small tumor specimens were obtained from surgically resected gastric cancer tissues and used with the permission and informed consent of patients or their families. They were immediately fixed in 10% formalin and embedded in paraffin. Four-µm-thick paraffin sections were mounted on aminoacyl silane-coated glass slides and used for immunohistochemical analysis of the LN 2, 3, and 3 chains. For immunohistochemical staining, the paraffin sections were dewaxed, rehydrated, and immersed in 0.3% hydrogen peroxide-containing methanol for inactivation of intrinsic peroxidase. All sections were treated with Protease XXIV (Sigma, St. Louis, MO) for 15 min at room temperature. The sections were then incubated with each antibody at 37°C for 1 h. The labeled antigen was detected by a HistoFine kit (Nichirei Pharmaceutical, Tokyo, Japan) and visualized by the 3,3'-diaminobenzidine reaction. Other experimental conditions were as described previously (24) .

Analysis of LN-5 and LN 2 Monomer Secreted from Cultured Cancer Cells.
A confluent culture of human gastric carcinoma cell line MKN-45 was incubated in serum-free medium for 2 days in the presence of 100 ng/ml of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate to stimulate LN-5 production, and the resulting conditioned medium was collected and concentrated as reported previously (8) . The concentrated conditioned medium was subjected to the two-dimensional SDS-PAGE essentially by the method of LeMosy et al. (25) . The one-dimensional SDS-PAGE was carried out on a 4% (v/w) polyacrylamide disc gel (2 mm in diameter and 70 mm in length) under nonreducing conditions. After electrophoresis, the gel was incubated in SDS sample buffer containing 5% (v/v) 2-mercaptoethanol at room temperature for 15 min, placed on a 6% (v/w) polyacrylamide slab gel (85 mm wide, 1 mm thick, and 70 mm long), and run for the two-dimensional SDS-PAGE under reducing conditions. The proteins separated on the slab gel were electrophoretically transferred onto a polyvinylidene difluoride membrane and probed sequentially with rabbit polyclonal antibody against the LN 3 chain, mouse monoclonal antibody against the LN 3 chain (kalinin B1), and mouse monoclonal antibody against the LN 2 chain (D4B5). The immunoreactive proteins were detected by the chemiluminescence method using an enhanced chemiluminescence kit (Amersham, Buckinghamshire, United Kingdom).

	RESULTS

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Distribution of the LN 2 Chain.
Human gastric adenocarcinoma tissues from 48 patients were classified as 24 differentiated carcinomas (or tubular adenocarcinomas), 19 poorly differentiated carcinomas, and 5 other carcinomas (2 signet ring carcinomas, 2 papillary adenocarcinomas, and 1 mucinous adenocarcinoma), based on the formation of neoplastic glandular structures or neoplastic tubules. When these tumor specimens were subjected to immunohistochemical analysis with the anti-2 monoclonal antibody D4B5, the immunostaining patterns were divided into two distinct types, extracellular staining and cytoplasmic staining. The results of the immunohistochemical analysis are summarized in Table 1 . Most typically in differentiated carcinomas, the basement membranes surrounding neoplastic glandular structures showed intense and continuous immunoreactivity for the 2 chain of LN-5 (Fig. 1, A and B) . This type of staining was essentially identical to that observed in adjacent normal gastric epithelia. In some cases, almost all parts of the neoplastic basement membranes were positive for the 2 chain (Fig. 1B) , whereas in other cases, only parts of the basement membranes, especially surface areas, were positive for the antigen (data not shown). The basement membrane staining of the LN 2 chain was less frequent in poorly differentiated carcinomas than in differentiated carcinomas (Table 1) . In another extracellular staining pattern, the immunoreactivity for the LN 2 chain was irregular and fibrous around tumor cells scattered in the stroma (Fig. 1C) . This staining pattern was focally observed in many cases of the solid type of poorly differentiated carcinomas (Table 1) . In signet ring carcinomas, the immunoreactivity showed small basement membrane-like structures surrounding a single tumor cell or several tumor cells (Fig. 1D) .

View this table: [in this window] [in a new window]   Table 1 Summary of immunohistochemical analysis of human gastric carcinomas for the LN 2 chain

View larger version (142K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Extracellular deposition of the LN 2 chain in gastric carcinomas. In two highly differentiated carcinomas (A and B), basement membranes surrounding neoplastic glandular structures are intensely and continuously stained for the LN 2 chain. In a poorly differentiated carcinoma (C), the immunoreactivity is diffuse around tumor cells. In a signet ring carcinoma (D), the immunoreactivity is localized around a single tumor cell or several tumor cells, showing basement membrane-like structures. Arrowheads, a positive signal for the LN 2 chain. Experimental conditions are described in the text. Magnification: x200 for A and C; x400 for B and D.

View larger version (142K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Cytoplasmic accumulation of the LN 2 chain in invading gastric carcinomas. In a highly differentiated carcinoma (A and B), only tumor cells budding or dissociating from the tumor nests show intense cytoplasmic immunostaining for the LN 2 chain. In another differentiated carcinoma (C), the positive signal is evident in tumor cells at the invading front in the stroma. In a poorly differentiated carcinoma (D), the immunoreactivity is seen in tumor cells scattered in the stroma. Arrowheads, positive signal for the LN 2 chain. Experimental conditions are described in the text. Magnification: x200 for A and C; x400 for B and D.

Distribution of the LN 3 and 3 Chains.

View larger version (154K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Expression of three LN-5 subunits in two highly differentiated gastric carcinomas. Close paraffin sections from two highly differentiated carcinomas were subjected to immunohistochemical staining for the LN 3 (A and D), 3 (B and E), and 2 (C and F) chains as described in the text. Tumor cells with positive basement membrane staining for the LN 2 chain (C) show positive cytoplasmic staining for the LN 3 (A) and 3 (B) chains. Invading tumor cells showing cytoplasmic staining for the LN 2 chain (F) are negative for cytoplasmic staining with the anti-3 (D) or anti-3 (E) antibody. Arrowheads, positive signal for each LN chain. Experimental conditions are described in the text. Magnification: x400 for A–C; x200 for C–E.

Secretion of the LN 2 Monomer by Human Gastric Carcinoma Cells in Culture.
To examine whether the LN 2 chain can be secreted as a monomer (or a single subunit), the three subunits of LN-5 secreted by human gastric carcinoma cell line MKN-45 were analyzed by two-dimensional SDS-PAGE (nonreducing SDS-PAGE in the first dimension and reducing SDS-PAGE in the second dimension) and subsequent immunoblotting with antibodies against the 3, 3, and 2 chains. MKN-45 cells were incubated in serum-free medium for 2 days in the presence of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate to stimulate LN-5 production, and the resulting conditioned medium was concentrated and subjected to the two-dimensional SDS-PAGE. During the 2 days of serum-free culture, few dead cells were observed under microscopic observation. As shown in Fig. 4 , the three immunoblots indicated the presence of two major LN-5 forms of approximately M_r 460,000 and M_r 400,000 in which the former was composed of M_r 160,000 3 chain, M_r 140,000 3 chain, and M_r 155,000 2 chain, and the latter was composed of M_r 160,000 3 chain, M_r 140,000 3 chain, and M_r 105,000 2 chain. In addition, LN-5 forms that migrated more slowly than the M_r 460,000 form in the one-dimensional SDS-PAGE were weakly detected. Furthermore, the immunoblot with the anti-2 antibody showed the most intense spot, which migrated to the position corresponding to M_r 155,000 in both the one- and two-dimensional SDS-PAGE. Neither anti-3 nor anti-3 antibody showed any spot in the position of M_r 140,000–160,000 in the one-dimensional SDS-PAGE. These results demonstrated that the gastric carcinoma cells secrete the LN 2 chain as a single subunit.

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Analysis of three subunits of LN-5 secreted from a human gastric carcinoma cell line by two-dimensional SDS-PAGE. Serum-free conditioned medium from human gastric carcinoma cell line MKN-45 was concentrated and analyzed by two-dimensional SDS-PAGE (nonreducing conditions were used for the one-dimensional SDS-PAGE, and reducing conditions were used for the two-dimensional SDS-PAGE) and subsequent immunoblotting with antibodies against the 3 (A), 3 (B), and 2 (C) chains, as described in the text. Arrowheads indicate the respective subunits derived from two major LN-5 forms of Mr 460,000 and Mr 400,000 (the former is composed of Mr 160,000 3 chain, Mr 140,000 3 chain, and Mr 155,000 2 chain, and the latter is composed of Mr 160,000 3 chain, Mr 140,000 3 chain, and Mr 105,000 2 chain). An arrow indicates the single Mr 155,000 2 chain (C). Experimental conditions are described in the text.

Some previous studies have shown preferential expression of the LN 2 chain in invading carcinoma cells (15 , 16 , 18) . However, most of these studies did not clarify whether LN 3 and 3 chains were coexpressed with the LN 2 chain in those tumor cells. As an exception, Sordat et al. (22) reported that the heterodimer of the LN 2 and LN 3 chains is accumulated in the cytoplasm of dissociating (or budding) tumor cells from neoplastic tubules of colon carcinomas. The LN 3 and 2 chains are found only in LN-5 among 11 LN species identified thus far. It has been reported that gene expression of the three subunits of LN-5 (3, 3, and 2) is regulated differently in cancer cell lines and in normal and malignant tissues (13 , 19 , 26) . In the present study, both normal gastric epithelial cells and tumor cells surrounded by basement membranes containing LN-5 showed no cytoplasmic immunoreactivity for the LN 2 chain. This suggests that in cells expressing all three subunits of LN-5, the 2 chain is secreted immediately as the complex of LN-5.

The marked cytoplasmic accumulation of the LN 2 chain in tumor cells that express neither the 3 nor 3 chain suggests that the 2 chain may not be secreted as the monomer. Indeed, it is known that in LN-1, the 1 chain can be secreted as a single subunit, whereas the 1 and 1 chains cannot (27) . When 1 and 1 chains are overexpressed separately or together, they remain intracellularly as the disulfide-linked dimer of 11or 11. Similarly, it has been reported that in LN-5, the 3 and 2 chains first form heterodimer 32 and then bind to the 3 chain to produce and secrete LN-5 (28) . No previous studies have shown the secretion of the LN 2 chain monomer. In this study, however, we showed that a high level of the LN 2 chain was secreted from cultured human gastric carcinoma cells as the monomer, in addition to the heterotrimer with the 3 and 3 chains. We have also obtained data that tumor cell lines expressing the 3 and 2 chains but not the 3 chain secrete the 2 chain as the monomer or the heterodimer with the 3 chain.⁴ Thus, the behavior of the LN 2 chain seems different from that of the LN 1 chain. It seems very likely that in the absence of the 3 and 3 chains, the LN 2 chain is accumulated intracellularly in gastric carcinoma, but a part of the overexpressed 2 chain is secreted.

What is the meaning of the sole expression of the LN 2 chain at the invasion fronts of tumors? There are several possibilities for positive roles of the LN 2 chain in tumor invasion. First, secreted LN 2 monomer or its proteolytic fragments might have some biological activities that promote tumor cell invasion. The LN 2 chain receives some proteolytic processing (8 , 29) . It has been reported that the limited proteolysis of the LN 2 chain by gelatinase A (MMP-2) increases the cell motility activity of LN-5, indicating the important role of the 2 chain (30) . It seems possible that the 2 chain acts directly on cells. Second, the LN 2 monomer may modulate the extracellular deposition and biological activity of LN-5 or other LNs. It is known that the NH₂-terminal region of the 2 chain contains sites to bind with fibulin-2 (31) and type VII collagen (32) . Therefore, the secretion of the 2 chain monomer may alter the extracellular location and interaction of LN-5 with target cells. Third, the cytoplasmic accumulation of the LN 2 chain might disturb the formation of other LN species such as LN-10 and LN-11. Multiple LN subunits are found in basement membranes surrounding differentiated gastric carcinoma cells and normal gastric epithelia (21 , 26) . It seems possible that the excess expression of the LN 2 chain may affect the association of the , , and chains in LN isoforms other than LN-5, although the interaction of the 2 chain with LN and chains other than the 3 and 3 chains has not been reported.

Gastric carcinoma cells secrete LN-5 at high levels in culture (8 , 13) . As shown here and in other studies (21 , 26) , tumor cells forming glandular structures deposit LN-5 on the basement membranes in well-differentiated gastric carcinomas. LN-5 appears to support tumor growth by maintaining the differentiated phenotype. In contrast to our initial expectation (8) , LN-5 may have a rather suppressive role for the invasive growth of poorly differentiated carcinoma cells. This appears to be consistent with the results of other groups showing that LN-5 is down-regulated in prostate, breast, and basal cell skin carcinomas (19 , 21) . Based on the results in this study, we suppose that the LN 2 monomer may play some role in the invasion and dissociation of tumor cells from the tumor cell nests.

	ACKNOWLEDGMENTS

 
We thank Dr. H. Yasumitsu for helpful discussions.

	FOOTNOTES

 
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.

¹ Supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists (to N. K.), a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (to K. Mi.), a grant from the Uehara Memorial Foundation, Japan (to K. Mi.), and the Haraguchi Memorial Cancer Research Fund of Japan (Y. N.).

² To whom requests for reprints should be addressed, at Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, 642-12 Maioka-cho, Totsuka-ku, Yokohama 244-0813, Japan. Phone: 81-45-820-1905; Fax: 81-45-820-1901; E-mail: miyazaki{at}yokohama-cu.ac.jp

³ The abbreviation used is: LN, laminin.

⁴ T. Hirosaki, H. Mizushima, and K. Miyazaki, unpublished observations.

Received 4/19/99. Accepted 9/ 3/99.

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